Liposomes as Nanovaccine Delivery Systems

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Current Topics in Medicinal Chemistry, 2014, 14, 1194-1208

Liposomes as Nanovaccine Delivery Systems Khairunnisa Abdul Ghaffar1, Ashwini Kumar Giddam1, Mehfuz Zaman1, Mariusz Skwarczynski1 and Istvan Toth1,2,* 1 2

The University of Queensland, School of Chemistry and Molecular Biosciences, St Lucia, Queensland 4072, Australia; The University of Queensland, Pharmacy Australia Centre of Excellence, Woolloongabba, Queensland 4102, Australia Abstract: Since the discovery of liposomes by Alec Bangham in mid-1960s, these phospholipid vesicles have been widely used as pharmaceutical carriers. Liposomes have been extensively studied in the vaccine delivery field as a carrier and an immune stimulating agent. Liposomes are usually formulated as nanoparticles, mimicking the properties of pathogens, and have the ability to induce humoral and cell-mediated immune responses. In this review, we focused on modern nanotechnology-based approaches for the improvement of liposomal vaccine delivery systems. Topics such as sizedependent uptake, processing and activation of antigen presenting cells, targeting liposomes and route of administration are discussed.

Keywords: Administration route, bilosome, liposome, niosome, size-dependent immunity, vaccine delivery, virosome. INTRODUCTION Since their discovery half a century ago, liposomes have been one of the most studied nanocarriers in the pharmaceutical industry [1]. The first liposome based therapeutic was approved by the Food and Drug Administration (FDA) in the 1990s [2]. Liposomes are widely used for drug delivery because they are biocompatible and biodegradable, concurrently increasing potency and reducing toxicity [3-5]. Drugs can be encapsulated into both the liposome core or membrane layer [6] as these microscopic vesicles have an aqueous core enclosed by one or more outer shell(s) made of phospholipids/sphingolipids bilayer membranes (Fig. 1 and Table 1) [7]. Liposomes are also good drug delivery carriers because they are able to entrap relatively large amounts of drug payload. These vesicles can be made up with different lipid compositions, enabling the optimization of pharmacokinetic properties through formulation [8]. Other advantages of liposomes as a drug/vaccine delivery system includes their ability to protect the active ingredient from degradation, improve the therapeutic index (TI) of drugs and also allow drug targeting [9, 10]. The clinical applications for liposomes are well known and many liposomal drugs have already reached the market, with many more in clinical trials [9]. However, there are many setbacks in liposome therapeutics. One of the major drawbacks is the rapid clearance from the blood by the mononuclear phagocytic system (MPS) [4, 11]. This observation prompted interest in development of liposome-based formulations that can deliver drugs/vaccines specifically into monocytes and macrophages [12]. It became the most studied delivery system for phagocyte-targeted therapeutics in diseases where inflammation is *Address correspondence to this author at the School of Chemistry & Molecular Biosciences, University of Queensland, Chemistry Blg #68 St. Lucia, Queensland 4072, Australia; Tel: (617) 33469892; Fax: (617) 3365 4273; E-mail: [email protected]

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a key driver of both pathogenesis and progression. This includes asthma, chronic obstructive pulmonary disease (COPD), atherosclerosis, cancer, [13-15] and pathogenic infections such as tuberculosis [16], human immunodeficiency virus (HIV), leishmaniasis etc. [17].

Fig. (1). Structure of typical liposomes.

Vaccination is considered the best strategy for eradicating infectious disease [18, 19]. An effective vaccine should stimulate the immune response and provide long lasting protection against the invading pathogen [20]. Traditional vaccines used live attenuated pathogens, killed/inactivated pathogens, or fragments of pathogens [21, 22]. One major drawback of using live/attenuated vaccines is that there is a possibility for the pathogen to revert or even mutate to become virulent, especially in immunocompromised patients [23, 24]. Other obstacles to the use of traditional vaccines include the possibility of allergic and autoimmune reactions, manufacturing difficulties, and instability [22]. Therefore, modern vaccines are usually designed based on the concept of a subunit vaccine. Subunit vaccines are usually based on recombinant, purified proteins or peptides. The use of subunit vaccines eliminates the risk of reversion from live attenuated microorganisms to virulent form and minimises allergic or autoimmune responses. They are usually easy to produce and relatively stable. The downside of using subunit vaccines is their poor immunogenicity, which decreases their © 2014 Bentham Science Publishers

Liposomes as Nanovaccine Delivery Systems

Table 1.

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Examples of phospholipids and hydrophobic chains used in liposomes.

R 2O

O O P O R1 R 3O O

Name of Phospholipid

Head, (R1)

Tail (R2 , R3 )

Dioleylphosphatidylcholine (DOPC)

Choline CH2CH2N +(CH3) 3

Oleyl CH3(CH 2)7CH=CH(CH2) 7C(O)

Dimyristoylphosphatidylcholine (DMPC)

Choline CH2CH2N +(CH3) 3

Myristoyl CH3(CH 2)12 C(O)

Dipalmitoylphosphatidylcholine (DPPC)

Choline CH2CH2N +(CH3) 3

Palmitoyl CH3(CH 2)14 C(O)

Distearoylphosphatidylcholine (DSPC)

Choline CH2CH2N +(CH3) 3

Stearoyl CH3(CH 2)16 C(O)

Dioleoylphosphatidylethanolamine (DOPE)

Ethanolamine CH2CH2NH3 +

Oleyl CH3(CH 2)7CH=CH(CH2) 7C(O)

Distearoylphosphatidylethanolamine (DSPE)

Ethanolamine CH2CH2NH3 +

Stearoyl CH3(CH 2)16 C-(O)

Dimyristoylphosphatidylglycerol (DMPG)

Glycerol CH2CHOHCH 2OH

Myristoyl CH3(CH 2)12 C(O)

Dipalmitoylphosphatidylglycerol (DPPG)

Glycerol CH2CHOHCH 2OH

Palmitoyl CH3(CH 2)14 C(O)

Dipalmitoylphosphatidylserine (DPPS)

Serine CH2CHNH 3+COO-

Palmitoyl CH3(CH 2)14 C(O)

Distearoylphosphatidylserine (DSPS)

Serine CH2CHNH 3+COO-

Stearoyl CH3(CH 2)16 C(O)

effectiveness in triggering long term immunological memory [25, 26]. To improve the immunogenicity of these vaccines various particulate delivery systems such as liposomes, microspheres, lipid-nanoparticles, dendrimers, polymericnanoparticles have been utilized. Liposomes have been a particularly promising method to overcome poor immunogenicity of subunit vaccines by acting as an immunestimulatory agent (adjuvant) and also allow targeted delivery of antigen by surface modification while maintaining general safety profile of liposomes [20, 27]. Targeting of specific sites/immune cells is easily achieved by embedding/coupling of targeting moieties onto the surface of liposomes thus improving internalization of the liposomes [28]. Natural ability of liposomes to protect encapsulated antigen from enzymatic degradation is also an important factor, especially in the case of peptide-based vaccines. Other advantages of liposomal vaccines over traditional vaccines include its ability to incorporate hydrophobic, amphipathic and hydrophillic agents and being able to act as sustained release depots [29]. However, a major concern regarding the use of liposomal vaccine is the stability of liposomes where there is a risk of leakage of encapsulated agents [30]. The mechanism of liposomes adjuvanting property is not well understood but it was sug-

gested that passive targeting and the ability to interact with the MPS are important factors [31]. This review highlights the various types of liposomes including surface-modified liposomes, virosomes, and bilosomes that are currently under study for the use in vaccine delivery. In addition, two important factors in vaccine delivery have been discussed, namely route of administration and size of liposomes which need to be considered when formulating vaccines. KEY IMMUNE FACTORS IN VACCINE DELIVERY When a foreign pathogen invades the body, it activates the innate immune system, which is the first line of internal body defence. It recognizes the pathogen or its fragment, mediates the initial protection against infections and activates adaptive immune responses. The adaptive immune system is responsible for the establishment of immunological memory [32]. Similar activation pathway is needed for an effective immune response triggered by vaccine where immunological memory is vital to achieve long term protection (Fig. 2).

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Fig. (2). Mechanism of dendritic cell uptake of liposomes.

Cells of the innate immunity recognize certain repeating motifs, pathogen-associated molecular patterns (PAMPs), on invading pathogens through pattern recognition receptors (PRRs) [32, 33]. Examples of PAMPs include double stranded RNA of viruses, and the cell walls of bacteria that are composed of a matrix of proteins, carbohydrates and lipids in a repetitive pattern [32]. PRRs are usually found on cells which are the first to encounter invading pathogens, i.e., effector cells of the innate immune system such as antigen presenting cells (APCs) located on the surface epithelia [32]. APCs such as are macrophages and dendritic cells (DCs) play a major role in innate immunity. DCs are key players in stimulating the appropriate immune responses to many infectious diseases and cancer. They play a key role in bridging the innate and adaptive immune systems [34]. Immature DCs which originate from progenitor cells in the bone marrow, leaves the bone marrow to circulate the peripheral tissues [35]. DCs migrate to peripheral tissues and become more competent at recognition, capturing and internalising self and non-self-antigens through endocytosis [36]. Once PRRs detect an infectious agent, immature DCs release soluble mediators such as inflammatory cytokines and type 1 interferon (IFN). The activated DCs internalize and process the antigen into short peptides and upregulates major histocompatibility complex (MHC) class I and II together with co-stimulatory molecules. These molecules stimulates the interactions between the DCs and naive CD4+/CD8+ T lymphocytes [37].

A differentiation process occurs when MHC II/antigen complex together with co-stimulatory molecules is presented to a CD4+ T cell. The differentiation stimulus influences the type of ensuing immune response, e.g., T-helper type 1 (Th1) versus Th2 types of CD4+ T cell responses [38]. Th1 and Th2 are distinguished by the cytokines they secrete. The Th2 cells secrete IL 4, 5, 10 and 13. B cells internalize, process and present peptide antigens on its own MHC II. Immunological memory is achieved once an activated T-helper recognizes the MHC II/antigen complex found on B cells. This triggers maturation of B cells and antibody secretion to mainly defend the body against extracellular pathogens. Activated Th1 cells in turn secrete IFN- and tumour necrosis factor-
(TNF-
). This process, together with antigen presentation by dendritic cells on MHC class I molecules, trigger CD8+ T cells mediated cellular immunity, which are responsible for killing of infected cells [39]. Antigens such as proteins (with the exception of large and lipidated proteins), peptides and carbohydrates are generally poor initiators of DC-mediated adaptive immunity. In addition, carbohydrate antigens are not able to induce immunological memory when they are not integrated into protein or peptide. [40]. Therefore, to stimulate strong immune responses against such antigens, the use of immunostimulatory molecules (adjuvants) is necessary. However, most adjuvants used in experimental applications are not approved for use in vaccine formulations for humans due to their toxicity. The only adjuvants that are approved for general human use are aluminium salts. A few other adjuvants have been approved for use only in the specific vaccine formulations: (a) oil in

Liposomes as Nanovaccine Delivery Systems

water emulsions (M59 and AS03) for influenza vaccines in Europe, (b) monophosporyl lipid A adsorbed to alum (AS04) for hepatitis B vaccine, and human papillomavirus, and (c) virosomes used for hepatitis A and influenza vaccines in [41, 42]. Non-toxic liposomes may provide a solution to this problem by acting as a carrier and immunostimulant, triggering innate immunity by activating the APCs, and subsequently adaptive immune responses [43]. Liposomes are also able to induce specific immune responses. For example, it was shown that liposomes with unsaturated lipids promoted the induction of T helper 2 (Th2) type immune responses whereas saturated lipids promoted T helper 1 (Th1) type immune responses [44, 45]. LIPOSOMES Liposomes are generally classified according to their size, number of bilayers and composition of lipids. Small unilamellar vesicles (SUVs) range from 20-40 nm while medium unilamellar vesicles (MUVs) are 40-80 nm and large unilamellar vesicles (LUVs) are greater than 100 nm [46]. Multilamellar vesicles (MLVs) are liposomes with several bilayers, usually in an ‘onion-like’ arrangement with very small space in between each layer. As the research has progressed on the vaccine delivery systems, liposomes have been modified tremendously and tailored according the needs of delivery in order to produce desired immune response. Thus, for vaccine delivery purpose liposomes are classified as conventional, surface modified liposomes, virosomes, bilosomes, etc.

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(TfRscFv) and cationic liposome-plasmid as a targeting moiety. The average size of the immunoliposomes complexed with DNA was 90 nm [53]. Immune responses in mice following vaccination with DNA/immunoliposome complexes and naked DNA showed that the delivery system was more efficient in stimulating immune responses with significantly higher levels of IFN-. As mice could not be infected with HCV, tests for T cell immune responses used chimpanzees as a model. Cationic Liposomes Cationic liposomes are made up of positively charged lipids. An advantage of using cationic liposomes in vaccine delivery is the increased potency of subunit vaccines without the need to increase the concentration of the active ingredient [54-56]. This phenomenon can be explained by the interaction between the positively charged liposome and the negatively charged cell membranes of APCs. As a consequence of this interaction positively charged liposomes are more readily uptaken by APC. Numerous studies show that cationic liposomes induce stronger antigen-specific immune responses than neutral or negatively charged liposomes [5759]. However, the targeting functions of these liposomes are limited as they also target other negatively charged cells and blood components.

Immunoliposomes

The influence of liposome surface charge on the depot effect after intramuscular administration was examined by Henriksen-Lacey and colleagues [60]. The depot effect is a characteristic of a vaccine formulation trapped at the injection site. A depot formulation slowly releases antigen/adjuvant to the circulation, allowing continuous activation of the immune system over a long period of time. In the study, cationic liposomes were composed of dimethyldioctadecyclammonium (DDA) and trehalose dibehenate (TDB). Subunit antigens for tuberculosis (TB), and Chlamydia Ag85B-ESAT-6 and CTH1 protein antigens were encapsulated in liposomes. As a comparison, neutral liposomes made of DSPC:TDB were used to investigate the effect of liposome charge on vaccine efficacy and depot effect when administered intramuscularly. Liposomes had an average size of 470 nm and cationic liposomes had a charge of 50 mV. Mice were immunized intramuscularly thrice with a 2 week interval between each immunization. Cationic liposome-associated antigens were retained at the site of injection longer than neutral ones, allowing prolonged antigen uptake by the APCs. The cationic liposomes also induced Th1 (IFN-) and Th17 (IL-17) type responses, aiding memory T cell responses in cell-mediated immunity [60].

Immunoliposomes are targeted liposomes with antibodies attached to their surface. These antibodies allow delivery of encapsulated vaccine to specific immune cells (Fig. 3b). Immunoglobulin (Ig) G and its fragments are the most commonly used targeting moieties [51]. These targeting molecules were either covalently bound to the surface of liposomes or anchored into the membrane [51]. For example, Zubkova et al. developed a vaccine strategy that employs immunoliposome complexes with DNA that expresses NS3NS5B proteins from Hepatitis C Virus (HCV) [52]. The immune-liposomal nanocomplex delivery system used antitransferrin receptor single chain antibody fragment

Barnier-Quer and colleagues concluded that the adjuvant effect of cationic liposomes was not solely dependent on charge, but also on the nature of the cationic compound and its concentration in the liposomal bilayer [54]. They studied cationic 3-
-[N-(N’-N’-dimethylaminoethane)-carbamoyl] (DC-Chol):DPPC liposomes loaded with a H3N2 influenza subunit vaccine based on purified hemagglutinin (HA). A study was carried out to examine the influence of the DCChol backbone in the cationic liposomal bilayer on HA immunogenicity by comparing the adjuvant effect of cationic liposome formulations (DDA, eDPPC, or DC-Chol). Liposomes had diameter of 100-170 nm with a positive

Conventional Liposomes Conventional liposomes are composed of mixture of lipids and phospholipids (Table 1) such as 1,2-distearoryl-snglycero-3-phosphatidyl choline (DSPC), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside and cholesterol [11]. Cholesterol is usually added to liposomes as a membrane sealer and stabiliser to improve liposomal rigidity [47]. Rigid liposomes released the bioactive compound from the liposome core into the surrounding environment more slowly than other liposomes. One of the major drawbacks of these liposomes is their instability in plasma [48, 49]. Conventional liposomes are poor vaccine delivery system [50]. This observation led to the extensive studies toward development of modified liposomes with improved pharmacokinetics and pharmacodynamics.

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charge of 40-50 mV. Despite similar charge and size, DCChol:DPPC liposomes significantly enhanced the overall immune response in the C57-BL/6 mice compare to other formulation, and resulting in enhanced levels of anti-HA IgG1 and IgG2a. They also showed superior protective immune responses when compared to HA formulated with aluminium hydroxide (Al(OH)3). A subsequent study carried out by the same group demonstrated that the presence of cholesterol in cationic liposomes significantly improve immune responses [55]. pH-sensitive Fusogenic Liposomes pH-Sensitive fusogenic liposomes (Fig. 3d) are liposomes that are stable at physiological pH 7.4) but destabilise at lower pH (acidic conditions) and acquire fusogenic properties [61]. These liposomes have the ability to fuse or merge with the membranes of APCs (mainly DCs) to deliver the antigen into the cells’ cytosols. This is a major advantage because antigen is direcly delivered to the cytosol, promoting MHC-I presentation [62]. To date, this fusion ability is mainly induced by viral proteins (e.g. Sendai virus protein) [63, 64]. However, viral proteins might trigger unfavourable and unexpected immune responses [62]. This could be avoided with the use of synthetic polymers for the delivery of antigens to DCs.

Ghaffar et al.

membrane destabilization and vesicle lysis, so that it would not release the contents prior to reaching the site of action. Bilosomes are able to protect an encapsulated agent from enzymatic degradation and may also act as an adjuvant. Thus bilosomes are attractive delivery system for oral immunization. Wilkhu et al. reported optimization of bilosomes formulated for vaccine delivery [68]. Wilkhu’s bilosomes were composed of lipids monopalmitoyl glycerol (MPG), cholesterol, dicetyl phosphate (DCP) and sodium deocycholate (bile salt) with protein antigens HA or H3N2. DCP concentration was the key regulator of zeta potential and pH of the vesicle suspension while the size was dependent on bile salt concentration. Bilosomes used in this study were larger than 4
m. The formulation that contained MPG:Chol:DCP in 5:4:1 ratio showed excellent antigen retention in gastric conditions. When exposed to simulated intestinal conditions, 28% of antigen was release after 1 hour. Mice were immunized with bilosomes that contained radio-labelled antigens. At a 30 minute interval, 8% of the bilosomes were found in stomach and 40% in the small intestine. After 1 h, a total of 39% free antigen was measured across the GIT when delivered without a carrier compared to 55% when antigens were formulated with bilosomes. The level of antigen delivered to Peyer’s patch and mesenteric lymph nodes were significantly higher when bilosomes were used as compared to free antigen. Ferrets were immunized with bilosomes containing HA, to examine the efficacy of the bilosome vaccine. There was a reduction in median temperature differential change when ferrets were immunized with bilosomes containing antigen compared to a dose of empty bilosomes which suggests that the antigen was efficient in providing protection from fever. A reduction in virus-infected cell count was seen when ferrets were immunized with bilosomes that contained the antigen compared to empty bilosomes. It was concluded that the bilosome formulation that contained the influenza vaccine promoted protection against fever and suppressed lung inflammation.

Kono et al. have developed new synthetic polymermodified liposomes with fusion ability under weak acidic conditions [65, 66]. It was expected that these liposomes would release their content into the cytoplasm by fusing with the endosomal membrane after internalization into cells through an endocytic pathway. Synthetic liposomes were produced by modifying egg yolk phosphatidylcholine (EYPC) with poly(glycidol) derivatives that contained carboxyl groups such as succinylated poly(glycidol) (SucPG) and 3-methyl-glutarylated poly(glycidol) (MGluPG). Liposomes modified with MGluPG exhibited higher fusion ability than to SucPG. pH-Sensitive fusogenic liposomes successfully delivered the antigen (ovalbumin, OVA) into the cytosol of DCs [67]. In a separate study to evaluate the efficiency of the fusogenic liposomes, Kono and colleagues compared the efficacy of SucPG modified liposomes (composed of SucPG:DPPC:DOPE:Monophosporyl Lipid A) and unmodified liposomes (composed of DPPC:DOPE: Monophosporyl Lipid A) encapsulating OVA antigen in a mouse model. The synthetic-polymer modified liposomes with fusion ability showed significantly higher cellular (Th2) and humoral (Th1) immune responses than to unmodified liposomes. Furthermore, the authors found that the SucPGmodified liposomes did not induce IgE production which may cause side effects such as allergy. This study concluded that the pH-sensitive fusogenic polymer, SucPG, is a potential vaccine delivery vehicle that can induce both cellular and humoral immune responses.

Recently, Premanand et al. found that oral administration of bilosomes with Baculovirus displaying VP1 protein (BacVP1) elicited high immune responses when compared to live Bac-VP1 alone or inactive Bac-VP1 in bilosomes. High levels of VP1-specific IgG and mucosal IgA antibodies were produced by the Bac-VP1 bilosome formulation. Also, BacVP1 bilosomes induced higher neutralising antibodies against both homologous and heterologous EV71 Baculovirus strains compared to the Bac-VP1 antigen alone. A passive protection assay using suckling mouse model of EV71 infection was also performed. Sera from mice that were orally immunized by bilosomes that contained live Bac-VP1 completely protected pups against EV71 infection. Thus bilosomes with Bac-VP1 efficiently induced humoral and mucosal immune responses against EV71 [69].

Bilosomes

Virosomes

Bilosomes are made of non-ionic surfactants and bile salts (Fig. 3c). In humans, bile salts are salts of steroid acids found in the small intestines which aids in breaking down dietary fat. In bilosomes, the bile salts act to stabilize the lipid vesicles in the gastrointestinal tract (GIT) by preventing

Developed by Crucell [70, 71], virosomes mimic empty viral lipid envelopes of viruses, such as influenza, which do not contain any DNA material from the viral source [72] (Fig. 3d). They are approximately 150 nm in size, and contain functional enveloped viral glycoproteins such as

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Fig. (3). Schematic of various types of liposomes. (a) Immunoliposome with antibody fragments attached to bilayer. (b) pH-sensitive fusogenic liposome with pH sensitive synthetic polymer attached. (c) Bilosomes made using lipids and the inclusion of bile salts. (d) Virosomes, empty viral lipid envelopes containing haemagglutinin and neuraminidase.

neuraminidase and haemagglutinin intercalated in the bilayer membrane, thus they are considered as surface-modified liposomes [73]. Neuraminidase assists virosomes to access epithelial cells by cleaving N-acetylneuraminic acid from bound sugar, decreasing mucus viscosity [74]. Haemagglutinin is responsible for the binding of these nanoparticles to the cell surface receptor that mediates pH-dependent fusion with endosomes [75]. There are two known vaccines against influenza on the market that uses this technology Epaxal® and Inflexal® V. Inflexal® V is composed of a mixture of 3 monovalent virosome pools that are distinct to individual strain’s specific haemagglutinin and neuraminidase glycoprotein [73]. The strains used in this vaccine were chosen according to World Health Organisation (WHO) and European Medicines Agency (EMEA) guidelines. A clinical study comparing a commercially available non-virosomal adjuvanted vaccine with Inflexal® V confirmed that Inflexal® V has a significant decrease rate of adverse effects which may be attributed due to its high purity and the biocompatible nature [73]. Inflexal® V is the only adjuvanted influenza vaccine that is approved for all age groups. Inflexal® V was found to be safe and well tolerated in children, adults, elderly and also immunocompromised patients. A study where children with human immunodeficiency virus (HIV) were immunised with Inflexal® V showed comparable immunogenicity results to studies with healthy children [76]. The vaccination did not induce any CD4+ cell activation nor had effect on HIV viral replication in patients with a weakened immune system. A study on the superior immunogenicity of Inflexal® V was carried out on the elderly. A significantly enhanced immunogenicity against two out of the three influenza strains for Inflexal® V was observed in elderly who did not possess protective antibody levels at baseline [77]. Another established virosomal vaccine is Epaxal®, which prevents hepatitis A infection (HAV) [78]. This vaccine uses virosomes as an adjuvant instead of alum-based salts. Formalin-inactivated and purified HAV virions are grown on MRC-5 human diploid cell culture and then adsorbed onto

the virosomal surface. A 100% seroprotection was achieved in adults less than 50 years of age following a secondary boost 12 months after primary immunization. However, lower immune responses were reported with elderly adults above 60 years of age [79]. In children and infants, a secondary boost also achieved 100% seroprotection. However, for infants below 12 months, there have been reports of interference with maternal antibodies, which is also seen in other hepatitis A infections [80, 81]. Therefore, a study was conducted to find out whether vaccine efficacy was affected by combination with other vaccines. It was found that the immunogenicity of Epaxal® was not compromised when people were simultaneously immunized with yellow fever vaccine [82]. Epaxal® was also found to elicit high antibody titres within 10 days after immunization, conferring long term protection against hepatitis A infection [83]. This makes it a perfect candidate when rapid vaccination is required. Cusi et al. found that respiratory syncytial virus-F antigen administered to mice induced a strong systemic and mucosal response with high levels of IgG and IgA, respectively, when delivered intranasally in a virosomal system. It was accompanied by a balanced Th1 and Th2 response, while without the virosome system, only a Th 2 response was observed [84]. Bernadis et al. used the virosome delivery system which contained a truncated recombinant aspartyl proteinase-2 of Candida albicans to protect against candidal vaginitis [85]. The vaccine (PEV7) was able to produce potent serum antibody responses in mice and rats following intramuscular administration. Additionally, IgG and IgA antibodies were found in rats following intravaginal administration. Bomsel and colleagues used a gp41-subunit protein antigen, derived from HIV-1, grafted on virosomes against HIV [86]. Monkeys were primarily immunized intramuscularly and given three boosts, either intranasally or intramuscularly. They were challenged intravaginally with a virulent strain of simian-HIV (SHIV). Six months post challenge, four out of the five monkeys remained HIV-1 free and the fifth monkey was only transiently infected.

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A virosome-formulated malarial vaccine candidate was recently evaluated in Phase Ib trials [87]. Cech and colleagues developed a peptide-phosphatidylethanolamine conjugate, UK-39 which resembles the natural conformation a major surface protein (circumsporozoite protein, CSP) of Plasmodium falciparum. A 49 amino acid long cyclised synthetic peptide, AMA49-Cl, was derived from the apical membrane antigen 1 (AMA-1) and was shown to induce asexual blood stage parasite growth inhibitory antibodies. Both, UK-39 and AMA49-Cl antigens were coupled to the surface of virosomes and used as a vaccine candidate, PEV3B. IgG antibody titres reached the highest level 30 days after the second immunization. This vaccine was also found to be protective against malaria in a human phase Ib trial. Niosomes Niosomes are similar to liposomes apart from their nonionic surfactant bilayer [88, 89]. The most commonly used non-ionic surfactants include alkyl ethers, alkyl esters, alkyl amides and sorbitan monoesters (Spans®) [89]. Niosomes range in size from 10 nm to 100 nm [90]. The uses of niosomes in medical applications has been extensively studied after its first appearance in the cosmetic industry. Niosomes offer the same advantages as liposomes, but are more chemically stable and less likely to oxidize [91]. The first study of niosomes for vaccine delivery proved that they were able to immunize Balb/c mice against bovine serum albumin (BSA) [89]. This study concluded that niosomes stimulate Th1 lymphocyte response faster than very potent Freund’s complete adjuvant (CFA). However, after 42 days post-secondary immunization the immune response was identical for both niosomes and CFA [92]. Pardakhty et al. studied autoclaved Leishmania major (ALM) encapsulated in niosomes as a vaccine candidate [93]. Various niosomal formulations were made using cholesterol, cetyl trimethyl ammonium bromide (CTAB) and non-ionic surfactants (sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan monooleate (Span 80), polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60) and polysorbate 80 (Tween 80)). Vesicle sizes ranged from 7-15
m depending on the formulation. ALM was composed of various soluble and insoluble components which allowed their entrapment in the aqueous core as well as the lipophilic bilayer. The entrapment efficiency varied between 15% and 37%. Four groups of mice (control, ALM (alone), niosomes without ALM and niosomes with ALM) were immunized thrice at 2 week intervals subcutaneously. Four-weeks after immunization, mice were challenged with culture media that contained stationary phase Leishmania promastigotes. Mouse group treated with control, ALM or niosomes without ALM developed lesions after 4 weeks. 5 out of 8 mice treated with niosomes that contained ALM developed lesions after 7 weeks. The mean lesion size in mice immunized with ALM-niosomes was significantly reduced in comparison to the other groups. For the mice group that received niosomes containing ALM, even though the formulation did delay the formation of lesions 5 out 8 mice have developed lesions. Thus, ALM-niosomes efficiently delay the disease symptoms but does not prevent infection.

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ROUTES OF ADMINISTRATION The administration route is a critical consideration in the development of liposome-based vaccines. Vaccines can be administered in three ways: systemic, mucosal and topical. Systemic administration is the most common method currently in use, where vaccines are delivered intramuscularly (i.m.) or subcutaneously (s.c.) [94]. Systemic delivery currently relies on the use of injectables, whereby professional staffs are prone to needlestick injuries and poor patient compliance. Vaccines administered by these routes are also not able to protect host from initial mucosal infection. These setbacks could be overcome by the use of mucosal delivery which may protect the host from infections even before the pathogen reaches the systemic distribution. In addition, mucosal vaccines may be more stable and thus more suitable for widespread vaccination programmes in developing countries [95]. Parenteral Administration Administration of vaccine to the systemic circulation offers several advantages including the direct administration of vaccine to the lymphatic circulatory system without the need to bypass physical/biological barriers (e.g. skin, intestinal membrane, harsh gastrointestinal environment) that may affect the liposomal formulation [96]. Absorption of the vaccine from subcutaneous tissue is influenced by the same factors that determine the rate of absorption from the muscle tissue when a vaccine is administered intramuscularly. However, vascularity of both tissues differs. The vascularity is poorer in the subcutaneous tissue, and therefore absorption of the vaccine may be slower in comparison to i.m. immunization [97]. I.m. and s.c. immunizations can exploit the depot effect (slow vaccine release into circulation system over a long period of time). Subcutaneous injections pierce the epidermal and dermal layers of the skin, delivering the vaccine to loose subcutaneous tissue. White et al. found that subcutaneous immunization with liposomes that encapsulated mannosylated lipid core peptide (LCP) with the minimal CD8 T cell epitope, SIINFEKL, together with Quil A adjuvant successfully induced strong cytotoxic and protective immune responses [98]. The LCP system contained a nonmicrobial lipopeptide adjuvant based on lipoaminoacids [94, 99]. Liposomes used in this study had an average size of 1
m and consisted of 5% w/w mannosylated LCP with 2% w/w Quil A. Mice were immunized twice subcutaneously at the back of the neck. Liposomal vaccine was well tolerated following s.c. immunization with no trace of redness or irritation at the injection site. It was concluded that liposomes containing mannosylated LCP and Quil A were as efficient as the positive control (mannosylated LCP plus ovalbumin protein in alum), in acting as a prophylactic cancer vaccine when administered subcutaneously. An example of i.m. immunization was reported by Amidi and colleagues. They used antigenexpressing immunostimulatory liposomes (AnExILs) to evaluate their ability to induce T cell responses following intramuscular immunization. A model CTL epitope was fused to the C-terminus of the reporter enzyme (luciferase or -galactosidase) to assess the antigen production and specific epitope T cell responses. Mice were immunized twice with

Liposomes as Nanovaccine Delivery Systems

three week intervals. Both humoral and T cell mediated immune responses were induced upon i.m. administration with AnExILs. Mucosal Administration Mucosal immunity is the first line of defence before pathogens enter systemic circulation [95]. Mucosal immunocytes, cells of the lymphoid series that are able to react with antigens to produce antibody or participate in cell-mediated immunity (pulmonary, nasal and oral), make up 80% of all immunocytes. These cells form the mucosal-associated lymphoid tissues (MALT), the largest mammalian lymphoid organ system [100]. Lymphocytes activated by the invading pathogen at the mucosal sites travel to regional lymph nodes and thoracic duct before migrating through the bloodstream to other mucosal sites. This migration leads to secretory mucosal antibody (IgA) production at other mucosal effector sites of the MALT. IgA antibodies are able to cross epithelial membranes, thus prevent future entry of pathogens [101104]. Enhanced antibody memory is usually seen at the mucosal priming site (localisation) as compared to other distal mucosal effector sites [105]. Meanwhile, cellular mediated immunity is also activated by the mucosal immune system. Generally, mucosal administration of vaccine also induces systemic antibody response (IgG antibody production). Thus, a “mucosal” vaccine triggers similar responses to vaccines administered systemically but also activates the production of mucosa-specific IgA. 1. Oral Administration Oral delivery is the most convenient route for mass vaccination and patient compliance. Oral delivery can also trigger mucosal and systemic immune responses [106]. Oral vaccines can induce an immune response at the gutassociated lymphoid tissues (GALT) which consists of follicle associated epithelia containing microfold cells (M cells). M cells act as an ‘antigen sampling system’ i.e. they have the ability to take up and present antigens to immune cells such as APCs [107]. However, the main drawback to the administration of vaccines via the oral route is the risk of inducing immunological tolerance [108]. Oral administration of liposomes is more complex than other routes of delivery. Orally delivered liposomes need to be stable under harsh conditions of gastrointestinal tract (GIT) [109]. This would mean that liposomes would have to withstand acidic conditions and bile. Conventional liposomal delivery systems would not have succeeded as liposomes are not stable and would experience enzymatic and chemical degradation before they reach their target. Polymer coating and other modification of liposomes were employed to increase stability, efficiency and retention time in the intestines [110]. Rescia et al. conducted a study that could potentially be of use in oral liposome vaccine delivery [111]. Liposomes were made using soy phosphatidylcholine (SPC) and cholesterol in a ratio of 3:1, coated with chitosan and stabilized with poly-vinylic alcohol (PVA). Chitosan was used because it is a mucoadhesive which may increase uptake and may act as a protective agent. The antigen, diphtheria toxoid (DT), was encapsulated in these liposomes. Liposome size was

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9

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approximately 400 nm with a negative zeta potential of -12.3 mV. Both chitosan and PVA delayed the liberation of encapsulated DT in liposomes when these vesicles were incubated under simulated gastric conditions more effectively than plain liposomes. Liberated DT retained its immunological activity. Vankatesan and Vyas conducted a study using polysaccharide (O-palmitoylpullulan; OPP) coated liposomes that encapsulated BSA [112]. Liposomes were composed of PC, cholesterol and PE with an average size of 4
m after coating with polysaccharide. Rats immunized with the coated liposomes showed similar IgA antibody titres when administered either orally or intraperitoneally. Significantly higher IgA antibody titres were seen in rats immunized with coated liposomes in comparison to rats immunized with alum adsorbed BSA or plain liposomes upon oral immunization. These results suggested that orally administered OPP coated liposomes were stable even in the GIT. Interestingly, Perrie and colleagues conducted a study using uncoated liposomes which incorporated plasmid DNA pRc/CMV-HBs which was derived from hepatitis B surface antigen [113]. These liposomes were approximately 700 nm to 1500 nm in size. Mice were orally immunized thrice with either PC:DOPE:DOTAP, DSPC:DOPE:DOTAP or DSPC:Chol: DOTAP in 4:2:1 molar ratios. Liposomes formulated with DSPC:DOPE:DOTAP induced the most robust immune responses, producing high levels of IgA. 2. Pulmonary Administration Pulmonary macrophages found in the alveoli together with DCs in the epithelial linings of conducting airways, below the airway epithelium, and within alveolar septal walls all contribute to the stimulation of both innate and adaptive immunity. The inhalation route for liposomal vaccine became possible once liposome drying methods were developed. Importantly, dried liposomes demonstrated increased shelf life in comparison of the liposome-based vaccine dispersed in aqueous solution as an added advantage. However, pulmonary administration may be difficult in people suffering from lung diseases where they may not be able to inhale adequately, resulting in variability of the dose upon administration. Aerosol vaccination is primarily dependent on the target pathogen and induction site. Particle size plays a major role in this route of administration. If the particle size is larger than 5
m, particles are mainly distributed in the upper and central airways due to “inertial impaction”, whereas particles below 3
m are deposited in the lower airways by sedimentation or diffusion [95]. Therefore, specific infection sites can be targeted with liposomes of particular sizes e.g. to target upper respiratory tract infections caused by pathogens such as Bordetella pertussis, liposomes greater than 5
m can be used [95]. Alternatively, liposomes below 3
m can be used to target lower respiratory tract infections caused by pathogens such as Streptococcus pneumoniae [114]. To date, most liposomal aerosols are composed of phosphatidylcholines (PC), phosphatidylethanoloamines (PE), phosphatidylglycerols (PG), cationic lipids, and cholesterol [95]. 3. Intranasal Administration Intranasal administration is preferred over other administration routes because it: (i) requires less antigen (compared

1202 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9

to the oral route); (ii) generates optimal secretory immunity against pathogens transmitted via aerosols in the respiratory lining [115, 116]; (iii) induces secretory IgA (sIgA) in saliva [117]; (iv) is an easy vaccination approach for large populations; (v) better systemic responses [118]. For example, Tiwari and colleagues sought to deliver viral protein HBsAg (Hepatitis B surface antigen) in haemagglutinin-(HA)complex liposomal constructs via intranasal delivery [119]. Liposomes in this study were composed of DSPC, cholesterol and N-glutarylphosphatidyl ethanolamines (NGPE) in a 6:3:1 molar ratio, which were then complexed with HA. Liposomes complexed with HA (712 ± 12 nm) were marginally bigger than plain liposomes (643 ± 10 nm), and the encapsulation efficacy of HBsAg was unaffected. The plain liposomes had a Zeta potential of value of
31 mV, where the anionic value is caused by anionic NGPE, and liposomes complexed with HA had a higher zeta potential (
42 mV). In vivo immunogenicity of the vaccine following intranasal administration was evaluated by measuring systemic (IgG and cytokine) and sIgA immune responses against the positive control, alum adsorbed formulation of antigen which was administered intramuscularly. Anti-HBsAg antibody titers were measured at various time points up to 8 weeks. HA-complexed liposomes showed the highest antibody titers following primary immunization. Cell mediated immunity is the prime requirement in eliminating hepatitis B virus. HA complexed liposomes produced the highest level of IL-2 and IFN- than any other formulations including the positive control. Furthermore, HA-complexed liposomes also produced high levels of sIgA in nasal, salivary and vaginal secretions which indicates successful mucosal immunity throughout the mucosal compartment. These results indicate that HA complexed liposomes are able to induce both mucosal and systemic immune responses following intranasal immunization. [119]. Baca-Estrada et al. developed a vaccine with liposomeformulated Yersinia pestis (Y. pestis) bacteria [120]. Positively charged multilamellar liposomal formulations were composed of PC:Chol:DDA in a molar ratio of 6.5:3.3:1. Authors used liposome-encapsulated formaldehyde-killed whole cells (KWC) of Y. pestis in comparison to bacteria alone. Mice were immunized twice either intranasally or subcutaneously. Bacterium-specific IgA antibody titres were found in mice that were immunized intranasally. Low antibody titres were produced in mice immunized with KWC in contrast to high antibody tires in mice immunized with liposome-KWC after intranasal administration. Also, lungs of mice immunized with liposome-KWC had a higher concentration of antigen-specific antibody-secreting cells than those treated with KWC alone. Mice immunized intranasally with liposome-KWC also showed elevated levels of antigenspecific IFN-, an indication that systemic immune response was also induced. Mice were challenged with Y. pestis intranasally following a subcutaneous primary immunization and intranasal secondary immunization. The group of mice that was vaccinated with liposome-KWC intranasally showed excellent protection compared to the group of mice that were vaccinated with the liposome-free formulation. Although vaccination via the intranasal route has numerous advantages, it is not without limitations. Vaccines that are administered into the nasal cavity are rapidly cleared

Ghaffar et al.

with a half life shorter than 30 minutes [121]. Due to the short residency time in the nasal cavity, the absorption of vaccine into the systemic circulation is low. Another cause of concern is the reproducibility of dosing in the nasal cavity which is dependent on a few factors such as the deposition and distribution pattern of the vaccine and the condition of nasal cavity and even the surrounding environment. As the nasal mucosa is highly sensitive, vaccines that may cause irritation or inflammation should not be administered via this route. Topical Administration The largest organ in the human body, the skin, offers an ideal route for vaccine administration as it consists of immunocompetent cells such as keratinocytes and Langerhans, which help stimulate both the innate and adaptive immune responses [122]. The major advantages of using liposomes in topical applications include: (i) reducing side effects from high systemic antigen/adjuvant absorption; and (ii) increased antigen accumulation at the site of administration due to the high substantivity of liposomes with the skin [5]. It was also suggested that topical application of vaccines to skin may be more efficacious as antigens expressed in the epidermis layer are more immunogenic due to the sentinel immune mechanisms on the borders of skin [122]. However, a disadvantage with topical administration is requirement of disrupting the first line barrier of defence, i.e. stratum corneum, which may need to be temporary/partially damaged to increase antigen presentation [123, 124]. It has been reported that size plays a part in penetration through the follicular epithelium. Smaller liposomes (i.e. 40 nm) are able to achieve a deeper penetration through to Langerhan cells compared to 200 nm particles [125]. A study by Li and colleagues used flexible liposomes (made of phosphatidylcholine, cholesterol and sodium deoxycholate surfactant) [126]. These flexible bilayers could mix with the lipids of the stratum corneum, disrupting the barrier function thus leading to enhanced drug disposition in the skin [127]. OVA was encapsulated in these flexible liposomes which ranged in size from 200-400 nm. To prove that flexible liposomes were able to penetrate the skin, a test was performed on Balb/c mice whereby one group of mice was stripped of the stratum corneum and the other had an intact layer. Results showed OVA-encapsulated flexible liposomes were able to penetrate the stratum corneum and induce anti-OVA IgG production. Li et al. also found that the OVA-encapsulated flexible liposomes exhibited stronger immune responses than OVA solution upon transcutaneous immunization. Additionally, Li also found that an enhanced vaccination effect could be observed when OVAencapsulated flexible liposomes were applied with imiquimod [126]. Two -galactosidase-encoding plasmids: pCMV-  and pCMV-sin-LacZ (pSIN-b) were used as a model system for vaccine delivery [128]. These plasmids were encapsulated into cationic liposomes made of DOTAP:DOPE in a 1:1 ratio. The liposomes had a positive charge of approximately 60 mV and size of 120 nm. The vaccine candidate was applied to middle-dorsum of the anaesthetized mouse where hair follicles were trimmed and plucked with a wax strip. Anti-

Liposomes as Nanovaccine Delivery Systems

body titres were compared to the positive control, in which mice were injected i.m. with naked DNA. It was found that anti--galactosidase IgG levels were significantly higher in groups that were immunized with pCMV-sin-LacZ (pSIN-b) compared to the group immunized with pCMV- , but lower than in the positive control. Interestingly, it was found that different methods of inducing hair growth had a significant effect on the uptake/retention of the plasmid. The highest amount of plasmid recovered from the skin was in the group of mice that had hair plucked using wax strips. Is Size Important? Although the majority of liposomes tested in vaccine delivery are nano-sized, few studies have examined the relationship between liposomal size and immune response. In general, liposomes with a particle size of more than 500 nm are easily engulfed by cells of the MPS via phagocytosis, whereas smaller particles between 20-200 nm are endocytosed by dendritic cells [129]. Therefore, it was concluded that larger liposomes will be more readily cleared by the MPS. For topical applications on the skin, small particles no bigger than 100 nm are carried to the lymph nodes via interstitial flow to interact with the resident DCs [130-133]. Larger liposomes (>500 nm) are usually trapped at the site of application and are internalized by skin DCs or monocytes before migrating to the efferent lymph nodes [134], whereas intermediate sized particles (100-500 nm) use both active and passive transportation to get to the lymph nodes. Oussoren and colleagues studied the effect of liposomal size on uptake and biodistribution following subcutaneous injection in rats [135]. Liposomes were made up of egg phosphatidylcholine (EPC):egg phosphatidylglycerol:cholesterol in a 10:1:4 ratio. The sizes of vesicles used in this study were 40 nm, 70 nm, 170 nm and 400 nm or
1000 nm. They found that the degree of lymphatic uptake was negatively correlated with an increase in liposome size. The smallest liposomes, 40 nm were rapidly taken up by the lymphatic system (74% of injected dose within 52 hours) while the largest liposomes (
1000 nm) where almost not detected in the lymphatics [135]. Epstein-Barash et al. recently performed a thorough comparative study on biphosphanates-based liposomes [136]. Liposomes ranging in size from 80 nm to 650 nm were analysed based on their internalization by monocytes. These liposomes were composed of neutral, positive or negative charges. The results supported the current understanding that larger liposomes exhibited increased internalization by monocytes and macrophages [12, 134]. The liposomes were tested in vitro using RAW264 macrophages, J774 murine monocytes, and THP-1 monocytes. Consequently, intravenous injections of the different sized liposomes into rats and rabbits showed reduction of monocytic activity for smaller liposomes. Liposomes smaller than 80 nm were found to be inactive both in vitro and in vivo whereas liposomes larger than 190 nm stimulated cytokine activation. Mann et al. demonstrated that 250 nm liposomes enhanced Th2 responses while those around 1 m increased Th1 responses, stimulating high levels of IFN- and IgG2 antibodies [137].

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Carstens and colleagues evaluated the influence of size on tissue localization of liposomal DNA [138]. Liposomes were composed of EPC, DOPE and DOTAP with encapsulated OVA-encoding plasmid DNA (pDNA). The liposomes were approximately either 500 nm or 140 nm in diameter. Mice were injected subcutaneously with the pDNAliposomes. The authors found that the larger pDNAliposomes had significantly stronger retention at the site of injection than the smaller pDNA-liposomes. OVA-specific IgG antibody levels were not detected in mice immunized with larger pDNA-liposomes even after 3 injections compared to mice immunized with smaller pDNA-liposomes which showed increased IgG antibody levels after the second injection. Three weeks following the last immunization, splenocytes were obtained to quantify the percentage of OVA-specific T cells. Large pDNA-liposomes induced negligible amounts of CD8+ T cells, however the small pDNAliposomes showed significantly higher induction of OVAspecific CD8+ T cells. IFN- production was also significantly higher in mice that received pDNA-liposomes. Thus, this study indicated that smaller liposomes were more efficient in delivery of DNA-based vaccine. Overall, the studies indicate that there is no single optimal size for a liposomes based vaccine. [139-141]. Other factors such as whether the liposomes require targeting moieties to actively promote uptake, inadvertently affecting the size of liposomes needs to be considered in addition to liposomal charge and lipid composition [142144]. Apart from size, various other factors such as liposome surface charge, bilayer composition, lamellarity, presence of targeting moiety, route of administration will also have significant impact on evoking immune response. For example, i) cationic liposomes were reported to induce higher immune response than neutral and anionic liposomes, ii) liposomes formulated with low phase transition temperature lipids are better suited for mucosal delivery and liposomes with higher phase transition temperature lipids (more rigid) are promising when administered via subcutaneous, intramuscular or intraperitoneal routes, iii) targeting moiety/ligand helps to deliver antigen intracellularly and evoke desired immune response iv) subcutaneous or intramuscular administration may provide depot effect and release the antigen for prolonged periods for better antigen sampling by APCs; mucosal administration induces enhanced mucosal immunity. Furthermore, the type of immune response (humoral or cellular) produced can be altered by manipulating these factors. More detailed information on how these factors influence the immune response can be found in the recent review by Giddam et al. [20]. In addition, results obtained from in vitro assays often differ from in vivo findings especially for parenterally administered vaccines, whereby liposomes may be cleared by the hepatocytes after interaction with other circulatory components [145]. CONCLUSION Over the past 40 years, thousands of articles have reported the use of liposomes in drug delivery and many liposome-based drug formulations are available on the market. Research into liposomal technology and its uses are ongoing and are unlikely to stop due to their diverse applica-

1204 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9

tions. Although the optimal size for liposomes cannot be concluded at this time, there are many factors that contribute to making liposomes a favourable vaccine delivery system. The optimal size remains under debate because different liposome sizes may be used to target different areas of the body and/or diseases. It is also possible that each individual vaccine candidate may have found discrete sizes optimal for its delivery. This delivery system is not trouble free and instability of conventional liposomes initially prevented their application for vaccine delivery. This led to research into the modification of conventional liposomes from the surfacemodified liposomes. From then on, new liposomal technologies expanded to further improve delivery of vaccines. Surface-modified liposomal vaccines are major delivery systems used for targeting various diseases such as malaria, HPV, and influenza. Virosomes were later invented, resulting in the first commercial liposomal vaccine against influenza and then expanded to include successful vaccine formulations against hepatitis A virus. Their success is most likely related to their nature which closely resembles natural pathogens (viruses). In addition to two currently approved liposomal vaccine formulations, there are numerous clinical trials in United Stated and Europe (more than ten clinical trials can be found on: http://clinicaltrials.gov; https://www.clinical trialsregister.eu). While most the liposomal vaccine are in the phase I of clinical trials, a vaccine against non-small cell lung cancer reached phase III trials and it has the highest chance of getting regulatory approval among tested liposomal formulations. In conclusion, a vast amount of liposomal vaccine research has produced encouraging results, thus it can be expected that more liposome-based vaccine will reach the market in the near future.

Ghaffar et al.

CFA

=

Freund’s Complete Adjuvant

CTAB

=

Cetyl Trimethyl Ammonium Bromide

DC-Chol

=

3-
-[N-(N’-N’-dimethylaminoethane)carbamoyl]

DCs

=

Dendritic Cells

DCP

=

Dicetyl Phosphate

DDA

=

Dimethyldioctadecyclammonium

DOPE

=

1,2-dioleoylphosphatidylethanolamine

DPPC

=

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

DSPC

=

1,2-distearoryl-sn-glycero-3-phosphatidyl choline

DT

=

Diphtheria Toxoid

EMEA

=

European Medicines Agency

EYPC

=

Egg Yolk Phosphatidylcholine

FDA

=

Food and drug administration

GIT

=

Gastrointestinal Tract

GALT

=

Gut-Associated Lymphoid Tissues

HA

=

Hemagglutinin

HAV

=

Hepatitis A Infection

HCV

=

Hepatitis C Virus

HIV

=

Human Immunodeficiency Virus

Ig

=

Immunoglobulin

i.m.

=

Intramuscular

CONFLICT OF INTEREST

IFN-

=

Interferon Gamma

The author(s) confirm that this article content has no conflicts of interest.

KWC

=

Formaldehyde-Killed Whole Cells

LCP

=

Lipid Core Peptide

ACKNOWLEDGEMENTS

LUVs

=

Large Unilamellar Vesicles

This work was supported by the National Health and Medical Research Council of Australia. We thank Thalia Guerin for her critical review of the manuscript.

M cells

=

Microfold Cells

MALT

=

Mucosal-Associated Lymphoid Tissues

MHC

=

Major Histocompatibility Complex

SUPPLEMENTARY MATERIALS

MGluPG

=

3-methyl-glutarylated poly(glycidol)

Supplementary material is available on the publishers web site along with the published article.

MLVS

=

Multilamellar Vesicles

MPG

=

Monopalmitoyl Glycerol

ABBREVIATIONS

MPS

=

Mononuclear Phagocytic System

ALM

=

Autoclaved Leishmania Major

MUVs

=

Medium Unilamellar Vesicles

AMA-1

=

Apical Membrane Antigen 1

OPP

=

O-palmitoylpullulan

AnExILs

=

Antigen-Expressing Immunostimulatory Liposomes

OVA

=

Ovalbumin

PAMPS

=

Pathogen-Associated Molecular Patterns

APCs

=

Antigen Presenting Cells

PRRs

=

Pattern Recognition Receptors

Bac-VP1

=

Baculovirus Displaying VP1 Protein

PVA

=

Poly-Vinylic Alcohol

BSA

=

Bovine Serum Albumin

SUVs

=

Small Unilamellar Vesicles

COPD

=

Chronic Obstructive Pulmonary Disease

TB

=

Tuberculosis

CSP

=

Circumsporozoite Protein

TDB

=

Trehalose Dibehenate

Liposomes as Nanovaccine Delivery Systems

TfRscFv

=

Anti-Transferrin Receptor Single Chain Antibody Fragment

Th

=

Thelper

TI

=

Therapeutic Index

s.c.

=

Subcutaneous

SPC

=

Soy Phosphatidylcholine

SucPG

=

Succinylated Poly(Glycidol)

WHO

=

World Health Organisation

Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 9 [18] [19]

[20]

[21]

[22]

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Received: December 12, 2013

Revised: February 07, 2014

Accepted: February 17, 2014

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