Abstract
Leishmaniasis is a parasitic disease that ranges in severity from skin lesions to fatality. Since long-lasting protection is induced upon recovery from cutaneous leishmaniasis, development of an effective vaccine is promising. However, there is no vaccine for use in humans yet. It seems limited efficacy in leishmaniasis vaccines is due to lack of an appropriate adjuvant or delivery system. Hence, the use of particulate adjuvants such as liposomes for effective delivery to the antigen presenting cells (APCs) is a valuable strategy to enhance leishmaniasis vaccine efficacy. The extraordinary versatility of liposomes because of their unique amphiphilic and biphasic nature allows for using antigens or immunostimulators within the core, on the surface or within the bilayer, and modulates both the magnitude and the T-helper bias of the immune response. In this review article, we attempt to summarize the role of liposomal adjuvants in the development of Leishmania vaccines and describe the main physicochemical properties of liposomes like phospholipid composition, surface charge, and particle size during formulation design. We also suggest potentially useful formulation strategies in order for future experiments to have a chance to succeed as liposomal vaccines against leishmaniasis.
Leishmaniasis vaccines
Leishmaniasis is caused by intracellular parasites of the Leishmania species and transmitted through the bite of sandflies. Visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), post-kala-azar dermal leishmaniasis (PKDL), and mucocutaneous leishmaniasis (MCL) are the four main types of the disease. The most common form is CL, which causes cutaneous lesions, while the most severe form is VL, which causes a fatal systemic disease. Available drugs for the treatment of leishmaniasis are toxic, need multiple injections, and have limited efficacy. Additionally, drug resistance emerges in some cases.1,2
Immunity to Leishmania spp. in animal models depends generally on development of the T-helper 1 (Th1)-mediated immune response characterized by high production of interleukin-12 (IL-12) and interferon-γ (IFN-γ). In mice, CD4+ Th1 cells mediate resistance in Leishmania major-infected mice whereas CD4+ Th2 cells promote susceptibility. 3 However, this is not always true in humans. Although, resistance to infection in humans has been associated with a Th1-mediated immune response, real protection from the disease requires the involvement of both a Th1-mediated and a Th2-mediated immune response from vaccination. Recent findings further support that a more complex cell-mediated immune response (CMI) influences the outcome of leishmaniasis, particularly CL. 4
Leishmaniasis vaccine candidates usually originate from Leishmania and can be categorized into three classes. These include: (a) live Leishmania, containing new genetically modified constructs; (b) first-generation vaccines consisting of killed Leishmania or parasite fractions; and (c) second-generation vaccines which are well-defined Leishmania molecules including recombinant proteins or DNA. 5 Many live Leishmania preparations were suggested as candidate vaccines against leishmaniasis but only a few of them reached clinical trials. An inoculation of live L. major promastigotes known as leishmanization (LZ) was used in several countries, including Iran. Although LZ was the most successful vaccine against CL, standardization and safety concerns have limited broad use of LZ. Development of the Leishmania vaccine using whole killed parasites have been attempted since the 1940s 6 and also reached clinical trials, but with limited efficacy.7 –9 In terms of second-generation vaccines, despite the proposal of several recombinant proteins or DNA molecules, only Leish-111f, Leish-F2, and Leish-F3 reached clinical trials. These vaccines contained recombinant polyproteins and were formulated with monophosphoryl lipid A (MPL) in an oil/water stable emulsion using squalene. The main drawback of using recombinant proteins as vaccine antigens is the lack of an appropriate adjuvant, since almost any Leishmania antigen induced protection in animal models when used with IL-12.10,11 DNA vaccination as another strategy in second-generation category against Leishmania is considered a promising technology, but no development has been reported for use in humans so far. 12
There are also vaccines of non-Leishmania origin from sandfly salivary glands which contain immunologically active molecules able to interfere with the host’s immune responses and markedly promote Leishmania infectivity. 13
Development of an effective vaccine against CL is promising because long-lasting protection is induced upon recovery from CL caused by natural infection or LZ.5,14 However, decades of studies on Leishmania vaccine development have resulted in only a few first-generation vaccines in phase III clinical trials that showed limited or no efficacy in humans. 15 Limited efficacy in vaccines against leishmaniasis is partly related to the lack of a suitable adjuvant.
The role of adjuvants in leishmaniasis vaccines
The use of an adjuvant or delivery system is necessary for almost any modern vaccine, particularly vaccines against leishmaniasis. In animal models, different Leishmania antigens induce a strong Th1 immune response and complete protection when combined with IL-12. However, in humans, the use of adjuvants is limited because of safety issues and therefore development of an effective anti-Leishmania vaccine will be a challenging area. For example, bacillus Calmette-Guérin (BCG) is an adjuvant safely used in humans and was used in several clinical trials; however, it displayed only limited efficacy. 5
A systematic approach to development of an effective vaccine requires precise information about the physicochemical properties of a selected antigen and adjuvant and knowledge of how to formulate adjuvant and antigen to achieve a stable, safe and still immunogenic vaccine. 16 Factors such as the nature of antigen, the route of administration, the immunization schedule, and the type of required immune response are critical to choice of an adjuvant. Moreover, optimum pharmaceutical parameters should be well defined in each case to develop an effective vaccine. 17
Many adjuvants with different properties and modes of action have been used in vaccination against leishmaniasis. Some of them are categorized as immunostimulatory adjuvants, such as monophosphoryl lipid A (MPL), muramyl di- or tripeptides and derivatives (MDP/MTP-PE), saponins (QuilA, QS-21), cytokines (IL-2, IL-12, GM-CSF), or CpG oligonucleotides. They are mostly pathogen-associated molecular patterns (PAMPs) and are highly conserved in a broad range of pathogens. 18 PAMPs, specifically those which bind to toll-like receptors (TLRs) or nucleotide-binding oligomerization domain-like receptors (NLRs), are the basis of many immunostimulatory adjuvants. 19 The most effective adjuvant in this category for leishmaniasis, at least in mice, is IL-12. However, it is not recommended for use in humans due to possible side effects, short in vivo half-life, the overall cost, and failure to induce long-term immunity.20 –22
The other category consists of particulate adjuvants including mineral-, lipid-, or polymer-based delivery systems.12,16,23 Particulate adjuvants selectively target antigens to the site of action, enhance and modulate the type of immune responses, and even enhance the potential to cross-present antigens in APCs. They also enhance and facilitate the absorption and uptake of antigens by APCs. From a formulation point of view, they increase the stability of antigens, reduce vaccine dose, and serve as a depot for controlled release of antigen. Moreover, they may be used to improve the solubility of insoluble antigens in aqueous medium to make them applicable for parenteral administration. 24 Particulate adjuvants such as emulsions, liposomes, and polymeric microspheres have been used successfully to deliver Leishmania antigens in preclinical models of leishmaniasis, as well as other infectious diseases. 25 An attractive and novel strategy for the rational design of potent adjuvants is the combination of immunostimulatory and particulate adjuvants, which has already reached clinical trials. A liposome-based vaccine adjuvant system containing two immunostimulants, that is, MPL and a QS-21 called AS01, is an example of the recent strategy. It has been used for malaria, tuberculosis, and herpes zoster in clinical trials.24,26
In this review article, we attempt to summarize the role of liposomal adjuvant in Leishmania vaccines and describe the main physicochemical properties of liposomes during formulation design. For more detailed information about the use of other adjuvants in leishmaniasis vaccines, we refer readers to articles elsewhere.16,18,25
Liposomes as a particulate vaccine adjuvant
Liposomes are spherical vesicles composed of amphipathic phospholipids. They are classified according to the number of lipids in the bilayer, as multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) with a size range between 0.02 to 10 µm in diameter. The size and morphology of liposomes are regulated by their composition and preparation method. 27 Compared with other particulate systems, liposomal adjuvants have several key advantages for vaccine development, such as safety and biodegradability. 28 Liposomes are often composed of lipids that occur naturally in cell membranes, such as phosphatidylcholine (PC) and cholesterol, which make these formulations completely biodegradable. The liposomal aqueous central phase is a compartment for encapsulation of hydrophilic molecules, while the lipid bilayer phase can be used for hydrophobic compounds. 29 Therefore, all types of antigens such as peptides, proteins, carbohydrates, nucleic acids, and small molecules can be used in liposomes. Moreover, immunostimulatory adjuvants, based on their physicochemical properties, can be readily inserted in liposomes containing antigen, as previously mentioned for AS01. Antigenic proteins delivered by conventional liposomes can be processed via the major histocompatibility complex class II (MHC II) pathway, while pH-sensitive liposomes can present antigen via MHC I molecules. 30
Influence of liposomal formulation parameters on immune responses
The formulation parameters of liposomes including phospholipid composition, bilayer fluidity, surface charge, particle size, lamellarity, liposome preparation, antigen attachment, and lamellar–hexagonal bilayer phase transition ability have significant impact on immune responses. They are often critical for incorporation strategies and the selection of antigen or immunostimulator. Most of these properties have been reviewed elsewhere for different diseases but in the following sections, the parameters that have had significant influence on liposomal adjuvanticity as anti-Leishmania vaccines will be reviewed. Table 1 presents the formulation details of liposomes that have been used as leishmaniasis vaccines to date and are reviewed in this article.
The formulation details of liposomes that have been used as leishmaniasis vaccines.
ALM, autoclaved Leishmania major; BALB/c, albino, laboratory-bred mouse; BCG, bacillus Calmette-Guérin; Chol, cholesterol; CpG ODN, cytosine phosphate guanine oligodeoxynucleotides; DCP, dicetyl phosphate; DDA, dimethyldioctadecylammonium; DDAB, dimethyldioctadecylammoniumbromide; DMPC, dimyristoyl phosphatidylcholine; DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane; DSPC, distearoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; EPC, egg phosphatidylcholine; FTP, freeze–thawed promastigote; GPI, glycosylphosphatidylinositol; LAg, Leishmania membrane antigen; LD, L. donovani promastigotes polypeptide; LmSTI1, Leishmania major stress-inducible protein 1; Mal-PEG2000-IgG: maleimide-polyethylene glycol (Mr 2000)-Immunoglobulin G; Man5-DPPE, mannopentaose-dipalmitoylphosphatidylethanolamine; MLV, multilamellar vesicles; MPL-TDM, monophosphoryl lipid−trehalose dicorynomycolate; NA, not applicable; ND, not determined; SA, stearylamine; SLA, soluble Leishmania antigen; sLAg: soluble Leishmania antigens; SM, sphingomyelin; PA, phosphatidic acid; PC, phosphatidylcholine; pDNA, plasmid DNA; PO CpG, nuclease sensitive phosphodiester CpG ODNs; PS CpG, nuclease-resistant phosphorothioate CpG oligodeoxynucleotides; rgp63, recombinant major surface glycoprotein; TDB, trehalose 6,6’-dibehenate.
Effect of phospholipid composition and bilayer fluidity
The lipid bilayer of liposomes can be in different physical phases depending on the specific lipid compositions and temperature. Lipids with a main-phase transition temperature (Tm) below 37ºC will be in a liquid-crystalline state in the body, while they will be in a gel state if the Tm is above 37ºC. The physical state of the bilayer can affect endocytosis, intracellular trafficking and processing of the vaccine components, which in turn may influence immune responses. 62 Generally, a correlation exists between the Tm of phospholipids and generated immune response for membrane-associated antigens. 63 However, a phospholipid composition that induces a strong immune response to a specific antigen may not necessarily induce the same immunity to others. Hence, there is a need for designing a liposomal adjuvant tailored for each antigen.64,65
Although the mechanism(s) of bilayer fluidity on liposomal adjuvanticity is still not clearly known, it is proposed that at least two mechanisms are involved at the same time: (a) the rate of antigen release from the vesicles at the site of injection and (b) the mode of liposomal interaction with APCs. In the first case, solid liposomes become unstable in vivo at a slower rate than fluid ones and so they present antigens more efficiently to APCs. Secondly, fluidity influences the extent of liposomal fusion, endocytosis, and processing by APCs that in the case of fusion and endocytosis, solid liposomes probably would not be a very suitable choice. Therefore, it seems that liposomes need to have an optimal fluidity for an antigen to induce the desired immune response. 28
Liposomal rgp63 composed of egg phosphatidylcholine (EPC, Tm <0°C), dipalmitoylphosphatidylcholine (DPPC, Tm 41°C), or distearoylphosphatidylcholine (DSPC, Tm 54°C) was prepared in a study to develop anti-Leishmania vaccine in albino, laboratory-bred (BALB/c) mice. 31 Liposomes composed of DPPC and DSPC elicit a Th1 immune response against L. major infection with no significant difference between them; however, liposomes prepared with EPC induced a Th2 response. Mazumdar and colleagues evaluated the effect of three phospholipids [DSPC, DPPC, and DMPC (Tm 23°C)] on the adjuvanticity and rate of protection of liposomal vaccines against VL in a hamster model. 32 All three formulations were similar in the entrapment of Leishmania donovani promastigote membrane antigens (LAg) and in the ability to stimulate a humoral response. The superiority of DSPC liposomes to potentiate CMI in comparison with DPPC and DMPC was observed in immunized mice. Badiee and colleagues prepared liposomes composed of a high Tm lipid, that is, DSPC, to optimize adjuvanticity of liposomes for rLmSTI1. 33 The results showed that subcutaneous (SC) immunization of BALB/c mice with liposomal rLmSTI1 induced a significant protection against challenge and a significantly lower parasite burden in the spleen up to 14 weeks after challenge compared with free antigen. Jaafari and colleagues studied the ability of liposomal rgp63 (Lip-rgp63) prepared with a low Tm lipid, that is, egg lecithin to induce protection against L. major infection in BALB/c mice. 34 The results showed that Lip-rgp63 conferred a partial protection against L. major infection. In another study, Kahl and colleagues evaluated efficacy of intravenous (IV) vaccination against CL in BALB/c mice by using radiation-attenuated promastigotes or colloidal antigen mixtures of L. major in three different liposomal formulations composed of DSPC, DPPC, or underivatized PC (Tm −10°C). 35 The DSPC formulation was found to be superior to DPPC and PC for inducing protective immunity to CL.
Colhone and colleagues prepared an extract of glycosylphosphatidylinositol (GPI)-anchored proteins from Leishmania amazonensis promastigotes and reconstituted them into proteoliposomes composed of DPPC and cholesterol. 36 BALB/c mice inoculated intraperitoneally with proteoliposomes displayed a complete protection (90%). This study suggests that liposomes promote the slow release of antigens at the site of injection and trigger the immune system. Altogether, liposomes prepared with higher Tm phospholipids are suitable formulations to induce the Th1 response and to protect mice against leishmaniasis. High Tm phospholipids or increasing the amount of cholesterol in the formulation increase the rigidity of vesicle bilayers or the packing of phospholipids at the body’s temperature and thus resist phospholipid loss to the extracellular matrix. Hence, liposomal integrity is preserved and entrapped antigens remain with the carrier for longer periods of time resulting in more adjuvanticity.
Beside the role of bilayer fluidity on the adjuvanticity, lipid composition of the liposome has an important effect on immune responses, particularly when these lipids have intrinsic immunostimulatory properties. Nonphosphatidylcholine (non-PC) liposomes (escheriosomes) prepared from E. coli lipids were used to deliver LAg to APCs. 37 The vaccine induced strong humoral, as well as CMI, both in hamsters and BALB/c mice. Immunization of BALB/c mice with escheriosomes elicited stronger cytotoxic T-lymphocyte (CTL) response as compared with LAg entrapped in egg PC/cholesterol liposomes or LAg administered with incomplete Freund’s adjuvant. In addition, the delivery of LAg via escheriosomes enhanced the expression of costimulatory signals (CD80 and CD86). The escheriosome-immunized hamsters were found to be better protected than those immunized with liposomal LAg. They concluded that escheriosomes have the potential to deliver antigens to the cytosol of APCs.
In another study, gp63 and Leishmania promastigote lipophosphoglycan were reconstituted into liposomes composed of isolated phospholipids from the trypanosomatid flagellate Crithidia fasciculate, and inoculated SC, Intraperitoneal Injection (IP), or IV into laboratory inbred (CBA/ca) or BALB/c mice. 66 Complete protection was obtained only in CBA/ca mice by SC inoculation of liposomal antigens. Interestingly, antigen-containing liposomes did not cause the disease exacerbation compared with nonliposomal crude parasite extracts. Additionally, Samiei and colleagues studied a way to construct an artificial liposome-based cell that contained fragments of L. major. 38 Because they were focussed on developing a new method for engrafting plasma membrane vesicles of parasites into liposomes, they didn’t evaluate immune responses in their study. They proposed that the presence of whole-cell membrane constituents of parasites (proteins, glycoproteins and glycol phospholipids) could be essential for obtaining a protective immune response. Moreover, adding a Th1-inducer adjuvant in the structure of engrafted liposomes could enhance the ability of prepared vaccines to protect against intracellular parasites that rely on Th1 responses. Finally, McConville and colleagues observed that the predominant cell surface glycoconjugated antigen of L. major contained a GPI-like membrane anchor. When this antigen was entrapped in multilamellar liposomes and injected into mice, the animals were protected from CL. 67
Effect of surface charge
The composition of lipids in liposome bilayers dictates the surface charge. Setting aside some exceptions, positively-charged liposomes are taken up by APCs to a much higher degree than negatively-charged or neutral liposomes. Since cationic entities interact with negatively charged molecules on the surface of APCs, they target antigens for endocytosis more efficiently than other types. In addition, positively charged liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a cationic lipid with fusogenic properties, induce the generation of CD8+ T cell responses which require antigen presentation in the context of the MHC I. 68 Most of the studies in the leishmaniasis field have used stearylamine (SA), DOTAP, or dimethyldioctadecylammonium bromide (DDAB) for liposomal formulation; however, other commonly used cationic lipids such as 3-β-[N-(N,N’-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N-[1-(2,3-dioleoyloxy)propel]-N,N,N-trimethylammonium (DOTMA), can be used.
Afrin and colleagues evaluated the immunogenicity of LAg alone or in association with positively charged liposomes. 40 The liposomes significantly protected the hamsters and BALB/c mice and the splenic parasite burden was reduced. Effective stimulation of all the immunoglobulin G (IgG) isotypes, particularly IgG2a, with liposomes, also occurred. Another study has compared negatively charged liposomes to enhance protection against LAg with control groups. 41 The level of protection by liposomal LAg was not significantly different from that induced by free LAg, and interestingly, anionic liposomes elicited strong antibody responses, particularly IgG1. They concluded that the Th2 response was dominant with anionic formulation. Bhowmick and colleagues showed that gp63 encapsulated in cationic liposomes consisting of DSPC induced significant protection against progressive VL in BALB/c mice. 43 The protective efficacy of liposomal gp63 vaccination was also dose dependent. The control of disease progression and parasitic burden in mice was associated with enhancement of IFN-γ and reduction of IL-4. Ravindran and colleagues compared the effectiveness of three adjuvants, cationic liposomes, BCG and MPL + trehalose dicorynomycolate (TDM), to confer protection against murine VL. 46 Although, all three formulations afforded significant protection against L. donovani, the highest level of protection was exhibited by liposomal LAg.
Thakur and colleagues determined the protective efficacy of freeze–thawed L. donovani (FTP) antigen in combination with different adjuvants 47 including alum, saponin, cationic liposomes or MPL. Cationic liposomes were composed of PC, SA and cholesterol. Maximum protection was achieved with liposome-encapsulated FTP antigen. Higher Delayed-type Hypersensitivity (DTH) response and maximum decline in parasite burden were induced by liposomal FTP over other adjuvants. Moreover, Bhowmick and colleagues compared the vaccine efficacy of four antigens including polypeptides 91(LD91), 72(LD72), 51(LD51) and 31(LD31)-kDa encapsulated in cationic liposomes composed of DSPC, cholesterol, and SA in BALB/c mice. 48 Results demonstrated that liposomal LD31 and LD51 immunization reduced parasite burden to the greatest degree over other formulations. Analysis of cytokine responses in immunized mice showed that all the vaccinated groups produced IFN-γ, IL-12 and IL-4.
Bhowmick and colleagues evaluated the protective efficacy of soluble Leishmania antigen in positive, neutral, and negative liposomes and compared the immune response for protection against L. donovani in a BALB/c model. 42 The liposomes were prepared with egg lecithin and cholesterol in the presence of either SA or phosphatidic acid (PA). The best vaccine formulation contained positively charged liposomes which were then used for immunotherapy. This vaccine induced more than 90% elimination of parasites from both liver and spleen. These findings suggest that an induced immune modulation by positively charged liposomes towards Th1 is effective for both successful vaccination and immunotherapy.
It seems that the type of cationic lipids and their physicochemical properties in combination with antigen have a key role in generated immune response. DDAB is usually used in liposome formulation to induce a Th1 response based on two main reasons. The first is to prepare positively charged liposomes and the second one to use the intrinsic adjuvanticity of synthetic quaternary ammonium compounds. However, it usually induces a mixed Th1/Th2 response for anti-Leishmania vaccines. The ability of rgp63-containing liposomes composed of neutral lipid (DPPC), negatively charged lipid (DCP), and positively charged lipid (DDAB) to elicit protection against leishmaniasis was compared in mice. 44 Interestingly, the DPPC liposomes without DCP or DDAB elicited the greatest IFN-γ production, the lowest parasite burden, and the least footpad swelling after infection. Positively charged liposomes showed a mixed Th1/Th2 immune response, which might be due to the inherent adjuvanticity of DDAB, not due to the positive surface charge of liposome. On the other hand, the influence of positively charged surface to interact with APCs might be masked by the adjuvanticity of DDAB. In another study, a cationic liposomal SLA vaccine was also developed using DDAB and cholesterol in the presence or absence of trehalose dibehenate (TDB). 39 The results showed that immunization with liposomal SLA composed of DDAB or TDB induces a mixed Th1/Th2 immune response and is not an appropriate strategy for induction of a Th1 response and protection against leishmaniasis.
In contrast to DDAB, another synthetic quaternary ammonium compound, that is, DOTAP, induced a potent Th1 response and protection in BALB/c mice against L. major infection. 49 Liposomal SLA showed significantly smaller footpad swelling and the lowest spleen and footpad parasite burden after the challenge. This group also showed the highest IFN-γ production, higher IgG2a antibody titer, and lower IL-4 levels. The protective efficacy of liposomal SLA vaccination was also dose dependent, with 50 µg of protein showing optimal protection. Collectively, the surface charge of liposomes has a crucial role in the type and extent of generated immune responses, and cationic lipids should be tailored to achieve a desired immune response in formulation with each antigen.
Egg sphingomyelin (SM), which is primarily composed of saturated acyl chains, was used in a study to produce a stable negatively charged liposome containing SLA. 50 Mice receiving these liposomes showed significantly large footpad swelling, higher parasite burden in the foot and higher IL-4 levels compared with the group immunized with buffer and were not protected against leishmaniasis. 50
The immune response not only depends on the surface charge of liposomes, but on liposome preparation method. Immune responses were significantly influenced by similar LAg-containing cationic liposomes prepared by three different methods, including conventional MLV, reverse-phase evaporation vesicles (REV) and Dehydration Rehydration Vesicles (DRV). 45 LAg in MLV or DRV, but not in REV induced almost complete protection. Analysis of immune response in MLV and DRV groups showed elicitation of humoral as well as CMI as evidenced by LAg-specific antibodies, DTH responses, and IFN-γ. Upregulation of IgG2a and low expression of IgG1 were observed in sera from BALB/c mice immunized with LAg either in MLV or DRV, suggesting that in these animals, the cellular immune response was primarily Th1. Cationic MLV, when used as adjuvant with protein antigens induced sustained Th1 immunity. In conclusion, positively charged liposomes induce stronger immune responses compared with equivalent neutral or negatively charged formulations. It should also be noted that extracellular matrix in the injection site which has a negative charge, can retain liposomes at the injection site for a longer time period. This may result in skewing the elicited immune response toward Th1, but this is not always true, at least in the case of DDAB liposomes, as discussed earlier.
Effect of liposome size and lamellarity
Vesicle size is one of the most significant factors in liposomal vaccine formulation. The role of particle size on uptake and trafficking of antigens are extensively reviewed elsewhere.69,70 Particle size may influence the draining kinetics of liposomes, as small liposomes are cleared faster from injection sites than large liposomes.71,72 Small particles, less than a few nanometers, are usually transported to the blood, whereas larger particles, up to 150 nm, are freely transported into the lymphatic capillaries. Liposomes over a few hundred nanometers in size will be trapped in the interstitial space for a longer period of time or transported by dendritic cells (DCs). 73
Badiee and colleagues studied the effect of liposome size on the rate of protection and type of immune response generated against leishmaniasis. 51 Liposomal rgp63 with different sizes (100, 400, 1000 nm) were prepared and inoculated into BALB/c mice. 51 The results showed that immunization with small size (100 nm) liposomes induces a Th2 response, but large size (⩾400 nm) liposomes induced a Th1 response and protection in mice. There was no significant difference in the type of induced immune response between 400 nm, 1000 nm, or unextruded liposomes.
The immune response against liposomal antigens may be influenced by liposome lamellarity. Bhowmick and colleagues compared the immune response in mice with LAg encapsulated in MLV (multilamellar, 405 nm), DRV (multilamellar, 882 nm), and REV (unilamellar, 897). 45 REV elicited higher IgG1, whereas DRV and MLV elicited higher IgG2a and IFN-γ. Hence, cationic MLV as well as DRV were considered as suitable adjuvants and antigen delivery systems for designing subunit vaccines.
To the best of our knowledge, there are no other studies showing the influence of liposome size and lamellarity on immune responses against leishmaniasis. However, the results of studies in other disease models suggest that liposomes with a size range of 250–700 nm in diameter shift the Th profile of the response toward Th1 through increasing both persistence at the injection site and transit to draining lymph nodes. It should be taken into account that this is usually applicable in the case of neutral or negatively charged liposomes but not for cationic liposomes. Cationic liposomes of any size range are partially immobilized at the injection site because of their electrostatic interaction with extracellular matrix contents.
Effect of codelivery of immunostimulator(s) with antigen
Liposomes may accommodate antigens, immunomodulators, and targeting ligands at the same time. Delivery of antigens and adjuvants into the same APCs at the same time efficiently enhances APCs’ activation. Moreover, formulation of a potent immunostimulatory adjuvant into the liposome limits adverse effects through restriction of systemic circulation of the adjuvant and protection from enzymatic degradation in the injection site. 74
Several studies have shown that liposomes can synergistically increase the immunogenic properties of many immunostimulators. The potential benefit of using liposomes composed of DSPC to enhance the adjuvanticity of cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) with LmSTI1 antigen was studied. 52 BALB/c mice were immunized subcutaneously with liposomal rLmSTI1 co-encapsulated with CpG ODN. The results of footpad swelling, parasite burden in spleen, IgG2a/IgG1 ratio showed that mice immunized with liposomal rLmSTI1 and CpG ODN can be protected against infection. The results also indicated the superiority of CpG ODN in its liposomal form over its soluble form to induce the Th1 response. Protection of CpG ODN from degradation by endonucleases in the injection site is very important for maximal vaccine efficacy. To evaluate whether liposomes provide nuclease protection, the nuclease-resistant phosphorothioate CpG ODN (PS CpG) or nuclease-sensitive phosphodiester CpG ODN (PO CpG) were used in liposomal form. 53 An efficient liposomal delivery system was developed to protect PO CpG from degradation. In this study, SLA as an antigen and DOTAP as a cationic lipid were used to protect PO CpG. DOTAP was also selected because of its unique adjuvanticity and electrostatic interaction with negatively charged CpG ODN. The groups of mice that received Lip-SLA–PO CpG or Lip-SLA–PS CpG (Liposomes containig soluble leishmania antigens and CpG ODN) showed a high protection rate compared with control groups and there was no significant difference between the liposomal form of PS CpG and PO CpG. They concluded liposomal PO CpG might be used instead of PS CpG in future vaccine formulations because of their higher safety and lower cost. Jaafari and colleagues evaluated immune response and protection in BALB/c mice against CL using CpG ODN co-encapsulated with rgp63 in cationic liposomes. 54 Positively charged liposomes containing both rgp63 and CpG ODN protected mice against L. major infection more effectively than liposomal rgp63 alone. The mice immunized with liposomal rgp63 and CpG ODN also showed the lowest spleen parasite burden and highest IFN-γ production. In another study, an efficient liposomal SLA co-encapsulated with CpG ODN was developed against CL in BALB/c mice. 56 The results showed that lesion size in mice immunized with liposomal SLA and CpG ODN was significantly smaller than the control groups. They concluded the liposomal form of CpG ODN induced more protection and for a longer period of time. Mazumder and colleagues evaluated the efficacy of noncoding plasmid DNA (pDNA) bearing immunostimulatory sequences (ISS) and SLA to protect against challenge with L. donovani infection. 55 In this study, immunization of BALB/c mice using cationic liposomal SLA and pDNA either mixed or in encapsulated form protected almost all of the mice and triggered strong immune responses against disease. A notable increase in the protective efficacy (~twofold) was reported when antigen and pDNA were codelivered within liposomes.
The immune response and protection induced by liposomal SLA mixed with MPL-TDM was evaluated through a subcutaneous route. 57 The liposomal SLA mixed (not co-encapsulated) with MPL-TDM induced significantly higher levels of protection in liver and spleen in mice. Protection conferred by this combination was sustained up to 12 weeks after immunization, and infection was controlled for at least 4 months of the challenge. A vaccine formulation was also developed by combining cationic liposomes with MPL-TDM using rgp63. 58 Formulation of rgp63 with either MPL-TDM, or entrapped within cationic liposomes or both, were developed. Combinatorial administration of liposomal rgp63 with MPL-TDM during priming confers activation of DCs, and induces an early, robust T-cell response. Boosting with the same combination resulted in higher IFN-γ production and splenocyte proliferation, and even better Th1 responses compared with mice boosted with liposomal rgp63.
Another study investigated whether liposomes containing autoclaved L. major (ALM) and mixed with BCG could selectively induce Th1 responses in resistant C57BL/6 mice. 59 The ALM formulation was prepared using egg lecithin and cholesterol mixed with BCG. The results indicated the superiority of liposomal ALM + BCG compared with control groups. A DTH test in mice that received liposomal ALM + BCG produced a significantly higher response than the other groups. It seems that neutral liposomes containing ALM mixed with BCG can be used to induce a Th1 response in C57BL/6 mice.
The role of liposomal CpG ODN as an immunotherapeutic agent during the course of L. major infection was studied in BALB/c mice. 75 Different groups of BALB/c mice were subcutaneously inoculated with L. major mixed with either liposomal CpG ODN composed of DSPC, or soluble CpG ODN. Interestingly, the results showed that co-administration of CpG ODN with L. major in both forms induced a significantly smaller lesion size with significantly lower death rate compared with control groups, and there was no significant difference between liposomal and soluble form. Because of the presence of DSPC in the liposome formulation, CpG ODN is not released easily from liposome into the phagosome and not bound effectively to its receptor that is, TLR9. The authors suggest that further studies are needed to identify a suitable lipid, probably a low Tm lipid, to formulate the liposomes, which releases CpG ODN at an appropriate time to interact properly with TLR9.
Effect of active targeting to antigen presenting cells
Liposomes deliver their contents into cells by passive or active targeting mechanisms. The physicochemical properties of liposomes, such as size and surface charge, play a pivotal role in passive targeting to APCs, 70 and active targeting aimed to increase the delivery of content-specific interactions with a target cell. DCs provide a critical link between innate and adaptive immunity. Among different types of APCs, the potent antigen presenting properties of DCs make them valuable targets for the delivery of vaccine components. Introduction of ligands or monoclonal antibodies like mannose, anti-DEC-205 Ab, anti-Clec9A Ab, or anti-DC-SIGN Ab for DC surface receptors improved the efficiency of targeted delivery of liposomes to DCs.76–78 A list of important DC surface receptors which can be used as a target to strongly enhance cellular and humoral immune responses can be found in the literature.79,80
A liposomal SLA formulation consisting of DPPC, cholesterol, and mannopentaose- dipalmitoylphosphatidylethanolamine (Man5-DPPE) was prepared to study its protective response in BALB/c mice against leishmaniasis. 60 The Man5-DPPE is a synthesized neoglycolipid which can interact with mannose receptors on the surface of APCs. Man5-DPPE-coated liposomes induced protection against leishmanial infection compared with noncoated liposomes. Both footpad swelling and parasite load at local lymph nodes decreased substantially in the mice immunized with Man5-DPPE-coated liposomes. The authors believed that actively targeted liposomes are a more suitable adjuvant than charged liposomes. As their adjuvanticity is due to an efficient carbohydrate–protein interaction through the mannose receptor, it is much stronger and more specific than interaction by charge. 60
The coupling of target-specific antibodies to the liposomal surface to create immunoliposomes has appeared as a promising way to achieve active targeting of liposomes. In a study, immunoliposomes were prepared by grafting nonimmune mouse IgG onto the liposomal surface. 61 The results indicated that although liposomes are effective adjuvants to induce protection against L. major challenge in mice, stronger CMI was induced when immunoliposomes were exploited. They concluded that DCs that endocytosed immunoliposomes via their FcγRs probably presented antigen on MHC I and MHC II molecules more efficiently, leading to effective stimulation of CD8+ as well as CD4+ T cells. 61 Altogether, it should be noted that active targeting to DCs using liposomes is an attractive approach to generate strong protective cellular immunity against leishmaniasis, but there are few studies so far that have evaluated this effect. Today, it is widely accepted that targeting antigens to DCs through some specific receptors elicits significant antigen-specific immune responses compared with nonreceptor-mediated antigen uptake by micropinocytosis or other types of endocytosis. Moreover, recent data suggest that combinatorial targeting of multiple DC subsets may significantly enhance the efficacy of DC targeting. 81 The development of such combinatorial approaches is possible in liposomal formulation and would allow the researchers to achieve a strong Th1 immune response against leishmaniasis.
Future perspective
Intensive development efforts have resulted in a number of different liposomal vaccines on the market against several different pathogens such as influenza and hepatitis A, but not for leishmaniasis. However, it should be noted that liposomal vaccines against leishmaniasis bring various perspectives beyond conventional vaccines because of the rapidly developing interdisciplinary field of liposome technology.
Since an effective prophylactic vaccine for leishmaniasis should, rationally, have a potent therapeutic effect, recent studies have showed that liposomes can be used in therapeutic vaccines as well. 82 Actually, many vaccine formulations designed for prophylaxis should also have been used for immunotherapy against leishmaniasis. In addition, immunotherapy alone or in combination with available chemotherapies is an option at least for patients with the nonhealing form of CL lesions. 83 However, there are still many barriers to overcome before extensive clinical use of liposomal vaccines in combination with antileishmanial drugs in humans can become available.
It seems that the method of association of antigen with liposome significantly influences the type and magnitude of generated immune response. Covalent surface phospholipid conjugation, noncovalent surface attachment, encapsulation, and surface adsorption are the most common modes of antigen association with liposomes. 28 Some studies showed that the highest CMI is generated when the antigen is conjugated to the liposome surface instead of encapsulated. However, in terms of humoral response, the difference is much more significant for antigens conjugated to the liposome surface. In contrast, surface conjugation and encapsulation for antigens were reported to induce similar CMI. 28 Since there is no study in the leishmaniasis field to compare these two types of association, this should be considered in the formulation step of new liposomal vaccines.
Fusogenicity of liposome formulation has an important role in cytosolic delivery of antigen that has not yet been considered during liposomal development of anti-Leishmania vaccines. Some lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), are usually used in pH-sensitive liposomes to deliver genetic materials into the cytosol. There are some studies that show they can also be used for antigens in liposome formulations. The majority of liposomal antigens, either pH sensitive or pH insensitive, are presented on MHC II. However, some fractions of liposomal antigens undoubtedly reach the cytosol when pH-sensitive liposomes are used and augment MHC I presentation, which is a favorable response for anti-Leishmania vaccines. 28 Positively charged liposomes are also used to induce cytosolic delivery of liposomal antigens through the disruption of endosomal membranes and result in a CD8+ T-cell response. Virosomes, as another liposome-based vaccine-delivery system consisting of functional viral envelope glycoproteins, may fuse with target cells and significantly contribute to the immunological response.
Recently, some lipids such as saturated and polyunsaturated fatty acids showed immunostimulatory properties. They can be used in liposomal vaccines because of their ability to formulate a similar structure to liposomes. 28 There is a study which shows phosphatidylserine and lysophosphatidylcholine induce DC maturation in vitro and produce CMI responses to co-administered antigens in vivo. 84 The adjuvanticity of these lysophospholipids has not been studied in the leishmaniasis field yet.
From the pharmaceutical point of view, the main challenges to developing liposomal vaccine formulations are optimization, standardization, scalability, and stability of final products. The translation of findings from basic research to clinical trials is a highly selective process for a pharmaceutical product, and technical challenges are common barriers to this translation. Since the physicochemical properties of liposomes (composition, structure, size, surface modification, and colloidal stability) all contribute to the clinical outcome, any small batch-to-batch variation in liposome characterization can cause significant changes in product performance. Therefore, the scalability process of liposome preparation must be reliable and consistent. Another technical challenge is to manufacture consistent liposomes at the industrial scale, which usually eliminates many lab-scale preparation methods. Most studies presented in this review showed promising results, but none have been reached clinical trial phases yet. The reason might be the formulation challenges, such as scalability issues and stability. It is critical to note that a close collaboration between parasitologists, immunologists, vaccinologists, and pharmaceutical scientists is needed to develop a stable, cost-effective, safe, and effective liposomal vaccine in the leishmaniasis field.
In summary, the versatility of liposomal adjuvants allows for the delivery of multiple active agents (such as antigens and targeting ligands) with the ability to target various types of APCs, as well as their receptors. Moreover, Pattern recognition receptor (PRR) agonists, such as CpG ODN can be easily included in liposomal formulations to modulate both the magnitude and the Th bias of the immune response. This leads to improved efficacy and developments of liposomal adjuvants. These unique properties make liposomes challenging to study, but at the same time, interesting to the scientific community aiming to improve anti-Leishmania vaccine efficacy in patients. It is worthy to mention that because of the intrinsic properties of the antigens themselves or the interdependence of the many factors involved in liposomal formulation, the formulation parameters must be always optimized carefully for each new liposomal vaccine candidate.
Footnotes
Acknowledgements
We would like to thank AP for English editing the manuscript.
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of interest statement
The authors declare that there is no conflict of interest.