Adjuvants and Antigens - How they Work

 

The term adjuvant comes from the Latin adjuvare, which means to help or aid [1]. Adjuvants can be defined as substances that increase immunogenicity of a vaccine formulation when added/mixed to it. 

https://www.newswise.com/articles/oregano-oil-kills-drug-resistant-bacteria

Oregano Oil Kills Drug-Resistant Bacteria

3-Oct-2001 12:00 AM EDT, by Journal of the American College of Nutrition Edit Institution

favorite_border

1

Oregano Oil Kills Drug-Resistant Bacteria

Oil from the common herb oregano may be an effective treatment against dangerous, and sometimes drug-resistant bacteria. Two types of experiments, one in test tubes and one on mice, have shown that oregano oil and a major component, carvacrol, appear to reduce infection as effectively as traditional antibiotics.

Dr. Harry G. Preuss (preusshg@georgetown.edu), and his research team, collaborating with Dr. Cass Ingram, of North American Herb and Spice, conducted research testing oregano oil on staphylococcus bacteria which is responsible for a variety of severe infections and is becoming increasingly resistant to many antibiotics. They compared the antibacterial effects of the oil on staphylococcus in a test tube with the standard antibiotics streptomycin, penicillin and vancomycin. The oregano oil, at relatively low doses, was found to inhibit the growth of staphylococcus in the test tubes as much as the standard antibiotics did.

The efficacy of oregano oil and one of its major components, a compound called carvarol, in 18 live mice infected with the staph bacteria showed that oregano oil was as effective as vancomycin and more effective than its constituent carvacrol in treating the staph infections. Evidently, oregano oil has more than one anti-bacterial component.

While this investigation was performed only in test tubes and on a small number of living mice, the preliminary results definitely warrant further study, Preuss said. The ability of oils from various spices to kill infectious organisms has been recognized since antiquity. Natural oils may turn out to be valuable adjuvants or even replacements for many anti-germicidals under a variety of conditions.

This study was sponsored by Waukegan, Ill - based North American Herb and Spice. The results were presented at the 42nd annual meeting of the American College of Nutrition held in Orlando, FL.

Georgetown University Medical Center Office of Communications 202.687.5100 (fax: 202.687.5213) (www.georgetown.edu/gumc)

27. Wendorf J.R., Singh M., O'Hagan D.T. Nanoparticles and Microparticles as Vaccine Adjuvants. In: Torchilin V.P., editor. Nanoparticulates as Drug Carriers. Imperial College Press; London, UK: 2006. pp. 675–696. [Google Scholar]

Nanoparticles and Microparticles as Vaccine Adjuvants

• Journal List
• HHS Author Manuscripts
• PMC2963573

Expert Rev Vaccines. Author manuscript; available in PMC 2011 Jul 1.

Published in final edited form as:

Expert Rev Vaccines. 2010 Sep; 9(9): 1095–1107.

doi: 10.1586/erv.10.89

PMCID: PMC2963573

NIHMSID: NIHMS236977

PMID: 20822351

Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses

Moses O Oyewumi,1,†* Amit Kumar,2 and Zhengrong Cui2,*

Author information Copyright and License information Disclaimer

The publisher's final edited version of this article is available at Expert Rev Vaccines

See other articles in PMC that cite the published article.

Go to:

Abstract

The development of novel immune adjuvants is emerging as a significant area of vaccine delivery based on the continued necessity to amplify immune responses to a wide array of new antigens that are poorly immunogenic. This article specifically focuses on the application of nanoparticles and microparticles as vaccine adjuvants. Many investigators are in agreement that the size of the particles is crucial to their adjuvant activities. However, reports on correlating the size of particle-based adjuvants and the resultant immune responses have been conflicting, with investigators on both sides of the fence with impressive data in support of the effectiveness of particles with small sizes (submicron) over those with larger sizes (micron) and vice versa, while other investigators reported data that showed submicron- and micron-sized particles are effective to the same degree as immune adjuvants. We have generated a list of biological, immunological and, more importantly, vaccine formulation parameters that may have contributed to the inconsistency from different studies and made recommendations on future studies attempting to correlate the size of particulate adjuvants and the immune responses induced. The information gathered could lead to strategies to optimize the performance of nano-microparticles as immune adjuvants.

Keywords: adjuvants, immune responses, microparticles, nanoparticles, particle size, vaccine formulation

The increasing attention on vaccine development is greatly justified based on the continuous emergence of deadly pathogens that are difficult to manage [1]. Over the years, it has become clear that vaccination is an effective and affordable measure to treat and prevent diseases/infections, which is achieved by the activation of innate, nonspecific defenses and the subsequent development of adaptive immune responses to fight intruding pathogens [2–4]. In order to ensure the quality and quantity of immune responses, it is fundamentally important that the immune systems are presented with antigens (from the pathogens in questions) at the right location and amount [5,6]. Ideally, the goal of vaccination is to ensure the production of strong and lasting immune responses after a single dose of antigen without the need for booster doses [7,8]. Success from recombinant DNA technology has afforded the production of antigens that are well preferred over traditional antigens based on safety reasons [4,9]. Traditional antigens obtained from whole or part of live, attenuated or killed pathogens are highly immunogenic, but can potentially pose health hazards when applied in immune-compromised individuals or if the antigens revert to virulent form [6,10]. A limiting factor with antigens made from recombinant DNA technology is that they are often weakly immunogenic on their own and require the inclusion of an immune adjuvant to enhance the resultant immune responses [11–13]. An immunologic adjuvant is defined as any substance that acts to accelerate, prolong or enhance antigen-specific immune responses, but is not immunogenic itself [14,15].

im·mu·no·gen·ic

/ˌimyənōˈjenik/

Learn to pronounce

adjective

1. relating to or denoting substances able to produce an immune response.

   "immunogenic vaccines"

Choose languageAfrikaansAlbanianAmharicArabicArmenianAzerbaijaniBanglaBasqueBelarusianBosnianBulgarianBurmeseCatalanCebuanoChinese (Simplified)Chinese (Traditional)CorsicanCroatianCzechDanishDutchEsperantoEstonianFilipinoFinnishFrenchGalicianGeorgianGermanGreekGujaratiHaitian CreoleHausaHawaiianHebrewHindiHmongHungarianIcelandicIgboIndonesianIrishItalianJapaneseJavaneseKannadaKazakhKhmerKinyarwandaKoreanKurdishKyrgyzLaoLatinLatvianLithuanianLuxembourgishMacedonianMalagasyMalayMalayalamMalteseMaoriMarathiMongolianNepaliNorwegianNyanjaOdiaPashtoPersianPolishPortuguesePunjabiRomanianRussianSamoanScottish GaelicSerbianShonaSindhiSinhalaSlovakSlovenianSomaliSouthern SothoSpanishSundaneseSwahiliSwedishTajikTamilTatarTeluguThaiTurkishTurkmenUkrainianUrduUyghurUzbekVietnameseWelshWestern FrisianXhosaYiddishYorubaZulu

Definitions from Oxford Languages

The immune response is how your body recognizes and defends itself against bacteria, viruses, and substances that appear foreign and harmful.Feb 2, 2020

Immune response: MedlinePlus Medical Encyclopedia

medlineplus.gov › Medical Encyclopedia

Feedback

About Featured Snippets

People also ask

What is a normal immune response?

Antigens may also exist on their own—for example, as food molecules or pollen. A normal immune response consists of the following: Recognizing a potentially harmful foreign antigen. Activating and mobilizing forces to defend against it.

Overview of the Immune System - Immune Disorders - Merck ...

www.merckmanuals.com › home › overview-of-the-im...

Search for: What is a normal immune response?

What are the 4 phases of the immune response?

This can be broken down into four stages: the lag, exponential, steady state, and declining phases. This is the time from initial antigen exposure to when antibodies are detected in the blood, and takes about a week. In this time, specialized B and T cells are activated by contact with the antigen.Jan 6, 2017

Clinically, the list of approved adjuvants is very limited. For decades, aluminum hydroxide or phosphates (alum) remained as the only approved adjuvants in the USA [16]. In late 2009, the US FDA approved the Cervarix® vaccine (GlaxoSmithKline Plc; Middlesex, UK), which contained both aluminum hydroxide and AS04 (3-O-desacyl-4′-monophosphoryl lipid A) as adjuvants. Although alum has a long track record of safety, it only improves the induction of humoral immune responses, and does not help cell-mediated immune responses. As such, there have been tremendous efforts to develop alternative adjuvants [8,9,17–20]. Materials that have been investigated as immune adjuvants may be divided into immune potentiators (e.g., mineral salts, immunostimulatory compounds, microorganism-derived compounds and poly-saccharides) and vaccine-delivery systems (e.g., particulates and liposomes) [14,21,22]. In this article, we have focused on particles with diameters in the nanometer or micrometer ranges that have been investigated as potential immune adjuvants [5,12,23].

In general, the performance of particulate carriers as vaccine adjuvants in the literature has been attributed to a number of functions, which include the following:

• Particulate carriers can serve as an effective antigen delivery system and, thus, enhance and/or facilitate the uptake of antigens by antigen-presenting cells (APCs) such as dendritic cells (DCs) or macrophages [24,25];

• Particle-based antigen carriers may serve as a depot for controlled release of antigen, thereby increasing the availability of antigens to the immune cells. It has been reported that extended antigen release may enhance not only the level, but also the quality of immune responses [26,27];

• Particle-based adjuvants may possess the ability to modulate the type of immune responses induced when used alone or in combination with other immunostimulatory compounds [22];

• Particulates have the ability to protect the integrity of antigens against degradation until delivered to the immune cells [28]. This is particularly important in oral vaccine formulations where antigens must be protected from the harsh acidic conditions of the stomach and enzymatic degradation in the GI tract [29]. However, it is important to caution that the right balance must be maintained between antigen protection and antigen release. Entrapment of the antigen of interest within the particle matrix may achieve satisfactory antigen protection, but the entrapped antigen may not be released at the right time, concentrations or location, which could lead to a weak immune response [15,30];

• Particulate vaccines can potentially cross-present antigen, and antigen cross-presentation is especially important to generate CD8+ T-cell responses against viral infections [31,32].

Considering the potential effectiveness of particulate-based immune adjuvants, a close review of literature in the field has shown areas of improvement or optimization if particulate-based adjuvants are to be used in vaccines. It is well reported that formulation and process parameters, such as particle size, methods of antigen loading and surface properties (e.g., surface charge), play important roles in influencing the activity of particle-based adjuvants [11,24,33]. However, there have been discrepancies from various studies on the nature of the influence of formulation and process parameters on the resultant immune response [26,34,35]. Taking the particle size as an example, reports on the correlation of sizes of particle-based adjuvants and the resultant immune responses have been conflicting, with investigators on both sides of the fence reporting excellent data either supporting the effectiveness of small particles over larger particles and vice versa [36,37]. Other investigators presented equally convincing data showing that submicron-sized and micron-sized particles were effective to the same degree as immune adjuvants [35,38,39]. The lack of consistency will bring into question the practicality and feasibility of potential clinical applications of particle-based immune adjuvants. Since the size of the particulate adjuvants is a central parameter, we have attempted to focus on the extent and nature of the effect of the size of particulate adjuvants on the resultant immune responses and offered a few possible reasons that may help in explaining the rather conflicting data in the literature. It is hoped that the information gathered will assist in the preparation of optimized vaccine formulations using reproducible processes so as to achieve and sustain the production of strong immune responses as desired. Application of standardized (optimized) vaccine formulations will hold great promise in achieving the translation of newly developed vaccine formulations from bench to clinic.

Go to:

Desired qualities of an ideal particle-based vaccine

Many investigators in the field are beginning to share the opinion that the success of vaccination is not only dependent on the nature of vaccine immunogens but also on the delivery system [24,40,41]. In order to design better vaccines and realize the full potential of particulate adjuvants, some of the ideal qualities of a good vaccine formulation are listed:

• The vaccine formulation must be safe and easy to administer;

• The vaccine formulation should be capable of eliciting the desired immune responses, humoral, cellular or both, after a single dose without the need for a booster dose(s);

• The vaccine preparation process should be simple, affordable, reproducible and easy to scale up. In this respect, it is important that all the components are commercially available, safe, affordable and nontoxic;

• The vaccine formulation should be stable with respect to size, surface morphology and size distribution throughout the process of preparation, storage and administration;

• The antigen should be chemically and physically stable throughout the process of antigen loading. There also should not be premature release/leakage of antigen;

• The vaccine preparation process should be amenable to secondary processes, which may include sterilization, drying (such as lyophilization, spray drying or vacuum drying), packaging and reconstitution of the dried powder. These processes should not distort the original particle size and size distribution of particulate vaccine formulations.

Go to:

Nano-microparticles as immune adjuvants

Examples of materials that have been used to prepare nano-microparticles as vaccine-delivery systems include polymers [42], copolymers [43] and lipids [44–46]. The choice of material in particle preparation is guided by many factors, such as biocompatibility, degradation rate, hydrophilicity or lipophilicity, and polarity. The effects of these factors can be grouped into two sections pertaining to the properties of resultant particles and the induced immune responses. The effects relating to properties of particles themselves will encompass the following properties: the size, stability, antigen loading and antigen-release kinetics [42], while the effects on the induced immune responses will include factors such as antigen stability, antigen release, particle interaction with APCs, antigen presentation and processing by APCs [47]. Polymers that have been used in the preparation of particles include, but are not limited to, poly(lactic acid) (PLA), poly(ortho esters) and the copolymer poly(lactic-co-glycolic acid) (PLGA), bioeliminable polyethylene glycol [48], and polyphosphazene [49]. In addition, a good number of natural polymers have been used in vaccine candidate formulations, such as albumin, gelatin [50], collagen, chitosan and alginate [51]. The attractiveness of some of these polymers in making particulate immune adjuvants is that they are biodegradable or biocompatible polymers with the US FDA’s approval for human use in suture material or in drug-delivery systems [52]. Solid lipid nanoparticles prepared with materials such as emulsifying wax [44,45] or lecithin-glyceryl monostearate have also been explored [46]. A number of techniques have been employed to prepare particles for application as immune adjuvants, including emulsification/solvent evaporation, spray drying, coacervation and (micro)-emulsification [53,54]. Although the best possible protection to antigens is offered when antigens are entrapped within particles [30], a limiting factor is that antigens added during the process of particle formation may be potentially unavailable upon administration (i.e., poorly released), and they are also subjected to physical or chemical degradation in the entrapment process [37]. In this respect, other investigators have applied antigen adsorption or conjugated antigen to particle surfaces [11,46,55]. Irrespective of the process employed to make particles, it is important to pay close attention to important factors relating to formulation and that will influence the performance as immune adjuvants.

Comparison of microparticles & nanoparticles as immune adjuvants

Correlating size of particulate adjuvants with the resultant immune responses

Many investigators have used the terms ‘nanoparticles’ and ‘microparticles’ interchangeably in the literature to describe various particles that have been used as vaccine adjuvants [37,56]. Theoretically, nanoparticles are solid particles ranging in size from 1 to 1000 nm (1 μm) while microparticles are particles that have sizes that range from 1 to 1000 μm [57]. For all practical purposes pertaining to targeted-delivery systems, small-sized particles are considered more effective than large-size ones [58]. This is because, compared with large-sized particles, small-sized particles are more efficient in permeating biological barriers, passing through capillaries after injection and achieving stability in blood circulation [59]. Thus, in targeted drug delivery, nanoparticles with a diameter of 100 nm or less are preferred [58,60,61]. However, for vaccine delivery, reports from studies are conflicting as to the optimum size ranges that will generate stronger and lasting immune responses [62]. A few examples are summarized in Table 1 and briefly discussed.

Table 1

Representative list of studies summarizing the effects of sizes of particulate adjuvants and the resultant immune responses.

Materials	Particle size (nm)	Route of administration	Immune responses measured	Comments	Ref.
Observations that small-sized particles were better immune adjuvants than large-sized particles
Polystyrene	40–49/93–123	id.	IFN-γ, IL-4, IgG1	Production of IgG1 was observed across all size ranges. IFN-γ was significantly higher with particles of 40–49 nm than 93–123-nm particles, but particles of 93–123 nm gave a higher IL-4 response	[77]
PVA-grafted PLG	100/500/1500	p.o.; ip.	IgG, IgA	Antibody titers were higher with particles of 100-nm size when compared with those of 500 nm. Particles of 1500 nm did not induce antibody titers	[37]
PLG	450–600/1000–3000/6000–32,000	ip.; sc.	CD8+	Particles of sizes less than 450–600 nm induced the strongest immune response	[102]
Chitosan	700–3000	in.; ip.	IgA	Particles of 400 and 1000 nm induced significantly higher IgA responses than particles of 3000 nm	[103]
PLA	7500–50,000	ip.	IgG	Particles of sizes 7500–15,000 nm gave higher antibody titers than particles of sizes 50,000 nm	[88]
PLGA	1000/5000	p.o.	IgG	Particles of 1000 nm were observed to give better immune response than particles of 5000 nm in less than 5 weeks of immunization	[104]
Observations that small-sized particles and large-sized particles were comparable immune adjuvants
PLG	110/800–900	ip.; im.	IgG, IgG1,IgG2a	Comparable immune responses were observed from particles of 110 and 998 nm	[39]
PLGA	200/500	p.o.; sc.	IgG, IgG1, IgG2a	The 200- and 500-nm particles elicited similar immune responses	[36]
Chitosan	700–3000	in.; ip.	IgG	No difference in IgG production between particles of different size groups	[103]
PVA-grafted PLG	100/500/1500	in.	IgG, IgA	Particles of 100- and 500-nm were observed as equal in the levels of induced immune responses	[37]
Observations that large-sized particles were more effective immune adjuvants than small-sized particles
PLGA	200/500/1000	in.; p.o.; sc.	IgG, IgG1, IgG2a	The immune responses elicited by 1000-nm particles were stronger than those induced by particles of 500 and 200 nm	[36]
PLGA	200/500	in.	IgG, IgG1, IgG2a	The 500-nm particles elicited a stronger immune response than the 200-nm particles	[36]
PLA	10,000–70,000/50,000–150,000	im.	IgG	The particles of 10,000–70,000 generated stronger immune responses than particles of 50,000–150,000 nm	[34]
PLA	200–600/2000–8000	im.	IgG, IFN-γ, IL-4,	Particles of 2000–8000 nm showed higher antibody titers than those of 200–600 nm. Particles of sizes 200–600 nm promoted IFN-γ production, while particles of sizes 2–8 μm promoted IL-4 secretion	[63]
Lipid vesicles	10–100/60–350/400–2500	p.o.	IgG1, IgG2a, IFN-γ	Particles of sizes 60–350 and 400–2500 nm generated significantly higher IgG2a titers and IFN-γ responses than particles of sizes 10–100 nm	[67]
Reports on the possibility of an optimum particle size range for immune responses
Polystyrene	20/40/100/500/1000/2000	id.	IFN-γ, IgG	Beads of 40 nm with OVA conjugated onto their surface induced the strongest immune responses	[55]
PLA	<2000/2000–8000/10,000–70,000/50,000–150,000	im.	IgG	At a particle size of 2000–8000 nm, the immune response was higher than for particles in other size ranges studied: <20,000, 10,000–70,000 and 50,000–150,000 nm	[34]
PLA	600–26,000	p.o.	IgG	The optimum particle size for induction of an IgG response was 4000 nm. Particles that were larger or smaller than 4000 nm could not enhance antibody production	[88]

Open in a separate window

id.: Intradermal; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; OVA: Ovalbumin; PLA: Poly(lactic acid); PLG: Poly(D,L-lactide-co-glycolide); PLGA: Poly(lactic-co-glycolic acid); p.o.: Oral; PVA: Polyvinyl alcohol; sc.: Subcutaneous.

For instance, using bovine serum albumin (BSA) as a model antigen entrapped into particles of different sizes (200, 500 and 1000 nm) prepared with PLGA, Gutierro et al. reported that the 1000-nm particles elicited a stronger serum IgG response than the 500- or 200-nm nanoparticles [36], and the immune response induced by the 500-nm particles was similar to that induced by the 200-nm particles by subcutaneous and oral routes [36]. Similarly, Kanchan and Panda reported that the hepatitis B surface antigen (HBsAg) entrapped in PLA particles of 2000–8000 nm induced a stronger anti-HBsAg antibody response than HBsAg entrapped in PLA particles of 200–600 nm [63]. On the contrary, data from other studies showed that the adjuvant activity of small nanoparticles was more potent than that of the large particles. For example, Jung et al. studied the effect of particle size on the immune responses induced by tetanus toxoid (TT) adsorbed onto particulates prepared from sulfobutylated poly(vinyl alcohol)-graft-PLGA and showed that small particles of 100 and 500 nm induced significantly higher antibody titers then larger ones (>1000 nm) after oral (p.o.) or intranasal (in.) administration [37]. Yet, Wendorf et al. reported that comparable levels of immune responses were induced in mice by protein antigens (Env from HIV-1 or MenB from Neisseria meningitidis) adsorbed onto anionic microparticles (~1 μm) and nanoparticles (110 nm) prepared with PLGA [39]. Others, however, suggested that there may be an optimal particle size in order to induce the strongest immune response (Table 1). For example, Fifis et al. conjugated ovalbumin (OVA) as a model antigen onto solid polystyrene beads of different size (i.e., 20, 40, 100 and 500 nm, and 1 and 2 μm) and reported that the particles of 40 nm were ideal in inducing both antibody and cellular immune responses when intradermally administered to mice [55]. Therefore, it was concluded that particles of 40–50 nm were ideal as immune adjuvants. Even for microparticles, there are reports showing that after a single-point intramuscular immunization with TT entrapped in different-sized microparticles prepared with PLA [34], antibody titers from particles in the size range of 2–8 μm were the highest, whereas 50–150-μm particles and particles with a size less than 2 μm generated weaker antibody titers [34]. Finally, data from other studies reported considerable antibody responses even from large microparticles of 10–90- and 15–60-μm size ranges [34,64,65].

To add another layer of complexity, it is becoming evident that the size of the particulate adjuvants may have different effects on the type of immune responses induced. There are data showing that microparticles promote humoral immune responses, whereas nanoparticles may favor the induction of cellular immune responses [7,63,66]. For example, data from Caputo et al.’s study showed that HIV TAT protein adsorbed on cationic polymeric nanoparticles of 220 or 630 nm induced a stronger TAT-specific cellular immune response and a weaker anti-TAT antibody response than the same TAT protein adsorbed on large microparticles (>2 μm) prepared with the same materials [66]. Similarly, using PLA particles with HBsAg entrapped inside, Kanchan and Panda showed that a single-point immunization with nanoparticles (200–600 nm) induced a lower antibody titer in comparison to microparticles (2–8 μm) [63]. Immunization with the 200–600-nm particles favored T-helper (Th) type 1 immune responses, whereas immunization with the 2–8-μm particles favored Th2 responses [63]. It was reasoned, with supporting data, that the nanoparticles (200–600 nm) can be efficiently taken up by APCs, such as macrophages, to induce cellular immune responses, whereas the macrophages cannot take up the large microparticles. Instead, microparticles simply attach to the surface of the macrophages and release the entrapped antigens. The macrophages then take up the antigens directly. On the contrary, data from Mann et al.’s recent study using oral bilosomes with influenza A antigens showed that the larger bilosome particles (400–2000 nm) elicited an immune response that was significantly biased towards Th1 rather than the smaller bilosomes (10–100 nm) [67]. However, data from Gutierro et al. using BSA entrapped into PLGA particles of 200, 500 and 1000 nm indicated that differences in the total serum IgG response induced by particles of different sizes do not result in differences in the IgG1- or IgG2a-type immune responses [36]. Overall, with the conflicting data, it is difficult to achieve an accurate prediction of particle size ranges that will dictate a Th1 or a mixed Th1/Th2 immune response outcome.

Biological & immunological parameters that may be responsible for the effect of particle size on the resultant immune responses

The influence of particle size on the type, level and quality of the immune response may be ascribed to differences in pathways and mechanisms for cell uptake, and antigen presentation and processing [24,56,68,69]. The publication by Xiang et al. is a good starting point for initial discussion, where it was reported that virus-sized particles in the size range of 20 to 200 nm are usually taken up by endocytosis, resulting in a cellular-based immune response. It was also explained that particles with sizes between 500 nm and 5 micron are mainly taken up by phagocytosis and/or macro-pinocytosis and are more likely to promote a humoral immune response [62].

Investigators that are in support of a potential superiority of small-sized particles over large-size particles have offered a number of explanations, which include:

• The expectation that antigen delivery across the mucosal surface will warrant permeability via biological barriers and nano-sized particles will be the most effective [70];

• Particles in the submicron size range are expected to be taken up efficiently by the APCs [24];

• The smaller the particle size, the larger the surface area for antigen loading [35].

Conversely, there was also outstanding evidence that demonstrated that large-sized particles are much more effective. One viewpoint was that the increased uptake of small-sized particles into APCs could be negated if the uptake is closely followed by extensive exocytosis [71], although the process of exocytosis is applicable to particles having sizes in both submicron and micron ranges. Another view point in favor of large particles as immune adjuvants was that large particles are preferentially attached to the macrophage surfaces, thus serving as an effective depot system for continuous antigen release [63,72]. Effective interaction with APCs may not be feasible in cases where the dimensions of the particles are much larger than a typical APC, such as in cases of particle sizes ranging from 50 to 100 μm [34]. Taken together, it is apparent that many factors that are involved in achieving strong and lasting immune response are interconnected, encompassing parameters relating to vaccine formulation, process of preparation, route of administration, antigen presentation and antigen-processing mechanisms.

Many investigators have shown that the entrapment of antigen in particles alters its acquisition and processing by APCs [26]. Targeting the APCs as a means to amplify, control and mediate the immunological consequences of prophylactic and/or therapeutic vaccines has been strongly propelled by encouraging results from ex vivo loading of DCs with antigen [61,73]. APCs are of critical importance to the transport of antigen from the periphery to local organized lymphoid tissues [74]. It is largely believed that the manner in which antigen reaches the lymph organs is crucial to the induction of the immune response [75]. Considering antigen presentation and processing, particulate adjuvants will influence the resultant immune responses in many ways, itemized as:

• Particulate form of antigen will enhance antigen uptake by APCs and subsequently the delivery of the antigen to lymphoid organs [24,68];

• Based on their size, particles taken up by APCs could rapidly escape from the endolysosomal compartment to the cytosol, thereby supporting the generation of CD8+ T-cell responses [55,76];

• Antigen-loaded particles could serve as a depot for constant antigen release to the APCs. This function will be dependent on the type of particulates (whether they are erodible or not), surface properties, biocompatibility (to facilitate interaction with APCs), size (in comparison to the size of typical APCs), antigen stability and release kinetics [26,63].

It has been reported that particles with a diameter of 500 nm or less are optimal for uptake by DCs or macrophages [77]. Particles of 20–200 nm are generally taken up via endocytosis with subsequent inducement of CD4 and CD8, and Th1-type immune responses [26]. For particles with a dimension greater than 500 nm, uptake is via phagocytosis or micropinocytosis, leading to a humoral immune response [77]. A number of reports have shown that biodegradable microparticles whose sizes allow them to be phagocytosed (size <10 μm) achieved prolonged antigen presentation to APCs [78,79]. Using polystyrene particles, Sharp et al. showed that particles of 430 nm and 1 μm in size were efficiently taken up by DCs, whereas there was limited uptake of 10-μm particles, and no uptake of particles of 32 μm [56]. In addition, Balasse et al. demonstrated phagocytosis of hyroxyethyl starch particles of 4–15-μm size range (average size 8.3 μm) loaded with BSA [79]. In addition to facilitating antigen uptake by APCs, the ability of particulate adjuvant to drain freely into the lymph nodes is highly desirable [80]. In this respect, it was shown that particles with small sizes of 20–200 nm can freely drain to the lymph nodes for antigen presentation, whereas large-sized particles (0.5–2 μm) made of the same materials were mostly dependent on DCs for transport to the lymph nodes [81].

Go to:

Possible formulation factors that contributed to the lack of consistency in the relationship between the adjuvant activity of particles & their size in literature

In addition to the biological and immunological parameters mentioned previously, various other formulation parameters are expected to affect the relationship between particle size and the adjuvant activity of the particulates. The formulation parameters can be in multiple folds, which may include, but are not limited to, materials used to prepare the particulates, nature of the antigens, method of antigen loading, particle size uniformity and distribution, and route of vaccine administration. It is important to emphasize that the effects of all aforementioned parameters are closely linked and should be viewed and evaluated in combination.

Materials used to prepare particulates

The material used to prepare particulate adjuvants is an important factor to consider in achieving reproducible immune responses. This is particularly important for materials that have adjuvant activity themselves. For example, ionic polyphosphazene was shown to have immune adjuvant activity [49]. The adjuvant activity of polyphosphazene appeared to be linked to its ability to form water-soluble noncovalent complexes with the antigen, which may enhance their interaction with APCs [49,82]. Moreover, recent evidence suggested that polyphosphazene activated innate immune cells to secrete IL-4 and IL-12 [82], and the cytokines may mediate the initiation of adaptive immune responses. Therefore, similar-sized particles prepared with polyphosphazene or other polymers, such as PLGA, may have different adjuvant activities.

Antigen & antigen dose

Another potential source of discrepancy is the variability that could arise from the nature of antigens, dose of the antigen and dosing frequency used in different studies even if other parameters pertaining to the immune adjuvants were kept constant. Considering plasmid DNA vaccines, parameters that may affect the performance may include the antigens encoded by the plasmids, the size of the plasmids, the amount of CpG motifs on the plasmids and the purity of the plasmids. There are even more variables that may be introduced by different protein antigens, which include the intrinsic immunogenicity of the proteins, size of the proteins, purity of the proteins and level of endotoxin. Many researchers used model antigens such as OVA, BSA, HBsAg, TT and HIV Tat to evaluate the adjuvant activity of the nanoparticles or microparticles. It is important that the level of endo-toxin in the antigen preparations is determined. Moreover, the intrinsic immunogenicity of the antigens used to evaluate the adjuvant activity of the nano-microparticles should be taken into consideration. For example, data in our previous studies showed that OVA as an antigen conjugated onto the surface of lecithin-based solid lipid nanoparticles induced stronger anti-OVA IgG responses in mice than when the OVA was adjuvanted with aluminum hydroxide [46]. However, when the OVA was replaced with the highly immunogenic Bacillus anthracis protective antigen (PA) protein, the PA-conjugated nanoparticles and the PA adjuvanted with aluminum hydroxide induced similar levels of anti-PA IgG in mouse serum samples after three doses [46]. It was interesting that when measured after a single injection, anti-PA IgG antibodies were detectable only in mice that received the PA-conjugated nanoparticles, but not in mice that received the PA adjuvanted with aluminum hydroxide [46]. Therefore, it is possible that when different antigens are used, one may draw different conclusions regarding the effect of particle size on the adjuvant activity of nano-microparticles. Also, it can be inferred from the aforementioned study that if a highly immunogenic antigen is dosed multiple times, one may find that within a certain range, particle size does not have any detectable effect on the adjuvant activity of particulate adjuvant [46].

Methods of antigen loading

At least three different methods have been used to load antigens of interest onto particulates. These include: entrapment, surface chemical conjugation and surface physical adsorption. In many studies, the antigens were entrapped into the particles [34,63,70]. However, antigens can also be chemically conjugated onto the surface of preformed particulates [46,55] or simply adsorbed onto the particle surface [37]. Entrapment of the antigens into the particulates could protect antigens from degradation. However, the release of antigens from the particulates could be problematic if not controlled well. In addition, the entrapment process could potentially cause chemical or physical damage to the antigens to be entrapped. Surface chemical conjugation is likely to provide less protection and the extra step of conjugation may not be ideal, and chemical conjugation may make certain critical epitopes on the antigens unavailable for presentation. The attractiveness of the physical adsorption of antigens onto particles is that it requires simpler processing. Similar to the use of alum suspension as an adjuvant, it will be convenient if the antigen of interest can be simply mixed with the particles before being given to the host. In fact, depending on the antigen and the particulate system used, there were data suggesting that plasmid DNA vaccine adsorbed on PLGA particles was more effective than when it was entrapped into the PLGA particles [83,84]. In addition, there were data showing that when a protein antigen was conjugated onto the surface of the particulates, it induced a much stronger immune response than when the same antigen was simply physically mixed with the same particulates [55]. Using OVA conjugated onto N-trimethyl chitosan (TMC–OVA), Slütter et al. recently showed that surface conjugation of antigen to particles will ensure that both the antigen and particles reach the APCs at the same time [28]. Mice immunized with TMC–OVA conjugate produced 1000-fold higher OVA-specific IgG titers than mice immunized with the physical mixture of TMC and OVA. It is important to note that antigens on the surface of the particles could stabilize or destabilize an otherwise unstable or stable particle suspension. For instance, particle aggregates and/or agglomerates after antigen conjugation were reported by Kalkanidis et al. [11]. Overall, the sizes of the particles before and after the antigen conjugation or adsorption should be monitored and reported.

Particle size distribution &/or uniformity

The wide size range of particulate adjuvants used in various studies could also be a potential source of variability. Just to list a few examples, Tabata et al. used OVA-loaded PLGA particles with average diameters of 600 nm and 1, 4, 7, 11, 15, 21 and 26 microns [70]. When given orally, the 4-micron particles induced the best serum antibody response. Katare et al. used TT-entrapped PLA particles of four different groups, less than 2 microns, 2–8 microns, 10–70 microns and 50–150 microns, and showed that the 2–8-micron particulates induced the strongest serum antibody response after a single-point intramuscular injection [34]. In another study from the same group, Kanchan and Panda used HBsAg-entrapped PLA particles of 200–600 nm and 2–8 microns, and reported that the 2–8-micron particulates induced a stronger antibody response, whereas the 0.2–0.6-microns particulates induced a stronger cellular immune response [63]. Mann et al. used biolosomes of 400–2000 nm [67]. Borges et al. reported that high polydispersity index is one of the major drawbacks in the application of chitosan particles generated by precipitation with sodium sulfate [85]. It is understandable that it is not easy to prepare particulates with high size uniformity, but it is possible that the immune system will not be able to differentiate particles of 1-, 4- and 7-micron sizes. Since particles of sizes 200 and 600 nm may have different adjuvant activities, it is likely that the size range in the aforementioned studies [63,67] of 400–2000 nm as well as 200–600 nm is too wide. To substantiate these points, it was shown that particulates of 20–200 nm can freely drain to local draining lymph nodes, whereas DCs were required for the transport of particles larger than 500 nm (0.5–2 microns) from the site of injection to the lymph nodes [81]. Moreover, it was reported that particulates of 500 nm or less were optimal for uptake by APCs [86]. Therefore, it is logical for one to reason that 500 nm should be used as a potential cutting point, with particles smaller than 500 nm and larger than 500 nm having totally different vaccine adjuvant activities. Fifis et al. used commercially available polystyrene beads of 20, 40, 100, 200 and 500 nm, and 1 and 2 microns [55]. OVA as a model antigen was chemically conjugated onto the beads and intradermally injected into mice. Their data showed that the 40-nm beads induced the strongest immune responses, suggesting that within the size range of 20 nm to 2 microns, the 40-nm size was optimal. For many reasons, we agree with the choice of particles and size ranges used by these investigators [55]. Firstly, the polystyrene beads were quite uniform in size (Polysciences Inc., Eppelheim, Germany). Secondly, beads of smaller than and larger than 500 nm were evaluated independently and, more importantly, the inclusion of 20-, 40- and 100-nm beads allowed the evaluation of adjuvant activities of particles with a diameter less than 100 nm. Unfortunately, in a subsequent paper from the same group of investigators, it was reported that the size of the OVA-conjugated 40–50-nm polystyrene beads was actually 232 nm with a polydispersity index of 0.384 when measured using a dynamic light scattering (DLS) particle sizer [11]. Therefore, it was the 232-nm OVA–polystyrene beads that actually induced the strongest immune responses [55]. Since it captures the hydrodynamic size of particles, the diameter measured from the DLS particle sizer may more closely resemble the size of the OVA–polystyrene beads that the immune cells have encountered. In that sense, it is encouraged that the size measured by DLS for particles dispersed in medium containing normal saline or serum proteins should be reported in all future studies.

Moreover, potential particle instability that can alter particle adjuvanticity could arise at different stages during particle preparation and application, such as during antigen loading, particle storage and upon particle contact with biological fluids. In addition to affecting antigen integrity, loading efficiency and release kinetics, particle instability could lead to changes in particle sizes involving an increase in sizes (as in particle aggregation/agglomeration and fusion) or decrease in sizes (as in polymer degradation). Kalkanidis et al. showed that vaccine formulations can undergo various degrees of aggregation during storage depending on the pH of the dispersing medium [11]. Therefore, it is important for investigators in the field to include studies on potential particle instability that can occur after storage and when dispersed in biologically relevant conditions. In other words, the physical stability or instability of the particles as an adjuvant before and after administration should be seriously considered when designing particle-based vaccine adjuvants.

Route of administration

It is known that the route of vaccine administration plays a significant role in shaping the induction of immune responses. In., p.o., intramuscular and subcutaneous (sc.) antigen delivery may encounter different subsets of DCs based on localization that may dictate the type, quantity and quality of immune responses [79]. For example, Newman et al. demonstrated that particles of the same size when dosed via different routes were taken up by different APCs, leading to different immune response [69]. Specifically, it was observed that intraperitoneal (ip.) immunization was associated with a predominant uptake of microparticles by macrophages in the peritoneal cavity, while intradermal immunization resulted in the uptake of microparticles by DCs [69]. In general, immunization by injection is most popular. However, considerable progress has been made with mucosal immunization over the years [87]. The following examples illustrate that the route of administration plays a significant role in the observed effect of the size of the particles on the resultant immune responses.

Using particles prepared with sulfobutylated poly(vinyl alcohol)-graft-PLGA with TT adsorbed, Jung et al. compared the serum anti-TT IgG titers induced by the particles of approximately 100, 450 and 1500 nm after p.o. or in. administration [37]. They reported that for the 450-nm particles, the in. route induced a stronger serum anti-TT IgG response than the p.o. route. However, for the 100-nm particles, the in. route was only slightly better than the p.o. route. Finally, for the 1.5-micron particles, the p.o. and in. routes did not differ, and only a very weak anti-TT IgG titer was observed as compared with that induced by the 100- and 400-nm particles.

In a study by Nakaoka et al., PLA microparticles containing OVA were prepared [88]. The resultant microspheres were fractionated into six different sizes ranging from 3.4 ± 2.2 to 50 μm. Microparticles were applied intraperitoneally or subcutaneously. It was observed that ip. immunization was much more influenced by the microsphere size, and higher serum levels of anti-OVA antibodies were induced when microparticles of small sizes were used. By contrast, sc. immunization showed comparable levels of serum antibodies with microparticles at all size ranges, indicating that the sc. route was not affected (or less likely affected) by changes in size. The investigators explained that there was the likelihood that particle instability occurred with sc. immunization [88]. Potential particle instability after injection leading to particle aggregation and/or agglomeration will most likely negate the influence of administering particles at various size ranges, further supporting the need for investigators to monitor the stability of the particulate vaccine formulations in simulated biological medium before injecting them in animals.

In a study by Gutierro et al., BSA was entrapped in particles of different sizes (200, 500 and 1000 nm) prepared from PLGA [36]. The particles were given either intranasally, orally or subcutaneously to BALB/c mice, and the serum IgG response elicited was compared. It was shown that the 1000-nm particles elicited a stronger serum IgG response than the 500- or 200-nm-sized nanoparticles. However, the immune response for 500-nm particles was similar to that obtained with the 200 nm administered by the sc. and the p.o. routes, but higher than the 200-nm particles by the in. route.

Go to:

Secondary formulation factors that may affect the size of particles & the resultant immune responses

It is important to note that the development of any vaccine delivery system from bench to clinics will involve secondary processes such as sterilization and lyophilization. It is recommended that at the early stages of any vaccine formulation design, the potential impact of secondary processes should be investigated, since these processes can influence the stability of vaccine formulations, particle sizes and size distribution, and thus the resultant immune responses.

Sterilization

All vaccine delivery systems for parenteral administration require sterilization. It is important to state that the aim of sterilization is to destroy or eliminate unwanted living microorganism contamination that may be present in the vaccine product [89]. This is to ascertain that the product is free of unwanted health hazards. Sterilization can be carried out by aseptic method/manufacture, filtration, γ-irradiation, heating, gassing with ethylene oxide and hydrostatic pressure [90]. Irrespective of the method of sterilization, it is desirable to ensure that the process of sterilization does not negatively affect the stability of the materials used in making nano-microparticles as well as particle sizes and size distribution. Furthermore, the sterilization process should preserve the integrity of the antigen and maintain antigen-loading efficiency in the particles. Among all the methods, the process of aseptic (sterile) filtration is simple and does not lead to direct degradation of materials used in preparing the particles [91–93]. It is important to note that sterile filtration will not be appropriate for particles with sizes larger than 200 nm [90,92]. Special attention should be paid to selecting suitable sterile filters to safeguard against antigen degradation or adsorption onto filters. The choice of γ-irradiation should be based on the chemical stability of components of the particles. Free radicals that are formed during γ-sterilization can initiate chemical modification of the materials used in making the particulate adjuvants [94]. Sterilization by heat has also been reported [89,90,95]. A major concern in the application of heat is the risk of triggering temperature-related changes resulting in: the degradation of antigens, chemical degradation of materials used to make the particulate adjuvants, and physical disruption of the integrity of antigens and particles. For instance, after moist heat sterilization of nanoparticles, an increase in size from 200 to 500 nm was reported where Miglyol was used as the oil phase [96].

Lyophilization (freeze-drying)

Vaccine formulations that will progress from bench to clinic are required to be stable during storage. While the primary consideration should be to maintain the immunogenicity from the time of preparation to the time of application, we believe that the stability of particle size and size distribution should also be evaluated. Instability during storage could lead to physical and chemical changes, antigen degradation or leakage from particulate adjuvants and particle size instability (aggregation or precipitation) [85,97,98]. One effective way to ensure storage stability is to lyophilize the vaccine formulation into a dry powder [89]. Freeze-drying is a well-known process used to produce stable proteins and polypeptides that are prone to instability in aqueous solutions [99]. Essentially, lyophilization is achieved in two major steps of freezing the sample and evaporation of water under vacuum. These two steps can potentially bring about a series of instabilities that are worth investigating while developing new vaccine formulations. Sizes of particles could significantly increase during the process of lyophilization [89,97]. A well-designed, freeze-drying cycle will ensure the physical and chemical stability of a product as well as increase the efficiency of the manufacturing process. Data obtained from lyophilization of vaccine formulations will be relevant to large-scale manufacturing, where it is necessary to convert particle-based vaccine formulations from suspensions to powders so as to reduce the bulk volume and to improve storage stability. The resultant lyophilized powder of the particulate adjuvants can be reconstituted to regenerate the original particle sizes using suitable cryoprotectants [89,97]. Examples of cryoprotectors that can be used are sorbitol, mannose, trehalose, sucrose, man-nitol, carboxymethylcellulose and polyvinylpyrrolidone. The type and amounts of cryoprotectants should be optimized for a given formulation.

Finally, due to the high cost associated with the storage and transfer of vaccines in cold chain and, in some situations, the lack of electricity to power freezers or refrigerators, the ability to store and transfer vaccines in ambient conditions is increasingly being incorporated into the early steps of vaccine formulations [100,101]. We recommend that future development of particulate adjuvant vaccine formulations should take into consideration the feasibility of preserving the particle size and, more importantly, the immunogenicity of the vaccine candidates while avoiding the cold chain at the early stage of formulation development.

Go to:

Expert commentary

Nanoparticles and/or microparticles are promising vaccine delivery systems with potential adjuvant activity. It is generally agreed that the adjuvanticity of nano-microparticles is affected by particle sizes, which in turn affect the type of immune responses (humoral or cellular) induced by antigens carried by particles. The desired translation from bench to clinics will be greatly hampered by the conflicting results from different studies in correlating the adjuvant activity and particle sizes. It is our belief that in future studies (attempting to correlate particle size and the adjuvant activity of the particles), there is a need to comprehensively evaluate and compare both humoral and cellular immune responses induced by very narrowly distributed particles prepared with the same materials, loaded with the same antigens by the proper method, and dosed via different routes to the same strain of animals. It is important to note that particle size as a parameter of interest herein cannot be separated from other parameters, such as particle surface properties or material type. Ultimately, it is possible that one may find the optimal particle size that favors strong humoral and cellular immune responses. In addition, one may have to mix particles of specific size ranges favoring either strong humoral responses or strong cellular immune responses in order to generate a desirable and balanced immune response. Another point to consider is the preservation of immunogenicity of antigens carried by particles while avoiding the cold chain in vaccine storage and transportation.

Go to:

Five-year view

Over the next few years, there is a need for comprehensive studies to define and understand the relationship between sizes of particulate adjuvants and the resultant immune responses. We expect more confirmative studies supporting the adjuvanticity of particle-based vaccine delivery systems. Importantly, more studies are warranted that will generate mechanistic data to clearly explain how and why the size of particle-based vaccine delivery systems significantly affects their adjuvant activities.

Key issues

• Particulates prepared with biocompatible materials hold great potential as vaccine delivery system with potent adjuvant activity.

• The adjuvant activities of different particulates are influenced by factors such as particle size, surface charges, method of antigen loading, route of administration, and soon.

• There were numerous attempts to correlate the size of particulate adjuvants and the resultant immune response, but the findings were conflicting and inconsistent.

• The size of the particulate adjuvants is not only related to the strength of the immune responses induced, but also the type of immune responses.

• Pharmaceutical formulation parameters can potentially affect the size of the particulate adjuvants and, thus, the resultant immune response.

• There is a critical need for comprehensive studies to identify the effect of the size of the particulate adjuvants on their adjuvant activity.

Go to:

Footnotes

Financial & competing interests disclosure

This work was supported, in part, by the NIH grants (AI070538 and AI078304) to Zhenrong Cui. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Go to:

References

Papers of special note have been highlighted as:

• of interest

1. Davis JP. Experience with hepatitis A and B vaccines. Am J Med. 2005;118(10 Suppl 1):7–15. [PubMed] [Google Scholar]

2. Look M, Bandyopadhyay A, Blum JS, Fahmy TM. Application of nanotechnologies for improved immune response against infectious diseases in the developing world. Adv Drug Deliv Rev. 2009;62(4–5):378–393. [PMC free article] [PubMed] [Google Scholar]

3. O’Hagan DT, Valiante NM. Recent advances in the discovery and delivery of vaccine adjuvants. Nat Rev Drug Discov. 2003;2(9):727–735. [PMC free article] [PubMed] [Google Scholar]

4. Perrie Y, Mohammed AR, Kirby DJ, McNeil SE, Bramwell VW. Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens. Int J Pharm. 2008;364(2):272–280. [PubMed] [Google Scholar]

5. Copland MJ, Baird MA, Rades T, et al. Liposomal delivery of antigen to human dendritic cells. Vaccine. 2003;21(9–10):883–890. [PubMed] [Google Scholar]

6. Schijns V. Immunological concepts for vaccine adjuvant activity. Curr Opin Immunol. 2000;12(4):456–463. [PubMed] [Google Scholar]

7. Caputo A, Sparnacci K, Ensoli B, Tondelli L. Functional polymeric nano/microparticles for surface adsorption and delivery of protein and DNA vaccines. Curr Drug Deliv. 2008;5(4):230–242. [PubMed] [Google Scholar]

8. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol. 2004;82(5):488–496. [PubMed] [Google Scholar]

9. Chang S, Warner J, Liang L, Fairman J. A novel vaccine adjuvant for recombinant flu antigens. Biologicals. 2009;37(3):141–147. [PMC free article] [PubMed] [Google Scholar]

10. Singh M, Briones M, Ott G, O’Hagan DT. Cationic microparticles: a potent delivety system for DNA vaccines. Proc Natl Acad Sci USA. 2000;97:811–816. [PMC free article] [PubMed] [Google Scholar]

11•. Kalkanidis M, Pietersz GA, Xiang SD, et al. Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity. Methods. 2006;40(1):20–29. Presents data showing that when ovalbumin was covalently conjugated onto the surface of polystyrene beads of 40–50 nm, the resultant ovalbumin–polystyrene beads have a particle size of 232 nm when measured using a dynamic light scattering particle sizer. [PubMed] [Google Scholar]

12. Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliv Rev. 2008;60(8):915–928. [PMC free article] [PubMed] [Google Scholar]

13. Scheerlinck J-PY, Greenwood DLV. Virus-sized vaccine delivery systems. Drug Discov Today. 2008;13(19–20):882–887. [PubMed] [Google Scholar]

14. Aguilar JC, Rodríguez EG. Vaccine adjuvants revisited. Vaccine. 2007;25(19):3752–3762. [PubMed] [Google Scholar]

15. O’Hagan DT, Singh M. Microparticles as vaccine adjuvants and delivery systems. Expert Rev Vaccines. 2003;2:269–283. [PubMed] [Google Scholar]

16. Schmidt CS, Morrow WJW, Sheikh NA. Smart adjuvants. Expert Rev Vaccines. 2007;6:391–400. [PubMed] [Google Scholar]

17. SayIn B, Somavarapu S, Li XW, Sesardic D, Senel S, Alpar OH. TMC-MCC (N-trimethyl chitosan-mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines. Eur J Pharm Sci. 2009;38(4):362–369. [PubMed] [Google Scholar]

18. Gómez S, Gamazo C, San Roman B, et al. A novel nanoparticulate adjuvant for immunotherapy with Lolium perenne. J Immunol Methods. 2009;348(1–2):1–8. [PubMed] [Google Scholar]

19. Gunaseelan S, Gunaseelan K, Deshmukh M, Zhang X, Sinko PJ. Surface modifications of nanocarriers for effective intracellular delivery of anti-HIV drugs. Adv Drug Deliv Rev. 2010;62(4–5):518–531. [PMC free article] [PubMed] [Google Scholar]

20. Reed SG, Bertholet S, Coler RN, Friede M. New horizons in adjuvants for vaccine development. Trends Immunol. 2009;30(1):23–32. [PubMed] [Google Scholar]

21. Jones KS. Biomaterials as vaccine adjuvants. Biotechnol Progress. 2008;24(4):807–814. [PubMed] [Google Scholar]

22. Mallapragada SK, Narasimhan B. Immunomodulatory biomaterials. Int J Pharm. 2008;364(2):265–271. [PubMed] [Google Scholar]

23. Thomas C, Gupta V, Ahsan F. Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses. Int J Pharm. 2009;379(1):41–50. [PubMed] [Google Scholar]

24. Reddy ST, Rehor A, Schmoekel HG, Hubbell JA, Swartz MA. In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles. J Controlled Release. 2006;112(1):26–34. [PubMed] [Google Scholar]

25. Walter E, Dreher D, Kok M, et al. Hydrophilic poly(D,L-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells. J Controlled Release. 2001;76(1–2):149–168. [PubMed] [Google Scholar]

26•. Rice-Ficht AC, Arenas-Gamboa AM, Kahl-McDonagh MM, Ficht TA. Polymeric particles in vaccine delivery. Curr Opin Microbiol. 2009;13(1):106–112. Reviews particle-based properties, such as surface chemistry and sizes, that will affect antigen presentation and processing by APCs. [PubMed] [Google Scholar]

27. Thomasin C, Corradin G, Men Y, Merkle HP, Gander B. Tetanus toxoid and synthetic malaria antigen containing poly(lactide)/poly(lactide-co-glycolide) microspheres: importance of polymer degradation and antigen release for immune response. J Controlled Release. 1996;41(1–2):131–145. [Google Scholar]

28. Slütter B, Soema PC, Ding Z, Verheul R, Hennink W, Jiskoot W. Conjugation of ovalbumin to trimethyl chitosan improves immunogenicity of the antigen. J Controlled Release. 2010;143(2):207–214. [PubMed] [Google Scholar]

29. O’Hagan DT. Microparticles and polymers for the mucosal delivery of vaccines. Adv Drug Deliv Rev. 1998;34(2–3):305–320. [PubMed] [Google Scholar]

30. Kazzaz J, Neidleman J, Singh M, Ott G, O’Hagan DT. Novel anionic microparticles are a potent adjuvant for the induction of cytotoxic T lymphocytes against recombinant p55 gag from HIV-1. J Controlled Release. 2000;67(2–3):347–356. [PubMed] [Google Scholar]

31. Jain S, Yap WT, Irvine DJ. Synthesis of protein-loaded hydrogel particles in an aqueous two-phase system for coincident antigen and CpG oligonucleotide delivery to antigen-presenting cells. Biomacromolecules. 2005;6(5):2590–2600. [PubMed] [Google Scholar]

32. Shen Z, Reznikoff G, Dranoff G, Rock K. Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol. 1997;158(6):2723–2730. [PubMed] [Google Scholar]

33•. Sharma S, Mukkur TKS, Benson HAE, Chen Y. Pharmaceutical aspects of intranasal delivery of vaccines using particulate systems. J Pharm Sci. 2009;98(3):812–843. Detailed review on pharmaceutical-based factors that will influence the performance of particles as adjuvants in intranasal immunization. [PubMed] [Google Scholar]

34. Katare YK, Muthukumaran T, Panda AK. Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int J Pharm. 2005;301(1–2):149–160. [PubMed] [Google Scholar]

35•. Wendorf J, Singh M, Chesko J, et al. A practical approach to the use of nanoparticles for vaccine delivery. J Pharm Sci. 2006;95(12):2738–2750. Focuses on the performance of particulate adjuvants and the influence of processes such as sterilization, lyophilization and particle resuspension on the adjuvant activity of the particles. [PubMed] [Google Scholar]

36. Gutierro I, Hernández RM, Igartua M, Gascón AR, Pedraz JL. Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres. Vaccine. 2002;21(1–2):67–77. [PubMed] [Google Scholar]

37. Jung T, Kamm W, Breitenbach A, Hungerer K-D, Hundt E, Kissel T. Tetanus toxoid loaded nanoparticles from sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide): evaluation of antibody response after oral and nasal application in mice. Pharm Res. 2001;18(3):352–360. [PubMed] [Google Scholar]

38. Vila A, Sanchez A, Evora C, Soriano I, Jato J, Alonso M. PEG-PLA nanoparticles as carriers for nasal vaccine delivery. J Aerosol Med. 2004;17:174–185. [PubMed] [Google Scholar]

39•. Wendorf J, Chesko J, Kazzaz J, et al. A comparison of anionic nanoparticles and microparticles as vaccine delivery systems. Hum Vaccin. 2008;4(1):44–49. In vivo comparison indicating that particles of 110- and 998-nm sizes induced comparable immune responses. [PubMed] [Google Scholar]

40. Manocha M, Pal PC, Chitralekha KT, et al. Enhanced mucosal and systemic immune response with intranasal immunization of mice with HIV peptides entrapped in PLG microparticles in combination with Ulex Europaeus-I lectin as M cell target. Vaccine. 2005;23(48–49):5599–5617. [PubMed] [Google Scholar]

41. Ying M, Gander B, Merkle HP, Corradin G. Induction of sustained and elevated immune responses to weakly immunogenic synthetic malarial peptides by encapsulation in biodegradable polymer microspheres. Vaccine. 1996;14(15):1442–1450. [PubMed] [Google Scholar]

42. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev. 2003;55(3):329–347. [PubMed] [Google Scholar]

43. Elamanchili P, Diwan M, Cao M, Samuel J. Characterization of poly(D,L,-lactic-co-glycolic acid) based nanoparticulate system for enhanced delivery of antigens to dendritic cells. Vaccine. 2004;22(19):2406–2412. [PubMed] [Google Scholar]

44. Cui Z, Mumper RJ. Topical immunization using nanoengineered genetic vaccines. J Controlled Release. 2002;81(1–2):173–184. [PubMed] [Google Scholar]

45. Cui Z, Mumper RJ. Genetic Immunization Using nanoparticles engineered from microemulsion precursors. Pharm Res. 2002;19(7):939–946. [PubMed] [Google Scholar]

46. Sloat BR, Sandoval MA, Hau AM, He Y, Cui Z. Strong antibody responses induced by protein antigens conjugated onto the surface of lecithin-based nanoparticles. J Controlled Release. 2010;141(1):93–100. [PMC free article] [PubMed] [Google Scholar]

47. Tobío M, Gref R, Sánchez A, Langer R, Alonso MJ. Stealth PLA-PEG nanoparticles as protein carriers for nasal administration. Pharm Res. 1998;15(2):270–275. [PubMed] [Google Scholar]

48. Rieger J, Freichels H, Imberty A, et al. Polyester nanoparticles presenting mannose residues: toward the development of new vaccine delivery systems combining biodegradability and targeting properties. Biomacromolecules. 2009;10(3):651–657. [PubMed] [Google Scholar]

49. Andrianov AK, Marin A, Roberts BE. Polyphosphazene polyelectrolytes: a link between the formation of noncovalent complexes with antigenic proteins and immunostimulating activity. Biomacromolecules. 2005;6(3):1375–1379. [PubMed] [Google Scholar]

50. Zwiorek K, Bourquin C, Battiany J, et al. Delivery by cationic gelatin nanoparticles strongly increases the immunostimulatory effects of CpG oligonucleotides. Pharm Res. 2008;25(3):551–562. [PubMed] [Google Scholar]

51. Coppi G, Iannuccelli V, Sala N, Bondi M. Alginate microparticles for polymyxin B Peyer’s patches uptake: microparticles for antibiotic oral administration. J Microencap Micro Nano Carriers. 2004;21(8):829–839. [PubMed] [Google Scholar]

52. Okada H, Toguchi H. Biodegradable microspheres in drug delivery. Crit Rev Ther Drug Carrier Syst. 1995;12(1):1–99. [PubMed] [Google Scholar]

53. Florindo HF, Pandit S, Lacerda L, Gonçalves LMD, Alpar HO, Almeida AJ. The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-[var ε]-caprolactone-based nanoparticles. Biomaterials. 2009;30(5):879–891. [PubMed] [Google Scholar]

54. Freitas S, Merkle HP, Gander B. Microencapsulation by solvent extraction/evaporation: reviewing the state of the art of microsphere preparation process technology. J Controlled Release. 2005;102(2):313–332. [PubMed] [Google Scholar]

55•. Fifis T, Gamvrellis A, Crimeen-Irwin B, et al. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol. 2004;173(5):3148–3154. The size of the particles (prior to antigen conjugation) chosen in this study was one of the ideal. [PubMed] [Google Scholar]

56. Sharp F, Ruane D, Claas B, et al. Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome. Proc Natl Acad Sci USA. 2009;106:870–875. [PMC free article] [PubMed] [Google Scholar]

57. Kreuter J. Nanoparticles and microparticles for drug and vaccine delivery. J Anat. 1996;189:503–505. [PMC free article] [PubMed] [Google Scholar]

58. Mumper RJ, Cui Z, Oyewumi MO. Nanotemplate engineering of cell specific nanoparticles. J Dispers Sci Technol. 2003;24(3):569–588. [Google Scholar]

59. Monfardini C, Veronese M. Stabilization of substances in circulation. Bioconjug Chem. 1998;9:418–450. [PubMed] [Google Scholar]

60. Kim GJ, Nie S. Targeted cancer nanotherapy. Mater Today. 2005;8(8 Suppl 1):28–33. [Google Scholar]

61. Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes to preferential accumulation of stealth liposomes in tumor tissue. Cancer Res. 1993;53(16):3765–3770. [PubMed] [Google Scholar]

62•. Xiang SD, Scholzen A, Minigo G, et al. Pathogen recognition and development of particulate vaccines: does size matter? Methods. 2006;40(1):1–9. Critical evaluation of the performance of particles of different sizes in antigen delivery. [PubMed] [Google Scholar]

63•. Kanchan V, Panda AK. Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response. Biomaterials. 2007;28(35):5344–5357. Provides evidence to explain why nanoparticles of 200–600 nm favor T helper (Th)1 immune responses, whereas microparticles of 2–8 microns generate a better antibody response. [PubMed] [Google Scholar]

64. Johansen P, Estevez F, Zurbriggen R, et al. Towards clinical testing of a single-administration tetanus vaccine based on PLA/PLGA microspheres. Vaccine. 2000;19(9–10):1047–1054. [PubMed] [Google Scholar]

65. Johansen P, Gander B, Merkle HP, Sesardic D. Ambiguities in the preclinical quality assessment of microparticulate vaccines. Trends Biotechnol. 2000;18(5):203–211. [PubMed] [Google Scholar]

66. Caputo A, Brocca-Cofano E, Castaldello A, et al. Characterization of immune responses elicited in mice by intranasal co-immunization with HIV-1 Tat, gp140 ΔV2Env and/or SIV Gag proteins and the nontoxicogenic heat-labile Escherichia coli enterotoxin. Vaccine. 2008;26(9):1214–1227. [PubMed] [Google Scholar]

67. Mann JFS, Shakir E, Carter KC, Mullen AB, Alexander J, Ferro VA. Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection. Vaccine. 2009;27(27):3643–3649. [PubMed] [Google Scholar]

68. Cruz LJ, Tacken PJ, Fokkink R, et al. Targeted PLGA nano- but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Controlled Release. 2010 In Press. [PubMed] [Google Scholar]

69. Newman KD, Elamanchili P, Kwon GS, Samuel J. Uptake of poly(D,L-lactic-co-glycolic acid) microspheres by antigen-presenting cells in vivo. J Biomed Mater Res. 2002;60(3):480–486. [PubMed] [Google Scholar]

70. Tabata Y, Inoue Y, Ikada Y. Size effect on systemic and mucosal immune responses induced by oral administration of biodegradable microspheres. Vaccine. 1996;14(17–18):1677–1685. [PubMed] [Google Scholar]

71. Panyam J, Labhasetwar V. Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm Res. 2003;20(2):212–220. [PubMed] [Google Scholar]

72. Horisawa E, Kubota K, Tuboi I, et al. Size-dependency of D,L-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharm Res. 2002;19(2):132–139. [PubMed] [Google Scholar]

73. Doe B, Selby M, Barnett S, Baenziger J, Walker CM. Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells. Proc Natl Acad Sci USA. 1996;16:8578–8583. [PMC free article] [PubMed] [Google Scholar]

74. Zinkernagel RM, Ehl S, Aichele P, Oehen S, Kündig T, Hengartner H. Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol Rev. 1997;156(1):199–209. [PubMed] [Google Scholar]

75. Bramwell VW, Perrie Y. Particulate delivery systems for vaccines: what can we expect? J Pharm Pharmacol. 2006;58:717–728. [PubMed] [Google Scholar]

76. Brewer JM, Pollock KGJ, Tetley L, Russell DG. Vesicle size influences the trafficking, processing, and presentation of antigens in lipid vesicles. J Immunol. 2004;173(10):6143–6150. [PubMed] [Google Scholar]

77. Mottram PL, Leong D, Crimeen-Irwin B, et al. Type 1 and 2 immunity following vaccination is influenced by nanoparticle size: formulation of a model vaccine for respiratory syncytial virus. Mol Pharm. 2006;4(1):73–84. [PubMed] [Google Scholar]

78. Audran R, Peter K, Dannull J, et al. Encapsulation of peptides in biodegradable microspheres prolongs their MHC class-I presentation by dendritic cells and macrophages in vitro. Vaccine. 2003;21(11–12):1250–1255. [PubMed] [Google Scholar]

79. Balasse E, Odot J, Gatouillat G, Andry M-C, Madoulet C. Enhanced immune response induced by BSA loaded in hydroxyethylstarch microparticles. Int J Pharm. 2008;353(1–2):131–138. [PubMed] [Google Scholar]

80. Reddy ST, van der Vlies AJ, Simeoni E, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotech. 2007;25(10):1159–1164. [PubMed] [Google Scholar]

81•. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann Martin F. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38(5):1404–1413. Using fluorescence-labeled particles and in vivo imaging, the authors showed that nanoparticles traffic to the draining lymph nodes in a size-dependent manner. Small particles (20–200 nm) drained freely to the lymph nodes, whereas dendritic cells (DCs) are strictly required for transport of large particles (500–2000 nm) from the injection site to the lymph nodes. [PubMed] [Google Scholar]

82. Mutwiri G, Benjamin P, Soita H, Babiuk LA. Co-administration of polyphosphazenes with CpG oligodeoxynucleotides strongly enhances immune responses in mice immunized with Hepatitis B virus surface antigen. Vaccine. 2008;26(22):2680–2688. [PubMed] [Google Scholar]

83. Briones M, Singh M, Ugozzoli M, et al. The preparation, characterization, and evaluation of cationic microparticles for DNA vaccine delivery. Pharm Res. 2001;18(5):709–712. [PubMed] [Google Scholar]

84. O’Hagan D, Singh M, Ugozzoli M, et al. Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines. J Virol. 2001;75(19):9037–9043. [PMC free article] [PubMed] [Google Scholar]

85. Borges O, Borchard G, Verhoef JC, de Sousa A, Junginger HE. Preparation of coated nanoparticles for a new mucosal vaccine delivery system. Int J Pharm. 2005;299(1–2):155–166. [PubMed] [Google Scholar]

86•. Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm. 2005;298(2):315–322. Presents cell culture data showing that human DCs took up particles of less than 500 nm more effectively than particles of more than 500 nm. [PubMed] [Google Scholar]

87. Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol. 2006;6(2):148–158. [PubMed] [Google Scholar]

88. Nakaoka R, Inoue Y, Tabata Y, Ikada Y. Size effect on the antibody production induced by biodegradable microspheres containing antigen. Vaccine. 1996;14(13):1251–1256. [PubMed] [Google Scholar]

89. Mohammed AR, Bramwell VW, Coombes AGA, Perrie Y. Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products. Methods. 2006;40(1):30–38. [PubMed] [Google Scholar]

90. Memisoglu-Bilensoy E, Hincal AA. Sterile, injectable cyclodextrin nanoparticles: Effects of γ irradiation and autoclaving. Int J Pharm. 2006;311(1–2):203–208. [PubMed] [Google Scholar]

91. Konan YN, Cerny R, Favet J, Berton M, Gurny R, Allémann E. Preparation and characterization of sterile sub-200 nm meso-tetra(4-hydroxylphenyl)porphyrin-loaded nanoparticles for photodynamic therapy. Eur J Pharm Biopharm. 2003;55(1):115–124. [PubMed] [Google Scholar]

92. Konan YN, Gurny R, Allémann E. Preparation and characterization of sterile and freeze-dried sub-200 nm nanoparticles. Int J Pharm. 2002;233(1–2):239–252. [PubMed] [Google Scholar]

93. Masson V, Maurin F, Fessi H, Devissaguet JP. Influence of sterilization processes on poly([var ε]-caprolactone) nanospheres. Biomaterials. 1997;18(4):327–335. [PubMed] [Google Scholar]

94. Sintzel MB, Merkli A, Tabatabay C, Gurny R. Influence of irradiation sterilization on polymers used as drug carriers: a review. Drug Develop Industrial Pharm. 1997;23(9):857–878. [Google Scholar]

95. Mehnert W, Mäder K. Solid lipid nanoparticles: production, characterization and applications. Adv Drug Deliv Rev. 2001;47(2–3):165–196. [PubMed] [Google Scholar]

96. Rollot JM, Couvreur P, Roblot-Treupel L, Puisieux F. Physicochemical and morphological characterization of polyisobutyl cyanoacrylate nanocapsules. J Pharm Sci. 1986;75(4):361–364. [PubMed] [Google Scholar]

97. Bozdag S, Dillen K, Vandervoort J, Ludwig A. The effect of freeze-drying with different cryoprotectants and γ-irradiation sterilization on the characteristics of ciprofloxacin HCl-loaded poly(D,L-lactide-glycolide) nanoparticles. J Pharm Pharmacol. 2005;57:699–708. [PubMed] [Google Scholar]

98. Quintanar-Guerrero D, Ganem-Quintanar A, Allemann E, Fessi H, Doelker E. Influence of the stabilizer coating layer on the purification and freeze-drying of poly(D,L-lactic acid) nanoparticles prepared by an emulsion-diffusion technique. J Microencapsul. 1998;15(1):107–119. [PubMed] [Google Scholar]

99. Glavas-Dodov M, Fredro-Kumbaradzi E, Goracinova K, et al. The effects of lyophilization on the stability of liposomes containing 5-FU. Int J Pharm. 2005;291(1–2):79–86. [PubMed] [Google Scholar]

100. Chen D, Kristensen D. Opportunities and challenges of developing thermostable vaccines. Expert Rev Vaccines. 2009;8:547–557. [PubMed] [Google Scholar]

101. Sloat BS, Sandoval MA, Cui Z. Towards preserving the immunogenicity of protein antigens carried by nanoparticles while avoiding the cold chain. Int J Pharm. 2010;393(1–2):197–202. [PMC free article] [PubMed] [Google Scholar]

102. Nixon DF, Hioe C, Chen P-D, et al. Synthetic peptides entrapped in microparticles can elicit cytotoxic T cell activity. Vaccine. 1996;14(16):1523–1530. [PubMed] [Google Scholar]

103. Nagamoto T, Hattori Y, Takayama K, Maitani Y. Novel chitosan particles and chitosan-coated emulsions inducing immune response via intranasal vaccine delivery. Pharm Res. 2004;21(4):671–674. [PubMed] [Google Scholar]

104. Igartua M, Hernández RM, Esquisabel A, Gascón AR, Calvo MB, Pedraz JL. Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres. J Controlled Release. 1998;56(1–3):63–73. [PubMed] [Google Scholar]

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963573/

Freeze drying, also known as lyophilisation or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure, then removing the ice by sublimation. This is in contrast to dehydration by most conventional methods that evaporate water using heat.

Freeze-drying - Wikipedia

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693489/#:~:text=Liposomal%20vaccines%20can%20be%20developed,(including%20tuberculosis%2C%20TB).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693489/#:~:text=Liposomal%20vaccines%20can%20be%20developed,(including%20tuberculosis%2C%20TB).

J Nanobiotechnology. 2017; 15: 83.

Published online 2017 Nov 17. doi: 10.1186/s12951-017-0319-9

PMCID: PMC5693489

PMID: 29149896

Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines

Luis O. De Serrano and David J. Burkhart

Author information Article notes Copyright and License information Disclaimer

This article has been cited by other articles in PMC.

Associated Data

Data Availability Statement

Go to:

Abstract

Vaccinology is one of the most important cornerstones in modern medicine, providing better quality of life. The human immune system is composed of innate and adaptive immune processes that interplay when infection occurs. Innate immunity relies on pathogen-associated molecular patterns which are recognized by pathogen recognition receptors localized in antigen presenting cells. After antigen processing and presentation, CD4+ T cell polarization occurs, further leading to B cell and CD8+ activation and humoral and cell-mediated adaptive immune responses. Liposomes are being employed as vaccine technologies and their design is of importance to ensure proper immune responses. Physicochemical parameters like liposome size, charge, lamellarity and bilayer fluidity must be completely understood to ensure optimal vaccine stability and efficacy. Liposomal vaccines can be developed to target specific immune cell types for the induction of certain immune responses. In this review, we will present promising liposomal vaccine approaches for the treatment of important viral, bacterial, fungal and parasitic infections (including tuberculosis, TB). Cationic liposomes are the most studied liposome types due to their enhanced interaction with the negatively charged immune cells. Thus, a special section on the cationic lipid dimethyldioctadecylammonium and TB is also presented.

Keywords: Liposomes, Vaccine formulations, CLR, TLR, Viral infections, Bacterial infections, Parasites

Go to:

Background

Vaccination is one of the most significant developments in modern science, improving the treatment and controlling the spread of diseases across communities. For formulation scientists and immunologists, there has been an interest in the development of liposomal vaccines for their prophylactic uses in infections (of viral, bacterial, fungal or parasitic origin) [1–3]. To develop safe and effective liposome-based vaccines, scientists should take into consideration several interconnected principles: (1) the design-dependent function of the liposomes (2) the characteristics of liposome-cell interactions when vaccine administration occurs and (3) the specific cell receptor and signaling involved once liposomal vaccines are administered. Each of these principles will affect vaccine efficacy and its potential development from bench to bedside applications. These three principles are also the basis for developing excellent subunit vaccine strategies, which have been of important interest for vaccine scientists for several years [4–7]. To comprehend how the potential liposomal vaccine might work in the host we must understand the immune responses involved in receptor signaling. The field of immunology plays a significant role that will help determine the utilization of vaccinology to the advantage of the patient by developing adequate prophylactic treatment approaches.

Immunologists investigate how our bodies defend themselves from pathogens by determining and describing the signaling mechanisms involved. Basically, when infection occurs, the innate arm of the immune system responds through recognition of distinct molecules on or in pathogens termed ‘pathogen associated molecular patterns (PAMPs)’. These PAMPs differ from host markers and as such are recognized by the first line of defense in the immune system, antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages and neutrophils. The PAMPs are recognized by pattern recognition receptors (PRRs) on the surface and in endosomes of APCs [8–11]. Vertebrates developed the adaptive immune system as a second line of defense that could ‘remember’ pathogens and fight back upon re-challenge. It is composed of cells such as T and B cells that employ de novo synthesized antigen-specific receptors (T- and B cell receptors, TCRs and BCRs). TCR and BCRs are able to recognize pathogen-specific antigens when presented complexed with major histocompatibility complexes (MHC) on the surface of an APC. But polarization of the T or B cells to ensure it acts appropriately against the pathogen, relies on signals provided by the APC (such as co-stimulatory molecules and precise cytokines) in response to the priming by PAMPs. These critical signals lead to differentiation of effector and eventually memory cells that are critical for driving the immune response against future infections with the same pathogen. While innate immune responses are broadly applicable and adaptive immune responses are antigen-specific, the two arms of the immune system work in close concert to mount an effective response to clear the pathogen.

The development of PRRs by the immune system represent a significant advancement in the fight for survival of the host. Therefore, an important question arises to that end: what type of PRRs have been discovered so far and which ligands (or PAMPs) do they tend to recognize? Some PRRs are secreted to the extracellular milieu and participate in pathogen opsonization. However, most PRRs are transmembrane (like C-type lectin and Toll-like receptors; CLRs and TLRs, respectively) or cytosolic (retinoic acid-inducible gene I and nucleotide-binding domain and leucine-rich repeat containing receptors; RLRs and NLRs, respectively). For the purposes of this review, we will discuss the uses of liposomes in vaccines that target CLRs and TLRs. The TLRs identified so far amount to 10 in humans, each one recognizing specific PAMPs from microbial pathogens (Table 1) [10–12]. TLR 10 has been recently described as a modulatory receptor; however, no known ligand has been linked to it [13]. Therefore, further research should be done on TLR 10 to uncover the ligand involved in innate immune responses.

Table 1

Pathogen-associated molecular patters (PAMPs) recognized by specific TLRs

Pathogen-associated molecular pattern (PAMP)	Microorganism or classification	TLRs
Lipoproteins	Bacteria	TLR 1
Peptidoglycan and lipoteichoic acid	Gram positive bacteria	TLR 2
β-glucans	Fungi
dsRNA	Double-stranded and negative-stranded viruses	TLR 3
Lipopolysacharide (LPS)	Gram negative bacteria	TLR 4
Flagelin	Bacteria	TLR 5
Profilin	Toxoplasma gondii
Lipoproteins	Mycoplasma	TLR 6
Imidazoquinolines and ssRNA	Single-stranded viruses	TLR 7 and 8
Unmethylated CpG DNA motifs	Prokaryotic genomes and viral DNA	TLR 9

ds double stranded, ss single stranded

Contrary to the transmembrane TLRs, CLRs are classified as soluble or membrane bound. Soluble CLRs (e.g. galectins and collectins) have been reviewed extensively [9, 14]. The mostly studied membrane-associated CLRs are DC-SIGN (DC-specific ICAM3-grabbing non-integrin), Dectin-1 (dendritic cell-associated C-type lectin 1), Dectin-2 (dendritic cell-associated C-type lectin 2), MCL (macrophage C-type lectin) and MINCLE (macrophage-inducible C-type lectin) receptors [8, 9]. These receptors play a significant role in immunomodulatory responses, triggering the differentiation of T-helper cells (TH cells) from naïve CD4+ T cells, through the assistance of an APC. CLRs not only recognize PAMPs but also the damaged-associated molecular patterns (DAMPs) and tumor-associated molecular patterns (TAMPs) from the host [15] during the processes of apoptosis and tumorigenesis, respectively. Glycans from a myriad of pathogens (parasitic, fungal, bacterial or viral) are recognized by CLRs (Table 2); the most common being mannan [16], ManLAM (mannose lipoarabinomannan) [17], mycobacterial cord factor [18], β-1,3-glucans [8] and α-1,2-mannose [8, 19]. In humans, DC-SIGN recognizes both mannan and ManLAM; Dectin-1 recognizes β-1,3-glucans and Dectin-2 recognizes α-1,2-mannose. In mice, mycobacterial cord factor is recognized by MINCLE and currently no glycans have been identified for the human MINCLE. We can observe from the literature review available, there is still more work to be done to determine ligands or PAMPs that interact with specific PRRs. Scientists must pay close attention to interspecies differences in PAMP recognition by PRRs, since we can obtain unwanted immune responses once we study them at the human level. Unwanted immunomodulatory responses can compromise the patient’s outcome from the disease.

Table 2

PAMPs recognized by specific CLRs

Pathogen-associated molecular pattern (PAMP)	Microorganism or classification	CLRs
Mannan	Fungi	DC-SIGN
Man-LAM	M. tuberculosis	DC-SIGN and Dectin 2
LeX	Schistosoma mansonii and tissue ligands	DC-SIGN
LeY + LPS	Helicobacter pylori
LDNF (SP)	Fasciola hepatica
β-1,3-glucans	Fungi	Dectin 1
gp120	HIV-1	DC-SIGN
α-1,2-mannose	Fungi	Dectin 2
Glucosyl and mannosyl glycolipids	Malassezia pachydermatis and M. furfur	MINCLE
Mycobacterial cord factor	M. bovis BCG and M. tuberculosis

Le X sialyl-Lewis X tetrasaccharide, Le Y Lewis Y tetrasaccharide, LPS lipopolysaccharide, LDNF fucosylated LacdiNAc

In the vaccine development field, we can identify the importance of collaboration between medicinal chemistry, immunology and formulation science. This article will present and discuss several parameters that play prominent roles in liposomal vaccine development. Liposome size, charge and bilayer composition are some of those parameters to be discussed. Evidence will be presented to the reader for understanding of the mechanisms or effects of such liposome physicochemical characteristics, which impact vaccine development, safety, integrity and efficacy. Furthermore, we present different applications of liposomal vaccine studies that have been published for the treatment of certain infections of distinct etiological origins (viral, bacterial, fungal and parasitic). Finally, the article presents a special section on the development of subunit cationic liposomal vaccines for the prophylactic treatment of tuberculosis infections, one of the most sought-after indications in contemporary vaccinology. 

Cationic | Definition of Cationic by Merriam-Webster

View all

1 : of, relating to, or being a cation. 2 : characterized by an active and especially surface-active cation a cationic dye.

Cationic surfactants have a positive charge on their hydrophilic end. The positive charge makes them useful in anti-static products, like fabric softeners. Cationic surfactants can also serve as antimicrobial agents, so they are often used in disinfectants. Cationic surfactants cannot be used with anionic surfactants.Jul 31, 2018

Understanding Surfactants: A Guide - International Products ...

The section for tuberculosis vaccine development is focused on DDA-based liposomes; and additional information is available that presents other lipids or phospholipids [20, 21]. The manuscript objective is to present liposomal formulations that are not currently commercially available and inform the scientific community about liposomal formulation for early vaccine development. To date, there are only two commercially approved liposomal vaccines Epaxal and Inflexal, both by Crucell/Berna Biotech. We recommend the reader to further their knowledge in commercially available liposome-based vaccines with another review [22], which discusses the topic extensively.

Go to:

Liposome design

When designing liposomal vaccines, we should take into consideration certain factors within the liposome structure and its physicochemical properties (Fig. 1). Previous reviews on the topic have discussed the different parameters that could affect the functions and efficacy of liposomes as vaccine agents [23, 24]. Liposomal subunit vaccines are safe, with low reactogenicity, biodegradable and versatile. Reactogenicity refers to the low incidence of expected immune responses, causing symptoms like allergies, fever or pain at injection site among others. This type of vaccine contains antigen(s) (either a protein, lipid, lipopeptide etc.) from the pathogen of interest that is incorporated (depending on the antigen physicochemical nature) in the lipid bilayer or core of the liposome. The liposome will serve as an adjuvant, which potentiates the immune responses of the vaccine, improving its efficacy. Antigen incorporation can be achieved by covalent lipid conjugation (at pre- or post-vesicle formation), non-covalent surface attachment (by antibody–epitopes interactions), encapsulation, electrostatic interactions (with lipids of opposite charge) or surface adsorption. 

Lipid–drug conjugates (LDCs) are drug molecules that have been covalently modified with lipids. The conjugation of lipids to drug molecules increases lipophilicity and also changes other properties of drugs. ... In this review, different lipids used for preparing LDCs and various conjugation strategies are summarized.Jan 24, 2017

Lipid–Drug Conjugate for Enhancing Drug Delivery - NCBI - NIH

co·va·lent

/ˌkōˈvālənt/

Learn to pronounce

adjective

CHEMISTRY

1. relating to or denoting chemical bonds formed by the sharing of electrons between atoms.

View all

10.1 Introduction. Adsorption is a surface phenomenon that causes accumulation of atoms, molecules, or ions at the interface of two phases like solid and liquid or solid and gas. The substance being adsorbed is the adsorbate and the solid on which adsorption occurs is the adsorbent.

Adsorption - an overview | ScienceDirect Topics

Seminal articles published earlier covered the effects of antigen encapsulation or adsorption on innate immune response differentiation [25–27]. Researchers reported that both incorporation methods dichotomously induced immune responses that enhanced T cell differentiation, when albumin was used as the incorporated model antigen in the liposomal formulations. However, when antigen size and complexity decreases (like in virus- or tumor-derived antigens), a surface adsorption incorporation method will induce better immune responses than encapsulation [28, 29].

Fig. 1

Physicochemical and morphological factors to consider in liposomal vaccine design

Liposomal vesicle size

The factors, or parameters, affecting the function and potential use of a liposome-based vaccine due to their influence in immune responses include liposome size, lamellarity, surface charge, fluidity of the bilayer, formation of lamellar-hexagonal bilayers and the addition of immunostimulatory lipids. Regarding size, it has been previously discussed that larger vesicles (> 2 µm) loaded with a tuberculosis (TB) antigen are likely to induce cell proliferation and low IL-10 induction, contrasting with vesicles around 500 nm that promoted a distinct set of cytokines (IL-1β and IFN-γ) [30]. A study by Brewer et al. presented the effects on immune response differentiation of large (> 225 nm) vs. small (< 155 nm) lipid vesicles [31]. Larger vesicles induced IL-12 cytokine production but smaller vesicles did not. Murine experiments revealed that large vesicles induced TH1 responses due to increased levels of IgG2a and IFN-γ. The smaller particles in this study induced a TH2 immune response due to increase of IL-5 and IgG1 levels. IL-1β triggers a TH17 immune response, whereas IFN-γ induces a TH1 immune response. Therefore, liposome size affects the differentiation of cellular immune responses, rendering this physical parameter a key role in liposomal formulation function.

Vesicle size could be affected by the storage period that the vaccine undergoes and by other environmental parameters. It is well recommended to perform stability studies in different environmental conditions to ensure that liposomal vaccine vesicles do not change over time [32]. Applying several methods during the formulation design and development (like spray drying, sterilization, cryoprotection and PEGylation) will lead to liposomal vesicle stabilization [33–35]. These methods or approaches mentioned earlier avoid unwanted changes in vesicle morphology that could subsequently affect the immune responses of the vaccine. Furthermore, vesicle size stability can be affected by the vesicle lipid composition [36]. An effect observed with unstable lipid vesicles is coalescence, but real-time methods have been developed to study the phenomenon and control stability [37]. Additional work has discussed vesicle stability extensively and we recommend the reader to review the appropriate literature [38–40].

Lamellarity nature of the liposome

The lamellar nature of the liposomal vesicle could also affect the immune system causing differential responses. Beck et al. studied the adjuvanted immune responses to a recombinant HIV protein, CN54 gp140, in small unilamellar (SUV) and large multilamellar vesicles (MLV) [41]. The liposomes were composed of different combinations of monophosphoryl lipid A (MPLA) and lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC; 1,2-dimyristoyl-sn-glycero-3-phospho-(1ʹ-rac-glycerol), DMPG; and cholesterol, Chol), with or without the addition of the saponin QS21. SUVs without QS21 could induce immune responses (characteristic of a TH2 cell-mediated response) to CN54 gp140 protein due to high antibody production, contrasting to MLVs. Adding the saponin QS21 restored immune responses in MLVs (higher IgG1 > IgG2a and IFN-γ titers), stimulating both TH1 and TH2 responses. The saponin did not influence SUVs immune response profile. Shek et al. presented one of the first experiments that compared vesicle lamellarity characteristics against antibody formation enhancement [42]. Liposomes composed of lecithin, dicetyl phosphate and cholesterol were prepared in the presence of bovine serum albumin (BSA). Animals injected with blank liposomes (no BSA) did not generate a significant immune response, as predicted. However, animals injected with BSA-loaded unilamellar vesicles (ULVs) generated strong immune responses compared with multilamellar vesicles (MLVs). Another seminal article presents the co-formulation or adsorption of bovine herpesvirus 1 proteins to large unilamellar (LUVs) and multilamellar (MLVs) liposomes composed of phosphatidylcholine (PC) as the main lipid component [43]. Strong antibody titers were detected in animals injected with LUVs prepared with virus proteins (both adsorbed and co-formulated) and egg PC. Recently, another study demonstrated the effects of the lamellar state for liposomes in subunit vaccines to induce immune responses [44]. SUVs with ovalbumin (OVA) induced greater levels of CD8+ IFN-γ responses against the protein in the spleen. Researchers added TLR3 and nine agonists, enhancing the immune responses in MLVs but not SUVs. Altogether, the studies demonstrate the effect of lamellarity of liposomes in immune responses, being SUVs the preferred state to potentiate innate and adaptive responses which improves vaccine efficacy.

Surface charge

The surface charge of liposomes would be of important consideration for appropriate vaccine design. The overall charge can determine the adsorption or antigen interaction with the liposomes (e.g. anionic antigens will prefer to interact with cationic lipids) which affects antigen loading in the vaccine [18]. Hussain et al. found that replacing the cationic lipid DDA with the neutral lipid distearoyl-sn-glycero-3-phosphocholine (DSPC) decreases the amount of the tuberculosis recombinant antigen H56 from 84 down to 15%. In addition, the function of the vaccine in relation to immune response induction is well documented. Joseph et al. were studying an intranasal influenza vaccine model based on the liposomal formulation of the HN antigen with the polycationic sphingolipid ceramide carbamoyl-spermine (CCS) or other monocationic, neutral and anionic lipids [45]. Neutral and anionic lipid-based formulations were not immunogenic upon intranasal administration in a murine model. However, two out of five monoccationic-based liposomal formulations (containing lipids 1,2-dimyristoyl-3-trimethylammonium-propane, DMTAP; and 1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) induced vigorous local and systemic immune responses (TH1 and TH2 type responses). Researchers compared the monocationic liposomal formulations with the CCS-based liposomal formulation and the only commercially available influenza vaccine, demonstrating the efficacy of the sphingolipid as an immunopotentiator with higher antibody titers and protective immunity for approximately 9 months. Another study with Newcastle disease virus compared the immunization effects in chickens of neutrally- (EPC-Lip), anionically- (PS-Lip) and cationically-charged (SA-Lip) liposomes [46]. Strong humoral responses (local and systemic) were observed in neutral formulations of EPC-Lip, contrasting to the cationic SA-Lip. The anionic formulation mainly composed of phosphatidyl serine (PS-Lip) elicited the higher hemagglutination titers. Recently, Hussain et al. determined that replacing the cationic lipid DDA with the neutral DSPC reduced the TH1-mediated immune response of the formulation [18]. Based on the studies presented above we can conclude that cationic formulations might be the most suitable option to elicit strong immune responses, increasing antibody titers. There is additional information available about cationic lipids, like DDA, which possess significant immunostimulatory and adjuvanting properties [47, 48] and further explanations will be provided ahead.

Bilayer fluidity

The bilayer fluidity, dependent on the lipid gel-liquid crystal transition temperature and its effects on immune responses, is of important interest when designing efficacious vaccines. Many published works have evaluated and described this phenomena, including the earliest work by Yasuda et al. [49]. The effects on immune responses of liposomes prepared with phospholipids of PC with different transition temperatures containing the hapten Dnp-Cap-PE were measured. DMPC, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPC (all with high transition temperatures Tm > 20 °C) were favorable in eliciting antibodies to the hapten, contrasting to results obtained with DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) and EPC (Tm < 0 °C). Another research team followed a similar experimental design preparing liposomes with low- (DOPC and DLPC, − 20–0 °C), intermediate- (DPPC and sphingomyelin, 25–40 °C) and high-transition temperature (DSPC, > 50 °C) lipids [50]. DMPC, DPPC and sphingomyelin induced immune responses as per a plaque-forming assay, but DSPC was a poor immunogen. Cholesterol was added to the liposomal formulations, inducing significant humoral immune responses. The results from these two groups produced different outcomes: Yasuda et al. [49] presenting that less fluid lipids are better immunogens than fluid phospholipids and van Houte et al. [50] establishing that intermediately fluid phospholipids (which have a phase transition temperature of 25–40 °C) are better immunostimulatory agents. This discrepancy might be attributed to other factors, like particle size and Zeta potential, but such factors were not reported or analyzed to determine their role in these studies.

Additionally, Mazumdar et al. revealed that liposome composition may have an effect on immune responses [51]. Here, researches incorporated a leishmanial antigen (LAg) in liposomes containing DMPC, DPPC or DSPC and described the immunization process and results in a hamster model. No significant delayed hypersensitivity was detected in DMPC- or DPPC-containing lipids, which contrasted with DSPC-containing lipids. Moreover, DSPC-containing lipids protected up to 95% of the hamsters against a leishmanial infection. Recently, Kaur et al. presented results on how cholesterol influences the bilayer fluidity [52]. For instance, a direct correlation of cholesterol and membrane fluidity was observed in DDA:TDB (trehalose dibehenate) liposomal formulations. However, less IgG was detected as cholesterol increased in the system after 12 days of immunization in mice. This effect might be due to the loss of antigen in more fluid (high cholesterol) liposomes as the authors pointed out. The cytokine IFN-γ was at elevated levels when cholesterol was not present in the lipid bilayer. Even with the compelling evidence of how transition temperature and lipid bilayer composition affects immune responses, we can find conflicting results in published data. For example, Hampl et al. found no significant influence of immune response induction based on liposomes containing phospholipids with different transition temperatures [53]. Although the data presented does not follow a clear pattern, the general conclusion is that the more fluid a liposome is the less immune response it will generate when administered in different animal models. Likely behind this phenomenon is that fluidity tends to increase the release of the antigen from the liposomes, affecting antigen presentation and consequently the strength of immune responses. We need to point out that cholesterol also participates as a bilayer stabilizer, increasing rigidity in biological membranes [54]. Therefore, we might consider that the increase in bilayer fluidity by cholesterol observed in the studies discussed above may occur in synthetic bilayers only. Further research must account for such correlation due to the complexities of biological membranes.

Immunostimulatory lipids and liposome deposition

Immunostimulatory lipids might be of value in the vaccine design process. The lipids can act as adjuvants in the vaccine formulation and enhance the immune response we are looking for (innate or adaptive) as previously reported and reviewed [2, 5, 6, 47]. In a study developed by Rao et al., two adjuvants (lipid A and CpG-containing oligodeoxynucleotides, CpG-ODN) were studied in a liposomal formulation based on the HIV envelop protein ogp140 [55]. Both lipid A and CpG-ODN-containing liposomes elicited six and threefold anti-ogp140 antibodies, respectively. Immunization of BALBc mice with the HIV antigen incorporated in lipid A-containing liposomes produced a mixed TH1/TH2 immune response. Combining both adjuvants in the liposomal formulation generated a TH1 immune response. Puangpetch et al. presented the effect of using zwitterionic or cationic lipids in vaccine liposomal formulations [2]. Researchers compared DOTAP (cationic phospholipid) vs. DOPC (neutral phospholid) containing the adjuvant CpG-ODN. DOTAP-based liposomes with adjuvant enhanced the immune response against Burkholderia pseudomallei, which suggest an alternative approach for the treatment of melioidosis. The published data available suggests that certain lipids can induce, or enhance, immune responses. Very important is the fact that cationic liposomes are the ideal model to design effective vaccines due to their immunostimulatory properties.

Finally, the immunostimulation of the vaccine can be affected by the deposition of liposomes at the site of injection which in turn is affected by particle size. Henriksen-Lacey et al. reported this in two separate articles [6, 56]. On Henriksen-Lacey et al. researchers utilized the phospholipid DDA as the building block for the liposomes containing the adjuvant TDB. The antigen for tuberculosis infection, Ag85B-ESAT-6, was co-administered or incorporated to liposomes in mice. Antigen administered alone to mice did not created a depot at the site of injection, causing the antigen to diffuse away from the site and reduce immune responses. The observations directed them to conclude that cationic liposomes made up of DDA promotes depot formation at the site of injection mainly due to size characteristics between antigen-loaded liposomes and antigen administered alone. Later, Henriksen-Lacey et al. [30] studied the effect of liposome composition [DOTAP, DDA or DC-Chol (dimethylaminoethane-carbamoyl-cholesterol)] and the depot formation and antigen distribution. DDA and DC-Chol represented the phospholipids where liposomes induced the migration of monocytes to the site of injection and a significant increase of IFN-γ levels. In general, larger liposomes will form a depot at the site of injection meanwhile smaller liposomes will migrate to lymphoid tissue for antigen presentation and processing.

Go to:

Liposome-immune cell interactions to improve vaccine effectiveness

Early vaccination strategies included the development of attenuated or inactivated vaccines, which are composed mainly of weakened or dead pathogens, respectively. Currently these kinds of vaccines are facing challenges with variabilities in immune response induction [57, 58]. Modern vaccination strategies are emphasizing the study of subunit vaccines and proving their effectiveness in immune response modulation [59, 60]. Subunit vaccines are characterized by the co-delivery of adjuvants and antigens for immunostimulatory purposes (Fig. 2). The antigen is either a natural or recombinant peptide, protein or molecule derived from the pathogen. The adjuvant will enhance the immune response of the vaccine and potentiate the effect of the antigen during the process. It is very important to know how to present the antigen incorporated in the liposomes to APCs because this will ensure their proper immune cell maturation, antigen presentation and eventual induction of adaptive immune responses as previously reviewed [61, 62]. Cell targeting studies have been investigated and produced significant data and observations of how antigen presentation plays a significant role. Aramaki et al. investigated the utilization of liposomes as carriers for antigens related to gut-associated lymphoid tissue and their uptake by rat Peyer’s patches [63]. Preferential uptake was observed for liposomes in the rat Peyer’s patches than non-patch tissues, specifically for DSPC:PS:Chol liposomes. This suggests that liposome composition affects their uptake. Fluorescently labeled liposomes were internalized by patch tissue in the lower ileum with size (> 374 nm) playing an important direct correlation with uptake. Another report looked at how cationic vesicles composed of DDA could interact with normal and transformed mouse fibroblasts cells [64]. Cell–cell adhesion was observed when DDA concentration was equal or greater to 50 µM. Investigators determined that normal cells were susceptible for DDA vesicles meanwhile transformed cells were resistant to DDA-mediated (> 1 mM) cell death. The interaction with cationic vesicles created a change in cell charge from anionic to cationic, making this system of cationic vesicles ideal for the delivery of negatively charged macromolecules like proteins and DNA.

Fig. 2

Co-delivery of antigen and adjuvant to APCs in subunit vaccines. Interaction of the adjuvant with the PRR results in upregulation of co-stimulatory molecules necessary for appropriate T cell stimulation

Vaccine interaction with neutrophils, monocytes and APCs

Several studies presented results on how liposome-based vaccines interact with neutrophils and monocytes, key players in inflammation and immune responses. First, Karathanasis et al. used previously characterized and purified peptides to target liposomal nanocarriers to some types of leukocyte cells [65]. Peptides were covalently attached through the carboxyl group of DSPE-PEG by utilizing the crosslinker N,N-dicyclohexylcarbodimide (DCC). Researchers found that targeted liposomes interacted better with monocytes and neutrophils, contrasting with results obtained by non-targeted liposomes. Moreover, the surface density of the peptide directly correlated with liposomes-cell interactions, making this parameter a new way to measure the effectiveness of the interactions and the potential induction of immune responses valuable in subunit vaccine development. Then, Johansen et al. investigated the targeting of monocytes and the delivery of a TLR agonist (TMX-202) using a cationic liposomes-based formulations (mainly POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine):DOTAP composition) [66]. After an hour incubation, the subset of monocytes targeted by the liposomes were lymphocytes and granulocytes (75–95%). A strong IL-6 and IL-12p40 induction was observed, accompanied by monocyte differentiation to CD14+/DC-SIGN+ DCs. Mainly found in the lymph nodes, lymphocytes include natural killer (NK), T and B cells, which are involved in innate immune responses, cell- and humoral-mediated immunities, respectively. Granulocytes, or polymorphonuclear (PMNs) leukocytes include basophils, neutrophils, mast cells and eosinophils that participate in allergic and inflammatory reactions. The information gathered by the researchers is important because it can determine future treatments and vaccine developments to focus on selected immune responses, depending on the cell or cell types that are being targeted, reducing or avoiding unwanted adverse reactions (e.g. allergies or chronic inflammation).

After interaction with the antigen, cytokines and/or interaction with their milieu, monocytes could differentiate into macrophages or dendritic cells. Both cell types will then migrate to lymph nodes to elicit the corresponding adaptive immune responses. Macrophages and dendritic cells are specialized APCs that reside in the blood stream and help in antigen uptake and antigen presentation to T and B cells, inducing cell-mediated and humoral immune responses, respectively. It is known that antigen-containing liposomes are internalized by pinocytosis in macrophages and then cross presented to CD8+ T cells (specialized T cells that attack and kill tumors), inducing antigen-specific cytotoxic T lymphocytes [67]. This cross-presentation occurs when exogenous antigen is up taken and presented via the class I major histocompatibility complex (MHC I), which classically only happens with endogenous antigens such as those from viruses. To cross-present and elicit a CD8+ T lymphocyte response, the antigen must be delivered to the cytosol APCs. This was studied by Owais et al. in which yeast-derived lipids liposomes and egg PC:Chol liposomes were tested against J774 A1 macrophages and the interactions measured [68]. The fusion rate for yeast lipid liposomes to macrophages was at 40–70%, compared to 1–8% for egg PC:Chol liposomes. Liposome contents were successfully delivered to the cytosol of macrophages. Ovalbumin was used as the model antigen for yeast-derived lipid liposomes and it was found that it elicited a strong CD8+ T cell response. Another group of researchers investigated the uptake mechanisms of liposomes in rat peritoneal macrophages (PM) [69]. Researchers determined that two uptake systems exist because of cholesterol content and size differences of liposomes. For high- (44% molar) and medium-cholesterol (33% molar) content liposomes, the complement receptor-mediated phagocytosis occurs. A complement-independent uptake pathway was suggested for low-cholesterol content liposomes since no inhibition of their internalization rate was observed by the anti-C3 antibody. Therefore, once again, lipid composition is a main player in liposome-cell interactions, potentially affecting the way immune responses develop.

Mannosylated liposomes

Mannosylated liposomes are becoming an alternative method to deliver antigens or antimicrobials to macrophages or DCs [70, 71]. This approach is due to the fact that mannose receptors can be found in these cell types, improving directed-targeting [16]. Other research teams have used cationic lipids like DDA, DOTAP and/or DC-Chol to enhance or potentiate the immune responses due to cell targeting strategies [48, 72, 73]. Korsholm et al. determine that DDA liposomes were minimally internalized by T cells in the mixed splenocyte cultures, contrasting to high uptake rates for APCs (bone marrow dendritic cells) through class II MHC (MHC II), leading to an enhanced OVA presentation. The immune responses described by Varypataki et al. contrasted to Korsholm et al. since DOTAP:DOPC liposomes bearing the peptide SIINFEKL and polyI:C assisted in the delivery of OVA to DCs through MHC I, inducing a CD8+ T cell response.

Understanding and applying innovative ways of cell targeting could improve the discovery pipeline for novel therapeutic agents, avoiding undesirable immune responses that can be detrimental to the patient. These therapies could treat inflammatory diseases [like arthritis or chronic obstructive pulmonary disease (COPD)], infections or cancer. The following sections will focus on how liposome-based vaccines are being utilized for the treatment of infections from viral, bacterial, fungal and parasitic origins. A special section will discuss liposomal vaccines that target the bacterium Mycobacterium tuberculosis to treat TB infections.

Go to:

Viral infections and liposomes-based vaccines

Viruses are ethiologic agents of different diseases in animals [74] (including humans [75, 76]), plants [77], parasites [78, 79] and bacteria [80, 81]. The basic definition of a virus is a non-living infectious agent that requires living cells for its replication and survival, which allows spreading of the disease. This cell lysis leads to inflammation and tissue damage which are detrimental for the host, enhancing the state of the disease. Additionally, some viruses, like HIV, destroy immune cells hindering effective immune responses when other infections (secondary infections in seropositive patients) occur. Viral infections can be treated by either antiviral therapeutic [82] or by prophylactic vaccine [83] approaches. Here, we will present several examples of vaccine applications based on liposomes for the prophylactic treatment of certain viral infections.

Hepatitis

One of the most significant viruses that affect human health are hepatitis viruses. Hepatitis is an inflammation of the liver tissue caused by the five types of hepatitis viruses (A, B, C, D and E). Hepatitis A and E are spread through contaminated food or water sources. Hepatitis B (HBV) is sexually transmitted or during pregnancy and birth. Both HBV and hepatitis C can be transmitted through blood (needle exchange by IV users) and hepatitis D can only infect people infected with HBV. Two seminal reports investigated novel approaches for the prophylactic treatment of HBV utilizing cationic lipids. Brunel et al. reported the effectiveness of recombinant hepatitis B surface antigen (HBsAg) presentation based on DC-Cholesterol liposomes or aluminum hydroxide (alum) adjuvants in a subcutaneous vaccine model [84]. The DC-Chol-based vaccine elicited antibody (IgG1 and IgG2a) titers in three mice lines (BALB/c, OF1 and B10.M). Compared to the alum-based vaccine, which demonstrated weak immunogenicity, DC-Chol liposomes induced controlled TH1 and TH2 immune responses characterized by normal, but significant levels of cytokines IL-2 and IFN-γ and IL-5, respectively. Controlled immune responses are important to avoid inflammation that could result in tissue damage. The researchers concluded that cationic lipids like DC-Chol could be used as adjuvants, enhancing the immunogenicity of previously non-immunogenic vaccines, specifically in the development of prophylactic vaccines against hepatitis B virus. Another group investigated the use of a transcutaneous vaccine for the treatment of HBV infections [85]. In that report, the vaccine presents some differences from the Brunel et al. article based on the antigen and carrier types. First, cationic transfersomes, a type of liposome, were prepared from DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) phospholipid and sodium deoxycholate (SDC) at different DOTMA weight ratios (75–95% w/w). Second, plasmid DNA encoding the HBsAg gene was loaded to the transfersomes instead of the antigen. The transfersomes were not cytotoxic to HepG2 cells and were stable at different temperatures (4 and 28 °C). Immunization studies included HBsAg DNA-loaded tranfersomes (topical), naked HBsAg DNA (topical and intramuscular) and pure HBsAg (intramuscular) administered to BALB/c mice. Significant levels of anti-HBsAg antibodies and cytokines (IL-2 and IFN-γ) were elicited in topical DNA-loaded transfersomes as compared to intramuscular naked DNA delivery. This antibody and cytokine profile confirmed the induction of TH1 and TH2 immune responses as observed in Brunel et al. Both studies represent great advances in the treatment of HBV infections with novel approaches and administration routes that could applied in the future as preventive vaccines.

Influenza

Influenza is an infectious disease caused by the influenza virus, affecting human health with a pandemic effect in several instances. The virus is divided in three types (A, B and C) and it is spread through the air from coughs and sneezes, therefore the importance for vaccine development studies for the infection. An intranasal (i.n.) liposomal influenza vaccine study was presented by Joseph et al. [45]. The vaccine formulation developed by the group was based in the polycationic sphingolipid N-palmitoyl-d-erythro-sphingosyl-carbamoyl-spermine (or ceramide carbamoyl-spermine, CCS) and was compared with other formulations containing monocationinc phospholipids (DC-Chol, DDA, DSTAP (1,2-stearoyl-3-trimethylammonium-propane), DMTAP and DOTAP). Cholesterol was added to increase liposome fluidity and the lipids DMPC and DMPG were included in neutral and anionic formulations, respectively. All formulations contained the influenza A antigens hemagglutinin and neuraminidase (HN). DMTAP- and DOTAP-based vaccines were the only monocationic lipid formulations to induce strong systemic (serum) and local (lung) TH1 and TH2 responses. Surprisingly, DDA-containing formulations did not induce strong, or significant, local or systemic immune responses. No specific reasons were provided by the team for such results but we can infer that vesicle morphology (multilamellar and oligolamellar cationic formulations), size (1–4 µm diameter) and encapsulation efficiencies (10–90% for non-CCS cationic formulations) would be responsible. The CCS-based vaccine formulation was the only formulation to be at the same or superior level of effectiveness compared to the commercially available vaccine with cholera toxin as the adjuvant. More recently, researchers investigated a triple co-culture model of the human respiratory tract to study the immunostimulatory responses of virosomes and liposomes and their internalization [86]. The epithelial cell line 16HBE was grown with monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) and exposed to liposomes and virosomes to evaluate the immune responses elicited by the nanocarriers. The virosomes were liposomes prepared with solubilized influenza A/Brisbane/59/2007 H1N1 membrane proteins and included the neutral lipids DOPC and OPPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE). Liposomes were prepared with previously mentioned neutral lipids and no influenza soluble membrane proteins were added. Virosomes were internalized more efficiently by all cell types in mono- and co-cultures, with APCs like MDMs and MDDCs presenting the highest internalization levels as per flow cytometry and laser scanning microscopy. MDDCs were moderately activated by liposomes and virosomes in monocultures, and inducing elevated levels of cytokine (IL-1β and IL-8) production in co-cultures. Virosomes were internalized at higher levels in epithelial cells in comparison to liposomes.

Respiratory syncytial virus

Another respiratory viral pathogen of interest is the respiratory syncytial virus (RSV). RSV causes respiratory tract infections, specifically lower respiratory tract infections in children and it presents high hospitalization incidence [87]. An i. n. study was developed by Klinguer et al. in which cationic DDA liposomes were mixed with the recombinant fragment of the RSV G protein (BBG2Na) and administered to BALB/c mice [88]. The DDA + BBG2Na liposomal formulation presented significant antibody (IgG and IgA) titers at systemic (serum) and local (nasal) levels. Cytokine production was higher for IL-2 and IFN-γ in cationic liposomes carrying the RSV recombinant antigen, confirming the protection after a viral challenge and the induction of TH1 immune response. These three reports on respiratory viruses and their prophylactic vaccine development confirms the importance of mucosal immunization as it elicits local and systemic B- and T-cell responses [89]. It is also clear that cationic phospholipids like DDA, DC-Chol or DOTAP play a leading role (Table 3) in the immunostimulation of subunit vaccines and more approaches should be investigated. Different administration routes were investigated in the studies for viral infections. However, we cannot verify and compared these administration routes to determine the best alternative due to experimental differences in the studies (liposomal composition and targeted virus). Future experiments should focus their efforts in comparing different administration routes with the same liposomal composition and prophylactic treatment of a particular virus.

Table 3

Promising vaccine formulations for the treatment of viral infections

Lipid(s) and sterol used	Virus type	Cell line/animal model used	Administration route	Promising liposome formulation	References
DOTMA	Hepatitis C	HepG2 cells and BALB/c mice	Transcutaneous	DOTMA	[85]
DC-Chol	Hepatitis B	Lymph node cells, BALB/c, OF1 and B10.M mice	Subcutaneous	DC-Chol	[84]
DMPC, DMPC/DMPG, DC-Chol/DOPE, DSTAP/Chol, DDA/Chol, DOTAP/Chol, DMTAP/Chol and CCS/Chol	Influenza H3N2	Splenocytes from BALB/c and C57BL/6 mice	Intranasal	DMTAP/Chol and DOTAP/Chol	[45]
DDA	Respiratory Syncytial Virus	BALB/c mice	Intranasal	DDA	[88]
DOPC/OPPE (Influenza virosome) and DOPC/OPPE (liposomes)	Influenza	MDMs, MDDCs, 16HBE14o cells, PHNECs and EPCam + cells	N/A	DOPC/OPPE (Influenza virosome)	[86]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

Bacterial infections and liposomal vaccines

Bacteria can be beneficial for humans, but also detrimental to our health when they harbor pathogenicity traits. Bacteria are divided in Gram positive (+) or negative (−) based on Gram staining, that surveys the peptidoglycan content in the cell wall. Gram (+) bacteria have a positive result in the Gram Stain method, which determines in a qualitative way the presence of the cell wall component: a thick peptidoglycan layer. In contrast, Gram (−) bacteria present a negative result in the stain, indicating a thin peptidoglycan layer, sandwiched between two cell membranes (plasma and outer membranes). Gram (−) bacteria also contain an important immunogenic molecule and pathogenicity factor, the lipopolysaccharide (LPS) [90, 91]. LPS has been used to increase the fusogenicity of cationic liposomes [92]. In that study, researchers developed a carrier system to incorporate LPS into mammalian cell membranes via DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine):DOTAP (1:1 wt. ratio) liposomes. The team of researchers demonstrated that high LPS concentrations on immortalized fibroblasts generated the activation of macrophages, starting the elimination of LPS-bearing cells.

Additionally, bacteria will have unique cell components, like genetic material, lipids or proteins, that could serve as adjuvants or antigen markers for subunit vaccine development, depending on the molecule chemical characteristics. Li et al. investigated the potential use of cationic (DDA-based) mannosylated liposomes to deliver the model DNA plasmid pGL4.10 (encoding luc2) [19]. The plasmid was protected from nuclease degradation by the liposomes. The cationic mannosylated liposomes showed high uptake and transfection, activating bone marrow DCs (BMDCs). BMDCs activation was characterized by the upregulation of CD80, CD86 and CD40. Another study by Nakanishi et al. studied the effects of positively, negatively and neutrally charged liposomes administered subcutaneously in the immune responses of the fragment A of diphtheria toxin (DTA) and ovalbumin (OVA) [93]. Cationic liposomes were composed of phosphatidylcholine:cholesterol:stearylamine (PC:Chol:SA, 4:5:1 molar ratio), anionic liposomes were composed of PC:Chol:l-α-dimirystoylphosphatidic acid (PC:Chol:DMPA, 4:5:1) and neutral liposomes were composed of PC:Chol (1:1). Positively charged liposomes could induce potent antigen-specific cytotoxic T cell responses. However, DTA-containing cationic liposomes were cytotoxic to macrophages. In contrast, empty cationic liposomes or DTA-loaded anionic and neutral liposomes were not cytotoxic. CD8+ OVA responses were highly induced by positively charged liposomal vaccines, potentially presenting the processed antigen through MHC I. Both research articles presented us the utilization of liposomes to investigate and test the effects on immune responses during vaccination. The immune responses measured varied, but making it clear that cationic liposomes induced the required cells (macrophages and DCs) to obtain the appropriate responses.

The liposomal vaccine studies mentioned above serve as the basis for the following articles which incorporate bacterial antigens for the development of prophylactic vaccines for certain infections. Puangpetch et al. developed a cationic-based liposomal formulations incorporating CpG ODN and to determine the prolongation and mechanisms of the immune responses [2]. The researchers employed the etiologic agent of melioidosis, Burkholderia pseudomalei, as the infection model in BALB/c mice. Cationic and not neutral liposomes administered intramuscularly granted protection against the bacterial challenge study. Prominent levels of IFN-γ were observed 2 days postinfection, but lowered by a CpG ODN-loaded cationic liposome pre-treatment. Neutrophils were not activated by the cationic liposomes with CpG ODN, but macrophages were stimulated by the formulation due to nitric acid production and low intracellular bacterial burden (30 days post vaccination). An additional study involving mannosylated liposomes containing the meningococcal PorA (from Neisseria meningitidis) focused its attention on cell interaction [94]. Anionic (PG- (phosphatidylglycerol) and PS-based) and cationic (DMTAP-based) liposomes were formulated, and one of the anionic formulations was mannosylated (PC:PG:Chol + Man-PE (mannosyl phosphatidylethanolammine). When exposing the formulations to human and murine DCs, researchers observed an increase of liposome-cell interaction in the anionic mannosylated liposomes and cationic liposomes when compared to anionic formulations alone. The result indicated that adding mannosyl moieties to liposomes generated a mannose receptor (MR)-mediated cell interaction. The murine DCs were confirmed to present the markers MHC II+, CD11c+ and CD11b+, meanwhile human DCs presented CD40+, CD1a+ and both MHC I and II. Researchers in the field should address studies that investigate route of administration effects on immune responses. With the studies presented here, it is difficult to determine what best route of administration we should follow for future prophylactic vaccine development. We recommend investigating the optimized formulations in different administration route studies.

The use of cationic liposomes seems of importance for the development of adequate vaccine formulations. The studies presented above demonstrate that by employing cationic phospholipids like DOTAP, DMTAP and DDA, would improve cell interaction levels, allow adequate antigen presentation and induce strong immune responses. These effects will insure that the vaccine will work properly and optimally. For the benefit of the reader, Table 4 presents a summary of the most relevant literature that optimized vaccine formulations for the treatment of bacterial infections. Additionally, previous research on cationic liposomes have discussed the cytotoxicity potential of such formulations. DDA has been determined to be safe as no relevant cytotoxic effects were determined in studies by Hilgers and Snippe and Gall [47, 95]. Only local inflammatory reactions manifested as swelling were observed in mice (when administered alone). Contrasting results are found for DOTAP and DOTMA cationic lipids. DOTAP has been found to be not cytotoxic to macrophages in a study by Jin et al. [96], but Romøren et al. determined that macrophage-derived cell lines were found to be affected by the cationic lipid [97]. The cytotoxic response differences might be due to structural and morphological characteristics in the formulations. Jin et al. employed Tween 20 and tricaprin (part of the solid core) to form solid lipid nanoparticles, meanwhile Romøren et al. just prepared liposomes. Further contradictory information is available for DOTMA, which can be cytotoxic for RAW 264.7 cells at all lipid ratios (DOTMA + DOPE), but not to human umbilical endothelial cells or mouse fibroblasts cells [98]. Kurosaki et al. determined that erythrocytes undergo agglutination and hemolysis when exposed to DOTMA-based liposomes [99]. However, an earlier study by Kurosaki et al. determined lower cytotoxicity for erythrocytes, showing no agglutination and hemolysis, when DOTMA was formulated with N-laurylsarcosine, and Chol, vitamin E and Chol or egg PC and Chol [100]. Future work should include the analysis of lipids alone and formulated with other lipids to elucidate the cytotoxic effects. Additional work should be done for these and other bacterial infections lacking proper prophylactic vaccines, leading to outcome improvement from the infection.

Table 4

Promising formulations for the treatment of bacterial infections

Lipid(s) and sterol used	Bacteria or disease	Cell line/animal model used	Administration route	Promising liposome formulation	References
PC/PG/Chol, PC/PG/Chol/Man-PE, PC/PS/Chol, PC/DMTAP/Chol	Meningitis	Monocyte-derived human DCs and murine bone marrow-derived DCs	N/A	PC/PG/Chol/Man-PE and PC/DMTAP/Chol	[94]
PC/Chol/SA, PC/Chol/PA and PC/Chol	Diphteria toxin	BALB/c, C57BL/6 and ddY mice; P815, P13.1 and CD8OVA cells	Subcutaneous	PC/Chol/SA	[93]
DOTAP and DOPC	Melioidosis	Neutrophils and splenocytes from BALB/c mice	Intramuscular	DOTAP	[2]
DOPE/DOTAP	E. coli	Mouse embryonic fibroblasts (MEF) and RAW 264.7 macrophages	N/A	DOPE/DOTAP	[92]
DDA/Chol/Man-C6-Chol and DDA/Chol	E. coli	DC 2.4 cells	N/A	DDA/Chol/Man-C6-Chol	[19]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

Liposomal-based vaccines in fungal infection treatment

Fungi are eukaryotic organisms that include yeasts, molds and mushrooms. Several yeasts and molds are common pathogenic agents, especially in immunocompromised patients [101, 102]. Antibiotic resistance is being detected, not only in isolates from the USA but also in other developed countries, in different fungal species (like Candida and Aspergillus) [103]. Due to the threat of antifungal resistance, other therapeutic approaches should be investigated, and liposomal vaccines may play a significant role. To the best of our knowledge, the literature review presents liposome-based vaccines for Candida sp. infections but limited information is available for other fungal pathogens. Studies investigating other fungal species with elevated infection prevalence, like Aspergillus, Fusarium, Coccidioidomyces and Zygomycetes sp., have been identified but their treatment approach does not include liposomal vaccines, and we invite further reading on the topic [101, 104, 105]. We will discuss ahead information available for liposomal vaccine development for the prophylactic treatment of Candida sp. infections (Table 5).

Table 5

Candidiasis treatment with potential liposomal vaccines

Lipid(s) and sterol used	Fungi or disease	Cell line/animal model used	Administration route	Promising liposome formulation	References
PC/Chol	C. albicans and C. tropicalis	BALB/cByJ mice	Intravenous	PC/Chol	[106]
PC/Chol		BALB/c mice	Intravenous	PC/Chol	[107]
DMPC/DMPG	C. albicans	ICR mice	Subcutaneous	DMPC/DMPG	[108]
EPD/DOGS-NTA-Ni		BALB/c mice	Intradermal	EPD/DOGS-NTA-Ni	[109]
		ICR mice	Intradermal		[110]
DDA:MO		Macrophages and BALB/c mice	Subcutaneous	DDA:MO	[111]
		BALB/c mice	Subcutaneous		[112]

The first report dealing with the study and development of a vaccine to treat candidiasis, encapsulated the mannan adhesin portion of Candida albicans [106]. The adhesin protion of the mannan of two C. albicans serotypes (A and B) were incorporated in PC:Chol liposomes (3.2:1 molar ratio). Mice were vaccinated (intravenously) during a period of 5–6 weeks and challenged with C. albicans infection, presenting increasing resistance to disseminated disease. Furthermore, antiserum agglutinins (IgM-type antibodies) from the immunized mice were studied for their humoral protective characteristics against C. tropicalis infection, demonstrating the efficacy of the vaccine. Subsequently, researchers decided to investigate the effectiveness of the monoclonal antibodies obtained from the previous study in a vaginal candidiasis model [107]. Similarly, the vaccine (L-mann) was prepared by the mannan adhesin fraction incorporation into PC:Chol liposomes. Mice were immunized intravenously once a week for 5 weeks prior vaginal inoculation. Vaccinated mice challenged with C. albicans presented lower CFUs (110 ± 38 × 103 CFU/g of vaginal tissue) when compared to non-mannan vaccine approach (240 ± 44 × 103 CFU/g of vaginal tissue). Both article demonstrated the capacity of the vaccine to protect at local or systemic infections of C. albicans.

Further studies investigated zwitterionic and cationic liposomes utilizing ribosomes and recombinant Hsp90 protein as antigens from C. albicans [108–110]. Eckstein et al. prepared anionic DMPC:DMPG liposomes co-lyophilized with RNA obtained from cell lysates of C. albicans cultures or by lipid film formation. Mice protection in a subcutaneous vaccination (60% survival) against the fungal challenge demonstrated the effectiveness of the vaccine prepared by the co-lyophilization method in the presence of C. albicans ribosomes. Subsequently, neutrally charged metalloliposomes with incorporated recombinant Hsp90 (heat shock) protein from C. albicans were developed. Nickel-chelating liposomes were prepared with EPC and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DOGS-NTS-Ni) at molar ratios of 95:5. A non-pyrogenic MDP was used as an adjuvant in the liposomal system and the protein was surface attached by metallochelating bonds. DCs interacted with the liposomes and phagocytosed the nanoparticles in vitro. TH1 and TH2 immune responses were induced after intradermal mice vaccination in comparable levels to the Freund’s complete adjuvant vaccine. Following the previous report, Knotigová et al. employed nickel-chelating liposomes with different MDP-derivatives (norAbuMDP/GMDPs) and tested their adjuvant vaccine potential [110]. Also, Hsp90 from C. albicans was employed as the model antigen. Adaptive and innate immune responses were induced by the developed vaccine systems in intradermal vaccinated rabbits and mice.

Recently, cationic liposomes studies incorporated cell wall surface proteins (CWSPs) of C. albicans and their immunostimulatory properties analyzed during subcutaneous administration [111, 112]. Both studies employed DDA:monooleoylglycerol (MO) at 33:67 molar ratio. Liposomes were not toxic to macrophages and were internalized within 20 min of exposure. In the first study, immunized mice displayed strong humoral- and cell-mediated immune responses. Antibodies were produced against cell wall proteins Cht3p and Xog1p. In the second report, two CWSP-loaded cationic liposomal formulation (ADS1 and ADS2) were tested against disseminated candidiasis. ADS1 immunized mice presented significantly higher levels of C. albicans antibodies, contrasting with the ADS2 formulation. This antibody titer production induced the phagocytosis of the fungus. Elevated levels of the cytokines IL-4, IL-17 and IL-10 were significantly higher than control groups, suggesting TH2, TH17 and anti-inflammatory immune responses, respectively.

Future studies on fungal infections and their prophylactic treatment with vaccines should be performed not only in C. albicans, but also other ethiological agents (e.g. Aspergillus). The literature review revealed a field with potential for growth and development of novel approaches to treat fungal infections, which benefit immunocompromised patients (elderly or HIV-seropositive patients). Additionally, further studies must employ further cationic lipids and their effects on immune response induction. Studies comparing physicochemical properties of liposomes to treat fungal infections must take place to optimize the vaccine strategy and avoid unwanted responses and results. In the studies presented above, the intravenous administration was investigated and revealed affirmative results (~ 60% survival of mice). However, we recommend further studies that compare immunomodulatory responses against mucosal vaccine administration for vaginal candidiasis infections. Similar survival rates were observed for subcutaneous and intradermal administration routes, but future studies should investigate a particular set of liposomes against the different administration to observe if survival rates are affected.

Go to:

Parasitic infections and liposomal vaccines

Parasitism and malaria

Parasitism is a non-mutual, biological interaction in which the parasite lives inside the host and derives its own nutrients at the host’s expense. Parasites can be classified as macroparasites (visible with the naked eye) like helminths, or microparasites (which are smaller) like viruses, bacteria or protozoa. A textbook example of a parasite is Plasmodium vinckei, causative agent of malaria. In malaria, the parasite (Plasmodium sp.) is transmitted by mosquito bites. The parasite’s sporozoites reside in the liver (in humans), developing into merozoites that infect human red blood cells, initiating the red blood cell cycle. When appropriate, the merozoites will developed into gametocytes that infect more red blood cells that are taken up by mosquitoes during the bites. This initiates the mosquito stages (gametes, ookinetes and oocysts). Oocysts are transmitted to the host, initiating the liver stage.

Postma et al. developed a novel desferrioxamine B (DFO) delivery system based on liposomes to treat malaria [113]. DFO is a siderophore that chelates ferric iron (iron is an vital component of red blood cells). Iron is an important nutrient for P. vinckei as it infects red blood cells. Contrasting to previous liposomal vaccine articles, the researchers investigated the lipid to drug composition and the bilayer fluidity effects on DFO delivery and protection from infection. The lipids were all anionic in charge due to the presence of egg PG. Three different treatments of DFO were analyzed in the study: (1) multiple free DFO subcutaneous injections, (2) intraperitoneal infusion of free DFO and (3) multiple subcutaneous DFO-loaded liposomes injections in C57B1/6J female mice. Parasitemia was suppressed by multiple subcutaneous injections of free DFO before and during infection, but injections prior to infection did not. Suppression of parasitemia and long-term survival was observed for intraperitoneal infusion of free DFO 1 day before infection or by subcutaneous injections of liposomal DFO prior to infection (day-1). Bilayer rigidity was studied by incorporating Chol (intermediate rigidity) and DSPC (high rigidity) in the liposomes, demonstrating that no relationship exists that affect the liposome antimalarial function. However, drug-to-lipid ratio affected the antimalarial activity of the liposomal-based vaccine, suggesting that low drug-to-lipid ratios are the best formulation parameter at combating the infection. When liposomal DFO was administered to mice, a long-term protection against malaria was observed (days 7 and 8, 400 mg/kg/day). This article proves that utilizing a common siderophore (DFO) as an iron chelator in combination with liposomes, improve the therapeutic and prophylactic effects of the vaccine. However, no immune response studies were performed, lacking essential information about the mechanisms of the vaccine when immunization occurs.

A pre-clinical report of the malaria vaccine RTS,S was published by Stewart et al. [114]. The RTS,S/AS02A vaccine utilizes the circumsporozoite protein as antigen. Investigators in this report evaluated the effects of certain adjuvants (AS01B, AS02A, AS05 and AS06) which vary in the concentration of MPL, QS21 or CpG and their formulation delivery system (emulsion vs. formulation). AS01B was the only liposomal formulation in the study. Rhesus macaques were immunized by intramuscular injection with the different RTS,S/adjuvant combinations and specific antibodies, IFN-γ and IL-5 levels were determined after weeks 14 and 34. All regimes were safe and presented elevated antibody titers (except for AS06-containing vaccine formulation). RTS,S/AS01B presented higher levels of IFN-γ at weeks 14 and 34, and the highest IFN-γ to IL-5 ratio when compared to RTS.S/AS02A. Leishmaniasis.

Another parasitic disease is leishmaniasis caused by the parasite Leishmania sp. It is transmitted by sandflies to mammals, transferring metacyclic promastigotes via feeding. The metacyclic promastigotes invade macrophages and granulocytes, developing into amastigotes which multiply by simple division, eventually causing macrophage lysis. Further, amastigotes infect new macrophages or are transferred to the sandflies during feeding. Amastigotes transform into procyclic promastigotes in the gut, maturing into metacyclic promastigotes by simple division. The disease is common in certain regions of Asia, Africa, South and Central America and even southern Europe. The disease is divided into three major syndromes, cutaneous, mucosal or visceral leishmaniasis. Visceral leishmaniasis poses a major risk of death incidence [115].

Three seminal articles provide valuable information regarding the studies and development efforts towards a prophylactic vaccine for leishmanial infections [116–118]. First, Bhowmick et al. presented the immunotherapy effects of leishmanial antigens in liposomes [116]. The researchers correlated the efficacy of soluble leishmanial antigens (SLAs) from Leishmania donovani promastigote membrane incorporated in neutral (lecithin:Chol), negative (lecithin:Chol:PA (phosphatidic acid)) and positively (lecithin:Chol:SA) charged liposomes intraperitoneally administered. L. donovani was eliminated from the liver and spleen when SLAs were present in cationic lipids. IL-4 and IL-10 were downregulated when SLA-cationic liposomes were administered to the mice and the immunomodulatory response presented the TH1 cytokines IFN-γ and IL-12. Subsequently, Banerjee et al. presented two articles which cover the study of cationic stearylamine liposomes for the development of a visceral leishmaniasis vaccine. In the first published article by the team of researchers, amphotericin B (AmB) is used in association with stearylamine (cationic) liposomes as a novel therapeutic approach [117]. When administered to BALBc mice, the leishmanial parasite was eliminated from the liver and spleen. Moreover, when comparing to the conventional liposomal formulation AmBisome, the intravenous administration of AmB-SA-PC liposomes induced the production of IFN-γ from CD8+ and CD4+ T cells. At the same time, the formulation reduced the toxicity effects of the drug by reducing TNF-α levels. In the splenic supernatant culture, IL-10 was downregulated, causing the production of IL-12 and nitric oxide during the AmB-SA-PC liposomes treatment. Also, Banerjee et al. investigated the effects of liposome charge in an antileishmanial assay [118]. Researchers provided evidence of membrane disruption caused by the cationic stearylamine liposomes in promastigotes and amastigotes. No toxicity in murine peritoneal macrophages and human erythrocytes was detected. These studies confirmed the prophylactic effect of SA-PC liposomes against leishmanial infections.

Amoebiasis

Finally, a research paper dealing with parasitic infections present us the incorporation of Entamoeba histolytica Gal/GalNAc lectin LecA antigen in liposomal, emulsion and alum formulations containing synthetic TLR agonists adjuvants [59]. E. histolytica is an anaerobic amoeba and is the etiological agent of amoebiasis, or diarrheal disease commonly transmitted through contaminated water and food sources [119]. The liposome formulation containing a TLR4 and TLR7/8 agonists was selected for further studies due to its ability to induce intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17a. A high mucosal IgA response hinder the ability of parasites to adhere to mammalian cells. The subcutaneous immunization regime success rate reached 55% efficacy.

Parasitic infections are common especially in underdeveloped regions, posing a notable risk for children, adults and the elderly. Further studies should address the use of adjuvants and their immunomodulatory mechanisms when administered in liposomes for vaccine development. Several studies previously discussed optimized certain formulations for vaccine development and we invite the reader to have a look at them (Table 6). Additionally, specific antigens should be employed to induce even more specific immune responses that will enhance the eradication of relevant parasitic diseases like leishmaniasis and amoebiasis. Furthermore, studies should consider administration routes as potential factors that may affect the immunomodulatory responses of vaccines for the treatment of parasitic infections. For instance, toxicity of L. donovani was reduced when administered intravenously and differences in the immune response cytokine profile were detected. These cytokine profiles must need to be addressed, conducting studies with the similar liposomal formulation and administering the vaccines through different routes.

Table 6

Liposomal vaccine formulations tested in parasitic infection models

Lipid(s) and sterol used	Parasite	Cell line/animal model used	Administration route	Promising liposome formulation	References
MPL/QS21 (liposome-based)	Plasmodium falciparum	Rhesus macaques	Intramuscular	MPL/QS21 (liposome-based)	[114]
Egg lecithin/Chol, egg lecithin/SA and egg lecithin/PA	Leishmania donovani	BALB/c mice	Intraperitoneal	Lecithin/Chol/SA	[116]
PC/Chol, PC/SA, PC/PA and PC/PS	L. donovani	–	Intravenous, N/A	PC/SA	[117, 118]
EPC/EPG, EPC/EPG/Chol and DSPC/DPPG/Chol	P. vinckei	Female C57BL/6J mice	Intraperitoneal	EPC/EPG, EPC/EPG/Chol and DSPC/DPPG/Chol	[113]

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

DDA-based cationic liposomal vaccines for the treatment of tuberculosis

The World Health Organization (WHO) estimated 1.8 million deaths in 2015 were related to tuberculosis infections (TBIs) [120]. The global estimate for latent tuberculosis infections (LTBI) was recently determined to be 23% (approximately 1.7 billion persons from the total population) [121]. Because of current tuberculosis (TB) vaccine efficacy variability, inefficiency and waning immunity (like Bacillus Calmette–Guerin, BCG) [57, 58], it is imperative to develop novel vaccines strategies that will improve prophylactic avenues for TBIs and reduce the overall death rate or latency associated with them. One of the principal improvements revealed in the last years is the development of adjuvanted subunit vaccines.

To diminish the detrimental effects of tuberculosis in humans, certain studies have focused on subunit vaccine development based in cationic lipid formulations (Table 7). Adjuvants are required for the development and efficacy of subunit vaccines, potentiating immune responses. Several adjuvants have been studied in the past 20 years including the adjuvanting properties of the lipid DDA [122]. Forty years ago, Snippe et al. investigated the effect of DDA in mice (via an intracutaneous administration) determining that delayed hypersensitivity occurred by the onset of footpad swelling 5 days after vaccination [123]. Subsequently, a study by van Houte et al., where low- (DOPC and DLPC) and high-transition temperature (DSPC) lipids were utilized with DDA as liposome bilayer components, discovered less immunogenicity of the liposomes as DDA concentration decreased [50]. Additionally, an earlier review discussed the immunostimulatory properties of DDA and its uses in different vaccines for veterinary and human infections [47]. We will present the reader with the most significant studies performed that cover the utilization of the cationic lipid DDA in subunit vaccine development.

Table 7

DDA-based TB vaccine formulation optimization studies

Lipid(s) and sterol used	Cell line/animal model used	Administration route	Promising liposome formulation	References
DDA, DDA/Tween 80, DDD/Span 85, DDA/Tween 80/Span 85, DDA/gelatin, DDA/Chol, DDA/Lecithin, DDA/β-Cyclodextrin and DDA/PLGA	C57BL/6 mice	Subcutaneous	DDA/Chol	[126]
DDA, DOTAP, DC-Chol, DOPE/PC and DOPE/PC/PG	Splenocytes from BALB/c and C57BL/6 mice	Subcutaneous	DDA	[125]
DDA/DSPC	Splenocytes from C57BL/6 mice	Intramuscular	DDA/DSPC	[18]
DDA	Splenocytes from BALB/c mice	Subcutaneous	DDA	[6]
DDA	Human/macrophage cell line THP-1 and splenocytes from BALB/c mice	Intramuscular	DDA	[30]
DDA	Inguinal lymph nodes or spleens	Subcutaneous	DDA	[128]
DDA	C57BL6; spleen and lung lymphocytes	Subcutaneous	DDA	[124]
DDA and DDA/Chol	Splenocytes from C57BL/6 mice and THP-1 cells	Intramuscular	DDA	[52]
DDA/MMG	Splenocytes from C57BL/6 mice	Subcutaneous	DDA/MMG	[134]
DDA	BALB/c mice	Intracutaneous	DDA	[122]
DDA	C57BL6 mice	Subcutaneous	DDA	[129]
DDA	BALB/c mice	Intracutaneous	DDA	[123]
DDA	Splenocytes from BALB/c mice	Intramuscular	DDA	[44]
LAM/PC/Chol/stearyl octaarginine	PBMCs	N/A	LAM/PC/Chol/stearyl octaarginine	[17]
DDA	DCs	Subcutaneous	DDA	[135]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Holten-Andersen et al. mixed the recombinant immunodominant M. tuberculosis antigens (ESAT-6 and Ag85B-ESAT6), DDA and different immunomodulators, analyzing the immune responses against BCG vaccination in mice (subcutaneous administration) [124]. The studied immunomodulators included saponin, calcitriol, β-glucan, n-hexadecane, TDB, muramyl dipeptide (MDP) and monophosphoryl lipid A (MPL). Investigators determined that the combination of the antigens with DDA and TDB generated a strong protective TH1 immune response against the mycobacterium, contrasting with BCG vaccination. In another study, M. bovis BCG lipid extracts were tested for their adjuvant characteristics [125]. BCG lipids were incorporated in DDA-based liposomes and administered subcutaneously to female BALB/c or C57BL/6 mice. BCG lipids coupled with the antigen Ag85B-ESAT-6 fusion protein in cationic liposomes induced significant levels of IFN-γ and relevant antibodies (IgG2A) titers, characteristic of TH1 immune responses. Antigens from other sources (Chlamydia muridarum and tetanus toxoid) were studied and relevant antibodies were detected when administered with the so called mycosomes (BCG lipids + cationic liposomes).

Subsequent studies on cationic liposomes bearing the antigen Ag85B-ESAT-6 (or its modifications) from M. tuberculosis were performed [6, 18, 52, 126]. Henriksen-Laceyet al. incorporated the antigen in DDA or DDA:TDB (8:1 molar ratio) liposomes and administered the vaccine formulation via intramuscular or subcutaneous injections to mice [6]. The antigen did not affect the liposome size, Zeta (ζ) potential or polydispersity index. The cationic lipid formulation was compared with antigen administered alone to mice. Investigators observed the rapid dissemination of the antigen administered alone in mice, contrasting with a depot formation at the injection site when administered in a liposomal formulation (up to 14 days post-vaccination). TDB allowed for the translocation of the liposomes form the site of injection to lymph nodes with the additional effect of monocyte infiltration to the injection site. Another study investigated the effects by which DDA plays a key role as an immunostimulatory lipid [18]. Mice were immunized intramuscularly with the proposed vaccine. Researchers concluded that removing or reducing DDA molar ratio in the liposome bilayer conduced to a reduction in TH1 immune responses against the antigen. Moreover, a team of researchers determined that the addition of cholesterol to the bilayer of DDA:TDB liposomes did not induced strong immune responses suggesting the prominent role of bilayer fluidity [52]. However, the previously mentioned study contrast with results obtained by Liu et al., were the TLR3 ligand Poly I:C was utilized as an adjuvant along with DDA and cholesterol liposomes (DPC liposomes) [126]. Researchers employed the TB fusion protein ESAT-6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) as the model antigen. DPC liposomes showed stability at size of 400 nm and ζ potential of 40 mV. Strong humoral and cell-mediated immune responses were detected by the production of antigen-specific antibodies after the subcutaneous administration and a markedly protection against a M. tuberculosis infection challenge than the traditional BCG vaccine.

With the advances in recombinant protein antigens from M. tuberculosis, other teams of researchers decided to observe at different antigenic proteins (like OVA) in DDA:TDB liposomes [44, 127], comparing different adjuvants and physicochemical properties in cationic liposomal formulations [30, 128] or investigating the mechanism of protein antigen adsorption [127]. OVA-containing SUVs liposomes composed of DDA:TDB with no TLR ligand showed a higher capacity to induce spleen CD8 IFN-γ responses against the antigen, contrasting to MLVs, administered intramuscularly [44]. Antigen-specific responses were higher on SUVs. Adding TLR3 and TLR9 agonists significantly increased the immune responses on MLVs carrying OVA, but that was not observed in SUVs. The study suggested that liposomes are an excellent delivery vehicle for antigen presentation and vaccine formulation, and we believe that this is the foundation for the subsequent studies based on cationic and neutral lipid formulations. Investigators selected DDA and the synthetic mycobacterial cord factor molecule, TDB, as a suitable model adjuvant (CAF01) for vaccine development [128]. This was based in that mice vaccination (subcutaneous) with CAF01 induced a strong antigen-specific cell- and humoral-mediated responses, contrasting with currently used adjuvants (e.g. alum and MPL). Furthermore, results were strongly supported by the protection from infection when mice were challenged by M. tuberculosis, C. trachomatis or malaria, revealing cell mediated (TB), cell-mediated/humoral (C. trachomatis) and humoral immune responses.

A study showed the significant role that liposome vesicle size plays in the cell-mediated immune responses [30]. Researchers determined that no differences in vaccine (administered intramuscularly) draining from the injection site occurred, but a size-dependent liposome movement (favored by large liposomes) was observed to the popliteal lymph node. Macrophage-like cells internalized liposomes in a size-independent pattern. Size did not affect the antigen-specific antibody response (IgG1/2) of Ag85B-ESAT-6-carrying liposomes, however larger liposomes induced highest cell proliferation and lowest IL-10 levels, which contrasted with smaller vesicles (inducing IFN-γ and IL-1β). Additionally, a study investigated the effects of charge, membrane fluidity and antigen-to-lipid ration on mechanism of protein antigen adsorption [127]. For this purpose, investigators compared cationic (CAF01) and neutral (NAF01) liposomal formulations, mainly composed of DDA or DSPC, respectively. α-Lactalbumin and lysozyme were used as antigen protein models to analyze the parameters. The anionic lactalbumin interacted with cationic liposomes by surface adsorption, and no interaction was observed with zwitterionic liposomes. However, the cationic lysozyme presented no detectable interaction with either type of liposomes. Adsorption of α-lactalbumin generated changes in its tertiary structure, neutralized liposome charge, affected lipid membrane packing (especially for CAF01 liposomes), resulting in a reduction of colloidal stability and liposome aggregation. The CAF01 formulation has completed multiple phase I safety trails in humans to date.

Other studies have focus their attention on cell components, synthetic lipid analogues or mycobacterial lipids as antigens for subunit vaccine development employing DDA as the principal component in the lipid bilayer. Mutant M. bovis BCG ΔmmaA4 strain was formulated in cationic liposomes of DDA:TDB (A4/Adj) and administered subcutaneously [129]. The mutation deletes the mmaA4 gene that encodes a S-adenosylmethionine-dependent methyltransferase involved in mycolic acid biosynthesis in the tubercle bacillus [130]. Immunocompromised mice (TCRδ−/−) immunized with A4/Adj were protected against an infection of M. tuberculosis (2 and 9 months post-vaccination), contrasting with non-adjuvanted mutant and non-vaccinated controls. It is important to note that the immunocompromised mice lack CD4+, CD8+ and NK1.1+ T cells but, due to immunization long-term results, an unconventional T cell population was responsible for the immune responses. Researchers observed CD4− CD8− double negative (DN) T cells and found that the cells accumulated in the lungs of A4/Adj-treated mice, with significant levels of IFN-γ production when comparing to nonvaccinated or nonadjuvanted BCG control test groups. In vitro studies revealed the antimycobacterial properties of DN T cells isolated from adjuvanted BCG-treated mice when compared to whole-spleen cells. The results of this study represent a milestone in medical research since tuberculosis affects dramatically immunocompromised patients, like in HIV infections [131–133].

Synthetic lipid analogues from monomycoloyl glycerol (MMG-1 to 6) from M. tuberculosis has been developed and their supramolecular structure and adjuvant efficacy tested via subcutaneous administration [134]. The analogues displayed longer (MMG-2) or shorter (MMG-3) alkyl chains, or stoichiometry variations of the polar head group (MMG-5) or the hydrophobic moiety (MMG-6). CryoTEM and synchrotron small-angle X-ray (SAX) experiments revealed the supramolecular organization varied from unilamellar and multilamellar (ULVs/MLVs) vesicles in DDA:MMG-1/2/5/6 liposomes to ULVs and hexosomes in DDA:MMG-3. TH1 and TH17 immune responses were induced by DDA:MMG-1/3/6 liposomal formulations in response to a chlamydial antigen, contrasting to different immunostimulatory properties of naked MMG-1 and MMG-6 analogues in vitro. We recommend further studies employing MMG analogues incorporated in liposomes with mycobacterial-derived antigens to assess the efficacy of this chlamydial model. In contrast, another study utilized natural mycobacterial lipids diacylated sulfoglycolipids (Ac2-SGL) and phosphatidyl-myo-inositol dimannosides (PIM2) as antigens in a liposomal vaccine formulated with DDA and TDB as adjuvants [135]. Researchers observed a reduction of bacterial load in the spleen of vaccinated animals via subcutaneous administration, contrasting with the unvaccinated group. The lipid antigen vaccine group showed a remarkable reduction of lung and spleen lesions when compared to the unvaccinated group. Comparison of lipid antigen vaccine with protein antigen vaccine regimes in a guinea pig model revealed no significant differences in the treatments.

From the articles discussed in this section we can count on promising advances in tuberculosis vaccine development. The approaches presented applied recombinant protein antigens, whole-mutant cell and synthetic and natural cell components formulations. Each approach could encounter some drawbacks: mass production of natural and synthetic products from M. tuberculosis and other mycobacteria; liposomal formulation instability and aggregation; unwanted immune responses. Most of the studies discussed above demonstrated that subcutaneous administration could be a successful route of administration and future research should direct efforts to determined other administration routes, like intranasal and intramuscular. By understanding the physicochemical parameters of liposomes and the effects of routes of administration on physiological and immune function requirements, scientists will be able to develop a novel tuberculosis vaccine.

Go to:

Conclusion

Vaccination represents the major advancement of modern medicine, second in effect only to clean water, in decreasing the spread of detrimental diseases and providing better quality of life. To develop effective and safe vaccines, collaboration between immunologists and formulation scientists must exist. This collaboration will ensure that the vaccine is designed adequately and that the therapy will not cause unwanted immune responses. Also, it is important to understand the basic concepts in immunology (innate vs. adaptive immunity) so we can target the corresponding receptors with the ligands and antigens employed in the vaccine. Many types of cell receptors participate in PAMPs recognition (innate immunity), namely NLRs, STING, RLRs, TLRs and CLRs. Each receptor contributes to significant responses that lead to T helper cell activation and differentiation, with eventual B cell (antibody-mediated) and CD8 T cell-mediated adaptive immune responses.

For liposomal vaccines, we must pay close attention to liposome size, surface charge (ζ potential), morphology (lamellarity) and lipid bilayer fluidity. Size may affect antigen presentation to APCs and consequently immune responses. Likewise, surface charge of the liposome may affect antigen adsorption on the liposome and could affect liposome-cell interactions (cationic liposomes interact better with the anionic membrane of immune cells). MLVs may reduce antigen presentation due to their concentric vesicle morphological nature, contrasting to ULVs which enhances antigen presentation. Antigen leakage and immune response reduction may occur on how the antigen was formulated (adsorption vs. absorption). Absortion is explained by the Tm of the lipids or phospholipids used when developing the vaccine. Lipids with lower Tm tend to be fluid meanwhile lipids with higher Tm would increase bilayer rigidity. During adsorption, the antigen is exposed in the outer layer of the lipid membrane, which could allow easy release or presentation of the antigen. Additionally, we must pay special attention to how liposomes would interact with immune cells and how we can target those cells, enhancing proper immune responses. Adjuvants play a significant role in APCs activation and maturation for eventual T and B cell activation. The adjuvants, properly selected, will interact with their corresponding TLRs or CLRs in a process called targeted cell vaccine delivery. This cell targeted process is also affected by the vaccine route of administration.

We observed in the sections previously discussed key developments in contemporary vaccine research for the treatment of viral, bacterial, fungal and parasitic infections. The studies presented to the reader demonstrated the collaborative and interdisciplinary nature of the field. Cell targeting and adjuvants dominated most of the approaches to develop effective prophylactic subunit vaccines for the treatment of detrimental diseases. In some instances, infection was hindered by the antimicrobial, antiviral, antifungal or antiparasitic activities of the vaccines due to the induction of adequate immune responses (cell- and antibody-mediated), leading to the survival of selected animal models. Additionally, we can conclude that cationic liposomal vaccines are of great interest for future vaccine development due to their enhanced interaction with the negatively charged immune cells. In specific, DDA-based liposomes are being tested in diverse vaccine studies which promise significant advances in the field. Such DDA applications as a building block of liposomal vaccines represent a step forward towards the prophylactic treatment of diverse infections. These developments will improve the current vaccine approaches and will provide better treatments for patients. Additional research efforts must be made towards the development of novel adjuvants that will contribute to the induction of significant immune responses. Both authors read and approved the final manunscript.

Go to:

Authors’ contributions

LODS and DB developed the concept of the manuscript. LODS reviewed the literature, drafted and wrote the manuscript. DB critically reviewed the manuscript and provided ideas and comments on the draft. Both authors approved the final manuscript.

Acknowledgements

Special thanks to the Formulation Team of the Center for Translational Medicine (CTM) at the University of Montana for their critical review of the manuscript. Special thanks to Dr. Alyson Smith, CTM’s Immunology Director, for her guidance in the immunology-related information discussed in the article.

Competing interests

LODS declares no competing interests. DB is the Chief Operating Officer and member of the Board of Directors, Inimmune, Inc. The authors are entirely responsible for the content of the review and the opinions contained within it.

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This review was written with the support of a NIAID Contract # HHSN272201400050C.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Go to:

Abbreviations

AmB	amphotericin B
APCs	antigen presenting cells
BCG	Bacillus Calmette–Guerin
BCR	B cell receptor
BMDCs	bone marrow dendritic cells
BSA	bovine serum albumin
CCS	ceramide carbamoyl-spermine
Chol	cholesterol
CLR	C-type lectin receptor
COPD	chronic obstructive pulmonary disease
CpG-ODN	CpG-containing oligodeoxynucleotides
CWSPs	cell wall surface proteins
DAMPs	damaged-associated molecular patterns
DCs	dendritic cells
DCC	N,N-dicyclohexylcarbodimide
DC-Chol	dimethylaminoethane-carbamoyl-cholesterol
DC-SIGN	DC-specific ICAM3-grabbing non-integrin
DDA	dimethyldioctadecylammonium
Dectin-1	dendritic cell-associated C-type lectin 1
Dectin-2	dendritic cell-associated C-type lectin 2
DFO or DFB	desferrioxamine B
DLPC	1,2-dilauroyl-sn-glycero-3-phosphocholine
DMPC	1,2-dimyristoyl-sn-glycero-3-phosphocholine
DMPG	1,2-dimyristoyl-sn-glycero-3-phospho-(1ʹ-rac-glycerol)
DMTAP	1,2-dimyristoyl-3-trimethylammonium-propane
DOGS-NTS-Ni	1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)
DOPC	1,2-dioleoyl-sn-glycero-3-phosphocholine
DOPE	1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DOTAP	1,2-dioleoyl-3-trimethylammonium-propane
DOTMA	1,2-di-O-octadecenyl-3-trimethylammonium propane
DPPC	1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPC	distearoyl-sn-glycero-3-phosphocholine
DSTAP	1,2-stearoyl-3-trimethylammonium-propane
DTA	diphtheria toxin
EPC	egg phosphatidylcholine
HBsAg	hepatitis B surface antigen
HBV	hepatitis B virus
HIV	human immunodeficiency virus
Hsp90	C. albicans heat shock protein 90
IFN-γ	gamma interferon
Ig	immunoglobulin
IL	interleukins
i.n.	intranasal
LAg	leishmanial antigen
Lip	liposome(s)
LPS	lipopolysaccharide
LTBIs	latent tuberculosis infections
LUVs	large unilamellar vesicles
ManLAM	mannose lipoarabinomannan
Man-PE	mannosyl phosphatidylethanolamine
MCL	macrophage C-type lectin
MDDCs	monocyte-derived dendritic cells
MDMs	monocyte-derived macrophages
MDP	muramyl dipeptide
MHC I	class I major histocompatibility complex
MHC II	class II major histocompatibility complex
MINCLE	macrophage-inducible C-type lectin
MLVs	multilamellar vesicles
MMG	monomycoloyl glycerol
MO	monooleoylglycerol
MPL or MPLA	monophosphoryl lipid A
MR	mannose receptor
NKs	natural killer cells
NLRs	nucleotide-binding domain and leucine-rich repeat receptor
OVA	ovabulmin
PA	phosphatidic acid
PAMPs	pathogen associated molecular patterns
PC	phosphatidylcholine
PG	phosphatidylglycerol
PMNs	polymorpho nuclear cells
POPC	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
POPE	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
PRRs	pathogen recognition receptors
PS	phosphatidylserine
RLRS	retinoic acid-inducible gene I receptor
RSV	respiratory syncytial virus
SA	stearylamine
SDC	sodium deoxycholate
SUVs	small unilamellar vesicles
TAMPs	tumor-associated molecular patters
TB	tuberculosis
TBIs	tuberculosis infections
TCR	T cell receptor
TDB	trehalose dibehenate
TH cells	T helper cells
TLR	toll-like receptor
TNF-α	tumor necrosis factor alpha
WHO	World Health Organization

Go to:

Contributor Information

Luis O. De Serrano, Email: moc.liamtoh@onarresedsiul.

David J. Burkhart, Email: ude.tmu.osm@trahkruB.divaD.

Go to:

References

1. Guy B, Pascal N, Francon A, Bonnin A, Gimenez S, Lafay-Vialon E, Trannoy E, Haensler J. Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine. Vaccine. 2001;19:1794–1805. [PubMed] [Google Scholar]

2. Puangpetch A, Anderson R, Huang YY, Sermswan RW, Chaicumpa W, Sirisinha S, Wongratanacheewin S. Cationic Liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice. Clin Vaccine Immunol. 2012;19:675–683. [PMC free article] [PubMed] [Google Scholar]

3. Capron A, Riveau G, Grzych J-M, Boulanger D, Capron M, Pierce R. Development of a vaccine strategy against human and bovine schistosomiasis: background and update. Trop Geogr Med. 1994;46:242–246. [PubMed] [Google Scholar]

4. Girard MP, Fruth U, Kieny MP. A review of vaccine research and development: tuberculosis. Vaccine. 2005;23:5725–5731. [PubMed] [Google Scholar]

5. Christensen D, Agger EM, Andreasen LV, Kirby D, Andersen P, Perrie Y. Liposome-based cationic adjuvant formulations (CAF): past, present, and future. J Liposome Res. 2009;19:2–11. [PubMed] [Google Scholar]

6. Henriksen-Lacey M, Bramwell VW, Christensen D, Agger EM, Andersen P, Perrie Y. Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen. J Control Release. 2010;142:180–186. [PubMed] [Google Scholar]

7. Kallerup RS, Madsen CM, Schioth ML, Franzyk H, Rose F, Christensen D, Korsholm KS, Foged C. Influence of trehalose 6,6ʹ-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes. Eur J Pharm Biopharm. 2015;90:80–89. [PubMed] [Google Scholar]

8. Geijtenbeek TBH, Gringhuis SI. C-type lectin receptors in the control of T helper cell differentiation. Nat Rev Immunol. 2016;16:433–448. [PubMed] [Google Scholar]

9. Goyal S, Klassert TE, Slevogt H. C-type lectin receptors in tuberculosis: what we know. Med Microbiol Immunol. 2016;205:513–535. [PubMed] [Google Scholar]

10. Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science. 2010;327:291–295. [PMC free article] [PubMed] [Google Scholar]

11. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect. 2004;6:1382–1387. [PubMed] [Google Scholar]

12. Yao C, Oh JH, Lee DH, Bae JS, Jin CL, Park CH, Chung JH. Toll-like receptor family members in skin fibroblasts are functional and have a higher expression compared to skin keratinocytes. Int J Mol Med. 2015;35:1443–1450. [PubMed] [Google Scholar]

13. Oosting M, Cheng SC, Bolscher JM, Vestering-Stenger R, Plantinga TS, Verschueren IC, Arts P, Garritsen A, van Eenennaam H, Sturm P, et al. Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc Natl Acad Sci USA. 2014;111:E4478–E4484. [PMC free article] [PubMed] [Google Scholar]

14. Cagnoni AJ, Saez JMP, Rabinovich GA, Marino KV. Turning-off signaling by siglecs, selectins, and galectins: chemical inhibition of glycan-dependent interactions in cancer. Front Oncol. 2016;6:21. [PMC free article] [PubMed] [Google Scholar]

15. Dambuza IM, Brown GD. C-type lectins in immunity: recent developments. Curr Opin Immunol. 2015;32:21–27. [PMC free article] [PubMed] [Google Scholar]

16. Opanasopit P, Higuchi Y, Kawakami S, Yamashita F, Nishikawa M, Hashida M. Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages. Biochim Biophys Acta Biomem. 2001;1511:134–145. [PubMed] [Google Scholar]

17. Kallert S, Zenk SF, Walther P, Grieshober M, Weil T, Stenger S. Liposomal delivery of lipoarabinomannan triggers Mycobacterium tuberculosis specific T-cells. Tuberculosis. 2015;95:452–462. [PubMed] [Google Scholar]

18. Hussain MJ, Wilkinson A, Bramwell VW, Christensen D, Perrie Y. Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants. J Pharm Pharmacol. 2014;66:358–366. [PubMed] [Google Scholar]

19. Li P, Chen S, Jiang Y, Jiang J, Zhang Z, Sun X. Dendritic cell targeted liposomes-protamine-DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine. Nanotechnology. 2013;24:295101. [PubMed] [Google Scholar]

20. Christensen D, Korsholm KS, Andersen P, Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines. 2011;10:513–521. [PubMed] [Google Scholar]

21. McNeil SE, Perrie Y. Gene delivery using cationic liposomes. Expert Opin Ther Pat. 2006;16:1371–1382. [Google Scholar]

22. Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics. 2017;9:12. [Google Scholar]

23. Watson DS, Endsley AN, Huang L. Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens. Vaccine. 2012;30:2256–2272. [PMC free article] [PubMed] [Google Scholar]

24. Perrie Y, Crofts F, Devitt A, Griffiths HR, Kastner E, Nadella V. Designing liposomal adjuvants for the next generation of vaccines. Adv Drug Deliv Rev. 2016;99:85–96. [PubMed] [Google Scholar]

25. Shahum E, Therien HM. Immunopotentiation of the humoral response by liposomes—encapsulation versus covalent linkage. Immunology. 1988;65:315–317. [PMC free article] [PubMed] [Google Scholar]

26. Shahum E, Therien HM. Liposomal adjuvanticity—effect of encapsulation and surface-linkage on antibody-production and proliferative response. Int J Immunopharmacol. 1995;17:9–20. [PubMed] [Google Scholar]

27. Therien HM, Shahum E. Immunopotentiation of the humoral response by liposomes—effect of a T-cell polyclonal activator. Cell Immunol. 1988;116:320–330. [PubMed] [Google Scholar]

28. Barnier-Quer C, Elsharkawy A, Romeijn S, Kros A, Jiskoot W. Adjuvant effect of cationic liposomes for subunit influenza vaccine: influence of antigen loading method, cholesterol and immune modulators. Pharmaceutics. 2013;5:392–410. [PMC free article] [PubMed] [Google Scholar]

29. Legrue SJ. Carrier and adjuvant properties of liposome-borne tumor-specific antigens. Cancer Immunol Immunother. 1984;17:135–141. [PubMed] [Google Scholar]

30. Henriksen-Lacey M, Devitt A, Perrie Y. The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production. J Control Release. 2011;154:131–137. [PubMed] [Google Scholar]

31. Brewer JM, Tetley L, Richmond J, Liew FY, Alexander J. Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen. J Immunol. 1998;161:4000–4007. [PubMed] [Google Scholar]

32. Jain S, Indulkar A, Harde H, Agrawal AK. Oral mucosal immunization using glucomannosylated bilosomes. J Biomed Nanotechnol. 2014;10:932–947. [PubMed] [Google Scholar]

33. Mohammed AR, Bramwell VW, Coombes AG, Perrie Y. Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products. Methods. 2006;40:30–38. [PubMed] [Google Scholar]

34. Ingvarsson PT, Schmidt ST, Christensen D, Larsen NB, Hinrichs WL, Andersen P, Rantanen J, Nielsen HM, Yang M, Foged C. Designing CAF-adjuvanted dry powder vaccines: spray drying preserves the adjuvant activity of CAF01. J Control Release. 2013;167:256–264. [PubMed] [Google Scholar]

35. Bakowsky H, Richter T, Kneuer C, Hoekstra D, Rothe U, Bendas G, Ehrhardt C, Bakowsky U. Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery. Biochim Biophys Acta. 2008;1778:242–249. [PubMed] [Google Scholar]

36. Dunnick JK, Badger RS, Takeda Y, Kriss JP. Vesicle interactions with antibody and peptide hormone: role of vesicle composition. J Nucl Med. 1976;17:1073–1076. [PubMed] [Google Scholar]

37. Perrie Y, Ali H, Kirby DJ, Mohammed AU, McNeil SE, Vangala A. Environmental scanning electron microscope imaging of vesicle systems. Methods Mol Biol. 2017;1522:131–143. [PubMed] [Google Scholar]

38. Cohen S, Alonso MJ, Langer R. Novel approaches to controlled-release antigen delivery. Int J Technol Assess Health Care. 1994;10:121–130. [PubMed] [Google Scholar]

39. Chen C, Han D, Cai C, Tang X. An overview of liposome lyophilization and its future potential. J Control Release. 2010;142:299–311. [PubMed] [Google Scholar]

40. Winterhalter M, Lasic DD. Liposome stability and formation: experimental parameters and theories on the size distribution. Chem Phys Lipids. 1993;64:35–43. [PubMed] [Google Scholar]

41. Beck Z, Matyas GR, Jalah R, Rao M, Polonis VR, Alving CR. Differential immune responses to HIV-1 envelope protein induced by liposomal adjuvant formulations containing monophosphoryl lipid A with or without QS21. Vaccine. 2015;33:5578–5587. [PubMed] [Google Scholar]

42. Shek PN, Yung BYK, Stanacev NZ. Comparison between multilamellar and unilamellar liposomes in enhancing antibody-formation. Immunology. 1983;49:37–44. [PMC free article] [PubMed] [Google Scholar]

43. Hampl J, Stepanek J, Franz J, Svoboda I. Incorporation of Bovine Herpesvirus-1 protein subunits into large unilamellar and multilamellar liposomes. Acta Veterinaria Brno. 1992;61:29–36. [Google Scholar]

44. Milicic A, Kaur R, Reyes-Sandoval A, Tang CK, Honeycutt J, Perrie Y, Hill AVS. Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses. PLoS ONE. 2012;7:10. [PMC free article] [PubMed] [Google Scholar]

45. Joseph A, Itskovitz-Cooper N, Samira S, Flasterstein O, Eliyahu H, Simberg D, Goldwaser I, Barenholz Y, Kedar E. A new intranasal influenza vaccine based on a novel polycationic lipid—ceramide carbamoyl-spermine (CCS)-I. Immunogenicity and efficacy studies in mice. Vaccine. 2006;24:3990–4006. [PubMed] [Google Scholar]

46. Tseng LP, Chiou CJ, Deng MC, Lin MH, Pan RN, Huang YY, Liu DZ. Evaluation of encapsulated newcastle disease virus liposomes using various phospholipids administered to improve chicken humoral immunity. J Biomed Mater Res Part B Appl Biomater. 2009;91B:621–625. [PubMed] [Google Scholar]

47. Hilgers LAT, Snippe H. DDA as an immunological adjuvant. Res Immunol. 1992;143:494–503. [PubMed] [Google Scholar]

48. Korsholm KS, Agger EM, Foged C, Christensen D, Dietrich J, Andersen CS, Geisler C, Andersen P. The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes. Immunology. 2007;121:216–226. [PMC free article] [PubMed] [Google Scholar]

49. Yasuda T, Dancey GF, Kinsky SC. Immunogenicity of liposomal model membranes in mice—dependence on phospholipid composition. Proc Natl Acad Sci USA. 1977;74:1234–1236. [PMC free article] [PubMed] [Google Scholar]

50. Van Houte AJ, Snippe H, Schmitz MGJ, Willers JMN. Characterization of immunogenic properties of haptenated liposomal model membranes in mice 5 effect of membrane composition on humoral and cellular immunogenicity. Immunology. 1981;44:561–568. [PMC free article] [PubMed] [Google Scholar]

51. Mazumdar T, Anam K, Ali N. Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens. J Parasitol. 2005;91:269–274. [PubMed] [Google Scholar]

52. Kaur R, Henriksen-Lacey M, Wilkhu J, Devitt A, Christensen D, Perrie Y. Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on bilayer properties, biodistribution, and immune responses. Mol Pharm. 2014;11:197–207. [PubMed] [Google Scholar]

53. Hampl J, Franz J, Jordanova K, Stepanek J. Effects of phospholipid-composition on adjuvant efficiency of liposomes. Acta Veterinaria Brno. 1995;64:163–169. [Google Scholar]

54. Kummerow FA, Przybylski R, Wasowicz E. Changes in arterial membrane lipid composition may precede growth factor influence in the pathogenesis of atherosclerosis. Artery. 1994;21:63–75. [PubMed] [Google Scholar]

55. Rao M, Matyas GR, Vancott TC, Birx DL, Alving CR. Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein. Immunol Cell Biol. 2004;82:523–530. [PubMed] [Google Scholar]

56. Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrom T, Agger EM, Andersen P, Perrie Y. Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3 beta Nʹ,Nʹ-dimethylaminoethane)carbomyl cholesterol (DC-Chol), and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses. Mol Pharm. 2011;8:153–161. [PubMed] [Google Scholar]

57. Andersen P, Doherty TM. The success and failure of BCG—implications for a novel tuberculosis vaccine. Nat Rev Microbiol. 2005;3:656–662. [PubMed] [Google Scholar]

58. Fine PEM. Variation in protection by BCG—implications of and for heterologous immunity. Lancet. 1995;346:1339–1345. [PubMed] [Google Scholar]

59. Abhyankar MM, Noor Z, Tomai MA, Elyecrog J, Fox CB, Petri WA., Jr Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica. Vaccine. 2017;35:916–922. [PMC free article] [PubMed] [Google Scholar]

60. Mohammed AR, Bramwell VW, Kirby DJ, McNeil SE, Perrie Y. Increased potential of a cationic liposome-based delivery system: enhancing stability and sustained immunological activity in pre-clinical development. Eur J Pharm Biopharm. 2010;76:404–412. [PubMed] [Google Scholar]

61. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu CZ, Middelberg APJ. Nanoparticle vaccines. Vaccine. 2014;32:327–337. [PubMed] [Google Scholar]

62. Lonez C, Vandenbranden M, Ruysschaert JM. Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res. 2008;47:340–347. [PubMed] [Google Scholar]

63. Aramaki Y, Tomizawa H, Hara T, Yachi K, Kikuchi H, Tsuchiya S. Stability of liposomes in-vitro and their uptake by rat peyer patches following oral-administration. Pharm Res. 1993;10:1228–1231. [PubMed] [Google Scholar]

64. CarmonaRibeiro AM, Ortis F, Schumacher RI, Armelin MCS. Interactions between cationic vesicles and cultured mammalian cells. Langmuir. 1997;13:2215–2218. [Google Scholar]

65. Karathanasis E, Geigerman CM, Parkos CA, Chan L, Bellamkonda RV, Jaye DL. Selective targeting of nanocarriers to neutrophils and monocytes. Ann Biomed Eng. 2009;37:1984–1992. [PMC free article] [PubMed] [Google Scholar]

66. Johansen PT, Zucker D, Parhamifar L, Pourhassan H, Madsen DV, Henriksen JR, Gad M, Barberis A, Maj R, Andresen TL, Jensent SS. Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist. Expert Opin Drug Deliv. 2015;12:1045–1058. [PubMed] [Google Scholar]

67. Tanaka Y, Taneichi M, Kasai M, Kakiuchi T, Uchida T. Liposome-coupled antigens are internalized by antigen-presenting cells via pinocytosis and cross-presented to CD8(+) T cells. PLoS ONE. 2010;5:e15225. [PMC free article] [PubMed] [Google Scholar]

68. Owais M, Gupta CM. Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development. Eur J Biochem. 2000;267:3946–3956. [PubMed] [Google Scholar]

69. Huong TM, Harashima H, Kiwada H. Complement dependent and independent liposome uptake by peritoneal macrophages: cholesterol content dependency. Biol Pharm Bull. 1998;21:969–973. [PubMed] [Google Scholar]

70. Song X, Lin Q, Guo L, Fu Y, Han J, Ke H, Sun X, Gong T, Zhang Z. Rifampicin loaded mannosylated cationic nanostructured lipid carriers for alveolar macrophage-specific delivery. Pharm Res. 2015;32:1741–1751. [PubMed] [Google Scholar]

71. White KL, Rades T, Furneaux RH, Tyler PC, Hook S. Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells. J Pharm Pharmacol. 2006;58:729–737. [PubMed] [Google Scholar]

72. Varypataki EM, van der Maaden K, Bouwstra J, Ossendorp F, Jiskoot W. cationic liposomes loaded with a synthetic long peptide and poly(I:C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity. Aaps J. 2015;17:216–226. [PMC free article] [PubMed] [Google Scholar]

73. Tada R, Hidaka A, Iwase N, Takahashi S, Yamakita Y, Iwata T, Muto S, Sato E, Takayama N, Honjo E, et al. Intranasal immunization with DOTAP cationic liposomes combined with DC-cholesterol induces potent antigen-specific mucosal and systemic immune responses in mice. PLoS ONE. 2015;10:e0139785. [PMC free article] [PubMed] [Google Scholar]

74. Meiklejohn G, Simpson TW, Stacy IB. experimental infection of domestic animals with Japanese B-encephalitis virus. Proc Soc Exp Biol Med. 1947;65:359–364. [PubMed] [Google Scholar]

75. Niklasson B, Hakansson C, Lowhagen GB, Jonsson R. Oral mucosal lesions associated with HIV-infection. Swed Dent J. 1986;10:258. [Google Scholar]

76. Greenberg HB, Pollard RB, Lutwick LI, Gregory PB, Robinson WS, Merigan TC. Effect of human leukocyte interferon on hepatitis B virus-infection in patients with chronic active hepatitis. N Engl J Med. 1976;295:517–522. [PubMed] [Google Scholar]

77. Allard HA. Some properties of the virus of the mosaic disease of tobacco. J Agric Res. 1916;6:649–674. [Google Scholar]

78. Brown M, Reed S, Levy JA, Busch M, McKerrow JH. Detection of HIV-1 in entamoeba-histolytica without evidence of transmission to human-cells. Aids. 1991;5:93–96. [PubMed] [Google Scholar]

79. Wang AL, Wang CC. Viruses of parasitic protozoa. Parasitol Today. 1991;7:76–80. [PubMed] [Google Scholar]

80. Applemans R, Wagemans J. The bacteriophages of different families. C R Seances Soc Biol Fil. 1922;86:738–739. [Google Scholar]

81. Maisin J. The bacteriophages. Arch Int Pharmacodyn Ther. 1922;26:215–245. [Google Scholar]

82. Piroth L, Wittkop L, Lacombe K, Rosenthal E, Gilbert C, Miailhes P, Carrieri P, Chas J, Poizot-Martin I, Gervais A, et al. Efficacy and safety of direct-acting antiviral regimens in HIV/HCV-co-infected patients—French ANRS CO13 HEPAVIH cohort. J Hepatol. 2017;67:23–31. [PubMed] [Google Scholar]

83. Sil A, Ravi MD, Patnaik BN, Dhingra MS, Dupuy M, Gandhi DJ, Dhaded SM, Dubey AP, Kundu R, Lalwani SK, et al. Effect of prophylactic or therapeutic administration of paracetamol on immune response to DTwP-HepB-Hib combination vaccine in Indian infants. Vaccine. 2017;35:2999–3006. [PubMed] [Google Scholar]

84. Brunel F, Darbouret A, Ronco J. Cationic lipid DC-Chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis B vaccine. Vaccine. 1999;17:2192–2203. [PubMed] [Google Scholar]

85. Mahor S, Rawat A, Dubey PK, Gupta PN, Khatri K, Goyal AK, Vyas SP. Cationic transfersomes based topical genetic vaccine against hepatitis B. Int J Pharm. 2007;340:13–19. [PubMed] [Google Scholar]

86. Blom RAM, Erni ST, Krempaska K, Schaerer O, van Dijk RM, Amacker M, Moser C, Hall SRR, von Garnier C, Blank F. A triple co-culture model of the human respiratory tract to study immune-modulatory effects of liposomes and virosomes. PLoS ONE. 2016;11:e0163539. [PMC free article] [PubMed] [Google Scholar]

87. Stein RT, Bont LJ, Zar H, Polack FP, Park C, Claxton A, Borok G, Butylkova Y, Wegzyn C. Respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis. Pediatr Pulmonol. 2017;52:556–569. [PMC free article] [PubMed] [Google Scholar]

88. Klinguer C, Beck A, De-Lys P, Bussat MC, Blaecke A, Derouet F, Bonnefoy JY, Nguyen TN, Corvaia N, Velin D. Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens. Vaccine. 2001;19:4236–4244. [PubMed] [Google Scholar]

89. Staats HF, McGhee JR. Application of basic principles of mucosal immunity to vaccine development. In: Kryono H, Ogra PL, McGhee JR (eds) Mucosal Vaccines. San Diego: Academic Press; 1996. p. 17–39. ISBN: 9780124105805.

90. Goldberg JB, Pier GB. Pseudomonas aeruginosa lipopolysaccharides and pathogenesis. Trends Microbiol. 1996;4:490–494. [PubMed] [Google Scholar]

91. Ketley JM. Pathogenesis of enteric infection by Campylobacter. Microbiol Uk. 1997;143:5–21. [PubMed] [Google Scholar]

92. Naumovska E, Ludwanowski S, Hersch N, Braun T, Merkel R, Hoffmann B, Csiszar A. Plasma membrane functionalization using highly fusogenic immune activator liposomes. Acta Biomater. 2014;10:1403–1411. [PubMed] [Google Scholar]

93. Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S, Nakanishi M, Tanaka K, Mayumi T. Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J Control Release. 1999;61:233–240. [PubMed] [Google Scholar]

94. Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine. 2004;22:1903–1913. [PubMed] [Google Scholar]

95. Gall D. The adjuvant activity of aliphatic nitrogenous bases. Immunology. 1966;11:369–386. [PMC free article] [PubMed] [Google Scholar]

96. Jin SE, Kim CK. Long-term stable cationic solid lipid nanoparticles for the enhanced intracellular delivery of SMAD3 antisense oligonucleotides in activated murine macrophages. J Pharm Pharm Sci. 2012;15:467–482. [PubMed] [Google Scholar]

97. Romøren K, Thu BJ, Bols NC, Evensen Ø. Transfection efficiency and cytotoxicity of cationic liposomes in salmonid cell lines of hepatocyte and macrophage origin. Biochim Biophys Acta. 2004;1663:127–134. [PubMed] [Google Scholar]

98. Sakurai F, Inoue R, Nishino Y, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, Hashida M. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression. J Control Release. 2000;66:255–269. [PubMed] [Google Scholar]

99. Kurosaki T, Kitahara T, Fumoto S, Nishida K, Yamamoto K, Nakagawa H, Kodama Y, Higuchi N, Nakamura T, Sasaki H. Chondroitin sulfate capsule system for efficient and secure gene delivery. J Pharm Pharm Sci. 2010;13:351–361. [PubMed] [Google Scholar]

100. Kurosaki T, Kitahara T, Teshima M, Nishida K, Nakamura J, Nakashima M, To H, Hukuchi H, Hamamoto T, Sasaki H. Exploitation of De Novo helper-lipids for effective gene delivery. J Pharm Pharm Sci. 2008;11:56–67. [PubMed] [Google Scholar]

101. Lehrnbecher T, Kalkum M, Champer J, Tramsen L, Schmidt S, Klingebiel T. Immunotherapy in invasive fungal infection—focus on invasive aspergillosis. Curr Pharm Des. 2013;19:3689–3712. [PubMed] [Google Scholar]

102. Mathew BP, Nath M. Recent approaches to antifungal therapy for invasive mycoses. ChemMedChem. 2009;4:310–323. [PubMed] [Google Scholar]

103. Jensen RH. Resistance in human pathogenic yeasts and filamentous fungi: prevalence, underlying molecular mechanisms and link to the use of antifungals in humans and the environment. Dan Med J. 2016;63(10). pii: B5288. [PubMed]

104. Hector RF, Rutherford GW, Tsang CA, Erhart LM, McCotter O, Anderson SM, Komatsu K, Tabnak F, Vugia DJ, Yang Y, Galgiani JN. The public health impact of Coccidioidomycosis in Arizona and California. Int J Environ Res Public Health. 2011;8:1150–1173. [PMC free article] [PubMed] [Google Scholar]

105. Krishnan-Natesan S, Chandrasekar PH. Current and future therapeutic options in the management of invasive aspergillosis. Drugs. 2008;68:265–282. [PubMed] [Google Scholar]

106. Han YM, Cutler JE. Antibody-response that protects against disseminated candidiasis. Infect Immun. 1995;63:2714–2719. [PMC free article] [PubMed] [Google Scholar]

107. Han YM, Morrison RP, Cutler JE. A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection. Infect Immun. 1998;66:5771–5776. [PMC free article] [PubMed] [Google Scholar]

108. Eckstein M, Barenholz Y, Bar LK, Segal E. Liposomes containing Candida albicans ribosomes as a prophylactic vaccine candidiasis in mice. Vaccine. 1997;15:220–224. [PubMed] [Google Scholar]

109. Masek J, Bartheldyova E, Turanek-Knotigova P, Skrabalova M, Korvasova Z, Plockova J, Koudelka S, Skodova P, Kulich P, Krupka M, et al. Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90. J Control Release. 2011;151:193–201. [PubMed] [Google Scholar]

110. Knotigova PT, Zyka D, Masek J, Kovalova A, Krupka M, Bartheldyova E, Kulich P, Koudelka S, Lukac R, Kauerova Z, et al. Molecular adjuvants based on nonpyrogenic lipophilic derivatives of norAbuMDP/GMDP formulated in nanoliposomes: stimulation of innate and adaptive immunity. Pharm Res. 2015;32:1186–1199. [PubMed] [Google Scholar]

111. Carneiro C, Correia A, Collins T, Vilanova M, Pais C, Gomes AC, Oliveira ME, Sampaio P. DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: a novel adjuvant and delivery system. Eur J Pharm Biopharm. 2015;89:190–200. [PubMed] [Google Scholar]

112. Carneiro C, Correia A, Lima T, Vilanova M, Pais C, Gomes AC, Real Oliveira MECD, Sampaio P. Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis. Acta Biomater. 2016;39:133–145. [PubMed] [Google Scholar]

113. Postma NS, Hermsen CC, Zuidema J, Eling WMC. Plasmodium vinckei: optimization of desferrioxamine B delivery in the treatment of murine malaria. Exp Parasitol. 1998;89:323–330. [PubMed] [Google Scholar]

114. Stewart VA, McGrath SM, Walsh DS, Davis S, Hess AS, Ware LA, Kester KE, Cummings JF, Burge JR, Voss G, et al. Pre-clinical evaluation of new adjuvant formulations to improve the immunogenicity of the malaria vaccine RTS, S/AS02A. Vaccine. 2006;24:6483–6492. [PubMed] [Google Scholar]

115. Ready PD. Epidemiology of visceral leishmaniasis. Clin Epidemiol. 2014;6:147–154. [PMC free article] [PubMed] [Google Scholar]

116. Bhowmick S, Ravindran R, Ali N. Leishmanial antigens in liposomes promote protective immunity and provide immunotherapy against visceral leishmaniasis via polarized Th1 response. Vaccine. 2007;25:6544–6556. [PubMed] [Google Scholar]

117. Banerjee A, De M, Ali N. Complete cure of experimental visceral leishmaniasis with amphotericin B in stearylamine-bearing cationic liposomes involves down-regulation of IL-10 and favorable T cell responses. J Immunol. 2008;181:1386–1398. [PubMed] [Google Scholar]

118. Banerjee A, Roychoudhury J, Ali N. Stearylamine-bearing cationic liposomes kill Leishmania parasites through surface exposed negatively charged phosphatidylserine. J Antimicrob Chemother. 2008;61:103–110. [PubMed] [Google Scholar]

119. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, Dopfer D, Fazil A, Fischer-Walker CL, Hald T, et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. Plos Med. 2015;12:21. [PMC free article] [PubMed] [Google Scholar]

120. World Health Organization . Global tuberculosis report 2016. Geneva: World Health Organization; 2016. [Google Scholar]

121. Houben R, Dodd PJ. The global burden of latent tuberculosis infection: a re-estimation using mathematical modelling. Plos Med. 2016;13:13. [PMC free article] [PubMed] [Google Scholar]

122. Tsuruta LR, Quintilio W, Costa MHB, CarmonaRibeiro AM. Interactions between cationic liposomes and an antigenic protein: the physical chemistry of the immunoadjuvant action. J Lipid Res. 1997;38:2003–2011. [PubMed] [Google Scholar]

123. Snippe H, Belder M, Willers JMN. Dimethyl dioctadecyl ammonium bromide as adjuvant for delayed-hypersensitivity in mice. Immunology. 1977;33:931–936. [PMC free article] [PubMed] [Google Scholar]

124. Holten-Andersen L, Doherty TM, Korsholm KS, Andersen P. Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines. Infect Immun. 2004;72:1608–1617. [PMC free article] [PubMed] [Google Scholar]

125. Rosenkrands I, Agger EM, Olsen AW, Korsholm KS, Andersen CS, Jensen KT, Andersen P. Cationic liposomes containing mycobacterial lipids: a new powerful Th1 adjuvant system. Infect Immun. 2005;73:5817–5826. [PMC free article] [PubMed] [Google Scholar]

126. Liu X, Da Z, Wang Y, Niu H, Li R, Yu H, He S, Guo M, Wang Y, Luo Y, et al. A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice. Vaccine. 2016;34:1370–1378. [PubMed] [Google Scholar]

127. Hamborg M, Rose F, Jorgensen L, Bjorklund K, Pedersen HB, Christensen D, Foged C. Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: effect of charge, fluidity and antigen-to-lipid ratio. Biochim Biophys Acta Biomembr. 2014;1838:2001–2010. [PubMed] [Google Scholar]

128. Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C, Werninghaus K, Kirschning C, Lang R, Christensen D, et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements. PLoS ONE. 2008;3:10. [PMC free article] [PubMed] [Google Scholar]

129. Derrick SC, Yabe I, Morris S, Cowley S. Induction of unconventional T cells by a mutant Mycobacterium bovis BCG strain formulated in cationic liposomes correlates with protection against Mycobacterium tuberculosis infections of immunocompromised mice. Clin Vaccine Immunol. 2016;23:638–647. [PMC free article] [PubMed] [Google Scholar]

130. Boissier F, Bardou F, Guillet VR, Uttenweiler-Joseph S, Daffe M, Quemard A, Mourey L. Further insight into S-adenosylmethionine-dependent methyltransferases—structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis. J Biol Chem. 2006;281:4434–4445. [PubMed] [Google Scholar]

131. Procop GW. HIV and mycobacteria. Semin Diagn Pathol. 2017;34:332–339. [PubMed] [Google Scholar]

132. Perronne C, Zahraoui M, Leport C, Salmon D, Pangon B, Bricaire F, Vilde JL. Tuberculosis in 30 patients with HIV infection. Ann Med Interne. 1988;139:375–376. [Google Scholar]

133. Haegi V. Septic tuberculosis in HIV-infection. Schweiz Med Wochenschr. 1987;117:1297–1301. [PubMed] [Google Scholar]

134. Martin-Bertelsen B, Korshohn KS, Roces CB, Nielsen MH, Christensen D, Franzyk H, Yaghmur A, Foged C. Nano-self-assemblies based on synthetic analogues of mycobacterial monomycoloyl glycerol and DDA: supramolecular structure and adjuvant efficacy. Mol Pharm. 2016;13:2771–2781. [PubMed] [Google Scholar]

135. Larrouy-Maumus G, Layre E, Clark S, Prandi J, Rayner E, Lepore M, de Libero G, Williams A, Puzo G, Gilleron M. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine. 2017;35:1395–1402. [PubMed] [Google Scholar]

---

Articles from Journal of Nanobiotechnology are provided here courtesy of BioMed Central

J Nanobiotechnology. 2017; 15: 83.

Published online 2017 Nov 17. doi: 10.1186/s12951-017-0319-9

PMCID: PMC5693489

PMID: 29149896

Liposomal vaccine formulations as prophylactic agents: design considerations for modern vaccines

Luis O. De Serrano and David J. Burkhart

Author information Article notes Copyright and License information Disclaimer

This article has been cited by other articles in PMC.

Associated Data

Data Availability Statement

Go to:

Abstract

Vaccinology is one of the most important cornerstones in modern medicine, providing better quality of life. The human immune system is composed of innate and adaptive immune processes that interplay when infection occurs. Innate immunity relies on pathogen-associated molecular patterns which are recognized by pathogen recognition receptors localized in antigen presenting cells. After antigen processing and presentation, CD4+ T cell polarization occurs, further leading to B cell and CD8+ activation and humoral and cell-mediated adaptive immune responses. Liposomes are being employed as vaccine technologies and their design is of importance to ensure proper immune responses. Physicochemical parameters like liposome size, charge, lamellarity and bilayer fluidity must be completely understood to ensure optimal vaccine stability and efficacy. Liposomal vaccines can be developed to target specific immune cell types for the induction of certain immune responses. In this review, we will present promising liposomal vaccine approaches for the treatment of important viral, bacterial, fungal and parasitic infections (including tuberculosis, TB). Cationic liposomes are the most studied liposome types due to their enhanced interaction with the negatively charged immune cells. Thus, a special section on the cationic lipid dimethyldioctadecylammonium and TB is also presented.

Keywords: Liposomes, Vaccine formulations, CLR, TLR, Viral infections, Bacterial infections, Parasites

Go to:

Background

Vaccination is one of the most significant developments in modern science, improving the treatment and controlling the spread of diseases across communities. For formulation scientists and immunologists, there has been an interest in the development of liposomal vaccines for their prophylactic uses in infections (of viral, bacterial, fungal or parasitic origin) [1–3]. To develop safe and effective liposome-based vaccines, scientists should take into consideration several interconnected principles: (1) the design-dependent function of the liposomes (2) the characteristics of liposome-cell interactions when vaccine administration occurs and (3) the specific cell receptor and signaling involved once liposomal vaccines are administered. Each of these principles will affect vaccine efficacy and its potential development from bench to bedside applications. These three principles are also the basis for developing excellent subunit vaccine strategies, which have been of important interest for vaccine scientists for several years [4–7]. To comprehend how the potential liposomal vaccine might work in the host we must understand the immune responses involved in receptor signaling. The field of immunology plays a significant role that will help determine the utilization of vaccinology to the advantage of the patient by developing adequate prophylactic treatment approaches.

Immunologists investigate how our bodies defend themselves from pathogens by determining and describing the signaling mechanisms involved. Basically, when infection occurs, the innate arm of the immune system responds through recognition of distinct molecules on or in pathogens termed ‘pathogen associated molecular patterns (PAMPs)’. These PAMPs differ from host markers and as such are recognized by the first line of defense in the immune system, antigen presenting cells (APCs) such as dendritic cells (DCs), macrophages and neutrophils. The PAMPs are recognized by pattern recognition receptors (PRRs) on the surface and in endosomes of APCs [8–11]. Vertebrates developed the adaptive immune system as a second line of defense that could ‘remember’ pathogens and fight back upon re-challenge. It is composed of cells such as T and B cells that employ de novo synthesized antigen-specific receptors (T- and B cell receptors, TCRs and BCRs). TCR and BCRs are able to recognize pathogen-specific antigens when presented complexed with major histocompatibility complexes (MHC) on the surface of an APC. But polarization of the T or B cells to ensure it acts appropriately against the pathogen, relies on signals provided by the APC (such as co-stimulatory molecules and precise cytokines) in response to the priming by PAMPs. These critical signals lead to differentiation of effector and eventually memory cells that are critical for driving the immune response against future infections with the same pathogen. While innate immune responses are broadly applicable and adaptive immune responses are antigen-specific, the two arms of the immune system work in close concert to mount an effective response to clear the pathogen.

The development of PRRs by the immune system represent a significant advancement in the fight for survival of the host. Therefore, an important question arises to that end: what type of PRRs have been discovered so far and which ligands (or PAMPs) do they tend to recognize? Some PRRs are secreted to the extracellular milieu and participate in pathogen opsonization. However, most PRRs are transmembrane (like C-type lectin and Toll-like receptors; CLRs and TLRs, respectively) or cytosolic (retinoic acid-inducible gene I and nucleotide-binding domain and leucine-rich repeat containing receptors; RLRs and NLRs, respectively). For the purposes of this review, we will discuss the uses of liposomes in vaccines that target CLRs and TLRs. The TLRs identified so far amount to 10 in humans, each one recognizing specific PAMPs from microbial pathogens (Table 1) [10–12]. TLR 10 has been recently described as a modulatory receptor; however, no known ligand has been linked to it [13]. Therefore, further research should be done on TLR 10 to uncover the ligand involved in innate immune responses.

Table 1

Pathogen-associated molecular patters (PAMPs) recognized by specific TLRs

Pathogen-associated molecular pattern (PAMP)	Microorganism or classification	TLRs
Lipoproteins	Bacteria	TLR 1
Peptidoglycan and lipoteichoic acid	Gram positive bacteria	TLR 2
β-glucans	Fungi
dsRNA	Double-stranded and negative-stranded viruses	TLR 3
Lipopolysacharide (LPS)	Gram negative bacteria	TLR 4
Flagelin	Bacteria	TLR 5
Profilin	Toxoplasma gondii
Lipoproteins	Mycoplasma	TLR 6
Imidazoquinolines and ssRNA	Single-stranded viruses	TLR 7 and 8
Unmethylated CpG DNA motifs	Prokaryotic genomes and viral DNA	TLR 9

ds double stranded, ss single stranded

Contrary to the transmembrane TLRs, CLRs are classified as soluble or membrane bound. Soluble CLRs (e.g. galectins and collectins) have been reviewed extensively [9, 14]. The mostly studied membrane-associated CLRs are DC-SIGN (DC-specific ICAM3-grabbing non-integrin), Dectin-1 (dendritic cell-associated C-type lectin 1), Dectin-2 (dendritic cell-associated C-type lectin 2), MCL (macrophage C-type lectin) and MINCLE (macrophage-inducible C-type lectin) receptors [8, 9]. These receptors play a significant role in immunomodulatory responses, triggering the differentiation of T-helper cells (TH cells) from naïve CD4+ T cells, through the assistance of an APC. CLRs not only recognize PAMPs but also the damaged-associated molecular patterns (DAMPs) and tumor-associated molecular patterns (TAMPs) from the host [15] during the processes of apoptosis and tumorigenesis, respectively. Glycans from a myriad of pathogens (parasitic, fungal, bacterial or viral) are recognized by CLRs (Table 2); the most common being mannan [16], ManLAM (mannose lipoarabinomannan) [17], mycobacterial cord factor [18], β-1,3-glucans [8] and α-1,2-mannose [8, 19]. In humans, DC-SIGN recognizes both mannan and ManLAM; Dectin-1 recognizes β-1,3-glucans and Dectin-2 recognizes α-1,2-mannose. In mice, mycobacterial cord factor is recognized by MINCLE and currently no glycans have been identified for the human MINCLE. We can observe from the literature review available, there is still more work to be done to determine ligands or PAMPs that interact with specific PRRs. Scientists must pay close attention to interspecies differences in PAMP recognition by PRRs, since we can obtain unwanted immune responses once we study them at the human level. Unwanted immunomodulatory responses can compromise the patient’s outcome from the disease.

Table 2

PAMPs recognized by specific CLRs

Pathogen-associated molecular pattern (PAMP)	Microorganism or classification	CLRs
Mannan	Fungi	DC-SIGN
Man-LAM	M. tuberculosis	DC-SIGN and Dectin 2
LeX	Schistosoma mansonii and tissue ligands	DC-SIGN
LeY + LPS	Helicobacter pylori
LDNF (SP)	Fasciola hepatica
β-1,3-glucans	Fungi	Dectin 1
gp120	HIV-1	DC-SIGN
α-1,2-mannose	Fungi	Dectin 2
Glucosyl and mannosyl glycolipids	Malassezia pachydermatis and M. furfur	MINCLE
Mycobacterial cord factor	M. bovis BCG and M. tuberculosis

Le X sialyl-Lewis X tetrasaccharide, Le Y Lewis Y tetrasaccharide, LPS lipopolysaccharide, LDNF fucosylated LacdiNAc

In the vaccine development field, we can identify the importance of collaboration between medicinal chemistry, immunology and formulation science. This article will present and discuss several parameters that play prominent roles in liposomal vaccine development. Liposome size, charge and bilayer composition are some of those parameters to be discussed. Evidence will be presented to the reader for understanding of the mechanisms or effects of such liposome physicochemical characteristics, which impact vaccine development, safety, integrity and efficacy. Furthermore, we present different applications of liposomal vaccine studies that have been published for the treatment of certain infections of distinct etiological origins (viral, bacterial, fungal and parasitic). Finally, the article presents a special section on the development of subunit cationic liposomal vaccines for the prophylactic treatment of tuberculosis infections, one of the most sought-after indications in contemporary vaccinology. The section for tuberculosis vaccine development is focused on DDA-based liposomes; and additional information is available that presents other lipids or phospholipids [20, 21]. The manuscript objective is to present liposomal formulations that are not currently commercially available and inform the scientific community about liposomal formulation for early vaccine development. To date, there are only two commercially approved liposomal vaccines Epaxal and Inflexal, both by Crucell/Berna Biotech. We recommend the reader to further their knowledge in commercially available liposome-based vaccines with another review [22], which discusses the topic extensively.

Go to:

Liposome design

When designing liposomal vaccines, we should take into consideration certain factors within the liposome structure and its physicochemical properties (Fig. 1). Previous reviews on the topic have discussed the different parameters that could affect the functions and efficacy of liposomes as vaccine agents [23, 24]. Liposomal subunit vaccines are safe, with low reactogenicity, biodegradable and versatile. Reactogenicity refers to the low incidence of expected immune responses, causing symptoms like allergies, fever or pain at injection site among others. This type of vaccine contains antigen(s) (either a protein, lipid, lipopeptide etc.) from the pathogen of interest that is incorporated (depending on the antigen physicochemical nature) in the lipid bilayer or core of the liposome. The liposome will serve as an adjuvant, which potentiates the immune responses of the vaccine, improving its efficacy. Antigen incorporation can be achieved by covalent lipid conjugation (at pre- or post-vesicle formation), non-covalent surface attachment (by antibody–epitopes interactions), encapsulation, electrostatic interactions (with lipids of opposite charge) or surface adsorption. Seminal articles published earlier covered the effects of antigen encapsulation or adsorption on innate immune response differentiation [25–27]. Researchers reported that both incorporation methods dichotomously induced immune responses that enhanced T cell differentiation, when albumin was used as the incorporated model antigen in the liposomal formulations. However, when antigen size and complexity decreases (like in virus- or tumor-derived antigens), a surface adsorption incorporation method will induce better immune responses than encapsulation [28, 29].

Fig. 1

Physicochemical and morphological factors to consider in liposomal vaccine design

Liposomal vesicle size

The factors, or parameters, affecting the function and potential use of a liposome-based vaccine due to their influence in immune responses include liposome size, lamellarity, surface charge, fluidity of the bilayer, formation of lamellar-hexagonal bilayers and the addition of immunostimulatory lipids. Regarding size, it has been previously discussed that larger vesicles (> 2 µm) loaded with a tuberculosis (TB) antigen are likely to induce cell proliferation and low IL-10 induction, contrasting with vesicles around 500 nm that promoted a distinct set of cytokines (IL-1β and IFN-γ) [30]. A study by Brewer et al. presented the effects on immune response differentiation of large (> 225 nm) vs. small (< 155 nm) lipid vesicles [31]. Larger vesicles induced IL-12 cytokine production but smaller vesicles did not. Murine experiments revealed that large vesicles induced TH1 responses due to increased levels of IgG2a and IFN-γ. The smaller particles in this study induced a TH2 immune response due to increase of IL-5 and IgG1 levels. IL-1β triggers a TH17 immune response, whereas IFN-γ induces a TH1 immune response. Therefore, liposome size affects the differentiation of cellular immune responses, rendering this physical parameter a key role in liposomal formulation function.

Vesicle size could be affected by the storage period that the vaccine undergoes and by other environmental parameters. It is well recommended to perform stability studies in different environmental conditions to ensure that liposomal vaccine vesicles do not change over time [32]. Applying several methods during the formulation design and development (like spray drying, sterilization, cryoprotection and PEGylation) will lead to liposomal vesicle stabilization [33–35]. These methods or approaches mentioned earlier avoid unwanted changes in vesicle morphology that could subsequently affect the immune responses of the vaccine. Furthermore, vesicle size stability can be affected by the vesicle lipid composition [36]. An effect observed with unstable lipid vesicles is coalescence, but real-time methods have been developed to study the phenomenon and control stability [37]. Additional work has discussed vesicle stability extensively and we recommend the reader to review the appropriate literature [38–40].

Lamellarity nature of the liposome

The lamellar nature of the liposomal vesicle could also affect the immune system causing differential responses. Beck et al. studied the adjuvanted immune responses to a recombinant HIV protein, CN54 gp140, in small unilamellar (SUV) and large multilamellar vesicles (MLV) [41]. The liposomes were composed of different combinations of monophosphoryl lipid A (MPLA) and lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC; 1,2-dimyristoyl-sn-glycero-3-phospho-(1ʹ-rac-glycerol), DMPG; and cholesterol, Chol), with or without the addition of the saponin QS21. SUVs without QS21 could induce immune responses (characteristic of a TH2 cell-mediated response) to CN54 gp140 protein due to high antibody production, contrasting to MLVs. Adding the saponin QS21 restored immune responses in MLVs (higher IgG1 > IgG2a and IFN-γ titers), stimulating both TH1 and TH2 responses. The saponin did not influence SUVs immune response profile. Shek et al. presented one of the first experiments that compared vesicle lamellarity characteristics against antibody formation enhancement [42]. Liposomes composed of lecithin, dicetyl phosphate and cholesterol were prepared in the presence of bovine serum albumin (BSA). Animals injected with blank liposomes (no BSA) did not generate a significant immune response, as predicted. However, animals injected with BSA-loaded unilamellar vesicles (ULVs) generated strong immune responses compared with multilamellar vesicles (MLVs). Another seminal article presents the co-formulation or adsorption of bovine herpesvirus 1 proteins to large unilamellar (LUVs) and multilamellar (MLVs) liposomes composed of phosphatidylcholine (PC) as the main lipid component [43]. Strong antibody titers were detected in animals injected with LUVs prepared with virus proteins (both adsorbed and co-formulated) and egg PC. Recently, another study demonstrated the effects of the lamellar state for liposomes in subunit vaccines to induce immune responses [44]. SUVs with ovalbumin (OVA) induced greater levels of CD8+ IFN-γ responses against the protein in the spleen. Researchers added TLR3 and nine agonists, enhancing the immune responses in MLVs but not SUVs. Altogether, the studies demonstrate the effect of lamellarity of liposomes in immune responses, being SUVs the preferred state to potentiate innate and adaptive responses which improves vaccine efficacy.

Surface charge

The surface charge of liposomes would be of important consideration for appropriate vaccine design. The overall charge can determine the adsorption or antigen interaction with the liposomes (e.g. anionic antigens will prefer to interact with cationic lipids) which affects antigen loading in the vaccine [18]. Hussain et al. found that replacing the cationic lipid DDA with the neutral lipid distearoyl-sn-glycero-3-phosphocholine (DSPC) decreases the amount of the tuberculosis recombinant antigen H56 from 84 down to 15%. In addition, the function of the vaccine in relation to immune response induction is well documented. Joseph et al. were studying an intranasal influenza vaccine model based on the liposomal formulation of the HN antigen with the polycationic sphingolipid ceramide carbamoyl-spermine (CCS) or other monocationic, neutral and anionic lipids [45]. Neutral and anionic lipid-based formulations were not immunogenic upon intranasal administration in a murine model. However, two out of five monoccationic-based liposomal formulations (containing lipids 1,2-dimyristoyl-3-trimethylammonium-propane, DMTAP; and 1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) induced vigorous local and systemic immune responses (TH1 and TH2 type responses). Researchers compared the monocationic liposomal formulations with the CCS-based liposomal formulation and the only commercially available influenza vaccine, demonstrating the efficacy of the sphingolipid as an immunopotentiator with higher antibody titers and protective immunity for approximately 9 months. Another study with Newcastle disease virus compared the immunization effects in chickens of neutrally- (EPC-Lip), anionically- (PS-Lip) and cationically-charged (SA-Lip) liposomes [46]. Strong humoral responses (local and systemic) were observed in neutral formulations of EPC-Lip, contrasting to the cationic SA-Lip. The anionic formulation mainly composed of phosphatidyl serine (PS-Lip) elicited the higher hemagglutination titers. Recently, Hussain et al. determined that replacing the cationic lipid DDA with the neutral DSPC reduced the TH1-mediated immune response of the formulation [18]. Based on the studies presented above we can conclude that cationic formulations might be the most suitable option to elicit strong immune responses, increasing antibody titers. There is additional information available about cationic lipids, like DDA, which possess significant immunostimulatory and adjuvanting properties [47, 48] and further explanations will be provided ahead.

Bilayer fluidity

The bilayer fluidity, dependent on the lipid gel-liquid crystal transition temperature and its effects on immune responses, is of important interest when designing efficacious vaccines. Many published works have evaluated and described this phenomena, including the earliest work by Yasuda et al. [49]. The effects on immune responses of liposomes prepared with phospholipids of PC with different transition temperatures containing the hapten Dnp-Cap-PE were measured. DMPC, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DSPC (all with high transition temperatures Tm > 20 °C) were favorable in eliciting antibodies to the hapten, contrasting to results obtained with DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) and EPC (Tm < 0 °C). Another research team followed a similar experimental design preparing liposomes with low- (DOPC and DLPC, − 20–0 °C), intermediate- (DPPC and sphingomyelin, 25–40 °C) and high-transition temperature (DSPC, > 50 °C) lipids [50]. DMPC, DPPC and sphingomyelin induced immune responses as per a plaque-forming assay, but DSPC was a poor immunogen. Cholesterol was added to the liposomal formulations, inducing significant humoral immune responses. The results from these two groups produced different outcomes: Yasuda et al. [49] presenting that less fluid lipids are better immunogens than fluid phospholipids and van Houte et al. [50] establishing that intermediately fluid phospholipids (which have a phase transition temperature of 25–40 °C) are better immunostimulatory agents. This discrepancy might be attributed to other factors, like particle size and Zeta potential, but such factors were not reported or analyzed to determine their role in these studies.

Additionally, Mazumdar et al. revealed that liposome composition may have an effect on immune responses [51]. Here, researches incorporated a leishmanial antigen (LAg) in liposomes containing DMPC, DPPC or DSPC and described the immunization process and results in a hamster model. No significant delayed hypersensitivity was detected in DMPC- or DPPC-containing lipids, which contrasted with DSPC-containing lipids. Moreover, DSPC-containing lipids protected up to 95% of the hamsters against a leishmanial infection. Recently, Kaur et al. presented results on how cholesterol influences the bilayer fluidity [52]. For instance, a direct correlation of cholesterol and membrane fluidity was observed in DDA:TDB (trehalose dibehenate) liposomal formulations. However, less IgG was detected as cholesterol increased in the system after 12 days of immunization in mice. This effect might be due to the loss of antigen in more fluid (high cholesterol) liposomes as the authors pointed out. The cytokine IFN-γ was at elevated levels when cholesterol was not present in the lipid bilayer. Even with the compelling evidence of how transition temperature and lipid bilayer composition affects immune responses, we can find conflicting results in published data. For example, Hampl et al. found no significant influence of immune response induction based on liposomes containing phospholipids with different transition temperatures [53]. Although the data presented does not follow a clear pattern, the general conclusion is that the more fluid a liposome is the less immune response it will generate when administered in different animal models. Likely behind this phenomenon is that fluidity tends to increase the release of the antigen from the liposomes, affecting antigen presentation and consequently the strength of immune responses. We need to point out that cholesterol also participates as a bilayer stabilizer, increasing rigidity in biological membranes [54]. Therefore, we might consider that the increase in bilayer fluidity by cholesterol observed in the studies discussed above may occur in synthetic bilayers only. Further research must account for such correlation due to the complexities of biological membranes.

Immunostimulatory lipids and liposome deposition

Immunostimulatory lipids might be of value in the vaccine design process. The lipids can act as adjuvants in the vaccine formulation and enhance the immune response we are looking for (innate or adaptive) as previously reported and reviewed [2, 5, 6, 47]. In a study developed by Rao et al., two adjuvants (lipid A and CpG-containing oligodeoxynucleotides, CpG-ODN) were studied in a liposomal formulation based on the HIV envelop protein ogp140 [55]. Both lipid A and CpG-ODN-containing liposomes elicited six and threefold anti-ogp140 antibodies, respectively. Immunization of BALBc mice with the HIV antigen incorporated in lipid A-containing liposomes produced a mixed TH1/TH2 immune response. Combining both adjuvants in the liposomal formulation generated a TH1 immune response. Puangpetch et al. presented the effect of using zwitterionic or cationic lipids in vaccine liposomal formulations [2]. Researchers compared DOTAP (cationic phospholipid) vs. DOPC (neutral phospholid) containing the adjuvant CpG-ODN. DOTAP-based liposomes with adjuvant enhanced the immune response against Burkholderia pseudomallei, which suggest an alternative approach for the treatment of melioidosis. The published data available suggests that certain lipids can induce, or enhance, immune responses. Very important is the fact that cationic liposomes are the ideal model to design effective vaccines due to their immunostimulatory properties.

Finally, the immunostimulation of the vaccine can be affected by the deposition of liposomes at the site of injection which in turn is affected by particle size. Henriksen-Lacey et al. reported this in two separate articles [6, 56]. On Henriksen-Lacey et al. researchers utilized the phospholipid DDA as the building block for the liposomes containing the adjuvant TDB. The antigen for tuberculosis infection, Ag85B-ESAT-6, was co-administered or incorporated to liposomes in mice. Antigen administered alone to mice did not created a depot at the site of injection, causing the antigen to diffuse away from the site and reduce immune responses. The observations directed them to conclude that cationic liposomes made up of DDA promotes depot formation at the site of injection mainly due to size characteristics between antigen-loaded liposomes and antigen administered alone. Later, Henriksen-Lacey et al. [30] studied the effect of liposome composition [DOTAP, DDA or DC-Chol (dimethylaminoethane-carbamoyl-cholesterol)] and the depot formation and antigen distribution. DDA and DC-Chol represented the phospholipids where liposomes induced the migration of monocytes to the site of injection and a significant increase of IFN-γ levels. In general, larger liposomes will form a depot at the site of injection meanwhile smaller liposomes will migrate to lymphoid tissue for antigen presentation and processing.

Go to:

Liposome-immune cell interactions to improve vaccine effectiveness

Early vaccination strategies included the development of attenuated or inactivated vaccines, which are composed mainly of weakened or dead pathogens, respectively. Currently these kinds of vaccines are facing challenges with variabilities in immune response induction [57, 58]. Modern vaccination strategies are emphasizing the study of subunit vaccines and proving their effectiveness in immune response modulation [59, 60]. Subunit vaccines are characterized by the co-delivery of adjuvants and antigens for immunostimulatory purposes (Fig. 2). The antigen is either a natural or recombinant peptide, protein or molecule derived from the pathogen. The adjuvant will enhance the immune response of the vaccine and potentiate the effect of the antigen during the process. It is very important to know how to present the antigen incorporated in the liposomes to APCs because this will ensure their proper immune cell maturation, antigen presentation and eventual induction of adaptive immune responses as previously reviewed [61, 62]. Cell targeting studies have been investigated and produced significant data and observations of how antigen presentation plays a significant role. Aramaki et al. investigated the utilization of liposomes as carriers for antigens related to gut-associated lymphoid tissue and their uptake by rat Peyer’s patches [63]. Preferential uptake was observed for liposomes in the rat Peyer’s patches than non-patch tissues, specifically for DSPC:PS:Chol liposomes. This suggests that liposome composition affects their uptake. Fluorescently labeled liposomes were internalized by patch tissue in the lower ileum with size (> 374 nm) playing an important direct correlation with uptake. Another report looked at how cationic vesicles composed of DDA could interact with normal and transformed mouse fibroblasts cells [64]. Cell–cell adhesion was observed when DDA concentration was equal or greater to 50 µM. Investigators determined that normal cells were susceptible for DDA vesicles meanwhile transformed cells were resistant to DDA-mediated (> 1 mM) cell death. The interaction with cationic vesicles created a change in cell charge from anionic to cationic, making this system of cationic vesicles ideal for the delivery of negatively charged macromolecules like proteins and DNA.

Fig. 2

Co-delivery of antigen and adjuvant to APCs in subunit vaccines. Interaction of the adjuvant with the PRR results in upregulation of co-stimulatory molecules necessary for appropriate T cell stimulation

Vaccine interaction with neutrophils, monocytes and APCs

Several studies presented results on how liposome-based vaccines interact with neutrophils and monocytes, key players in inflammation and immune responses. First, Karathanasis et al. used previously characterized and purified peptides to target liposomal nanocarriers to some types of leukocyte cells [65]. Peptides were covalently attached through the carboxyl group of DSPE-PEG by utilizing the crosslinker N,N-dicyclohexylcarbodimide (DCC). Researchers found that targeted liposomes interacted better with monocytes and neutrophils, contrasting with results obtained by non-targeted liposomes. Moreover, the surface density of the peptide directly correlated with liposomes-cell interactions, making this parameter a new way to measure the effectiveness of the interactions and the potential induction of immune responses valuable in subunit vaccine development. Then, Johansen et al. investigated the targeting of monocytes and the delivery of a TLR agonist (TMX-202) using a cationic liposomes-based formulations (mainly POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine):DOTAP composition) [66]. After an hour incubation, the subset of monocytes targeted by the liposomes were lymphocytes and granulocytes (75–95%). A strong IL-6 and IL-12p40 induction was observed, accompanied by monocyte differentiation to CD14+/DC-SIGN+ DCs. Mainly found in the lymph nodes, lymphocytes include natural killer (NK), T and B cells, which are involved in innate immune responses, cell- and humoral-mediated immunities, respectively. Granulocytes, or polymorphonuclear (PMNs) leukocytes include basophils, neutrophils, mast cells and eosinophils that participate in allergic and inflammatory reactions. The information gathered by the researchers is important because it can determine future treatments and vaccine developments to focus on selected immune responses, depending on the cell or cell types that are being targeted, reducing or avoiding unwanted adverse reactions (e.g. allergies or chronic inflammation).

After interaction with the antigen, cytokines and/or interaction with their milieu, monocytes could differentiate into macrophages or dendritic cells. Both cell types will then migrate to lymph nodes to elicit the corresponding adaptive immune responses. Macrophages and dendritic cells are specialized APCs that reside in the blood stream and help in antigen uptake and antigen presentation to T and B cells, inducing cell-mediated and humoral immune responses, respectively. It is known that antigen-containing liposomes are internalized by pinocytosis in macrophages and then cross presented to CD8+ T cells (specialized T cells that attack and kill tumors), inducing antigen-specific cytotoxic T lymphocytes [67]. This cross-presentation occurs when exogenous antigen is up taken and presented via the class I major histocompatibility complex (MHC I), which classically only happens with endogenous antigens such as those from viruses. To cross-present and elicit a CD8+ T lymphocyte response, the antigen must be delivered to the cytosol APCs. This was studied by Owais et al. in which yeast-derived lipids liposomes and egg PC:Chol liposomes were tested against J774 A1 macrophages and the interactions measured [68]. The fusion rate for yeast lipid liposomes to macrophages was at 40–70%, compared to 1–8% for egg PC:Chol liposomes. Liposome contents were successfully delivered to the cytosol of macrophages. Ovalbumin was used as the model antigen for yeast-derived lipid liposomes and it was found that it elicited a strong CD8+ T cell response. Another group of researchers investigated the uptake mechanisms of liposomes in rat peritoneal macrophages (PM) [69]. Researchers determined that two uptake systems exist because of cholesterol content and size differences of liposomes. For high- (44% molar) and medium-cholesterol (33% molar) content liposomes, the complement receptor-mediated phagocytosis occurs. A complement-independent uptake pathway was suggested for low-cholesterol content liposomes since no inhibition of their internalization rate was observed by the anti-C3 antibody. Therefore, once again, lipid composition is a main player in liposome-cell interactions, potentially affecting the way immune responses develop.

Mannosylated liposomes

Mannosylated liposomes are becoming an alternative method to deliver antigens or antimicrobials to macrophages or DCs [70, 71]. This approach is due to the fact that mannose receptors can be found in these cell types, improving directed-targeting [16]. Other research teams have used cationic lipids like DDA, DOTAP and/or DC-Chol to enhance or potentiate the immune responses due to cell targeting strategies [48, 72, 73]. Korsholm et al. determine that DDA liposomes were minimally internalized by T cells in the mixed splenocyte cultures, contrasting to high uptake rates for APCs (bone marrow dendritic cells) through class II MHC (MHC II), leading to an enhanced OVA presentation. The immune responses described by Varypataki et al. contrasted to Korsholm et al. since DOTAP:DOPC liposomes bearing the peptide SIINFEKL and polyI:C assisted in the delivery of OVA to DCs through MHC I, inducing a CD8+ T cell response.

Understanding and applying innovative ways of cell targeting could improve the discovery pipeline for novel therapeutic agents, avoiding undesirable immune responses that can be detrimental to the patient. These therapies could treat inflammatory diseases [like arthritis or chronic obstructive pulmonary disease (COPD)], infections or cancer. The following sections will focus on how liposome-based vaccines are being utilized for the treatment of infections from viral, bacterial, fungal and parasitic origins. A special section will discuss liposomal vaccines that target the bacterium Mycobacterium tuberculosis to treat TB infections.

Go to:

Viral infections and liposomes-based vaccines

Viruses are ethiologic agents of different diseases in animals [74] (including humans [75, 76]), plants [77], parasites [78, 79] and bacteria [80, 81]. The basic definition of a virus is a non-living infectious agent that requires living cells for its replication and survival, which allows spreading of the disease. This cell lysis leads to inflammation and tissue damage which are detrimental for the host, enhancing the state of the disease. Additionally, some viruses, like HIV, destroy immune cells hindering effective immune responses when other infections (secondary infections in seropositive patients) occur. Viral infections can be treated by either antiviral therapeutic [82] or by prophylactic vaccine [83] approaches. Here, we will present several examples of vaccine applications based on liposomes for the prophylactic treatment of certain viral infections.

Hepatitis

One of the most significant viruses that affect human health are hepatitis viruses. Hepatitis is an inflammation of the liver tissue caused by the five types of hepatitis viruses (A, B, C, D and E). Hepatitis A and E are spread through contaminated food or water sources. Hepatitis B (HBV) is sexually transmitted or during pregnancy and birth. Both HBV and hepatitis C can be transmitted through blood (needle exchange by IV users) and hepatitis D can only infect people infected with HBV. Two seminal reports investigated novel approaches for the prophylactic treatment of HBV utilizing cationic lipids. Brunel et al. reported the effectiveness of recombinant hepatitis B surface antigen (HBsAg) presentation based on DC-Cholesterol liposomes or aluminum hydroxide (alum) adjuvants in a subcutaneous vaccine model [84]. The DC-Chol-based vaccine elicited antibody (IgG1 and IgG2a) titers in three mice lines (BALB/c, OF1 and B10.M). Compared to the alum-based vaccine, which demonstrated weak immunogenicity, DC-Chol liposomes induced controlled TH1 and TH2 immune responses characterized by normal, but significant levels of cytokines IL-2 and IFN-γ and IL-5, respectively. Controlled immune responses are important to avoid inflammation that could result in tissue damage. The researchers concluded that cationic lipids like DC-Chol could be used as adjuvants, enhancing the immunogenicity of previously non-immunogenic vaccines, specifically in the development of prophylactic vaccines against hepatitis B virus. Another group investigated the use of a transcutaneous vaccine for the treatment of HBV infections [85]. In that report, the vaccine presents some differences from the Brunel et al. article based on the antigen and carrier types. First, cationic transfersomes, a type of liposome, were prepared from DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) phospholipid and sodium deoxycholate (SDC) at different DOTMA weight ratios (75–95% w/w). Second, plasmid DNA encoding the HBsAg gene was loaded to the transfersomes instead of the antigen. The transfersomes were not cytotoxic to HepG2 cells and were stable at different temperatures (4 and 28 °C). Immunization studies included HBsAg DNA-loaded tranfersomes (topical), naked HBsAg DNA (topical and intramuscular) and pure HBsAg (intramuscular) administered to BALB/c mice. Significant levels of anti-HBsAg antibodies and cytokines (IL-2 and IFN-γ) were elicited in topical DNA-loaded transfersomes as compared to intramuscular naked DNA delivery. This antibody and cytokine profile confirmed the induction of TH1 and TH2 immune responses as observed in Brunel et al. Both studies represent great advances in the treatment of HBV infections with novel approaches and administration routes that could applied in the future as preventive vaccines.

Influenza

Influenza is an infectious disease caused by the influenza virus, affecting human health with a pandemic effect in several instances. The virus is divided in three types (A, B and C) and it is spread through the air from coughs and sneezes, therefore the importance for vaccine development studies for the infection. An intranasal (i.n.) liposomal influenza vaccine study was presented by Joseph et al. [45]. The vaccine formulation developed by the group was based in the polycationic sphingolipid N-palmitoyl-d-erythro-sphingosyl-carbamoyl-spermine (or ceramide carbamoyl-spermine, CCS) and was compared with other formulations containing monocationinc phospholipids (DC-Chol, DDA, DSTAP (1,2-stearoyl-3-trimethylammonium-propane), DMTAP and DOTAP). Cholesterol was added to increase liposome fluidity and the lipids DMPC and DMPG were included in neutral and anionic formulations, respectively. All formulations contained the influenza A antigens hemagglutinin and neuraminidase (HN). DMTAP- and DOTAP-based vaccines were the only monocationic lipid formulations to induce strong systemic (serum) and local (lung) TH1 and TH2 responses. Surprisingly, DDA-containing formulations did not induce strong, or significant, local or systemic immune responses. No specific reasons were provided by the team for such results but we can infer that vesicle morphology (multilamellar and oligolamellar cationic formulations), size (1–4 µm diameter) and encapsulation efficiencies (10–90% for non-CCS cationic formulations) would be responsible. The CCS-based vaccine formulation was the only formulation to be at the same or superior level of effectiveness compared to the commercially available vaccine with cholera toxin as the adjuvant. More recently, researchers investigated a triple co-culture model of the human respiratory tract to study the immunostimulatory responses of virosomes and liposomes and their internalization [86]. The epithelial cell line 16HBE was grown with monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) and exposed to liposomes and virosomes to evaluate the immune responses elicited by the nanocarriers. The virosomes were liposomes prepared with solubilized influenza A/Brisbane/59/2007 H1N1 membrane proteins and included the neutral lipids DOPC and OPPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE). Liposomes were prepared with previously mentioned neutral lipids and no influenza soluble membrane proteins were added. Virosomes were internalized more efficiently by all cell types in mono- and co-cultures, with APCs like MDMs and MDDCs presenting the highest internalization levels as per flow cytometry and laser scanning microscopy. MDDCs were moderately activated by liposomes and virosomes in monocultures, and inducing elevated levels of cytokine (IL-1β and IL-8) production in co-cultures. Virosomes were internalized at higher levels in epithelial cells in comparison to liposomes.

Respiratory syncytial virus

Another respiratory viral pathogen of interest is the respiratory syncytial virus (RSV). RSV causes respiratory tract infections, specifically lower respiratory tract infections in children and it presents high hospitalization incidence [87]. An i. n. study was developed by Klinguer et al. in which cationic DDA liposomes were mixed with the recombinant fragment of the RSV G protein (BBG2Na) and administered to BALB/c mice [88]. The DDA + BBG2Na liposomal formulation presented significant antibody (IgG and IgA) titers at systemic (serum) and local (nasal) levels. Cytokine production was higher for IL-2 and IFN-γ in cationic liposomes carrying the RSV recombinant antigen, confirming the protection after a viral challenge and the induction of TH1 immune response. These three reports on respiratory viruses and their prophylactic vaccine development confirms the importance of mucosal immunization as it elicits local and systemic B- and T-cell responses [89]. It is also clear that cationic phospholipids like DDA, DC-Chol or DOTAP play a leading role (Table 3) in the immunostimulation of subunit vaccines and more approaches should be investigated. Different administration routes were investigated in the studies for viral infections. However, we cannot verify and compared these administration routes to determine the best alternative due to experimental differences in the studies (liposomal composition and targeted virus). Future experiments should focus their efforts in comparing different administration routes with the same liposomal composition and prophylactic treatment of a particular virus.

Table 3

Promising vaccine formulations for the treatment of viral infections

Lipid(s) and sterol used	Virus type	Cell line/animal model used	Administration route	Promising liposome formulation	References
DOTMA	Hepatitis C	HepG2 cells and BALB/c mice	Transcutaneous	DOTMA	[85]
DC-Chol	Hepatitis B	Lymph node cells, BALB/c, OF1 and B10.M mice	Subcutaneous	DC-Chol	[84]
DMPC, DMPC/DMPG, DC-Chol/DOPE, DSTAP/Chol, DDA/Chol, DOTAP/Chol, DMTAP/Chol and CCS/Chol	Influenza H3N2	Splenocytes from BALB/c and C57BL/6 mice	Intranasal	DMTAP/Chol and DOTAP/Chol	[45]
DDA	Respiratory Syncytial Virus	BALB/c mice	Intranasal	DDA	[88]
DOPC/OPPE (Influenza virosome) and DOPC/OPPE (liposomes)	Influenza	MDMs, MDDCs, 16HBE14o cells, PHNECs and EPCam + cells	N/A	DOPC/OPPE (Influenza virosome)	[86]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

Bacterial infections and liposomal vaccines

Bacteria can be beneficial for humans, but also detrimental to our health when they harbor pathogenicity traits. Bacteria are divided in Gram positive (+) or negative (−) based on Gram staining, that surveys the peptidoglycan content in the cell wall. Gram (+) bacteria have a positive result in the Gram Stain method, which determines in a qualitative way the presence of the cell wall component: a thick peptidoglycan layer. In contrast, Gram (−) bacteria present a negative result in the stain, indicating a thin peptidoglycan layer, sandwiched between two cell membranes (plasma and outer membranes). Gram (−) bacteria also contain an important immunogenic molecule and pathogenicity factor, the lipopolysaccharide (LPS) [90, 91]. LPS has been used to increase the fusogenicity of cationic liposomes [92]. In that study, researchers developed a carrier system to incorporate LPS into mammalian cell membranes via DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine):DOTAP (1:1 wt. ratio) liposomes. The team of researchers demonstrated that high LPS concentrations on immortalized fibroblasts generated the activation of macrophages, starting the elimination of LPS-bearing cells.

Additionally, bacteria will have unique cell components, like genetic material, lipids or proteins, that could serve as adjuvants or antigen markers for subunit vaccine development, depending on the molecule chemical characteristics. Li et al. investigated the potential use of cationic (DDA-based) mannosylated liposomes to deliver the model DNA plasmid pGL4.10 (encoding luc2) [19]. The plasmid was protected from nuclease degradation by the liposomes. The cationic mannosylated liposomes showed high uptake and transfection, activating bone marrow DCs (BMDCs). BMDCs activation was characterized by the upregulation of CD80, CD86 and CD40. Another study by Nakanishi et al. studied the effects of positively, negatively and neutrally charged liposomes administered subcutaneously in the immune responses of the fragment A of diphtheria toxin (DTA) and ovalbumin (OVA) [93]. Cationic liposomes were composed of phosphatidylcholine:cholesterol:stearylamine (PC:Chol:SA, 4:5:1 molar ratio), anionic liposomes were composed of PC:Chol:l-α-dimirystoylphosphatidic acid (PC:Chol:DMPA, 4:5:1) and neutral liposomes were composed of PC:Chol (1:1). Positively charged liposomes could induce potent antigen-specific cytotoxic T cell responses. However, DTA-containing cationic liposomes were cytotoxic to macrophages. In contrast, empty cationic liposomes or DTA-loaded anionic and neutral liposomes were not cytotoxic. CD8+ OVA responses were highly induced by positively charged liposomal vaccines, potentially presenting the processed antigen through MHC I. Both research articles presented us the utilization of liposomes to investigate and test the effects on immune responses during vaccination. The immune responses measured varied, but making it clear that cationic liposomes induced the required cells (macrophages and DCs) to obtain the appropriate responses.

The liposomal vaccine studies mentioned above serve as the basis for the following articles which incorporate bacterial antigens for the development of prophylactic vaccines for certain infections. Puangpetch et al. developed a cationic-based liposomal formulations incorporating CpG ODN and to determine the prolongation and mechanisms of the immune responses [2]. The researchers employed the etiologic agent of melioidosis, Burkholderia pseudomalei, as the infection model in BALB/c mice. Cationic and not neutral liposomes administered intramuscularly granted protection against the bacterial challenge study. Prominent levels of IFN-γ were observed 2 days postinfection, but lowered by a CpG ODN-loaded cationic liposome pre-treatment. Neutrophils were not activated by the cationic liposomes with CpG ODN, but macrophages were stimulated by the formulation due to nitric acid production and low intracellular bacterial burden (30 days post vaccination). An additional study involving mannosylated liposomes containing the meningococcal PorA (from Neisseria meningitidis) focused its attention on cell interaction [94]. Anionic (PG- (phosphatidylglycerol) and PS-based) and cationic (DMTAP-based) liposomes were formulated, and one of the anionic formulations was mannosylated (PC:PG:Chol + Man-PE (mannosyl phosphatidylethanolammine). When exposing the formulations to human and murine DCs, researchers observed an increase of liposome-cell interaction in the anionic mannosylated liposomes and cationic liposomes when compared to anionic formulations alone. The result indicated that adding mannosyl moieties to liposomes generated a mannose receptor (MR)-mediated cell interaction. The murine DCs were confirmed to present the markers MHC II+, CD11c+ and CD11b+, meanwhile human DCs presented CD40+, CD1a+ and both MHC I and II. Researchers in the field should address studies that investigate route of administration effects on immune responses. With the studies presented here, it is difficult to determine what best route of administration we should follow for future prophylactic vaccine development. We recommend investigating the optimized formulations in different administration route studies.

The use of cationic liposomes seems of importance for the development of adequate vaccine formulations. The studies presented above demonstrate that by employing cationic phospholipids like DOTAP, DMTAP and DDA, would improve cell interaction levels, allow adequate antigen presentation and induce strong immune responses. These effects will insure that the vaccine will work properly and optimally. For the benefit of the reader, Table 4 presents a summary of the most relevant literature that optimized vaccine formulations for the treatment of bacterial infections. Additionally, previous research on cationic liposomes have discussed the cytotoxicity potential of such formulations. DDA has been determined to be safe as no relevant cytotoxic effects were determined in studies by Hilgers and Snippe and Gall [47, 95]. Only local inflammatory reactions manifested as swelling were observed in mice (when administered alone). Contrasting results are found for DOTAP and DOTMA cationic lipids. DOTAP has been found to be not cytotoxic to macrophages in a study by Jin et al. [96], but Romøren et al. determined that macrophage-derived cell lines were found to be affected by the cationic lipid [97]. The cytotoxic response differences might be due to structural and morphological characteristics in the formulations. Jin et al. employed Tween 20 and tricaprin (part of the solid core) to form solid lipid nanoparticles, meanwhile Romøren et al. just prepared liposomes. Further contradictory information is available for DOTMA, which can be cytotoxic for RAW 264.7 cells at all lipid ratios (DOTMA + DOPE), but not to human umbilical endothelial cells or mouse fibroblasts cells [98]. Kurosaki et al. determined that erythrocytes undergo agglutination and hemolysis when exposed to DOTMA-based liposomes [99]. However, an earlier study by Kurosaki et al. determined lower cytotoxicity for erythrocytes, showing no agglutination and hemolysis, when DOTMA was formulated with N-laurylsarcosine, and Chol, vitamin E and Chol or egg PC and Chol [100]. Future work should include the analysis of lipids alone and formulated with other lipids to elucidate the cytotoxic effects. Additional work should be done for these and other bacterial infections lacking proper prophylactic vaccines, leading to outcome improvement from the infection.

Table 4

Promising formulations for the treatment of bacterial infections

Lipid(s) and sterol used	Bacteria or disease	Cell line/animal model used	Administration route	Promising liposome formulation	References
PC/PG/Chol, PC/PG/Chol/Man-PE, PC/PS/Chol, PC/DMTAP/Chol	Meningitis	Monocyte-derived human DCs and murine bone marrow-derived DCs	N/A	PC/PG/Chol/Man-PE and PC/DMTAP/Chol	[94]
PC/Chol/SA, PC/Chol/PA and PC/Chol	Diphteria toxin	BALB/c, C57BL/6 and ddY mice; P815, P13.1 and CD8OVA cells	Subcutaneous	PC/Chol/SA	[93]
DOTAP and DOPC	Melioidosis	Neutrophils and splenocytes from BALB/c mice	Intramuscular	DOTAP	[2]
DOPE/DOTAP	E. coli	Mouse embryonic fibroblasts (MEF) and RAW 264.7 macrophages	N/A	DOPE/DOTAP	[92]
DDA/Chol/Man-C6-Chol and DDA/Chol	E. coli	DC 2.4 cells	N/A	DDA/Chol/Man-C6-Chol	[19]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

Liposomal-based vaccines in fungal infection treatment

Fungi are eukaryotic organisms that include yeasts, molds and mushrooms. Several yeasts and molds are common pathogenic agents, especially in immunocompromised patients [101, 102]. Antibiotic resistance is being detected, not only in isolates from the USA but also in other developed countries, in different fungal species (like Candida and Aspergillus) [103]. Due to the threat of antifungal resistance, other therapeutic approaches should be investigated, and liposomal vaccines may play a significant role. To the best of our knowledge, the literature review presents liposome-based vaccines for Candida sp. infections but limited information is available for other fungal pathogens. Studies investigating other fungal species with elevated infection prevalence, like Aspergillus, Fusarium, Coccidioidomyces and Zygomycetes sp., have been identified but their treatment approach does not include liposomal vaccines, and we invite further reading on the topic [101, 104, 105]. We will discuss ahead information available for liposomal vaccine development for the prophylactic treatment of Candida sp. infections (Table 5).

Table 5

Candidiasis treatment with potential liposomal vaccines

Lipid(s) and sterol used	Fungi or disease	Cell line/animal model used	Administration route	Promising liposome formulation	References
PC/Chol	C. albicans and C. tropicalis	BALB/cByJ mice	Intravenous	PC/Chol	[106]
PC/Chol		BALB/c mice	Intravenous	PC/Chol	[107]
DMPC/DMPG	C. albicans	ICR mice	Subcutaneous	DMPC/DMPG	[108]
EPD/DOGS-NTA-Ni		BALB/c mice	Intradermal	EPD/DOGS-NTA-Ni	[109]
		ICR mice	Intradermal		[110]
DDA:MO		Macrophages and BALB/c mice	Subcutaneous	DDA:MO	[111]
		BALB/c mice	Subcutaneous		[112]

The first report dealing with the study and development of a vaccine to treat candidiasis, encapsulated the mannan adhesin portion of Candida albicans [106]. The adhesin protion of the mannan of two C. albicans serotypes (A and B) were incorporated in PC:Chol liposomes (3.2:1 molar ratio). Mice were vaccinated (intravenously) during a period of 5–6 weeks and challenged with C. albicans infection, presenting increasing resistance to disseminated disease. Furthermore, antiserum agglutinins (IgM-type antibodies) from the immunized mice were studied for their humoral protective characteristics against C. tropicalis infection, demonstrating the efficacy of the vaccine. Subsequently, researchers decided to investigate the effectiveness of the monoclonal antibodies obtained from the previous study in a vaginal candidiasis model [107]. Similarly, the vaccine (L-mann) was prepared by the mannan adhesin fraction incorporation into PC:Chol liposomes. Mice were immunized intravenously once a week for 5 weeks prior vaginal inoculation. Vaccinated mice challenged with C. albicans presented lower CFUs (110 ± 38 × 103 CFU/g of vaginal tissue) when compared to non-mannan vaccine approach (240 ± 44 × 103 CFU/g of vaginal tissue). Both article demonstrated the capacity of the vaccine to protect at local or systemic infections of C. albicans.

Further studies investigated zwitterionic and cationic liposomes utilizing ribosomes and recombinant Hsp90 protein as antigens from C. albicans [108–110]. Eckstein et al. prepared anionic DMPC:DMPG liposomes co-lyophilized with RNA obtained from cell lysates of C. albicans cultures or by lipid film formation. Mice protection in a subcutaneous vaccination (60% survival) against the fungal challenge demonstrated the effectiveness of the vaccine prepared by the co-lyophilization method in the presence of C. albicans ribosomes. Subsequently, neutrally charged metalloliposomes with incorporated recombinant Hsp90 (heat shock) protein from C. albicans were developed. Nickel-chelating liposomes were prepared with EPC and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DOGS-NTS-Ni) at molar ratios of 95:5. A non-pyrogenic MDP was used as an adjuvant in the liposomal system and the protein was surface attached by metallochelating bonds. DCs interacted with the liposomes and phagocytosed the nanoparticles in vitro. TH1 and TH2 immune responses were induced after intradermal mice vaccination in comparable levels to the Freund’s complete adjuvant vaccine. Following the previous report, Knotigová et al. employed nickel-chelating liposomes with different MDP-derivatives (norAbuMDP/GMDPs) and tested their adjuvant vaccine potential [110]. Also, Hsp90 from C. albicans was employed as the model antigen. Adaptive and innate immune responses were induced by the developed vaccine systems in intradermal vaccinated rabbits and mice.

Recently, cationic liposomes studies incorporated cell wall surface proteins (CWSPs) of C. albicans and their immunostimulatory properties analyzed during subcutaneous administration [111, 112]. Both studies employed DDA:monooleoylglycerol (MO) at 33:67 molar ratio. Liposomes were not toxic to macrophages and were internalized within 20 min of exposure. In the first study, immunized mice displayed strong humoral- and cell-mediated immune responses. Antibodies were produced against cell wall proteins Cht3p and Xog1p. In the second report, two CWSP-loaded cationic liposomal formulation (ADS1 and ADS2) were tested against disseminated candidiasis. ADS1 immunized mice presented significantly higher levels of C. albicans antibodies, contrasting with the ADS2 formulation. This antibody titer production induced the phagocytosis of the fungus. Elevated levels of the cytokines IL-4, IL-17 and IL-10 were significantly higher than control groups, suggesting TH2, TH17 and anti-inflammatory immune responses, respectively.

Future studies on fungal infections and their prophylactic treatment with vaccines should be performed not only in C. albicans, but also other ethiological agents (e.g. Aspergillus). The literature review revealed a field with potential for growth and development of novel approaches to treat fungal infections, which benefit immunocompromised patients (elderly or HIV-seropositive patients). Additionally, further studies must employ further cationic lipids and their effects on immune response induction. Studies comparing physicochemical properties of liposomes to treat fungal infections must take place to optimize the vaccine strategy and avoid unwanted responses and results. In the studies presented above, the intravenous administration was investigated and revealed affirmative results (~ 60% survival of mice). However, we recommend further studies that compare immunomodulatory responses against mucosal vaccine administration for vaginal candidiasis infections. Similar survival rates were observed for subcutaneous and intradermal administration routes, but future studies should investigate a particular set of liposomes against the different administration to observe if survival rates are affected.

Go to:

Parasitic infections and liposomal vaccines

Parasitism and malaria

Parasitism is a non-mutual, biological interaction in which the parasite lives inside the host and derives its own nutrients at the host’s expense. Parasites can be classified as macroparasites (visible with the naked eye) like helminths, or microparasites (which are smaller) like viruses, bacteria or protozoa. A textbook example of a parasite is Plasmodium vinckei, causative agent of malaria. In malaria, the parasite (Plasmodium sp.) is transmitted by mosquito bites. The parasite’s sporozoites reside in the liver (in humans), developing into merozoites that infect human red blood cells, initiating the red blood cell cycle. When appropriate, the merozoites will developed into gametocytes that infect more red blood cells that are taken up by mosquitoes during the bites. This initiates the mosquito stages (gametes, ookinetes and oocysts). Oocysts are transmitted to the host, initiating the liver stage.

Postma et al. developed a novel desferrioxamine B (DFO) delivery system based on liposomes to treat malaria [113]. DFO is a siderophore that chelates ferric iron (iron is an vital component of red blood cells). Iron is an important nutrient for P. vinckei as it infects red blood cells. Contrasting to previous liposomal vaccine articles, the researchers investigated the lipid to drug composition and the bilayer fluidity effects on DFO delivery and protection from infection. The lipids were all anionic in charge due to the presence of egg PG. Three different treatments of DFO were analyzed in the study: (1) multiple free DFO subcutaneous injections, (2) intraperitoneal infusion of free DFO and (3) multiple subcutaneous DFO-loaded liposomes injections in C57B1/6J female mice. Parasitemia was suppressed by multiple subcutaneous injections of free DFO before and during infection, but injections prior to infection did not. Suppression of parasitemia and long-term survival was observed for intraperitoneal infusion of free DFO 1 day before infection or by subcutaneous injections of liposomal DFO prior to infection (day-1). Bilayer rigidity was studied by incorporating Chol (intermediate rigidity) and DSPC (high rigidity) in the liposomes, demonstrating that no relationship exists that affect the liposome antimalarial function. However, drug-to-lipid ratio affected the antimalarial activity of the liposomal-based vaccine, suggesting that low drug-to-lipid ratios are the best formulation parameter at combating the infection. When liposomal DFO was administered to mice, a long-term protection against malaria was observed (days 7 and 8, 400 mg/kg/day). This article proves that utilizing a common siderophore (DFO) as an iron chelator in combination with liposomes, improve the therapeutic and prophylactic effects of the vaccine. However, no immune response studies were performed, lacking essential information about the mechanisms of the vaccine when immunization occurs.

A pre-clinical report of the malaria vaccine RTS,S was published by Stewart et al. [114]. The RTS,S/AS02A vaccine utilizes the circumsporozoite protein as antigen. Investigators in this report evaluated the effects of certain adjuvants (AS01B, AS02A, AS05 and AS06) which vary in the concentration of MPL, QS21 or CpG and their formulation delivery system (emulsion vs. formulation). AS01B was the only liposomal formulation in the study. Rhesus macaques were immunized by intramuscular injection with the different RTS,S/adjuvant combinations and specific antibodies, IFN-γ and IL-5 levels were determined after weeks 14 and 34. All regimes were safe and presented elevated antibody titers (except for AS06-containing vaccine formulation). RTS,S/AS01B presented higher levels of IFN-γ at weeks 14 and 34, and the highest IFN-γ to IL-5 ratio when compared to RTS.S/AS02A. Leishmaniasis.

Another parasitic disease is leishmaniasis caused by the parasite Leishmania sp. It is transmitted by sandflies to mammals, transferring metacyclic promastigotes via feeding. The metacyclic promastigotes invade macrophages and granulocytes, developing into amastigotes which multiply by simple division, eventually causing macrophage lysis. Further, amastigotes infect new macrophages or are transferred to the sandflies during feeding. Amastigotes transform into procyclic promastigotes in the gut, maturing into metacyclic promastigotes by simple division. The disease is common in certain regions of Asia, Africa, South and Central America and even southern Europe. The disease is divided into three major syndromes, cutaneous, mucosal or visceral leishmaniasis. Visceral leishmaniasis poses a major risk of death incidence [115].

Three seminal articles provide valuable information regarding the studies and development efforts towards a prophylactic vaccine for leishmanial infections [116–118]. First, Bhowmick et al. presented the immunotherapy effects of leishmanial antigens in liposomes [116]. The researchers correlated the efficacy of soluble leishmanial antigens (SLAs) from Leishmania donovani promastigote membrane incorporated in neutral (lecithin:Chol), negative (lecithin:Chol:PA (phosphatidic acid)) and positively (lecithin:Chol:SA) charged liposomes intraperitoneally administered. L. donovani was eliminated from the liver and spleen when SLAs were present in cationic lipids. IL-4 and IL-10 were downregulated when SLA-cationic liposomes were administered to the mice and the immunomodulatory response presented the TH1 cytokines IFN-γ and IL-12. Subsequently, Banerjee et al. presented two articles which cover the study of cationic stearylamine liposomes for the development of a visceral leishmaniasis vaccine. In the first published article by the team of researchers, amphotericin B (AmB) is used in association with stearylamine (cationic) liposomes as a novel therapeutic approach [117]. When administered to BALBc mice, the leishmanial parasite was eliminated from the liver and spleen. Moreover, when comparing to the conventional liposomal formulation AmBisome, the intravenous administration of AmB-SA-PC liposomes induced the production of IFN-γ from CD8+ and CD4+ T cells. At the same time, the formulation reduced the toxicity effects of the drug by reducing TNF-α levels. In the splenic supernatant culture, IL-10 was downregulated, causing the production of IL-12 and nitric oxide during the AmB-SA-PC liposomes treatment. Also, Banerjee et al. investigated the effects of liposome charge in an antileishmanial assay [118]. Researchers provided evidence of membrane disruption caused by the cationic stearylamine liposomes in promastigotes and amastigotes. No toxicity in murine peritoneal macrophages and human erythrocytes was detected. These studies confirmed the prophylactic effect of SA-PC liposomes against leishmanial infections.

Amoebiasis

Finally, a research paper dealing with parasitic infections present us the incorporation of Entamoeba histolytica Gal/GalNAc lectin LecA antigen in liposomal, emulsion and alum formulations containing synthetic TLR agonists adjuvants [59]. E. histolytica is an anaerobic amoeba and is the etiological agent of amoebiasis, or diarrheal disease commonly transmitted through contaminated water and food sources [119]. The liposome formulation containing a TLR4 and TLR7/8 agonists was selected for further studies due to its ability to induce intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17a. A high mucosal IgA response hinder the ability of parasites to adhere to mammalian cells. The subcutaneous immunization regime success rate reached 55% efficacy.

Parasitic infections are common especially in underdeveloped regions, posing a notable risk for children, adults and the elderly. Further studies should address the use of adjuvants and their immunomodulatory mechanisms when administered in liposomes for vaccine development. Several studies previously discussed optimized certain formulations for vaccine development and we invite the reader to have a look at them (Table 6). Additionally, specific antigens should be employed to induce even more specific immune responses that will enhance the eradication of relevant parasitic diseases like leishmaniasis and amoebiasis. Furthermore, studies should consider administration routes as potential factors that may affect the immunomodulatory responses of vaccines for the treatment of parasitic infections. For instance, toxicity of L. donovani was reduced when administered intravenously and differences in the immune response cytokine profile were detected. These cytokine profiles must need to be addressed, conducting studies with the similar liposomal formulation and administering the vaccines through different routes.

Table 6

Liposomal vaccine formulations tested in parasitic infection models

Lipid(s) and sterol used	Parasite	Cell line/animal model used	Administration route	Promising liposome formulation	References
MPL/QS21 (liposome-based)	Plasmodium falciparum	Rhesus macaques	Intramuscular	MPL/QS21 (liposome-based)	[114]
Egg lecithin/Chol, egg lecithin/SA and egg lecithin/PA	Leishmania donovani	BALB/c mice	Intraperitoneal	Lecithin/Chol/SA	[116]
PC/Chol, PC/SA, PC/PA and PC/PS	L. donovani	–	Intravenous, N/A	PC/SA	[117, 118]
EPC/EPG, EPC/EPG/Chol and DSPC/DPPG/Chol	P. vinckei	Female C57BL/6J mice	Intraperitoneal	EPC/EPG, EPC/EPG/Chol and DSPC/DPPG/Chol	[113]

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Go to:

DDA-based cationic liposomal vaccines for the treatment of tuberculosis

The World Health Organization (WHO) estimated 1.8 million deaths in 2015 were related to tuberculosis infections (TBIs) [120]. The global estimate for latent tuberculosis infections (LTBI) was recently determined to be 23% (approximately 1.7 billion persons from the total population) [121]. Because of current tuberculosis (TB) vaccine efficacy variability, inefficiency and waning immunity (like Bacillus Calmette–Guerin, BCG) [57, 58], it is imperative to develop novel vaccines strategies that will improve prophylactic avenues for TBIs and reduce the overall death rate or latency associated with them. One of the principal improvements revealed in the last years is the development of adjuvanted subunit vaccines.

To diminish the detrimental effects of tuberculosis in humans, certain studies have focused on subunit vaccine development based in cationic lipid formulations (Table 7). Adjuvants are required for the development and efficacy of subunit vaccines, potentiating immune responses. Several adjuvants have been studied in the past 20 years including the adjuvanting properties of the lipid DDA [122]. Forty years ago, Snippe et al. investigated the effect of DDA in mice (via an intracutaneous administration) determining that delayed hypersensitivity occurred by the onset of footpad swelling 5 days after vaccination [123]. Subsequently, a study by van Houte et al., where low- (DOPC and DLPC) and high-transition temperature (DSPC) lipids were utilized with DDA as liposome bilayer components, discovered less immunogenicity of the liposomes as DDA concentration decreased [50]. Additionally, an earlier review discussed the immunostimulatory properties of DDA and its uses in different vaccines for veterinary and human infections [47]. We will present the reader with the most significant studies performed that cover the utilization of the cationic lipid DDA in subunit vaccine development.

Table 7

DDA-based TB vaccine formulation optimization studies

Lipid(s) and sterol used	Cell line/animal model used	Administration route	Promising liposome formulation	References
DDA, DDA/Tween 80, DDD/Span 85, DDA/Tween 80/Span 85, DDA/gelatin, DDA/Chol, DDA/Lecithin, DDA/β-Cyclodextrin and DDA/PLGA	C57BL/6 mice	Subcutaneous	DDA/Chol	[126]
DDA, DOTAP, DC-Chol, DOPE/PC and DOPE/PC/PG	Splenocytes from BALB/c and C57BL/6 mice	Subcutaneous	DDA	[125]
DDA/DSPC	Splenocytes from C57BL/6 mice	Intramuscular	DDA/DSPC	[18]
DDA	Splenocytes from BALB/c mice	Subcutaneous	DDA	[6]
DDA	Human/macrophage cell line THP-1 and splenocytes from BALB/c mice	Intramuscular	DDA	[30]
DDA	Inguinal lymph nodes or spleens	Subcutaneous	DDA	[128]
DDA	C57BL6; spleen and lung lymphocytes	Subcutaneous	DDA	[124]
DDA and DDA/Chol	Splenocytes from C57BL/6 mice and THP-1 cells	Intramuscular	DDA	[52]
DDA/MMG	Splenocytes from C57BL/6 mice	Subcutaneous	DDA/MMG	[134]
DDA	BALB/c mice	Intracutaneous	DDA	[122]
DDA	C57BL6 mice	Subcutaneous	DDA	[129]
DDA	BALB/c mice	Intracutaneous	DDA	[123]
DDA	Splenocytes from BALB/c mice	Intramuscular	DDA	[44]
LAM/PC/Chol/stearyl octaarginine	PBMCs	N/A	LAM/PC/Chol/stearyl octaarginine	[17]
DDA	DCs	Subcutaneous	DDA	[135]

Open in a separate window

Cell cultures and no vaccination in animal models were employed

N/A not applicable

Holten-Andersen et al. mixed the recombinant immunodominant M. tuberculosis antigens (ESAT-6 and Ag85B-ESAT6), DDA and different immunomodulators, analyzing the immune responses against BCG vaccination in mice (subcutaneous administration) [124]. The studied immunomodulators included saponin, calcitriol, β-glucan, n-hexadecane, TDB, muramyl dipeptide (MDP) and monophosphoryl lipid A (MPL). Investigators determined that the combination of the antigens with DDA and TDB generated a strong protective TH1 immune response against the mycobacterium, contrasting with BCG vaccination. In another study, M. bovis BCG lipid extracts were tested for their adjuvant characteristics [125]. BCG lipids were incorporated in DDA-based liposomes and administered subcutaneously to female BALB/c or C57BL/6 mice. BCG lipids coupled with the antigen Ag85B-ESAT-6 fusion protein in cationic liposomes induced significant levels of IFN-γ and relevant antibodies (IgG2A) titers, characteristic of TH1 immune responses. Antigens from other sources (Chlamydia muridarum and tetanus toxoid) were studied and relevant antibodies were detected when administered with the so called mycosomes (BCG lipids + cationic liposomes).

Subsequent studies on cationic liposomes bearing the antigen Ag85B-ESAT-6 (or its modifications) from M. tuberculosis were performed [6, 18, 52, 126]. Henriksen-Laceyet al. incorporated the antigen in DDA or DDA:TDB (8:1 molar ratio) liposomes and administered the vaccine formulation via intramuscular or subcutaneous injections to mice [6]. The antigen did not affect the liposome size, Zeta (ζ) potential or polydispersity index. The cationic lipid formulation was compared with antigen administered alone to mice. Investigators observed the rapid dissemination of the antigen administered alone in mice, contrasting with a depot formation at the injection site when administered in a liposomal formulation (up to 14 days post-vaccination). TDB allowed for the translocation of the liposomes form the site of injection to lymph nodes with the additional effect of monocyte infiltration to the injection site. Another study investigated the effects by which DDA plays a key role as an immunostimulatory lipid [18]. Mice were immunized intramuscularly with the proposed vaccine. Researchers concluded that removing or reducing DDA molar ratio in the liposome bilayer conduced to a reduction in TH1 immune responses against the antigen. Moreover, a team of researchers determined that the addition of cholesterol to the bilayer of DDA:TDB liposomes did not induced strong immune responses suggesting the prominent role of bilayer fluidity [52]. However, the previously mentioned study contrast with results obtained by Liu et al., were the TLR3 ligand Poly I:C was utilized as an adjuvant along with DDA and cholesterol liposomes (DPC liposomes) [126]. Researchers employed the TB fusion protein ESAT-6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) as the model antigen. DPC liposomes showed stability at size of 400 nm and ζ potential of 40 mV. Strong humoral and cell-mediated immune responses were detected by the production of antigen-specific antibodies after the subcutaneous administration and a markedly protection against a M. tuberculosis infection challenge than the traditional BCG vaccine.

With the advances in recombinant protein antigens from M. tuberculosis, other teams of researchers decided to observe at different antigenic proteins (like OVA) in DDA:TDB liposomes [44, 127], comparing different adjuvants and physicochemical properties in cationic liposomal formulations [30, 128] or investigating the mechanism of protein antigen adsorption [127]. OVA-containing SUVs liposomes composed of DDA:TDB with no TLR ligand showed a higher capacity to induce spleen CD8 IFN-γ responses against the antigen, contrasting to MLVs, administered intramuscularly [44]. Antigen-specific responses were higher on SUVs. Adding TLR3 and TLR9 agonists significantly increased the immune responses on MLVs carrying OVA, but that was not observed in SUVs. The study suggested that liposomes are an excellent delivery vehicle for antigen presentation and vaccine formulation, and we believe that this is the foundation for the subsequent studies based on cationic and neutral lipid formulations. Investigators selected DDA and the synthetic mycobacterial cord factor molecule, TDB, as a suitable model adjuvant (CAF01) for vaccine development [128]. This was based in that mice vaccination (subcutaneous) with CAF01 induced a strong antigen-specific cell- and humoral-mediated responses, contrasting with currently used adjuvants (e.g. alum and MPL). Furthermore, results were strongly supported by the protection from infection when mice were challenged by M. tuberculosis, C. trachomatis or malaria, revealing cell mediated (TB), cell-mediated/humoral (C. trachomatis) and humoral immune responses.

A study showed the significant role that liposome vesicle size plays in the cell-mediated immune responses [30]. Researchers determined that no differences in vaccine (administered intramuscularly) draining from the injection site occurred, but a size-dependent liposome movement (favored by large liposomes) was observed to the popliteal lymph node. Macrophage-like cells internalized liposomes in a size-independent pattern. Size did not affect the antigen-specific antibody response (IgG1/2) of Ag85B-ESAT-6-carrying liposomes, however larger liposomes induced highest cell proliferation and lowest IL-10 levels, which contrasted with smaller vesicles (inducing IFN-γ and IL-1β). Additionally, a study investigated the effects of charge, membrane fluidity and antigen-to-lipid ration on mechanism of protein antigen adsorption [127]. For this purpose, investigators compared cationic (CAF01) and neutral (NAF01) liposomal formulations, mainly composed of DDA or DSPC, respectively. α-Lactalbumin and lysozyme were used as antigen protein models to analyze the parameters. The anionic lactalbumin interacted with cationic liposomes by surface adsorption, and no interaction was observed with zwitterionic liposomes. However, the cationic lysozyme presented no detectable interaction with either type of liposomes. Adsorption of α-lactalbumin generated changes in its tertiary structure, neutralized liposome charge, affected lipid membrane packing (especially for CAF01 liposomes), resulting in a reduction of colloidal stability and liposome aggregation. The CAF01 formulation has completed multiple phase I safety trails in humans to date.

Other studies have focus their attention on cell components, synthetic lipid analogues or mycobacterial lipids as antigens for subunit vaccine development employing DDA as the principal component in the lipid bilayer. Mutant M. bovis BCG ΔmmaA4 strain was formulated in cationic liposomes of DDA:TDB (A4/Adj) and administered subcutaneously [129]. The mutation deletes the mmaA4 gene that encodes a S-adenosylmethionine-dependent methyltransferase involved in mycolic acid biosynthesis in the tubercle bacillus [130]. Immunocompromised mice (TCRδ−/−) immunized with A4/Adj were protected against an infection of M. tuberculosis (2 and 9 months post-vaccination), contrasting with non-adjuvanted mutant and non-vaccinated controls. It is important to note that the immunocompromised mice lack CD4+, CD8+ and NK1.1+ T cells but, due to immunization long-term results, an unconventional T cell population was responsible for the immune responses. Researchers observed CD4− CD8− double negative (DN) T cells and found that the cells accumulated in the lungs of A4/Adj-treated mice, with significant levels of IFN-γ production when comparing to nonvaccinated or nonadjuvanted BCG control test groups. In vitro studies revealed the antimycobacterial properties of DN T cells isolated from adjuvanted BCG-treated mice when compared to whole-spleen cells. The results of this study represent a milestone in medical research since tuberculosis affects dramatically immunocompromised patients, like in HIV infections [131–133].

Synthetic lipid analogues from monomycoloyl glycerol (MMG-1 to 6) from M. tuberculosis has been developed and their supramolecular structure and adjuvant efficacy tested via subcutaneous administration [134]. The analogues displayed longer (MMG-2) or shorter (MMG-3) alkyl chains, or stoichiometry variations of the polar head group (MMG-5) or the hydrophobic moiety (MMG-6). CryoTEM and synchrotron small-angle X-ray (SAX) experiments revealed the supramolecular organization varied from unilamellar and multilamellar (ULVs/MLVs) vesicles in DDA:MMG-1/2/5/6 liposomes to ULVs and hexosomes in DDA:MMG-3. TH1 and TH17 immune responses were induced by DDA:MMG-1/3/6 liposomal formulations in response to a chlamydial antigen, contrasting to different immunostimulatory properties of naked MMG-1 and MMG-6 analogues in vitro. We recommend further studies employing MMG analogues incorporated in liposomes with mycobacterial-derived antigens to assess the efficacy of this chlamydial model. In contrast, another study utilized natural mycobacterial lipids diacylated sulfoglycolipids (Ac2-SGL) and phosphatidyl-myo-inositol dimannosides (PIM2) as antigens in a liposomal vaccine formulated with DDA and TDB as adjuvants [135]. Researchers observed a reduction of bacterial load in the spleen of vaccinated animals via subcutaneous administration, contrasting with the unvaccinated group. The lipid antigen vaccine group showed a remarkable reduction of lung and spleen lesions when compared to the unvaccinated group. Comparison of lipid antigen vaccine with protein antigen vaccine regimes in a guinea pig model revealed no significant differences in the treatments.

From the articles discussed in this section we can count on promising advances in tuberculosis vaccine development. The approaches presented applied recombinant protein antigens, whole-mutant cell and synthetic and natural cell components formulations. Each approach could encounter some drawbacks: mass production of natural and synthetic products from M. tuberculosis and other mycobacteria; liposomal formulation instability and aggregation; unwanted immune responses. Most of the studies discussed above demonstrated that subcutaneous administration could be a successful route of administration and future research should direct efforts to determined other administration routes, like intranasal and intramuscular. By understanding the physicochemical parameters of liposomes and the effects of routes of administration on physiological and immune function requirements, scientists will be able to develop a novel tuberculosis vaccine.

Go to:

Conclusion

Vaccination represents the major advancement of modern medicine, second in effect only to clean water, in decreasing the spread of detrimental diseases and providing better quality of life. To develop effective and safe vaccines, collaboration between immunologists and formulation scientists must exist. This collaboration will ensure that the vaccine is designed adequately and that the therapy will not cause unwanted immune responses. Also, it is important to understand the basic concepts in immunology (innate vs. adaptive immunity) so we can target the corresponding receptors with the ligands and antigens employed in the vaccine. Many types of cell receptors participate in PAMPs recognition (innate immunity), namely NLRs, STING, RLRs, TLRs and CLRs. Each receptor contributes to significant responses that lead to T helper cell activation and differentiation, with eventual B cell (antibody-mediated) and CD8 T cell-mediated adaptive immune responses.

For liposomal vaccines, we must pay close attention to liposome size, surface charge (ζ potential), morphology (lamellarity) and lipid bilayer fluidity. Size may affect antigen presentation to APCs and consequently immune responses. Likewise, surface charge of the liposome may affect antigen adsorption on the liposome and could affect liposome-cell interactions (cationic liposomes interact better with the anionic membrane of immune cells). MLVs may reduce antigen presentation due to their concentric vesicle morphological nature, contrasting to ULVs which enhances antigen presentation. Antigen leakage and immune response reduction may occur on how the antigen was formulated (adsorption vs. absorption). Absortion is explained by the Tm of the lipids or phospholipids used when developing the vaccine. Lipids with lower Tm tend to be fluid meanwhile lipids with higher Tm would increase bilayer rigidity. During adsorption, the antigen is exposed in the outer layer of the lipid membrane, which could allow easy release or presentation of the antigen. Additionally, we must pay special attention to how liposomes would interact with immune cells and how we can target those cells, enhancing proper immune responses. Adjuvants play a significant role in APCs activation and maturation for eventual T and B cell activation. The adjuvants, properly selected, will interact with their corresponding TLRs or CLRs in a process called targeted cell vaccine delivery. This cell targeted process is also affected by the vaccine route of administration.

We observed in the sections previously discussed key developments in contemporary vaccine research for the treatment of viral, bacterial, fungal and parasitic infections. The studies presented to the reader demonstrated the collaborative and interdisciplinary nature of the field. Cell targeting and adjuvants dominated most of the approaches to develop effective prophylactic subunit vaccines for the treatment of detrimental diseases. In some instances, infection was hindered by the antimicrobial, antiviral, antifungal or antiparasitic activities of the vaccines due to the induction of adequate immune responses (cell- and antibody-mediated), leading to the survival of selected animal models. Additionally, we can conclude that cationic liposomal vaccines are of great interest for future vaccine development due to their enhanced interaction with the negatively charged immune cells. In specific, DDA-based liposomes are being tested in diverse vaccine studies which promise significant advances in the field. Such DDA applications as a building block of liposomal vaccines represent a step forward towards the prophylactic treatment of diverse infections. These developments will improve the current vaccine approaches and will provide better treatments for patients. Additional research efforts must be made towards the development of novel adjuvants that will contribute to the induction of significant immune responses. Both authors read and approved the final manunscript.

Go to:

Authors’ contributions

LODS and DB developed the concept of the manuscript. LODS reviewed the literature, drafted and wrote the manuscript. DB critically reviewed the manuscript and provided ideas and comments on the draft. Both authors approved the final manuscript.

Acknowledgements

Special thanks to the Formulation Team of the Center for Translational Medicine (CTM) at the University of Montana for their critical review of the manuscript. Special thanks to Dr. Alyson Smith, CTM’s Immunology Director, for her guidance in the immunology-related information discussed in the article.

Competing interests

LODS declares no competing interests. DB is the Chief Operating Officer and member of the Board of Directors, Inimmune, Inc. The authors are entirely responsible for the content of the review and the opinions contained within it.

Availability of data and materials

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This review was written with the support of a NIAID Contract # HHSN272201400050C.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Go to:

Abbreviations

AmB	amphotericin B
APCs	antigen presenting cells
BCG	Bacillus Calmette–Guerin
BCR	B cell receptor
BMDCs	bone marrow dendritic cells
BSA	bovine serum albumin
CCS	ceramide carbamoyl-spermine
Chol	cholesterol
CLR	C-type lectin receptor
COPD	chronic obstructive pulmonary disease
CpG-ODN	CpG-containing oligodeoxynucleotides
CWSPs	cell wall surface proteins
DAMPs	damaged-associated molecular patterns
DCs	dendritic cells
DCC	N,N-dicyclohexylcarbodimide
DC-Chol	dimethylaminoethane-carbamoyl-cholesterol
DC-SIGN	DC-specific ICAM3-grabbing non-integrin
DDA	dimethyldioctadecylammonium
Dectin-1	dendritic cell-associated C-type lectin 1
Dectin-2	dendritic cell-associated C-type lectin 2
DFO or DFB	desferrioxamine B
DLPC	1,2-dilauroyl-sn-glycero-3-phosphocholine
DMPC	1,2-dimyristoyl-sn-glycero-3-phosphocholine
DMPG	1,2-dimyristoyl-sn-glycero-3-phospho-(1ʹ-rac-glycerol)
DMTAP	1,2-dimyristoyl-3-trimethylammonium-propane
DOGS-NTS-Ni	1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)
DOPC	1,2-dioleoyl-sn-glycero-3-phosphocholine
DOPE	1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DOTAP	1,2-dioleoyl-3-trimethylammonium-propane
DOTMA	1,2-di-O-octadecenyl-3-trimethylammonium propane
DPPC	1,2-dipalmitoyl-sn-glycero-3-phosphocholine
DSPC	distearoyl-sn-glycero-3-phosphocholine
DSTAP	1,2-stearoyl-3-trimethylammonium-propane
DTA	diphtheria toxin
EPC	egg phosphatidylcholine
HBsAg	hepatitis B surface antigen
HBV	hepatitis B virus
HIV	human immunodeficiency virus
Hsp90	C. albicans heat shock protein 90
IFN-γ	gamma interferon
Ig	immunoglobulin
IL	interleukins
i.n.	intranasal
LAg	leishmanial antigen
Lip	liposome(s)
LPS	lipopolysaccharide
LTBIs	latent tuberculosis infections
LUVs	large unilamellar vesicles
ManLAM	mannose lipoarabinomannan
Man-PE	mannosyl phosphatidylethanolamine
MCL	macrophage C-type lectin
MDDCs	monocyte-derived dendritic cells
MDMs	monocyte-derived macrophages
MDP	muramyl dipeptide
MHC I	class I major histocompatibility complex
MHC II	class II major histocompatibility complex
MINCLE	macrophage-inducible C-type lectin
MLVs	multilamellar vesicles
MMG	monomycoloyl glycerol
MO	monooleoylglycerol
MPL or MPLA	monophosphoryl lipid A
MR	mannose receptor
NKs	natural killer cells
NLRs	nucleotide-binding domain and leucine-rich repeat receptor
OVA	ovabulmin
PA	phosphatidic acid
PAMPs	pathogen associated molecular patterns
PC	phosphatidylcholine
PG	phosphatidylglycerol
PMNs	polymorpho nuclear cells
POPC	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
POPE	1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
PRRs	pathogen recognition receptors
PS	phosphatidylserine
RLRS	retinoic acid-inducible gene I receptor
RSV	respiratory syncytial virus
SA	stearylamine
SDC	sodium deoxycholate
SUVs	small unilamellar vesicles
TAMPs	tumor-associated molecular patters
TB	tuberculosis
TBIs	tuberculosis infections
TCR	T cell receptor
TDB	trehalose dibehenate
TH cells	T helper cells
TLR	toll-like receptor
TNF-α	tumor necrosis factor alpha
WHO	World Health Organization

Go to:

Contributor Information

Luis O. De Serrano, Email: moc.liamtoh@onarresedsiul.

David J. Burkhart, Email: ude.tmu.osm@trahkruB.divaD.

Go to:

References

1. Guy B, Pascal N, Francon A, Bonnin A, Gimenez S, Lafay-Vialon E, Trannoy E, Haensler J. Design, characterization and preclinical efficacy of a cationic lipid adjuvant for influenza split vaccine. Vaccine. 2001;19:1794–1805. [PubMed] [Google Scholar]

2. Puangpetch A, Anderson R, Huang YY, Sermswan RW, Chaicumpa W, Sirisinha S, Wongratanacheewin S. Cationic Liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice. Clin Vaccine Immunol. 2012;19:675–683. [PMC free article] [PubMed] [Google Scholar]

3. Capron A, Riveau G, Grzych J-M, Boulanger D, Capron M, Pierce R. Development of a vaccine strategy against human and bovine schistosomiasis: background and update. Trop Geogr Med. 1994;46:242–246. [PubMed] [Google Scholar]

4. Girard MP, Fruth U, Kieny MP. A review of vaccine research and development: tuberculosis. Vaccine. 2005;23:5725–5731. [PubMed] [Google Scholar]

5. Christensen D, Agger EM, Andreasen LV, Kirby D, Andersen P, Perrie Y. Liposome-based cationic adjuvant formulations (CAF): past, present, and future. J Liposome Res. 2009;19:2–11. [PubMed] [Google Scholar]

6. Henriksen-Lacey M, Bramwell VW, Christensen D, Agger EM, Andersen P, Perrie Y. Liposomes based on dimethyldioctadecylammonium promote a depot effect and enhance immunogenicity of soluble antigen. J Control Release. 2010;142:180–186. [PubMed] [Google Scholar]

7. Kallerup RS, Madsen CM, Schioth ML, Franzyk H, Rose F, Christensen D, Korsholm KS, Foged C. Influence of trehalose 6,6ʹ-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes. Eur J Pharm Biopharm. 2015;90:80–89. [PubMed] [Google Scholar]

8. Geijtenbeek TBH, Gringhuis SI. C-type lectin receptors in the control of T helper cell differentiation. Nat Rev Immunol. 2016;16:433–448. [PubMed] [Google Scholar]

9. Goyal S, Klassert TE, Slevogt H. C-type lectin receptors in tuberculosis: what we know. Med Microbiol Immunol. 2016;205:513–535. [PubMed] [Google Scholar]

10. Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science. 2010;327:291–295. [PMC free article] [PubMed] [Google Scholar]

11. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect. 2004;6:1382–1387. [PubMed] [Google Scholar]

12. Yao C, Oh JH, Lee DH, Bae JS, Jin CL, Park CH, Chung JH. Toll-like receptor family members in skin fibroblasts are functional and have a higher expression compared to skin keratinocytes. Int J Mol Med. 2015;35:1443–1450. [PubMed] [Google Scholar]

13. Oosting M, Cheng SC, Bolscher JM, Vestering-Stenger R, Plantinga TS, Verschueren IC, Arts P, Garritsen A, van Eenennaam H, Sturm P, et al. Human TLR10 is an anti-inflammatory pattern-recognition receptor. Proc Natl Acad Sci USA. 2014;111:E4478–E4484. [PMC free article] [PubMed] [Google Scholar]

14. Cagnoni AJ, Saez JMP, Rabinovich GA, Marino KV. Turning-off signaling by siglecs, selectins, and galectins: chemical inhibition of glycan-dependent interactions in cancer. Front Oncol. 2016;6:21. [PMC free article] [PubMed] [Google Scholar]

15. Dambuza IM, Brown GD. C-type lectins in immunity: recent developments. Curr Opin Immunol. 2015;32:21–27. [PMC free article] [PubMed] [Google Scholar]

16. Opanasopit P, Higuchi Y, Kawakami S, Yamashita F, Nishikawa M, Hashida M. Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages. Biochim Biophys Acta Biomem. 2001;1511:134–145. [PubMed] [Google Scholar]

17. Kallert S, Zenk SF, Walther P, Grieshober M, Weil T, Stenger S. Liposomal delivery of lipoarabinomannan triggers Mycobacterium tuberculosis specific T-cells. Tuberculosis. 2015;95:452–462. [PubMed] [Google Scholar]

18. Hussain MJ, Wilkinson A, Bramwell VW, Christensen D, Perrie Y. Th1 immune responses can be modulated by varying dimethyldioctadecylammonium and distearoyl-sn-glycero-3-phosphocholine content in liposomal adjuvants. J Pharm Pharmacol. 2014;66:358–366. [PubMed] [Google Scholar]

19. Li P, Chen S, Jiang Y, Jiang J, Zhang Z, Sun X. Dendritic cell targeted liposomes-protamine-DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine. Nanotechnology. 2013;24:295101. [PubMed] [Google Scholar]

20. Christensen D, Korsholm KS, Andersen P, Agger EM. Cationic liposomes as vaccine adjuvants. Expert Rev Vaccines. 2011;10:513–521. [PubMed] [Google Scholar]

21. McNeil SE, Perrie Y. Gene delivery using cationic liposomes. Expert Opin Ther Pat. 2006;16:1371–1382. [Google Scholar]

22. Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics. 2017;9:12. [Google Scholar]

23. Watson DS, Endsley AN, Huang L. Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens. Vaccine. 2012;30:2256–2272. [PMC free article] [PubMed] [Google Scholar]

24. Perrie Y, Crofts F, Devitt A, Griffiths HR, Kastner E, Nadella V. Designing liposomal adjuvants for the next generation of vaccines. Adv Drug Deliv Rev. 2016;99:85–96. [PubMed] [Google Scholar]

25. Shahum E, Therien HM. Immunopotentiation of the humoral response by liposomes—encapsulation versus covalent linkage. Immunology. 1988;65:315–317. [PMC free article] [PubMed] [Google Scholar]

26. Shahum E, Therien HM. Liposomal adjuvanticity—effect of encapsulation and surface-linkage on antibody-production and proliferative response. Int J Immunopharmacol. 1995;17:9–20. [PubMed] [Google Scholar]

27. Therien HM, Shahum E. Immunopotentiation of the humoral response by liposomes—effect of a T-cell polyclonal activator. Cell Immunol. 1988;116:320–330. [PubMed] [Google Scholar]

28. Barnier-Quer C, Elsharkawy A, Romeijn S, Kros A, Jiskoot W. Adjuvant effect of cationic liposomes for subunit influenza vaccine: influence of antigen loading method, cholesterol and immune modulators. Pharmaceutics. 2013;5:392–410. [PMC free article] [PubMed] [Google Scholar]

29. Legrue SJ. Carrier and adjuvant properties of liposome-borne tumor-specific antigens. Cancer Immunol Immunother. 1984;17:135–141. [PubMed] [Google Scholar]

30. Henriksen-Lacey M, Devitt A, Perrie Y. The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production. J Control Release. 2011;154:131–137. [PubMed] [Google Scholar]

31. Brewer JM, Tetley L, Richmond J, Liew FY, Alexander J. Lipid vesicle size determines the Th1 or Th2 response to entrapped antigen. J Immunol. 1998;161:4000–4007. [PubMed] [Google Scholar]

32. Jain S, Indulkar A, Harde H, Agrawal AK. Oral mucosal immunization using glucomannosylated bilosomes. J Biomed Nanotechnol. 2014;10:932–947. [PubMed] [Google Scholar]

33. Mohammed AR, Bramwell VW, Coombes AG, Perrie Y. Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products. Methods. 2006;40:30–38. [PubMed] [Google Scholar]

34. Ingvarsson PT, Schmidt ST, Christensen D, Larsen NB, Hinrichs WL, Andersen P, Rantanen J, Nielsen HM, Yang M, Foged C. Designing CAF-adjuvanted dry powder vaccines: spray drying preserves the adjuvant activity of CAF01. J Control Release. 2013;167:256–264. [PubMed] [Google Scholar]

35. Bakowsky H, Richter T, Kneuer C, Hoekstra D, Rothe U, Bendas G, Ehrhardt C, Bakowsky U. Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery. Biochim Biophys Acta. 2008;1778:242–249. [PubMed] [Google Scholar]

36. Dunnick JK, Badger RS, Takeda Y, Kriss JP. Vesicle interactions with antibody and peptide hormone: role of vesicle composition. J Nucl Med. 1976;17:1073–1076. [PubMed] [Google Scholar]

37. Perrie Y, Ali H, Kirby DJ, Mohammed AU, McNeil SE, Vangala A. Environmental scanning electron microscope imaging of vesicle systems. Methods Mol Biol. 2017;1522:131–143. [PubMed] [Google Scholar]

38. Cohen S, Alonso MJ, Langer R. Novel approaches to controlled-release antigen delivery. Int J Technol Assess Health Care. 1994;10:121–130. [PubMed] [Google Scholar]

39. Chen C, Han D, Cai C, Tang X. An overview of liposome lyophilization and its future potential. J Control Release. 2010;142:299–311. [PubMed] [Google Scholar]

40. Winterhalter M, Lasic DD. Liposome stability and formation: experimental parameters and theories on the size distribution. Chem Phys Lipids. 1993;64:35–43. [PubMed] [Google Scholar]

41. Beck Z, Matyas GR, Jalah R, Rao M, Polonis VR, Alving CR. Differential immune responses to HIV-1 envelope protein induced by liposomal adjuvant formulations containing monophosphoryl lipid A with or without QS21. Vaccine. 2015;33:5578–5587. [PubMed] [Google Scholar]

42. Shek PN, Yung BYK, Stanacev NZ. Comparison between multilamellar and unilamellar liposomes in enhancing antibody-formation. Immunology. 1983;49:37–44. [PMC free article] [PubMed] [Google Scholar]

43. Hampl J, Stepanek J, Franz J, Svoboda I. Incorporation of Bovine Herpesvirus-1 protein subunits into large unilamellar and multilamellar liposomes. Acta Veterinaria Brno. 1992;61:29–36. [Google Scholar]

44. Milicic A, Kaur R, Reyes-Sandoval A, Tang CK, Honeycutt J, Perrie Y, Hill AVS. Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses. PLoS ONE. 2012;7:10. [PMC free article] [PubMed] [Google Scholar]

45. Joseph A, Itskovitz-Cooper N, Samira S, Flasterstein O, Eliyahu H, Simberg D, Goldwaser I, Barenholz Y, Kedar E. A new intranasal influenza vaccine based on a novel polycationic lipid—ceramide carbamoyl-spermine (CCS)-I. Immunogenicity and efficacy studies in mice. Vaccine. 2006;24:3990–4006. [PubMed] [Google Scholar]

46. Tseng LP, Chiou CJ, Deng MC, Lin MH, Pan RN, Huang YY, Liu DZ. Evaluation of encapsulated newcastle disease virus liposomes using various phospholipids administered to improve chicken humoral immunity. J Biomed Mater Res Part B Appl Biomater. 2009;91B:621–625. [PubMed] [Google Scholar]

47. Hilgers LAT, Snippe H. DDA as an immunological adjuvant. Res Immunol. 1992;143:494–503. [PubMed] [Google Scholar]

48. Korsholm KS, Agger EM, Foged C, Christensen D, Dietrich J, Andersen CS, Geisler C, Andersen P. The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes. Immunology. 2007;121:216–226. [PMC free article] [PubMed] [Google Scholar]

49. Yasuda T, Dancey GF, Kinsky SC. Immunogenicity of liposomal model membranes in mice—dependence on phospholipid composition. Proc Natl Acad Sci USA. 1977;74:1234–1236. [PMC free article] [PubMed] [Google Scholar]

50. Van Houte AJ, Snippe H, Schmitz MGJ, Willers JMN. Characterization of immunogenic properties of haptenated liposomal model membranes in mice 5 effect of membrane composition on humoral and cellular immunogenicity. Immunology. 1981;44:561–568. [PMC free article] [PubMed] [Google Scholar]

51. Mazumdar T, Anam K, Ali N. Influence of phospholipid composition on the adjuvanticity and protective efficacy of liposome-encapsulated Leishmania donovani antigens. J Parasitol. 2005;91:269–274. [PubMed] [Google Scholar]

52. Kaur R, Henriksen-Lacey M, Wilkhu J, Devitt A, Christensen D, Perrie Y. Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on bilayer properties, biodistribution, and immune responses. Mol Pharm. 2014;11:197–207. [PubMed] [Google Scholar]

53. Hampl J, Franz J, Jordanova K, Stepanek J. Effects of phospholipid-composition on adjuvant efficiency of liposomes. Acta Veterinaria Brno. 1995;64:163–169. [Google Scholar]

54. Kummerow FA, Przybylski R, Wasowicz E. Changes in arterial membrane lipid composition may precede growth factor influence in the pathogenesis of atherosclerosis. Artery. 1994;21:63–75. [PubMed] [Google Scholar]

55. Rao M, Matyas GR, Vancott TC, Birx DL, Alving CR. Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein. Immunol Cell Biol. 2004;82:523–530. [PubMed] [Google Scholar]

56. Henriksen-Lacey M, Christensen D, Bramwell VW, Lindenstrom T, Agger EM, Andersen P, Perrie Y. Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3 beta Nʹ,Nʹ-dimethylaminoethane)carbomyl cholesterol (DC-Chol), and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses. Mol Pharm. 2011;8:153–161. [PubMed] [Google Scholar]

57. Andersen P, Doherty TM. The success and failure of BCG—implications for a novel tuberculosis vaccine. Nat Rev Microbiol. 2005;3:656–662. [PubMed] [Google Scholar]

58. Fine PEM. Variation in protection by BCG—implications of and for heterologous immunity. Lancet. 1995;346:1339–1345. [PubMed] [Google Scholar]

59. Abhyankar MM, Noor Z, Tomai MA, Elyecrog J, Fox CB, Petri WA., Jr Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica. Vaccine. 2017;35:916–922. [PMC free article] [PubMed] [Google Scholar]

60. Mohammed AR, Bramwell VW, Kirby DJ, McNeil SE, Perrie Y. Increased potential of a cationic liposome-based delivery system: enhancing stability and sustained immunological activity in pre-clinical development. Eur J Pharm Biopharm. 2010;76:404–412. [PubMed] [Google Scholar]

61. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu CZ, Middelberg APJ. Nanoparticle vaccines. Vaccine. 2014;32:327–337. [PubMed] [Google Scholar]

62. Lonez C, Vandenbranden M, Ruysschaert JM. Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res. 2008;47:340–347. [PubMed] [Google Scholar]

63. Aramaki Y, Tomizawa H, Hara T, Yachi K, Kikuchi H, Tsuchiya S. Stability of liposomes in-vitro and their uptake by rat peyer patches following oral-administration. Pharm Res. 1993;10:1228–1231. [PubMed] [Google Scholar]

64. CarmonaRibeiro AM, Ortis F, Schumacher RI, Armelin MCS. Interactions between cationic vesicles and cultured mammalian cells. Langmuir. 1997;13:2215–2218. [Google Scholar]

65. Karathanasis E, Geigerman CM, Parkos CA, Chan L, Bellamkonda RV, Jaye DL. Selective targeting of nanocarriers to neutrophils and monocytes. Ann Biomed Eng. 2009;37:1984–1992. [PMC free article] [PubMed] [Google Scholar]

66. Johansen PT, Zucker D, Parhamifar L, Pourhassan H, Madsen DV, Henriksen JR, Gad M, Barberis A, Maj R, Andresen TL, Jensent SS. Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist. Expert Opin Drug Deliv. 2015;12:1045–1058. [PubMed] [Google Scholar]

67. Tanaka Y, Taneichi M, Kasai M, Kakiuchi T, Uchida T. Liposome-coupled antigens are internalized by antigen-presenting cells via pinocytosis and cross-presented to CD8(+) T cells. PLoS ONE. 2010;5:e15225. [PMC free article] [PubMed] [Google Scholar]

68. Owais M, Gupta CM. Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development. Eur J Biochem. 2000;267:3946–3956. [PubMed] [Google Scholar]

69. Huong TM, Harashima H, Kiwada H. Complement dependent and independent liposome uptake by peritoneal macrophages: cholesterol content dependency. Biol Pharm Bull. 1998;21:969–973. [PubMed] [Google Scholar]

70. Song X, Lin Q, Guo L, Fu Y, Han J, Ke H, Sun X, Gong T, Zhang Z. Rifampicin loaded mannosylated cationic nanostructured lipid carriers for alveolar macrophage-specific delivery. Pharm Res. 2015;32:1741–1751. [PubMed] [Google Scholar]

71. White KL, Rades T, Furneaux RH, Tyler PC, Hook S. Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells. J Pharm Pharmacol. 2006;58:729–737. [PubMed] [Google Scholar]

72. Varypataki EM, van der Maaden K, Bouwstra J, Ossendorp F, Jiskoot W. cationic liposomes loaded with a synthetic long peptide and poly(I:C): a defined adjuvanted vaccine for induction of antigen-specific T cell cytotoxicity. Aaps J. 2015;17:216–226. [PMC free article] [PubMed] [Google Scholar]

73. Tada R, Hidaka A, Iwase N, Takahashi S, Yamakita Y, Iwata T, Muto S, Sato E, Takayama N, Honjo E, et al. Intranasal immunization with DOTAP cationic liposomes combined with DC-cholesterol induces potent antigen-specific mucosal and systemic immune responses in mice. PLoS ONE. 2015;10:e0139785. [PMC free article] [PubMed] [Google Scholar]

74. Meiklejohn G, Simpson TW, Stacy IB. experimental infection of domestic animals with Japanese B-encephalitis virus. Proc Soc Exp Biol Med. 1947;65:359–364. [PubMed] [Google Scholar]

75. Niklasson B, Hakansson C, Lowhagen GB, Jonsson R. Oral mucosal lesions associated with HIV-infection. Swed Dent J. 1986;10:258. [Google Scholar]

76. Greenberg HB, Pollard RB, Lutwick LI, Gregory PB, Robinson WS, Merigan TC. Effect of human leukocyte interferon on hepatitis B virus-infection in patients with chronic active hepatitis. N Engl J Med. 1976;295:517–522. [PubMed] [Google Scholar]

77. Allard HA. Some properties of the virus of the mosaic disease of tobacco. J Agric Res. 1916;6:649–674. [Google Scholar]

78. Brown M, Reed S, Levy JA, Busch M, McKerrow JH. Detection of HIV-1 in entamoeba-histolytica without evidence of transmission to human-cells. Aids. 1991;5:93–96. [PubMed] [Google Scholar]

79. Wang AL, Wang CC. Viruses of parasitic protozoa. Parasitol Today. 1991;7:76–80. [PubMed] [Google Scholar]

80. Applemans R, Wagemans J. The bacteriophages of different families. C R Seances Soc Biol Fil. 1922;86:738–739. [Google Scholar]

81. Maisin J. The bacteriophages. Arch Int Pharmacodyn Ther. 1922;26:215–245. [Google Scholar]

82. Piroth L, Wittkop L, Lacombe K, Rosenthal E, Gilbert C, Miailhes P, Carrieri P, Chas J, Poizot-Martin I, Gervais A, et al. Efficacy and safety of direct-acting antiviral regimens in HIV/HCV-co-infected patients—French ANRS CO13 HEPAVIH cohort. J Hepatol. 2017;67:23–31. [PubMed] [Google Scholar]

83. Sil A, Ravi MD, Patnaik BN, Dhingra MS, Dupuy M, Gandhi DJ, Dhaded SM, Dubey AP, Kundu R, Lalwani SK, et al. Effect of prophylactic or therapeutic administration of paracetamol on immune response to DTwP-HepB-Hib combination vaccine in Indian infants. Vaccine. 2017;35:2999–3006. [PubMed] [Google Scholar]

84. Brunel F, Darbouret A, Ronco J. Cationic lipid DC-Chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis B vaccine. Vaccine. 1999;17:2192–2203. [PubMed] [Google Scholar]

85. Mahor S, Rawat A, Dubey PK, Gupta PN, Khatri K, Goyal AK, Vyas SP. Cationic transfersomes based topical genetic vaccine against hepatitis B. Int J Pharm. 2007;340:13–19. [PubMed] [Google Scholar]

86. Blom RAM, Erni ST, Krempaska K, Schaerer O, van Dijk RM, Amacker M, Moser C, Hall SRR, von Garnier C, Blank F. A triple co-culture model of the human respiratory tract to study immune-modulatory effects of liposomes and virosomes. PLoS ONE. 2016;11:e0163539. [PMC free article] [PubMed] [Google Scholar]

87. Stein RT, Bont LJ, Zar H, Polack FP, Park C, Claxton A, Borok G, Butylkova Y, Wegzyn C. Respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis. Pediatr Pulmonol. 2017;52:556–569. [PMC free article] [PubMed] [Google Scholar]

88. Klinguer C, Beck A, De-Lys P, Bussat MC, Blaecke A, Derouet F, Bonnefoy JY, Nguyen TN, Corvaia N, Velin D. Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens. Vaccine. 2001;19:4236–4244. [PubMed] [Google Scholar]

89. Staats HF, McGhee JR. Application of basic principles of mucosal immunity to vaccine development. In: Kryono H, Ogra PL, McGhee JR (eds) Mucosal Vaccines. San Diego: Academic Press; 1996. p. 17–39. ISBN: 9780124105805.

90. Goldberg JB, Pier GB. Pseudomonas aeruginosa lipopolysaccharides and pathogenesis. Trends Microbiol. 1996;4:490–494. [PubMed] [Google Scholar]

91. Ketley JM. Pathogenesis of enteric infection by Campylobacter. Microbiol Uk. 1997;143:5–21. [PubMed] [Google Scholar]

92. Naumovska E, Ludwanowski S, Hersch N, Braun T, Merkel R, Hoffmann B, Csiszar A. Plasma membrane functionalization using highly fusogenic immune activator liposomes. Acta Biomater. 2014;10:1403–1411. [PubMed] [Google Scholar]

93. Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S, Nakanishi M, Tanaka K, Mayumi T. Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins. J Control Release. 1999;61:233–240. [PubMed] [Google Scholar]

94. Foged C, Arigita C, Sundblad A, Jiskoot W, Storm G, Frokjaer S. Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition. Vaccine. 2004;22:1903–1913. [PubMed] [Google Scholar]

95. Gall D. The adjuvant activity of aliphatic nitrogenous bases. Immunology. 1966;11:369–386. [PMC free article] [PubMed] [Google Scholar]

96. Jin SE, Kim CK. Long-term stable cationic solid lipid nanoparticles for the enhanced intracellular delivery of SMAD3 antisense oligonucleotides in activated murine macrophages. J Pharm Pharm Sci. 2012;15:467–482. [PubMed] [Google Scholar]

97. Romøren K, Thu BJ, Bols NC, Evensen Ø. Transfection efficiency and cytotoxicity of cationic liposomes in salmonid cell lines of hepatocyte and macrophage origin. Biochim Biophys Acta. 2004;1663:127–134. [PubMed] [Google Scholar]

98. Sakurai F, Inoue R, Nishino Y, Okuda A, Matsumoto O, Taga T, Yamashita F, Takakura Y, Hashida M. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression. J Control Release. 2000;66:255–269. [PubMed] [Google Scholar]

99. Kurosaki T, Kitahara T, Fumoto S, Nishida K, Yamamoto K, Nakagawa H, Kodama Y, Higuchi N, Nakamura T, Sasaki H. Chondroitin sulfate capsule system for efficient and secure gene delivery. J Pharm Pharm Sci. 2010;13:351–361. [PubMed] [Google Scholar]

100. Kurosaki T, Kitahara T, Teshima M, Nishida K, Nakamura J, Nakashima M, To H, Hukuchi H, Hamamoto T, Sasaki H. Exploitation of De Novo helper-lipids for effective gene delivery. J Pharm Pharm Sci. 2008;11:56–67. [PubMed] [Google Scholar]

101. Lehrnbecher T, Kalkum M, Champer J, Tramsen L, Schmidt S, Klingebiel T. Immunotherapy in invasive fungal infection—focus on invasive aspergillosis. Curr Pharm Des. 2013;19:3689–3712. [PubMed] [Google Scholar]

102. Mathew BP, Nath M. Recent approaches to antifungal therapy for invasive mycoses. ChemMedChem. 2009;4:310–323. [PubMed] [Google Scholar]

103. Jensen RH. Resistance in human pathogenic yeasts and filamentous fungi: prevalence, underlying molecular mechanisms and link to the use of antifungals in humans and the environment. Dan Med J. 2016;63(10). pii: B5288. [PubMed]

104. Hector RF, Rutherford GW, Tsang CA, Erhart LM, McCotter O, Anderson SM, Komatsu K, Tabnak F, Vugia DJ, Yang Y, Galgiani JN. The public health impact of Coccidioidomycosis in Arizona and California. Int J Environ Res Public Health. 2011;8:1150–1173. [PMC free article] [PubMed] [Google Scholar]

105. Krishnan-Natesan S, Chandrasekar PH. Current and future therapeutic options in the management of invasive aspergillosis. Drugs. 2008;68:265–282. [PubMed] [Google Scholar]

106. Han YM, Cutler JE. Antibody-response that protects against disseminated candidiasis. Infect Immun. 1995;63:2714–2719. [PMC free article] [PubMed] [Google Scholar]

107. Han YM, Morrison RP, Cutler JE. A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection. Infect Immun. 1998;66:5771–5776. [PMC free article] [PubMed] [Google Scholar]

108. Eckstein M, Barenholz Y, Bar LK, Segal E. Liposomes containing Candida albicans ribosomes as a prophylactic vaccine candidiasis in mice. Vaccine. 1997;15:220–224. [PubMed] [Google Scholar]

109. Masek J, Bartheldyova E, Turanek-Knotigova P, Skrabalova M, Korvasova Z, Plockova J, Koudelka S, Skodova P, Kulich P, Krupka M, et al. Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90. J Control Release. 2011;151:193–201. [PubMed] [Google Scholar]

110. Knotigova PT, Zyka D, Masek J, Kovalova A, Krupka M, Bartheldyova E, Kulich P, Koudelka S, Lukac R, Kauerova Z, et al. Molecular adjuvants based on nonpyrogenic lipophilic derivatives of norAbuMDP/GMDP formulated in nanoliposomes: stimulation of innate and adaptive immunity. Pharm Res. 2015;32:1186–1199. [PubMed] [Google Scholar]

111. Carneiro C, Correia A, Collins T, Vilanova M, Pais C, Gomes AC, Oliveira ME, Sampaio P. DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: a novel adjuvant and delivery system. Eur J Pharm Biopharm. 2015;89:190–200. [PubMed] [Google Scholar]

112. Carneiro C, Correia A, Lima T, Vilanova M, Pais C, Gomes AC, Real Oliveira MECD, Sampaio P. Protective effect of antigen delivery using monoolein-based liposomes in experimental hematogenously disseminated candidiasis. Acta Biomater. 2016;39:133–145. [PubMed] [Google Scholar]

113. Postma NS, Hermsen CC, Zuidema J, Eling WMC. Plasmodium vinckei: optimization of desferrioxamine B delivery in the treatment of murine malaria. Exp Parasitol. 1998;89:323–330. [PubMed] [Google Scholar]

114. Stewart VA, McGrath SM, Walsh DS, Davis S, Hess AS, Ware LA, Kester KE, Cummings JF, Burge JR, Voss G, et al. Pre-clinical evaluation of new adjuvant formulations to improve the immunogenicity of the malaria vaccine RTS, S/AS02A. Vaccine. 2006;24:6483–6492. [PubMed] [Google Scholar]

115. Ready PD. Epidemiology of visceral leishmaniasis. Clin Epidemiol. 2014;6:147–154. [PMC free article] [PubMed] [Google Scholar]

116. Bhowmick S, Ravindran R, Ali N. Leishmanial antigens in liposomes promote protective immunity and provide immunotherapy against visceral leishmaniasis via polarized Th1 response. Vaccine. 2007;25:6544–6556. [PubMed] [Google Scholar]

117. Banerjee A, De M, Ali N. Complete cure of experimental visceral leishmaniasis with amphotericin B in stearylamine-bearing cationic liposomes involves down-regulation of IL-10 and favorable T cell responses. J Immunol. 2008;181:1386–1398. [PubMed] [Google Scholar]

118. Banerjee A, Roychoudhury J, Ali N. Stearylamine-bearing cationic liposomes kill Leishmania parasites through surface exposed negatively charged phosphatidylserine. J Antimicrob Chemother. 2008;61:103–110. [PubMed] [Google Scholar]

119. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, Dopfer D, Fazil A, Fischer-Walker CL, Hald T, et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. Plos Med. 2015;12:21. [PMC free article] [PubMed] [Google Scholar]

120. World Health Organization . Global tuberculosis report 2016. Geneva: World Health Organization; 2016. [Google Scholar]

121. Houben R, Dodd PJ. The global burden of latent tuberculosis infection: a re-estimation using mathematical modelling. Plos Med. 2016;13:13. [PMC free article] [PubMed] [Google Scholar]

122. Tsuruta LR, Quintilio W, Costa MHB, CarmonaRibeiro AM. Interactions between cationic liposomes and an antigenic protein: the physical chemistry of the immunoadjuvant action. J Lipid Res. 1997;38:2003–2011. [PubMed] [Google Scholar]

123. Snippe H, Belder M, Willers JMN. Dimethyl dioctadecyl ammonium bromide as adjuvant for delayed-hypersensitivity in mice. Immunology. 1977;33:931–936. [PMC free article] [PubMed] [Google Scholar]

124. Holten-Andersen L, Doherty TM, Korsholm KS, Andersen P. Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines. Infect Immun. 2004;72:1608–1617. [PMC free article] [PubMed] [Google Scholar]

125. Rosenkrands I, Agger EM, Olsen AW, Korsholm KS, Andersen CS, Jensen KT, Andersen P. Cationic liposomes containing mycobacterial lipids: a new powerful Th1 adjuvant system. Infect Immun. 2005;73:5817–5826. [PMC free article] [PubMed] [Google Scholar]

126. Liu X, Da Z, Wang Y, Niu H, Li R, Yu H, He S, Guo M, Wang Y, Luo Y, et al. A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice. Vaccine. 2016;34:1370–1378. [PubMed] [Google Scholar]

127. Hamborg M, Rose F, Jorgensen L, Bjorklund K, Pedersen HB, Christensen D, Foged C. Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems: effect of charge, fluidity and antigen-to-lipid ratio. Biochim Biophys Acta Biomembr. 2014;1838:2001–2010. [PubMed] [Google Scholar]

128. Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C, Werninghaus K, Kirschning C, Lang R, Christensen D, et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements. PLoS ONE. 2008;3:10. [PMC free article] [PubMed] [Google Scholar]

129. Derrick SC, Yabe I, Morris S, Cowley S. Induction of unconventional T cells by a mutant Mycobacterium bovis BCG strain formulated in cationic liposomes correlates with protection against Mycobacterium tuberculosis infections of immunocompromised mice. Clin Vaccine Immunol. 2016;23:638–647. [PMC free article] [PubMed] [Google Scholar]

130. Boissier F, Bardou F, Guillet VR, Uttenweiler-Joseph S, Daffe M, Quemard A, Mourey L. Further insight into S-adenosylmethionine-dependent methyltransferases—structural characterization of Hma, an enzyme essential for the biosynthesis of oxygenated mycolic acids in Mycobacterium tuberculosis. J Biol Chem. 2006;281:4434–4445. [PubMed] [Google Scholar]

131. Procop GW. HIV and mycobacteria. Semin Diagn Pathol. 2017;34:332–339. [PubMed] [Google Scholar]

132. Perronne C, Zahraoui M, Leport C, Salmon D, Pangon B, Bricaire F, Vilde JL. Tuberculosis in 30 patients with HIV infection. Ann Med Interne. 1988;139:375–376. [Google Scholar]

133. Haegi V. Septic tuberculosis in HIV-infection. Schweiz Med Wochenschr. 1987;117:1297–1301. [PubMed] [Google Scholar]

134. Martin-Bertelsen B, Korshohn KS, Roces CB, Nielsen MH, Christensen D, Franzyk H, Yaghmur A, Foged C. Nano-self-assemblies based on synthetic analogues of mycobacterial monomycoloyl glycerol and DDA: supramolecular structure and adjuvant efficacy. Mol Pharm. 2016;13:2771–2781. [PubMed] [Google Scholar]

135. Larrouy-Maumus G, Layre E, Clark S, Prandi J, Rayner E, Lepore M, de Libero G, Williams A, Puzo G, Gilleron M. Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis. Vaccine. 2017;35:1395–1402. [PubMed] [Google Scholar]

---

Articles from Journal of Nanobiotechnology are provided here courtesy of BioMed Central

preferentially preparing zwitterionic liposomes comprised of

PH, the reason being that biological surfactants can be

arranged in decreasing order of toxicity according to the com-

monly observed trend for oral and parenteral applications:

cationic >anionic >amphoteric ≥nonionic [–]. When

initial testing revealed inconveniences in using only PH,

a combination of PH and anionic DMPG was introduced,

with emphasis being placed on using the minimal amount

of DMPG needed to resolve the inconvenience. Following

more iteration, a : PH/DMPG ratio (L) proved itself

to be the liposome of choice for conducting subsequent

extract-encapsulation studies, as this liposome displayed

favorable mean particle size, zeta potential, and PDI traits.

Table  is a formulation summary of some of the nalized

liposome-based candidates used in this study. e subsequent

internalization of Origanum onites L. was successfully carried

out via the method of Fricker et al. using empty liposomes

L and L to yield extract-loaded liposomes LE, LE,

LE, and LE. Several iterations were necessary and minor

protocol variations were implemented to arrive at cluster-

free, reasonably uniform particles and a good level of extract

internalization []

https://www.researchgate.net/publication/326590395_Liposomal_Incorporation_to_Improve_Dissolution_and_Stability_of_Rosmarinic_Acid_and_Carvacrol_Extracted_from_Oregano_O_onites_L

https://www.researchgate.net/publication/326590395_Liposomal_Incorporation_to_Improve_Dissolution_and_Stability_of_Rosmarinic_Acid_and_Carvacrol_Extracted_from_Oregano_O_onites_L

The potential antimicrobial benefit of high levels of rosmarinic acid (RA) and carvacrol (CA) in oregano ( O. onites L.) extract has been limited until now by poor bioavailability arising from the low aqueous-phase solubility and slow dissolution behaviour of the lyophilized extract (E). To address this issue, various ratios of phospholipon 90H (P90H) and 1,2-dimyristoyl- sn -glycero-3-phospho-(1'-rac-glycerol), sodium salt (DMPG) were sonicated, yielding four empty liposomes (L1, L2, L3, and L90). After an initial selection process, Turkish oregano extract was internalized into the more promising candidates. Each empty liposome, extract-loaded liposome (LE1, LE2, and LE3), and freeze-dried control (E) was assessed in terms of structure, composition, RA and CA dissolution profile, storage stability, and, when relevant, zeta potential. Empty liposome L1, which was prepared using P90H and DMPG in a 1:1 ratio, displayed the most convenient encapsulation traits among the four unloaded types. Loaded liposome LE1, obtained by combining oregano extract and L1 in a 1:1 ratio, proved superior as a vehicle to deliver RA & CA when compared against control freeze-dried E and test liposomes LE2 and LE3. Dissolution profiles of the active compounds RA and CA in loaded liposomes were determined using a semi-automated dissolution tester. The basket method was applied using artificial gastric juice without pepsin (AGJ, 50rpm, 500mL). The pH value was maintained at 1.5 (37 ± 0.5°C). Aliquots (5ml) were manually extracted from parallel dissolution vessels at 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, and 60-minute time points. Dissolution tests, run to completion on LE1, showed that approximately 99% of loaded CA and 88% of RA had been released. Shorter dissolution times were also noted in using LE1. In particular, the release profile of CA and RA had levelled off after only 25 minutes, respectively, depicting an impressive 3.0–3.3 and 2.3-2.6 rate increase compared to the freeze-dried control extract. The improved dispersibility of RA and CA in the form of LE1 was supported by particle size and zeta potential measurements of the liposome, yielding 234.3nm and −30.9mV, respectively. The polydispersity index value was 0.35, indicating a reasonable particle size distribution. To study storage stability, liposomes were stored (4°C, 6 months) in amber coloured glass containers (4 oz.). Each container held 30 capsules, which were stored according to the ICH guidelines prescribed for long-term storage ( 25°C±2°C ; 60%±5% RH). Triplicate samples were withdrawn after 0, 3, 6, 9, and 12 months for analysis. Lastly, LE1 displayed good storage stability. The results imply that RA and CA can be conveniently and routinely delivered via oral and mucosal routes by first internalizing oregano extracts into appropriately engineered liposomes.

preferentially preparing zwitterionic liposomes comprised of

PH, the reason being that biological surfactants can be

arranged in decreasing order of toxicity according to the com-

monly observed trend for oral and parenteral applications:

cationic >anionic >amphoteric ≥nonionic [–]. When

initial testing revealed inconveniences in using only PH,

a combination of PH and anionic DMPG was introduced,

with emphasis being placed on using the minimal amount

of DMPG needed to resolve the inconvenience. Following

more iteration, a : PH/DMPG ratio (L) proved itself

to be the liposome of choice for conducting subsequent

extract-encapsulation studies, as this liposome displayed

favorable mean particle size, zeta potential, and PDI traits.

Table  is a formulation summary of some of the nalized

liposome-based candidates used in this study. e subsequent

internalization of Origanum onites L. was successfully carried

out via the method of Fricker et al. using empty liposomes

L and L to yield extract-loaded liposomes LE, LE,

LE, and LE. Several iterations were necessary and minor

protocol variations were implemented to arrive at cluster-

free, reasonably uniform particles and a good level of extract

internalization [].

ioMed Research International 

T : Characteristics of various empty and Oregano extract-loaded liposomes.

Formulation Mean Size (nm) Zeta potential (mV) PDI (polydispersity index)

L ±.++ .±.++ .±.

L .±. -.±. .±. 

L .±. -.±. .±.

L .±. -.±. .±.

E.±. -.±. .±.

LE ±. .±. ++ .±. 

LE .±. + -.±. .±. 

LE .±. + -.±. + .±.

LE .±. + -.±. + .±. 

∗RA: rosmarinic acid.

∗∗CA: carvacrol.

+p≤. vs L, L, and L.

++ p ≤.vsL,L,L,E,LE,LE,andLE.

⧫p≤. vs E

1. nature  
2. scientific reports  
3. articles  
4. article

• Article
• Open Access
• Published: 08 August 2019

Clinical and Experimental Immunomodulation 2016

View this Special Issue

---

Review Article | Open Access

Volume 2016 |Article ID 1459394 | https://doi.org/10.1155/2016/1459394

Adjuvants: Classification, Modus Operandi, and Licensing

---

Juliana de Souza Apostólico,¹ Victória Alves Santos Lunardelli,¹ Fernanda Caroline Coirada,¹ Silvia Beatriz Boscardin,² and Daniela Santoro Rosa^1,3

Academic Editor: Lenin Pavon

Received18 Feb 2016

Revised02 Apr 2016

Accepted11 Apr 2016

Published04 May 2016

Abstract

Vaccination is one of the most efficient strategies for the prevention of infectious diseases. Although safer, subunit vaccines are poorly immunogenic and for this reason the use of adjuvants is strongly recommended. Since their discovery in the beginning of the 20th century, adjuvants have been used to improve immune responses that ultimately lead to protection against disease. The choice of the adjuvant is of utmost importance as it can stimulate protective immunity. Their mechanisms of action have now been revealed. Our increasing understanding of the immune system, and of correlates of protection, is helping in the development of new vaccine formulations for global infections. Nevertheless, few adjuvants are licensed for human vaccines and several formulations are now being evaluated in clinical trials. In this review, we briefly describe the most well known adjuvants used in experimental and clinical settings based on their main mechanisms of action and also highlight the requirements for licensing new vaccine formulations.

1. Introduction

Vaccination is one of the most efficient strategies for infectious diseases prevention. According to the World Health Organization (WHO), vaccination saves 5 lives every minute and will save over 25 million lives from 2011 to 2020. Traditional vaccine approaches like inactivated or live-attenuated viruses, although highly effective and immunogenic, present safety concerns. Despite being safer, subunit vaccines are normally less immunogenic/effective and need to be delivered together with an adjuvant. Hence, adjuvants are essential for enhancing and directing the adaptative immune response to vaccine antigens.

The term adjuvant comes from the Latin adjuvare, which means to help or aid [1]. Adjuvants can be defined as substances that increase immunogenicity of a vaccine formulation when added/mixed to it. The choice of the adjuvant is of utmost importance as it can stimulate strong humoral and cell mediated immunity indispensable for protection against some pathogens. In addition, the balance between the adjuvant properties and adverse effects plays a critical role in the selection.

The history of adjuvant discovery begins with Gaston Ramon, a veterinary working at the Pasteur Institute in 1920, that described the term adjuvant after he observed that higher specific antibody titers were detected in horses that developed abscesses at the injection site [2]. To confirm the hypothesis, he induced sterile abscesses at the injection site with starch or breadcrumbs together with inactivated toxin and confirmed that substances capable of inducing inflammation at the injection site also improved the production of antisera [3]. About the same time, Glenny et al. discovered the adjuvant effect of aluminum salts [4], and since then billions of alum-based vaccine doses have been administered to people. Jules Freund developed, in 1930, a powerful adjuvant composed of a water-in-mineral oil emulsion that also contained heat-killed mycobacteria (Mycobacterium tuberculosis or others) [5]. Although highly effective, complete Freund’s adjuvant (CFA) is also reactogenic and frequently induces granulomas, sterile abscesses, and ulcerative necrosis at the site of inoculation, which precludes it from being used in human vaccines. Figure 1 shows a timeline of adjuvant discovery.

Figure 1 

Timeline of vaccine adjuvants discovery.

A variety of compounds with adjuvant properties currently exist, and they seem to exert their functions through different mechanisms of action. Mineral salts, emulsions, microparticles, saponins, cytokines, microbial components/products, and liposomes have all been evaluated as adjuvants [6–8]. Nevertheless, few adjuvants are licensed for human use and several formulations are now being evaluated in clinical trials. In many cases, their use is empirical. Over the past years, many efforts have been made to investigate how and why adjuvants work. Recent advances have shown that adjuvants can (i) increase the biological half-life of vaccines, (ii) increase antigen uptake by antigen presenting cells (APCs), (iii) activate/mature APCs (e.g., dendritic cells), (iv) induce the production of immunoregulatory cytokines, (v) activate inflammasomes, and (vi) induce local inflammation and cellular recruitment [3, 9].

Independently of their mechanism of action, adjuvants have been traditionally used in the formulation of vaccines in an attempt to (i) decrease the amount of antigen, (ii) reduce the number of doses required to induce protective immunity, (iii) induce protective responses more rapidly, and (iv) increase the rate of seroconversion in special populations (the elderly, immunocompromised individuals, individuals with chronic disease, neonates and infants) [9].

2. Classification of Adjuvants

Different criteria may be used to group adjuvants in order to allow a rational comparison. Adjuvants can be classified according to their physicochemical properties, origin, and mechanisms of action [10]. Based on their mechanisms of action, adjuvants can be divided into delivery systems (particulate) and immune potentiators (immunostimulatory) [11]. Mucosal adjuvants are a class of compounds that can fit in both of the previously described categories (Table 1).

Table 1 

Classification of adjuvants.

Delivery systems can function as carriers to which antigens can be associated. Also, they create local proinflammatory responses that recruit innate immune cells to the site of injection [12]. Hence, it has been proposed that this type of adjuvants can activate innate immunity.

In a simplistic definition, the role of immune potentiators is to activate innate immune responses through pattern-recognition receptors (PRRs) or directly (e.g., cytokines). Pattern-recognition receptors (PRRs) consist of different classes of receptors [Toll-like receptors (TLRs), nucleotide-binding oligomerization domain- (NOD-) like receptors (NLRs), and the retinoic acid-inducible gene-I- (RIG-I-) like receptors (RLRs)] that are widely expressed on immune cells. Their engagement by pathogen-associated molecular patterns (PAMPs) triggers the activation of such innate cells that can ultimately mature/migrate to other tissues and produce cytokines and chemokines [13].

2.1. Delivery Systems

2.1.1. Mineral Salts

Delivery systems (particulate adjuvants) cover a wide range of materials such as aluminum salts (alum), lipid particles, and microparticles. Alum is by far the most widely used adjuvant since its introduction in the 1920s [14]. This adjuvant is in the formulation of licensed vaccines against Hepatitis A (HAV), Hepatitis B (HBV), diphtheria/tetanus/pertussis (DTP), human papillomavirus (HPV), Haemophilus influenza type B (HiB), and Pneumococcus.

Until recently, alum was believed to owe its adjuvant properties to the slow release of the antigen associated with it [15]. However, several reports demonstrated that if “antigen-alum depot” was removed after immunization, the immune response remained unaltered [16, 17], demonstrating that the depot effect and slow release of the antigen were not responsible for its adjuvant activity. Indeed, recent evidence showed that alum can activate the innate immune response [18, 19]. Aluminum-containing adjuvants are a class of adjuvants that do not use the classical TLRs and MyD88 or TRIF signaling pathways to activate innate immunity. Instead, they are sensed by NOD-like receptors (NLRs) through direct activation of NLRP3/NALP3 inflammasome complex or by the release of uric acid [18, 20, 21]. Another feature of alum is its ability to reduce antigen degradation [22].

However, for some vaccine formulations, alum does not elicit protective and sustained immune responses. This is because aluminum-containing adjuvants preferentially induce Th2 responses (characterized by antibody production), and for some pathogens a Th1 immune response (including cytotoxic CD8 T cells) is required [14, 23]. Hence, for such vaccines alum should not be used, at least not alone.

2.1.2. Emulsion Adjuvants

Freund’s Adjuvants. Complete Freund’s adjuvant (CFA) is a water-in-oil emulsion that contains heat-killed mycobacteria and is a classic “gold standard” representative of this group of adjuvants. In general, CFA is used to evaluate the immunogenicity of antigens in mice and on the induction of autoimmune diseases like uveitis and experimental autoimmune encephalomyelitis. In order to induce autoimmunity, evidence suggests that the components of mycobacteria direct T-lymphocytes to acquire a Th1 pattern that mediates delayed type hypersensitivity (DTH). One of the major concerns regarding the use of CFA is the induction of strong long-lasting local inflammation that may be painful to the animal often leading to ulcer at the site of injection [24]. Hence, there are numerous regulatory guidelines to work with CFA in experimental animals [25, 26].

Incomplete Freund’s adjuvant (IFA) is also a water-in-oil emulsion, but without mycobacteria. In the 50s, the use of IFA as an adjuvant in a human influenza vaccine led to higher long-lived antibody titers when compared to the same formulation without the adjuvant [27]. Its adjuvant activity is the result of a continuous release of the antigen from the oily deposit, an increased antigen lifetime, and the stimulation of local innate immunity, as it enhances phagocytosis, leukocyte infiltration, and cytokine production [28]. Although there is a consensus that the use of IFA in humans is hampered by the strong side effects, a survey conducted by the WHO reported that immunization of one million individuals with IFA showed severe side effects, such as sterile abscesses, in 40,000 [29]. Hence, due to the balance between potency and side effects, there are several completed clinical trials using IFA in vaccine candidates for HIV infection (see https://clinicaltrials.gov/, access number: NCT00381875), melanoma (NCT00003224,  NCT00706992, and NCT00085189), renal carcinoma (NCT00001703), and also multiple sclerosis (NCT02200718).

MF59. MF59 is a water-in-oil squalene based emulsion that is currently licensed as part of a flu vaccine (Fluad™, Seqirus) for individuals >65 years old. Initially, the vaccine focused on elderly subjects but was later tested in the second major flu risk group, young children and infants, and was successful in both cases [30, 31]. In addition, it was also approved for the H1N1 pandemic vaccine for pregnant woman and young children [32]. Moreover, infants vaccinated with MF59-adjuvant trivalent inactivated influenza vaccine (TIV) presented higher antibody titers and polyfunctional cytokine producing CD4+ T cells than children immunized with the nonadjuvant TIV [33, 34]. The inclusion of MF59 enhanced the low effectiveness of this influenza vaccine in children under 2 years of age. Thereafter, MF59 was tested as an adjuvant for an HBV vaccine, and it was able to induce an immune response one hundred times more potent than the one induced with alum [35].

As with the majority of adjuvants, the mechanisms of action of MF59 are not fully understood. Similar to alum, MF59 effect does not rely on depot formation at the injection site, as its half-life is 42 hours [7, 36]. However, MF59 seems to be a powerful adjuvant due to its ability to induce cellular and humoral responses, including high titers of functional antibodies [37]. Indeed, MF59 is able to stimulate macrophages, resident monocytes, and DCs to secrete several chemokines like CCL4, CCL2, CCL5, and CXCL8 that in turn induce leukocyte recruitment and antigen uptake leading to migration to lymph nodes and triggering the adaptative immune response [32, 38, 39]. Systems biology studies also revealed that MF59 increases expression of the leukocyte transendothelial migration gene cluster and recruitment of MHCII+CD11b+ cells at injection site and this profile may be predictive of robust immune responses [40]. Moreover, an elegant paper by Vono and colleagues showed that transient ATP release is required for innate and adaptive immune responses induced by MF59 [41].

AS03. AS03 is an oil-in-water adjuvant emulsion that contains -tocopherol, squalene, and polysorbate 80 and was developed by GlaxoSmithKline Biologicals [42]. The addition of α-tocopherol to the formulation differentiated AS03 from other oil-in-water emulsion adjuvants [43]. Its first use in humans was together with a malaria vaccine [44]. More recently, this adjuvant has been included for use in human vaccines especially for influenza. Recent clinical trials have showed that oil-in-water adjuvants as AS03 administered with influenza vaccine induced a more robust immune response [45]. Indeed, children aged from 6 to 35 months immunized with one dose of AS03 adjuvant vaccine developed strong immune response that was observed even 6 months after vaccination [46].

AS03 stimulates the immune system by the activation of NF-B, proinflammatory cytokine and chemokine production, recruitment of immune cells, mainly monocytes and macrophages, and induction of high antibody titers. An important issue is to administer AS03 with the antigen at the same injection site at the same time to avoid diminished response [42].

2.1.3. Microparticles

Virus-Like Particles. Virus-like particles (VLPs) are formed by structural viral proteins such as capsid or envelope that mimic intact virus size, shape, and molecule organization with self-assembly properties [47]. Although highly immunogenic because of their self-adjuvant properties, VLPs are noninfective and nonreplicative [48]. The structure of VLPs can be enveloped or nonenveloped depending on the parental virus. Nonenveloped VLPs are only composed by pathogen components with the ability to self-assemble (e.g., HPV) while enveloped VLPs consist of the host cell membrane (an envelope) in combination with the antigen of interest [49]. Other components such as TLRs agonists can also be incorporated into VLPs.

VLPs can induce direct B cell activation, proliferation, and upregulation of genes involved in class switch recombination and somatic hypermutation [50]. In addition, VLPs can bind, activate, and be captured by DCs [51, 52] which in turn lead to T cell immunity. They can also induce cross-presentation to CD8+ T cells [53]. Hence, VLPs are able to induce broad humoral and cellular immune responses including neutralizing antibodies and specific helper CD4+ and cytotoxic CD8+ T cells [54, 55]. There are a few commercially available vaccines that are based on VLPs including Engerix®/Recombivax® (Hepatitis B), Cervarix®/Gardasil® (HPV), and Mosquirix® (malaria) [49]. Currently, several enveloped and nonenveloped VLPs are in clinical development (Table 2).

Table 2 

Adjuvants in clinical development (for details see https://www.clinicaltrials.gov/).

Virosomes. Virosomes are a type of VLP platform that is composed of reconstituted viral envelopes with membrane lipids and viral glycoproteins that work as a carrier system for antigens or as adjuvants. Although composed of viral proteins, virosomes are not virulent since the genetic material of the native virus is absent and does not replicate [56]. Virosomes are produced by dissolving the envelope of the virus with a detergent followed by a complete removal of the genetic material of the virus and the nonmembranous proteins. The most used virosomal system is the immunopotentiating reconstituted influenza virosome (IRIV) [57, 58] that contains both the hemagglutinin (HA) and neuraminidase (NA) proteins intercalated within a lipid membrane. Currently, there are five licensed vaccines based on this approach: Inflexal® V, Nasalflu®, and Invivac® for influenza and Epaxal® and Epaxal Junior for Hepatitis A virus [58].

Virosomal HA and sialic acid can interact with APCs and induce particle endocytosis. After the acidification of the endosome, HA changes conformation and the fused antigen can either be released into the cytosol and be processed via MHCI or stay in the endosome and be processed via MHCII pathway. Concomitantly, virosomes increase the expression of costimulatory molecules (CD80, CD86, and CD40) on the APC surface. The whole process leads to CD8+ and CD4+ T cell activation and cytokine production such as IFNγ, TNFα, and GM-CSF [59].

PLA/PLGA. Poly(lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA) are biodegradable and biocompatible polymeric micro/nanoparticles that function as a delivery system by encapsulating an antigen or antigen plus adjuvant in the same particle [60, 61]. These particles are produced using techniques such as emulsification/solvent evaporation. Ligands against surface receptors (PRRs, CD1d) have also been loaded in PLGA nanoparticles as an adjuvant to trigger signaling pathways of innate immune responses [62, 63].

The particles are internalized by pinocytosis and clathrin-mediated endocytosis and can rapidly be localized into the cytosol [64]. PLGA can efficiently reach MHCI molecules and cross-present antigens to CD8+ T cells [65]. PLGA nanoparticle delivery system enhances the uptake by APCs [66] allowing prolonged release of the antigen and induces higher immune responses [67] when compared with the soluble counterpart.

PLGA has been used to deliver antigens from different pathogens including Bacillus anthracis [68], Plasmodium vivax [69], and Hepatitis B virus (HBV) [70].

2.2. Immune Potentiators

As stated before, immune potentiators target innate immunity signaling pathways through PRRs like TLRs, RLRs, and NLRs. In general, activation of PRRs by their agonists induces APC activation/maturation and cytokine/chemokine production that ultimately leads to adaptive immune responses. Examples of PRRs agonists include, but are not limited to, poly(I:C), MPL, flagellin, imiquimod, resiquimod, CpG ODN, and MDP (Figure 2).

Figure 2 

Adjuvants activate different immune innate receptors. TLRs (Toll-like receptors) and NLRs (NOD-like receptors).

2.2.1. TLR3 Agonists

Poly(I:C) (polyinosinic:polycytidylic acid) is a synthetic double strand RNA (dsRNA) that mimics viral RNAs and activates TLR3 located within endosomes [71, 72]. Poly(I:C) can also bind to the melanoma differentiation associated gene 5 (MDA5), a cytoplasmic protein that contains two caspase-recruitment domains (CARDs) and a DExD/H-box helicase domain. Results using knockout mice indicate that MDA5 is essential for poly(I:C)-induced IFN production, while TLR3 signaling is critical for IL-12 production. Both seem to regulate IL-6 production [73]. The administration of poly(I:C) activates DCs that quickly produce IL-12 and type I IFN and upregulate MHC II expression [74, 75]. In response to IL-12, NK cells produce IFN that in turn enhances T and B cell immunity. Type I IFN plays a critical role in the induction of Th1 responses and is also associated with cross-presentation [76]. Hence, poly(I:C) impacts APC maturation, antigen processing, and ultimately T and B cell immunity.

Poly(I:C) is the most TLR3 agonist tested as adjuvant against diseases including HIV [77, 78], dengue [79], malaria [80], and cancer [81, 82].

Poly-ICLC (Hiltonol®) is a poly(I:C) synthetic derivative stabilized with poly-L-lysine that is more resistant to RNAses [74, 83]. Several ongoing clinical trials (Table 2) are evaluating poly-ICLC for immunotherapy in patients with cancer [58]. More recently, poly-ICLC was also nasally delivered with a chimeric antibody containing HIV-p24 protein in mice and induced gastrointestinal immune responses [84].

2.2.2. TLR4 Agonists

Monophosphoryl lipid A (MPL) is the detoxified derivative of lipopolysaccharide (LPS) from Gram-negative bacteria (Salmonella minnesota R595). Removal of a phosphate residue from LPS renders MPL just 0.1% of the toxicity from the parental molecule. MPL mediates immune activation by interacting with TLR4 similarly to LPS [72]. MPL preferentially activates the TRIF signaling pathway [85] that triggers different cytokine production when compared to LPS that activates MyD88 and produces high amounts of TNF. Indeed, MPL is able to induce IL-12 and IFN production that promote Th1 responses.

MPL is approved for use in some countries as part of a vaccine against allergy (Pollinex Quattro®) [86] and in Canada for stage IV melanoma (Melacine®) [87]. Ongoing clinical trials evaluate MPL as a potential adjuvant for leishmaniasis, malaria, and Herpes antigens (Table 2).

2.2.3. TLR5 Agonists

Flagellin is the main component of bacterial flagella from both Gram-positive and Gram-negative bacteria and is recognized by the cell surface TLR5. Engagement of TLR5 induces TNF production but flagellin, when administered together with a vaccine antigen of interest, is also able to induce high antibody titers and mixed Th1/Th2 responses [88, 89]. Flagellin can simultaneously target inflammasomes [90] through NLRC4 phosphorylation [91, 92] and NAIP5 [93].

Flagellin can also be fused to the antigen of interest allowing its codelivery to the same APC. Influenza vaccines composed of fused flagellin-hemagglutinin (VAX128 and VAX125) and flagellin-matrix protein (VAX102) completed initial clinical trials [94, 95]. Results demonstrated that immunization with flagellin-fused proteins induced high antibody titers, seroconversion, and protection. Moreover, flagellin was also evaluated as a potent adjuvant to prevent rhinitis in mice [96].

2.2.4. TLR7/8 Agonists

Imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) and resiquimod (R848, 4-amino-2-(etoximetil)-a,a-dimethyl-1H-imidazo [4,5-c]quinoline-1-ethanol) are imidazoquinolines with antiviral properties [97–99]. Imidazoquinolines mimic single stranded RNAs (ssRNAs) that are recognized by TLR7/8 on endosomes triggering signaling through MyD88 [100–102]. Imiquimod is able to activate TLR7, while resiquimod actives TLR7 and TLR8. An important issue is the different TLR7 and TLR8 expression/function between human and mouse cells [103]. In mice, TLR7 is expressed by CD8− DC subsets but not by CD8+ DCs [104]. Nevertheless, in both species TLR7 is expressed on plasmacytoid DCs (pDC), B cells, and neutrophils. In contrast, TLR8 is nonfunctional in mice whereas in humans it is expressed by myeloid DCs (mDC) and monocytes [105]. Activation of both DC subsets in humans (mDCs and pDCs) facilitates type I IFN and IL-12 production [106] and enhances expression of costimulatory molecules, inducing direct and cross-presentation to CD8+ T cells [107], while it also induces NK cell activation [108]. Activation of Th1 cellular immune response can control viral replication, reactivation, and clearance [105]. Furthermore, resiquimod directly stimulates B cell proliferation by mimicking CD40 signal both in humans and in mice that ultimately stimulates antibody and cytokine production [109].

Imiquimod (Aldara) is approved for topical use in humans for treatment of actinic keratosis [110], basal cell carcinoma [111, 112], and genital warts caused by HPV 1, HPV 2, HPV 4, and HPV 7 [113, 114]. Resiquimod was tested in clinical trials to treat lesions caused by human Herpes virus (HSV) [115, 116]. Besides the use in therapy against established infections, these adjuvants are being evaluated for their ability to increase vaccine immunogenicity [78] and also in allergy and tumor therapy such as basocellular carcinoma and central nervous system tumors (Table 2) [117, 118].

Besides imiquimod and resiquimod, other TLR7/8 agonists have also been tested. Among them, we can cite the imidazoquinoline immune response modifier 3M-052 [119], the benzazepine TLR8 agonist, VTX-294 [120], and two benzonaphthyridines compounds SMIP.7-7 and SMIP.7-8 that bind to TLR7 [121].

2.2.5. TLR9 Agonists

CpG ODNs are 18–25 base synthetic oligodeoxynucleotides (ODN) composed of unmethylated CG motifs (cytosine phosphate guanidine) recognized by endosomal TLR9 [122–124]. Murine TLR9 is preferentially activated by GACGTT motif while the ideal sequence for human is GTCGTT [125]. TLR9 engagement signals through MyD88, IRAK, and TRAF-6 that ultimately leads to upregulation of costimulatory molecules (CD40, CD80, and CD86) and proinflammatory cytokines (IL-6, IL-12, IL-18, and TNF) [125, 126].

Three different types of CpG ODNs have been identified: A, B, and C [127]. Type A CpG ODNs contain a central phosphodiester palindromic motif in a phosphorothioate backbone and induce type I IFN production by pDCs. B type CpG ODNs have an entire phosphorothioate backbone that protects from degradation by nucleases and stimulates proliferation, IL-6/IgM production by B cells, and IL-6/TNF production by DCs [100, 126]. Type C CpG ODNs combine features of types A and B since they are composed of phosphorothioate backbone with palindromic motif and induce B cell responses as well as type I IFN production by pDCs [128, 129]. In general, CpG ODNs increase antibody responses and polarize to Th1 profile.

One of the most promising clinical results showed that commercial Hepatitis B vaccine administered together with CpG induced higher protective antibody titers after fewer doses both in healthy and in hyporesponsive individuals [130, 131]. Moreover, CpG ODNs have also been used in combination with conventional treatments for cancer [132].

2.2.6. NOD Agonists

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine) is a peptidoglycan biologically potent motif found on all bacteria cell walls. MDP was discovered in 1974 as the minimum component of mycobacteria’s cell wall required for the efficacy of complete Freund’s adjuvant [133].

MDP is able to activate NOD2 [134] leading to NF-κB transcription that results in the production of proinflammatory cytokines (TNF, IL-1, IL-6, and IL-8) as well as Th2 cytokines, nitric oxide secretion, enhanced cytotoxicity, and upregulation of adhesion molecules (CD11a, CD11b, CD11c/CD18, CD54) [135]. Studies have focused on the use of MDP for solid tumor therapy based on its ability to stimulate cellular as well as the cytokine response, eliciting antibody production [136].

2.3. Combination of Adjuvants

A recent approach to optimize vaccine immune responses is the use of different adjuvant combinations that could trigger different signaling pathways [137]. Such observation comes from studies using effective live-attenuated vaccines such as yellow fever that induce activation of different PRRs [138].

Based on this observation, one strategy is to use different TLR agonists to trigger activation of different signaling pathways (e.g., MyD88 and TRIF). Previous work tested different TLR agonist combinations in human PBMCs and evaluated cytokine and chemokine production [139]. Combinations of TLR7+TLR9 agonists induced type I IFN whereas TLR4+TLR7/8 synergistically upregulated IFN and IL2; TLR2+TLR7/8 synergistically upregulated IFN and others. MF59 and Carbopol-971P in combination were able to increase specific anti-HIV antibody titers [140]. However, not all combinations increase the magnitude of immune responses. For example, mice immunized with a recombinant HIV gp140 together with MPL plus alum or MDP exert synergic effects on the magnitude and quality of humoral response. However, when the mixture contained MDP plus poly(I:C) or resiquimod, no impact on antibody titers was observed but a significant difference was observed in IgG subclasses [78]. Another study showed that immunization of mice with nanoparticles containing antigens plus TLR4 and TLR7 ligands induced synergistic increases in antigen-specific, neutralizing antibodies when compared to immunization with nanoparticles containing antigens plus a single TLR ligand [141]. DCs activation by different combinations of TLR ligands was also evaluated. Results showed that, in human DCs, agonists of TLR3 and TLR4 potently acted in synergy with a TLR8 agonist and induced higher amounts of IL-12 and IL-23 than those induced by optimal concentrations of single agonists. This synergism led to enhanced and sustained Th1-polarizing capacity [142].

2.3.1. AS01 and AS02

Adjuvant System 01 (AS01) and Adjuvant System 02 (AS02) were the first in this type to be developed and tested in the RTS,S (Plasmodium falciparum circumsporozoite protein) vaccine candidate against malaria [143]. They are composed of MPL and the saponin QS21, but AS01 contains a liposomal suspension while AS02 is an oil-in-water emulsion [144]. When the trial began, AS02 was primarily tested and showed protection against controlled human malaria infection (CHMI) by the bite of infected mosquitoes [143]. However, when AS01 was included a higher production of specific antibody and improved efficacy was observed when compared to AS02 [145, 146]. Several clinical trials are in progress with AS01 and AS02 as vaccine adjuvants against HIV, tuberculosis, and malaria.

2.3.2. AS04

AS04 is composed of a combination of MPL and aluminum salts. Currently, two adjuvant vaccines are licensed: against HPV (Cervarix) [147, 148] and HBV (Fendrix®) [149].

This adjuvant also leads to activation of NF-B, production of proinflammatory cytokines and chemokines, and recruitment of monocytes and macrophages to the injection site, but specifically DCs. It is important to emphasize the need for AS04 and the antigen to be colocalized at the moment of antigen presentation on lymph nodes [144]. The advantage of AS04 for human vaccines is the induction of specific Th1 immune response and production of IL-2 and IFN, a profile weakly induced when alum is used alone [88].

2.4. Mucosal Adjuvants

The first immunization through mucosal surface was accomplished with attenuated poliovirus in 1962. Thereafter, other mucosal vaccines based on Salmonella typhi, Vibrio cholerae [150], rotavirus [151], and influenza virus were developed [152]. Administration by mucosal route has some advantages as needle-free delivery, lower costs, few adverse effects, and induction of local mucosal immunity, an important feature when infection occurs at mucosal routes [150, 153].

The most promising adjuvants for mucosal immunization are bacterial toxins extracted from Escherichia coli (heat-labile enterotoxin, LT) and Vibrio cholerae (cholera toxin, CT), TLRs agonists [flagellin, poly(I:C), CpG ODNs], and novel small molecules (-galactosylceramide, chitosan, etc.). To avoid development of cholera and travellers’ diarrhea symptoms, these toxins have been genetically modified to generate less toxic derivatives (LTK3, LTR-72, and CTB) [154, 155]. Alternative mucosal routes have been evaluated with LT mutants and CT, including nasal, intravaginal, and intrarectal. LTK3 and LTR-72 were shown to induce potent immune responses against influenza virus after oral immunization [156]. Oral immunization with LT was also efficient in protection against H. pylori infection in mice after challenge [157]. Studies that used intranasal delivery of LT as an adjuvant showed that immunization was able to induce strong immune response and protection against Herpes simplex virus [158], S. pneumonia [159], and B. pertussis [160].

Mucosal adjuvants CT and LT amplify B and T responses and stimulate isotype switching to IgA and mixed Th1/Th2 profile [161]. Further studies also demonstrated their ability to increase antigen uptake/presentation and DCs maturation/activation due to antigen permeation across epithelial barriers [162].

Mice intranasally immunized with Plasmodium vivax merozoite surface protein 1 (MSP119) in the presence of the adjuvants CT or LT presented high and long-lasting specific antibody titers. In the same study mice immunized with MSP119 fused to a T cell epitope (PADRE) in the presence of CpG ODN developed lower IgG titers when compared to mice that received CpG ODN plus CT [163]. In a recent study, an anti-HIV chimeric antibody (DEC205-p24) nasally delivered in combination with polyICLC induced polyfunctional immune responses within nasopulmonary lymphoid sites and mucosal gastrointestinal tract [164].

Chitosan is a biopolymer based on glucosamine extracted from a crustacean shell and is a mucosal adjuvant commonly used for intranasal delivery. The adjuvant acts in vitro by the translocation of “tight junctions” that improve transepithelial antigen transport and reduces the mucociliary clearance rate that facilitates antigen phagocytosis [165]. A study using a nontoxic mutant (CRM197) of diphtheria toxin in combination with chitosan showed that intranasal immunization was able to increase Th2 responses and, after a boost with the conventional diphtheria toxoid vaccine, enhanced antigen-specific IFN production [166]. Another study showed that intranasal administration of chitosan and CRM197 was as immunogenic as intramuscular immunization with the conventional diphtheria vaccine adsorbed to alum [167]. Furthermore, H. pylori vaccine with chitosan was used successfully in a therapeutic setting in mice with an equivalent performance as the traditional vaccine adjuvant, cholera toxin (CT). In addition, when infection was not fully eradicated, chitosan immunized mice presented lower bacteria density in the gastric mucosa when compared to CT groups [168].

3. Licensing

The introduction of an adjuvant in a new (or already licensed) vaccine formulation is still a challenge and may take several years. It is of utmost importance to test the compatibility of each component of the vaccine alone and in combination before any trials start [169]. Due to the urgent need to develop vaccines against infectious diseases, the Center for Biologics Evaluation and Research (CBER), a division of the US Food and Drug Administration, launched an important guide to facilitate the development of new formulations [170].

It is recommended that evaluation of safety/immunogenicity of a formulation begins with preclinical tests using an appropriate animal model (Figure 3). At this stage, the evaluation of adjuvant effect on the immune response is also recommended [171]. Of note, control groups composed of adjuvant and the antigen alone should also be included to provide evidence for adjuvant effect. The immunogenicity evaluation may include humoral (e.g., antibody titers, subclasses, avidity, and neutralization) and cellular (e.g., cytokine production, proliferation assays, and cell phenotyping) responses. If an animal model for the disease is available, initial protective efficacy information can be obtained [3].

Figure 3 

The different stages of vaccine development.

After preclinical testing and GMP (good manufacturing practice) production of the vaccine formulation, human clinical trials begin. Phase I vaccine studies are conducted in healthy individuals () to evaluate safety—to minimize adverse events and potential risks—and the dosage. Safety concerns include, but are not limited to, pain, granuloma formation, fever, sterile abscess formation, nausea, headache, malaise, and other local or systemic events. Initial immunogenicity information can be obtained from Phase I.

Phase IIa trials are designed to evaluate immunogenicity, tolerability, and safety and typically involve hundreds of volunteers. When tests reach Phases IIb/III, an important goal is to ascertain the immunogenicity and efficacy in the vaccine target population (e.g., children). Another difference is based on the number of volunteers and the study duration; the more the people involved, the longer the trial duration (several years).

After the process that confirms safety and efficacy of the vaccine, it can be licensed and marketed. After that, the formulation undergoes a postmarket safety monitoring, Phase IV, to evaluate additional rare adverse reactions.

4. Concluding Remarks

Adjuvants have been used to increase the immunogenicity of vaccines for almost a century. Until recently, adjuvant selection was empirical, but considerable advances in the field have allowed a rational/targeted use. This information together with an increasing understanding of the immune system will allow the development of effective vaccine formulations. Currently, only few adjuvant vaccines are licensed, but several ones are on clinical development and expected to reach approval in the near future. Finally, we believe that adjuvant selection could highly impact on rational vaccine design.

Competing Interests

The authors declare that they have no competing interests.

About 10,100,000 results (0.52 seconds) 

Open results in new tab

Search Results

Featured snippet from the web

View all

Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. In other words, immunogenicity is the ability to induce a humoral and/or cell-mediated immune responses.

Immunogenicity - Wikipedia

preferentially preparing zwitterionic liposomes comprised of

PH, the reason being that biological surfactants can be

arranged in decreasing order of toxicity according to the com-

monly observed trend for oral and parenteral applications:

cationic >anionic >amphoteric ≥nonionic [–]. When

initial testing revealed inconveniences in using only PH,

a combination of PH and anionic DMPG was introduced,

with emphasis being placed on using the minimal amount

of DMPG needed to resolve the inconvenience. Following

more iteration, a : PH/DMPG ratio (L) proved itself

to be the liposome of choice for conducting subsequent

extract-encapsulation studies, as this liposome displayed

favorable mean particle size, zeta potential, and PDI traits.

Table  is a formulation summary of some of the nalized

liposome-based candidates used in this study. e subsequent

internalization of Origanum onites L. was successfully carried

out via the method of Fricker et al. using empty liposomes

L and L to yield extract-loaded liposomes LE, LE,

LE, and LE. Several iterations were necessary and minor

protocol variations were implemented to arrive at cluster-

free, reasonably uniform particles and a good level of extract

internalization []