Recombinant protein vaccines

Traditional vaccines have comprised live-attenuated microbes, inactivated microorganisms, purified microbial components, polysaccharide-carrier protein conjugates or recombinant proteins. The first of the latter type of vaccine was derived from the diphtheria and tetanus toxoids, and was developed in the first half of the twentieth century. The two toxins were chemically detoxified to produce the non-toxic toxoids. Conventional approaches to vaccine development have been based on biochemical, immunological and microbiological methods that have been labourious, time-consuming and have allowed identification of the most abundant antigens of any given pathogen. Recent progress in DNA sequencing and subsequently in bioinformatics have resulted in advances in vaccine development. When the whole sequence of a bacterial genome became available, the genomic information was used to discover novel antigens that had been missed by conventional methods of vaccine development. This novel approach, now termed reverse vaccinology, involved the in silico analysis of the microbial genome sequence^{1, 2, 3}. This approach has already resulted in the identification of immunogenic antigens as potential candidates for a vaccine against Neisseria meningitidis⁴. This approach holds great promise for future vaccine development.

Immunopotentiating adjuvants for protein vaccines

Mutants of heat labile enterotoxin from Escherichia coli and cholera toxin from Vibrio cholera

Genetically detoxified mutants of heat labile enterotoxin (LT) have been shown to be potent adjuvants for inducing mucosal and systemic immune responses. To retain the adjuvanticity of these molecules but reduce their toxicity, several mutants have been generated by site directed mutagenesis. Of these, two mutants of the enzymatic A subunit, LTK63 and LTR72, maintain a high degree of adjuvanticity. LTK63 results from the substitution of serine 63 with a lysine in the A subunit, which renders it enzymatically inactive and non-toxic^{5, 6, 7, 8, 9}. LTR72 is derived from a substitution of alanine 72 with an arginine in the A subunit, and has approximately 0.6% of the enzymatic activity of wild-type LT. LTR72 is shown to be 100 000 times less toxic than wild-type LT in Y1 cells in vitro and 25–100 times less toxic than wild-type LT in the rabbit ileal loop assay¹⁰.

In a recent study, we showed that an influenza vaccine given intranasally (i.n.) together with LTK63 in a novel bioadhesive microsphere delivery system, induced enhanced serum IgG as well as nasal IgA responses in mice and pigs¹¹. Thus, combination of LT mutant adjuvants with a microparticle system enhanced local and systemic humoral responses. Collectively, these data show that LT mutants are effective mucosal adjuvants in small and larger animal models, and can be used in combination with microparticle formulations to enhance immune responses.

We recently described the ability of LT mutants to induce CTL responses against HIV-1 p55 gag, following i.n., oral or i.m. immunization¹². Interestingly, we found evidence that LTK63 and LTR72 had diverse effects when used as mucosal adjuvants for oral versus i.n. immunization. We found that LTK63 induced stronger CTL responses following i.n. immunization with p55 compared with LTR72. In contrast, LTR72 induced stronger CTL responses against p55 when given orally, and it also induced local CTL responses. Thus, it appears that some ADP-ribosyl-transferase activity of the LT mutant may be required for oral, but not for i.n. immunization, if induction of CTL responses is the objective. These studies showed that i.n. immunization with protein vaccines and LT mutant adjuvants can be an effective means to induce cell mediated immunity. However, some antigens may require optimization of the delivery systems as well as inclusion of adjuvants.

Because LT mutants are very potent antigens following mucosal immunization, there is a concern that immunity to LT might affect the potency of these molecules when used as adjuvants. Therefore, we recently evaluated the potency of LTK63 in mice and pigs with pre-existing immunity to the adjuvant. We found that pre-existing immunity to LTK63 did not affect its potency as an adjuvant, when used for i.n. immunization with a second vaccine soon after the first. In addition, these studies showed the potency of LT mutants for a protein polysaccharide conjugate vaccine (Neisseria meningitidis group C CRM conjugate), and extended their use into a larger animal model, the pig¹³.

Another important genetically detoxified immunopotentiating adjuvant is the CTA1-DD adjuvant derived from the genetic fusion of the enzymatically active CTA1 gene from whole cholera toxin from Vibrio cholera, to a gene encoding a synthetic analogue of Staphylococcus aureus protein A with a high affinity towards B cells. Studies have shown that this adjuvant has at least 100–1000 times reduced toxicity compared to the whole wild-type cholera toxin, and that up to 500 g injected intraperitoneally to mice did not show any toxic effects in the spleen, liver or kidney. Moreover, this adjuvant appeared to give protection against rotavirus, comparable to LTR192 (another mutant of the heat labile enterotoxin) and CpG (immunostimulatory dinucleotides, see below)¹⁴.

MPL

Monophosphoryl lipid A (MPL) is derived from the lipopolysaccharide (LPS) of Salmonella minnesota, a Gram-negative bacteria, and therefore, is an archetypal pathogen associated molecular patterns (PAMP) adjuvant. Like LPS, MPL is thought to interact with TLR4 on APC, resulting in the release of pro-inflammatory cytokines. In a number of preclinical studies, MPL has been shown to induce the synthesis and release of IL-2 and IFN-, which promote the generation of Th1 responses^{15, 16}. Clinically, MPL has often been used in complex formulations, including liposomes and emulsions, and has also been used in combination with alum and QS21¹⁷. Overall, MPL has been extensively evaluated in the clinic (>12 000 subjects immunized) for cancer (melanoma and breast) and infectious disease vaccines (genital herpes, hepatitis B virus, malaria and human papilloma virus), and for allergies, with an acceptable profile of adverse effects. In addition, MPL has been approved in Europe for use in combination with allergy vaccines¹⁸. MPL has been used most successfully as a mucosal adjuvant when formulated with delivery systems such as liposomes^{19, 20, 21}.

CpG

In the last few years, a whole new class of adjuvant actives have been identified, following the demonstration that bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on immune cells in vitro^{22, 23}. The immunostimulatory effect was due to the presence of unmethylated CpG dinucleotides²⁴, which are under-represented and methylated in vertebrate DNA. Unmethylated CpG in the context of selective flanking sequences are thought to be recognized by cells of the innate immune system to allow discrimination of pathogen-derived DNA from self-DNA²⁵. It has been shown that cellular responses to CpG DNA are dependent on the presence of TLR9²⁶. In addition, it has been reported that CpG are taken up by non-specific endocytosis and that endosomal maturation is necessary for cell activation and the release of pro-inflammatory cytokines²⁷. The Th1 adjuvant effect of CpG appears to be maximized by their conjugation to protein antigens²⁸. Importantly, CpG also appear to have potential for modulating pre-existing immune responses, which may be useful in various clinical settings, including allergies²⁹. A recent review discusses in detail the interaction of CpG with TLR on dendritic cells³⁰. CpG has been shown to function as a mucosal adjuvant in a number of studies^{31, 32, 33, 34}.

QS21

Another group of immunostimulatory adjuvants are the triterpenoid glycosides, or saponins, derived from Quil A, which is fractionated from the bark of a Chilean tree, Quillaja saponaria. Saponins appear to function mainly through the induction of cytokines. Saponins have been widely used as adjuvants for many years and have been included in several veterinary vaccines. QS21, a highly purified fraction from Quil A, has been shown to be a potent adjuvant for Th1 cytokines (IL-2 and IFN-) and antibodies of the IgG2a isotype, which indicates a Th1 response in mice³⁵. Saponins have been shown to intercalate into cell membranes, through interaction with structurally similar cholesterol, forming 'holes' or pores³⁶. It is currently unknown if the adjuvant effect of saponins is related to pore formation, which may allow antigens to gain access to the endogenous pathway of antigen presentation, promoting a CTL response. A number of clinical trials have been performed, using QS21 as an adjuvant, initially for cancer vaccines (melanoma, breast and prostate cancer), and subsequently for infectious diseases, including HIV-1, influenza, herpes simplex, malaria and hepatitis B³⁷ and more than 3500 people have been immunized with QS21. Doses of 200 g or higher of QS21 have been associated with significant local reactions³⁷, but lower doses appear to be better tolerated. To date, relatively few studies have addressed the use of QS21 as a mucosal adjuvant; however, these studies do show that QS21 can serve as a mucosal adjuvant^{38, 39}.

Cytokines

As an alternative to the use of cytokine inducing adjuvants, cytokines may also be used directly. Most cytokines have the ability to modify and re-direct the immune response. The cytokines that have been evaluated most extensively as adjuvants include IL-1, IL-2, IFN-, IL-12 and GM-CSF⁴⁰. However, all of these molecules show dose related toxicity. In addition, since they are proteins, they have stability problems and a short in vivo half-life, and they are relatively expensive. Therefore, it is unlikely that cytokines will prove acceptable for use as adjuvants in vaccines designed to protect against infectious diseases. Nevertheless, considerable progress has been made in the use of cytokines for the immunotherapy of cancer⁴¹. Microparticles have been used as a delivery system for encapsulated cytokines, including GM-CSF⁴² and IL-12⁴³, mostly for use in oncology settings. Various cytokines, particularly IL-12, have been used as effective mucosal adjuvants, either as soluble proteins or as plasmid DNA^{44, 45, 46, 47, 48}.

Delivery systems for protein vaccines

The use of particulate antigen delivery systems as alternatives to immunostimulatory adjuvants has been evaluated by many groups. Particulate adjuvants (e.g. emulsions, microparticles, iscoms, liposomes) have comparable dimensions to the pathogens that the immune system has evolved to combat. Immunostimulatory adjuvants may also be included in delivery systems to enhance the level of response, or to focus the response through a desired pathway. In addition, formulating potent immunostimulatory adjuvants into delivery systems may limit adverse events, through restricting the systemic circulation of the adjuvant.

PLG microparticles

Particulate delivery systems present multiple copies of antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. In addition, antigen uptake by APC is enhanced by association of antigen with particles, or by the use of polymers or proteins which self-assemble into particles. The biodegradable and biocompatible polyesters, the polylactide-coglycolides (PLG) are the primary candidates for the development of microparticles as adjuvants, as they have been used in humans for many years as resorbable suture material and as controlled release drug delivery systems^{49, 50}. The adjuvant effect achieved through the encapsulation of antigens into PLG microparticles was first demonstrated by several groups in the early 1990s^{51, 52, 53, 54}. In contrast to alum, PLG microparticles were shown to be effective for the induction of CTL responses in rodents^{55, 56, 57}.

The uptake of microparticles (<5 m) both in vitro and in vivo by phagocytic cells has been demonstrated on many occasions. For example, in an early paper⁵⁸, the uptake of microparticles (1–3 m) by macrophages was described, but it was shown that microparticles of 12 m were not taken up, while Tabata and Ikada showed that maximal uptake of microparticles into macrophages occurred with particles of <2 m^{59, 60}. In addition, surface charge and hydrophobicity of the microparticles also modified uptake⁶⁰. It has also been reported that macrophages that carry microparticles to lymph nodes can mature into dendritic cells (DC)⁶¹. In addition, uptake of PLG microparticles into DC in vitro⁶² and in vivo has been demonstrated⁶³. It is assumed that the uptake of microparticles into APC underpins the ability of the particles to perform as vaccine delivery systems/adjuvants. More recently, we developed a novel approach to using PLG microparticles as adjuvants, in which the antigen is adsorbed onto the surface of the particles, which have been modified to have enhanced surface charge to promote adsorption⁶⁴. This approach allowed the induction of significantly enhanced antibody titres in mice with adsorbed p55 gag from HIV-1. A related approach has been described in which a novel charged polymer was used as a stabilizer to prepare PLG nanoparticles, which were able to adsorb tetanus toxoid for mucosal delivery⁶⁵.

Alternative microencapsulation approaches to PLG have also been made. Strong evidence for dissemination of antigen-specific antibody-secreting cells from nasal associated lymphoid tissue (NALT) to the cervical lymph nodes and spleen following i.n. immunization has been provided by Heritage et al⁶⁶. These local and systemic humoral responses were generated by entrapment of human serum albumin (HSA) in polymer-grafted microparticles (3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane [TS-PDMS]) with a size range of 1–100 m. McDermott et al. reported that polymer-grafted starch microparticles have been used as an alternative to PLG particles and were shown to effectively deliver antigens following oral or i.n. immunization and elicit local and systemic humoral responses⁶⁷. However, in comparison to PLG, these microparticles are poorly defined and their biocompatibility has not been tested in humans.

In another study, a single i.n. or oral immunization with a Schistosoma mansoni antigen entrapped in PLG or polycaprolactone (PCL) microparticles resulted in sustained serum IgG responses⁶⁸. However, this vaccine strategy failed to induce IgA responses in serum or broncho-alveolar lavage (BAL) fluids following oral immunization, although i.n. immunization resulted in both serum and BAL fluid IgA responses. Interestingly, only PLG-entrapped and not PCL-entrapped vaccine resulted in strong neutralizing antibody responses, following either i.n. or oral immunization. Moreover, the humoral responses were detectable earlier following PLG versus PCL immunization, presumably due to the physicochemical differences between the two polymers and different rates of antigen release.

Following i.n. immunization of anaesthetized mice with haemagglutinin (HA) from influenza virus entrapped in one of four microparticle resins (sodium polystyrene sulphonate, calcium polystyrene solfinate, polystyrene benzyltrimetylammonium chloride or polystyrene divinylbenzene) sized to 20–45 m enhanced serum as well as nasal wash IgA responses against HA⁶⁹. Importantly, this study showed that this immunization strategy reduced viral burden in the lungs following i.n. administration of virus to the lungs of anaesthetized mice. Interestingly, these resins induced enhanced serum IFN- levels following intra-tracheal (i.t.) immunization, while the levels of IL-4, IL-2 and IL-6 remained unchanged, suggesting a Th1-type response⁶⁹. More recently, Kim et al. demonstrated mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres⁷⁰.

Emulsions

A potent oil-in-water (o/w) adjuvant, the syntex adjuvant formulation (SAF)⁷¹, was developed using a biodegradable oil (squalene) in the 1980s, as a replacement for Freund's adjuvants which are potent, but toxic⁷². However, SAF contained a bacterial cell wall-based synthetic adjuvant, threonyl muramyl dipeptide (MDP), and a non-ionic surfactant, poloxamer L121, and proved too toxic for widespread use in humans⁷³. Therefore, a squalene o/w emulsion was developed (MF59) without the presence of additional immunostimulatory adjuvants, which proved to be a potent adjuvant with an acceptable safety profile⁷⁴. MF59 enhanced the immunogenicity of influenza vaccine in small animal models^{75, 76, 77} and was shown to be a more potent adjuvant than alum for HBV in baboons⁷⁸ and in humans⁷⁹. Subsequently, the safety and immunogenicity of MF59 adjuvanted influenza vaccine (Fluad; Chiron Vaccines, Siena, Italy) was confirmed in elderly subjects in clinical trials^{80, 81}, and this data allowed the approval of this product for licensure in Europe in 1997.

Liposomes

Liposomes are phospholipid vesicles that have been evaluated both as adjuvants and as delivery systems for antigens and adjuvants^{82, 83}. Liposomes have been commonly used in complex formulations, often including MPL, which makes it difficult to determine the contribution of the liposome to the overall adjuvant effect. Nevertheless, several liposomal vaccines based on viral membrane proteins (virosomes) without additional immunostimulators have been extensively evaluated in the clinic and are approved as products in Europe for hepatitis A and influenza⁸⁴.

Iscoms

The immunostimulatory fractions from Quillaja saponaria (Quil A) have been incorporated into lipid particles comprising cholesterol, phospholipids and cell membrane antigens, which are called iscoms⁸⁵. In a study in macaques, an influenza iscom vaccine was shown to be more immunogenic than a classical subunit vaccine and induced enhanced protective efficacy⁸⁶. A similar formulation has been evaluated in human clinical trials and was shown to induce CTL responses⁸⁷. The principal advantage of the preparation of iscoms is to allow a reduction in the dose of the haemolytic Quil A adjuvant and to target the formulation directly to APC. In addition, within the iscom structure, the Quil A is bound to cholesterol and is not free to interact with cell membranes. Therefore, the haemolytic activity of the saponins is significantly reduced^{85, 88}. However, a potential problem with iscoms is that inclusion of antigens into the adjuvant is often difficult, and may require extensive antigen modification⁸⁹.

DNA as a vaccine candidate

Although traditional vaccines have comprised proteins, live attenuated viruses, or killed bacteria, much attention has recently been focused on DNA vaccines. Immunization with DNA has several advantages over immunization with protein, including the induction of potent CTL responses in human and non-human primates^{90, 91}. The ruggedness and simplicity of DNA offers the potential for improved vaccine stability and reduced costs for vaccine production. Moreover, compared to attenuated viruses as delivery vehicles for HIV genes, plasmid DNA offers a safe alternative. Clinical trials involving intramuscular (i.m.) immunization with DNA vaccines have already been performed in humans, and they appear to be safe and well-tolerated at the doses tested^{92, 93}. However, although DNA vaccines have proven potent in small animal models, their potency in larger primates, including humans, has been disappointing. Consequently, there is a clear need to improve the potency of DNA vaccines for human immunization.

Immunopotentiation and adjuvants for DNA vaccines

There are several distinct possible approaches to increasing the potency of DNA vaccines. The first approach is modification of the plasmid DNA vector to increase expression levels; this has resulted in increased immunogenicity in vivo. Changing the nucleotide sequence of certain genes to better reflect preferential codon usage in mammalian cells can result in markedly higher levels of expression in eukaryotic cells in vitro⁹⁴ and, when incorporated into a DNA vaccine vector, can increase immunogenicity substantially^{95, 96, 97}. For example, our modification of the HIV gag in this way has yielded a very potent DNA vaccine that expresses 100- to 1000-fold higher levels of protein compared to the wild-type gene in vitro, and induces robust immune responses in vivo in non-human primates⁹⁵. In two separate investigations using lethal-challenge models of viral infection, plasmid DNA-based alphavirus replicons were shown to be significantly more efficacious in vaccinated mice compared to conventional plasmid DNA expression vectors^{98, 99}. In both investigations, the immune correlates of protection against lethal virus challenge included humoral and cellular responses. The CD4⁺ T-cell immune response in the alphavirus plasmid immunized mice was primarily of the Th1 type, as demonstrated by a high IgG2a : IgG1 ratio⁹⁸. For Sindbis alphavirus based-plasmid (SIN), CTL precursors were induced by replicons expressing HSV glycoprotein B at 1000-fold lower dosage levels of DNA, compared to animals immunized with conventional plasmid⁹⁹. Recently, we have evaluated pSIN encoding HIV gag (SF2) in mice and rhesus macaques. pSINgag was very potent, even when delivered as naked DNA, and comparable to pCMV for priming cellular immune responses (M. Vajdy et al., unpubl. data).

A second general approach to improving DNA vaccines is the use of adjuvants. These include proteins, small molecule compounds or DNA plasmids encoding immunologically active proteins, such as cytokines, chemokines and costimulatory molecules. The specific examples are too numerous to list here, but are reviewed elsewhere¹⁰⁰. We have shown that simple mixtures of DNA vaccines with adjuvants are sometimes effective, but appropriate formulation may be required. For example, certain aluminium gels, such as aluminium phosphate, when mixed with DNA vaccines, enhance antibody responses¹⁰¹, while others, such as aluminium hydroxide, inhibit responses, as a consequence of electrostatic interaction between the negatively charged DNA and positively charged adjuvant. This detrimental effect can be overcome with an appropriate formulation to prevent such binding. We have assessed the effects of aluminium phosphate, together with other DNA formulations such as PLG micro-particles, and found that additive enhancing effects can be achieved.

The third approach to increasing the potency of DNA vaccines is to facilitate DNA delivery into cells. Potential barriers to transfection include (i) lack of widespread distribution of DNA within the inoculated tissue; (ii) rapid degradation of unprotected DNA; (iii) inefficient uptake of DNA by cells (either directly through the plasma membrane or by endocytosis); (iv) degradation of DNA within the endosome/lysosome; and (v) inefficient uptake of DNA by the nucleus, particularly in non-dividing cells where the nuclear membrane remains intact. These limitations may explain why only a small fraction of muscle cells are detectably transfected¹⁰², and only approximately one in 10⁷ molecules of injected plasmid DNA can be recovered from a mouse muscle by 7 days after injection¹⁰³. While APC can also be transfected^{104, 105, 106, 107}, the efficiency appears to be even less than in muscle cells. Our studies with fluorescently tagged plasmid DNA have revealed that much of the inoculated DNA is phagocytosed by macrophages within the muscle, with much less DNA found in muscle cells, and little or none detected within the nuclei of any cells¹⁰⁸. Therefore, a means to facilitate egress of DNA out of endosomes or bypassing this pathway altogether should be beneficial.

Delivery systems for DNA vaccines

The early DNA encapsulation approaches of plasmid DNA into microparticles had several limitations^{109, 110}. The high shear required in generating the emulsion for microencapsulation induced plasmid damage and in addition, encapsulation efficiency was often low^{111, 112, 113}. To avoid these problems, we developed a novel cationic PLG microparticle formulation that had DNA adsorbed onto the surface¹¹⁰. Importantly, the cationic microparticles enhanced the responses in comparison to naked DNA, and this enhancement was apparent in all species evaluated, including non-human primates. In addition, the cationic microparticles efficiently adsorb DNA and can deliver several plasmids simultaneously on the same formulation, at a range of different loading levels^{114, 115}. The microparticles appear to be effective as a consequence of efficient delivery of the adsorbed plasmids into DC¹⁰⁵. A similar approach was recently described in which DNA was adsorbed to the surface of novel cationic emulsions¹¹⁶.

Antigens administered to the nasal cavity are believed to be taken up by M cells overlying the follicle-associated epithelium of the NALT^{117, 118}. It is well established that M cells are highly efficient in the uptake of particulate antigens and microparticles and delivery to underlying APC in the local lymphoid structure^{117, 119}. Following intratracheal (i.t.) delivery, it is likely that microparticles are engulfed by macrophages or DC and transported to bronchus-associated lymphoid tissue and then to local draining lymph nodes. Alternatively, microparticles may be cleared from the lung through the mucociliary elevator and swallowed. Nevertheless, delivery to the lungs is technically difficult and may be associated with potential toxicity issues, including hypersensitivity problems. Thus, in terms of vaccination strategies, the i.n. route appears more practical and feasible compared to delivery of vaccines to the lungs.

In a study designed to determine the inductive sites for ocular IgA responses, i.n. immunization of PLG-entrapped haptenated proteins led to uptake and distribution of microparticles in NALT and cervical lymph node (CLN) within minutes¹²⁰. In this study, no evidence of particle uptake into Peyer's patches (PP) was found following i.n. immunization. Conversely, following oral immunization, no antigen uptake was evident in NALT or CLN, although PP was shown to have taken up the PLG-entrapped antigen. Intranasal immunization led to induction of antigen-specific serum IgG as well as the presence of IgA- and IgG-secreting cells in CLN. Importantly, this study showed that i.n. immunization was superior to ocular immunization for the induction of antigen-specific IgA responses in tears¹²⁰.

Although some insight into vaccine uptake following i.n. immunization with protein antigens has been obtained, little information is available regarding the mechanisms of DNA uptake and expression following mucosal delivery. It is important to note that, although some studies have suggested an important role for DC for uptake and expression of encoded protein following i.m. DNA injection^{121, 122}, there are distinct differences in the anatomy, structure and cellular constituents of nasal and upper respiratory mucosa compared to muscle.

To investigate a possible mechanism for the enhanced immune responses induced following i.n. immunization with PLG-DNA, we localized and phenotypically identified the cells that expressed gag protein in local and systemic lymphoid tissues. Following a single i.n. immunization with PLG-DNA expressing HIV-1 gag, we localized and identified the cells that expressed the encoded gene by immunofluorescent staining (M. Vajdy, unpubl. data). In the immunostaining studies of CLN and spleen, the majority of gag-expressing cells were CD11b⁺, suggesting that this population is responsible for uptake and expression of DNA following i.n. immunization with PLG-DNA. Although CD11b is expressed by many cell populations, it is primarily considered a marker for tissue macrophages (Macs) and DC, which are both professional APC^{56, 123, 124}. However, compared to Macs, DC are more potent as APC^{125, 126}. Our data suggest that following i.n. immunization with DNA adsorbed onto PLG-microparticles, monocyte lineage cells, Macs and/or DC, are involved in the uptake and expression of gag-DNA, because we detected both CD11b⁺ and CD11c⁺ gag-expressing cells. Whether these cells also actively present gag peptides to neighbouring naïve T cells in vivo is an important question that needs further investigation. Our previous in vitro data showed that bone marrow-derived DC can take up PLG-DNA encoding HIV-1 gag and present it to a gag-specific T-cell hybridoma¹⁰⁵.

The prolonged expression of DNA following i.n. immunization with PLG-DNA may be in part due to protection of DNA from damage by tissue DNase, which has previously been reported in vitro¹¹⁰. In addition, the presence of the cationic surfactant cetyl trimethyl ammonium bromide (CTAB) on the surface of PLG microparticles may contribute to disruption of endosomes and subsequent release of DNA into the cytoplasm, to enhance the response. Elucidation of these and other possible factors that contribute to the enhanced responses observed following i.n. delivery of PLG-DNA is important and will facilitate efforts to design effective DNA vaccines.

Combinations of delivery systems and adjuvants

The potency of microparticle formulations with adsorbed antigens has been significantly improved by their coadministration with adsorbed adjuvants¹²⁷. Simultaneous delivery of antigens and adjuvants on microparticles ensures that both agents can be delivered into the same APC population. To illustrate this approach, we used cationic PLG microparticles to adsorb the poly anionic adjuvant CpG¹²⁷, which was coadministered with p55 gag protein adsorbed onto anionic PLG microparticles¹²⁷. In addition, we induced enhanced serum antibody responses against HIV-1 gp120 protein when it was administered in combination with PLG-CpG microparticles in mice. Microparticles can also be used to deliver entrapped adjuvants, to ensure long-term, controlled release of these agents. Tabata and Ikada first entrapped a synthetic adjuvant, muramyl dipeptide (MDP), in microspheres of gelatin¹²⁸, while Puri and Sinko¹²⁹ showed that MDP entrapped in microspheres induced enhanced immune responses. Importantly, Tabata and Ikada⁵⁹ had also shown that the pyrogenicity of MDP was reduced by microencapsulation, establishing that microparticles can improve potency and also reduce the reactogenicity of adjuvants. PLG microparticles have also been used for delivery of QS21 adjuvant in combination with gp120 antigen¹³⁰. However, although studies with an implantable osmotic pump showed that higher titres were obtained with a discontinuous rather than a continuous release profile for the adjuvant¹³⁰, these studies also showed that while the adjuvant was critical for the induction of high initial titres, it was not required for the secondary response. Therefore, the easiest approach was to suspend the microparticles containing the antigen in a solution containing the adjuvant, so that it was immediately available to enhance titres. In addition to the use of microparticles to deliver adjuvants, microparticles may also be used in conjunction with traditional adjuvants, including alum^{131, 132}. In addition, microparticles may also be combined with emulsion-based adjuvants to improve their potency^{133, 134}.

RNA-based vaccines

The physicochemical nature of RNA presents interesting challenges as a gene-based vaccine strategy. In contrast to plasmid DNA, RNA-based vaccines offer a degree of simplicity in their delivery directly to the cytoplasmic site of function, thus bypassing altogether any requirement for DNA or nuclear stages with dependence on cellular transcription machinery and transport of nucleic acids to and from the nucleus. This feature is particularly attractive as a means to eliminate the potential for integration into the host chromosome¹³⁵. However, RNA is relatively labile and is likely to be expensive to manufacture at a commercial scale.

Vaccines that directly utilize mRNA for antigen expression have found application primarily in the oncology field, where efficient transfection of DC with mRNA expressing tumour-associated antigens, followed by vaccination with the RNA-pulsed DC, has shown promise in murine models and more recently in humans¹³⁶. In this context, there appears to be no requirement for prior identification and characterization of individual gene sequences encoding the tumour-associated antigens, as preparations of total mRNA isolated directly from tumours may also be used^{135, 136}. Although the ex vivo RNA vaccine strategy may not be suitable for commercially advancing most infectious disease applications, the oncology studies do support further exploration of RNA as a vaccine modality.

The approach of using mRNA-based vaccines in vivo can be expected to encounter potency issues based simply on limitations in transfecting sufficient mRNA copies into each cell to ensure that protein antigen is expressed at high enough levels to stimulate the desired immune responses. In contrast, DNA vaccines typically rely on the use of promoters, such as a strong RNA polymerase II promoter, to transcribe multiple copies of a given mRNA within each transfected cell. An attractive strategy to increase the intracellular levels of mRNA following delivery of RNA-based vaccines is through the incorporation of replication elements derived from various RNA viruses. Such elements generally will include both the gene(s) coding for enzymatic 'replicase' functions, as well as corresponding cis recognition sequences for the replicase, which together program the cytoplasmic self-amplification of RNA within transfected cells. By specifically excluding essential virus genes, such as those encoding the structural 'coat' proteins, these modified RNA vaccine vectors are prevented from producing any undesirable, infectious virus. RNA expression vectors of this generic configuration have been termed 'replicons', and often are derived from one of three distinct groups of RNA viruses: alphaviruses, flaviviruses and picornaviruses¹³⁷.

The most widely studied replicon vectors, and those which are now in human trials, are derived from the alphaviruses. Alphaviruses are enveloped, positive-stranded RNA viruses. Three members of this group, Sindbis virus (SIN), Semliki Forest virus (SFV) and Venezuelan equine encephalitis virus (VEE), have been developed into replicon vectors for vaccine applications¹³⁸. Alphavirus-based replicon vectors are devoid of the viral structural protein genes, but maintain the replication elements necessary for cytoplasmic RNA self-amplification and expression of the inserted heterologous gene(s) via a highly active alphaviral RNA promoter¹³⁹. The absence of structural protein genes ensures that the replicons are completely defective and incapable of producing infectious virus. In addition to providing high-level antigen expression and the inherent safety advantages related to the absence of DNA or nuclear stages, other features of alphavirus replicon vectors may be advantageous as gene-based vaccines. Such features include immunostimulatory adjuvant effects resulting from double-stranded RNA amplification intermediates¹⁴⁰, and also the induction of apoptosis in transfected cells, which appears to enhance immunogenicity¹⁴¹.

Alphavirus RNA replicon vaccines face many of the same delivery issues discussed previously for plasmid DNA vaccines, although simple i.m. administration of the replicon RNA is clearly capable of inducing a significant immune response¹⁴². Delivery strategies for alphavirus and other replicon vectors have focused primarily on packaging of the RNA vector into replication-defective virus-like particles, thus exploiting the natural receptor-mediated entry process, similar to virus infection. Replicon particles harbouring the RNA vector also exhibit tropism for particular in vivo cell types based on the parental virus source of the structural coat proteins used for packaging, thus enabling exploitation of desirable properties such as mucosal delivery and the in vivo targeting of DC¹⁴³.

As alphavirus replicon vectors do not encode the viral structural proteins necessary for packaging, production of replicon particles is achieved by providing these proteins in trans, in suitable cultured cells^{138, 139}. Typically, the necessary complement of alphavirus structural proteins is provided either by the transient cotransfection of in vitro transcribed replicon and 'helper' RNA encoding the structural proteins, or by introducing the replicons into packaging cell lines (PCL) that express the structural proteins from one or more DNA expression cassettes. Production of replicon particles in this manner preserves the replication-defective nature of the vectors, as the genetic information for the structural proteins remains absent¹³⁸.

The alphavirus replicon particle strategy for RNA vaccines has been evaluated extensively using many diverse antigens, in a variety of animal models. Publications in this field are far too numerous to list in the present context, but collectively demonstrate the potent induction of cellular, humoral and mucosal immune responses by alphavirus replicon particles, following a variety of immunization routes, including mucosal delivery. As with other vaccine strategies, the delivery of alphavirus particles via mucosal routes may be the most efficient means to generate local, mucosal responses. However, even parenteral immunization regimens such as i.m. or s.c. clearly have been observed to induce mucosal immunity^{144, 145, 146, 147, 148}.

In a number of studies, VEE-based alphavirus vectors have been shown to elicit strong mucosal and systemic immune responses following systemic immunization of mice and larger animals¹⁴⁷. Balasuriya et al.¹⁴⁵ used VEE replicon particles expressing the major envelope proteins of equine arteritis virus (EAV) to immunize horses by the s.c. route and demonstrate protection from intranasal or intrauterine EAV challenge. For the SFV replicon particle system, systemic or mucosal immunization with SFV particles expressing the respiratory syncytial virus (RSV) F and G proteins resulted in protection from i.n. challenge with RSV, with i.n. immunization inducing higher neutralizing antibody and pulmonary IFN- T-cell responses¹⁴⁶. In other studies, SIN replicon particles expressing HIV-1 gag induced potent cell mediated (CTL) immune responses in mucosal, as well as in systemic, lymphoid tissues following mucosal immunization. The SIN-gag particles also conferred protection against vaginal viral challenge in mice with vaccinia virus expressing HIV-1 gag¹⁴⁴. Thus, these and other data suggest that alphavirus replicon particles are strong candidates for mucosal gene delivery, with the potential to protect against mucosally transmitted pathogens.

Concluding remarks

It may be concluded that most protein-based vaccines induce better humoral than cellular responses, although inclusion in or on delivery systems and/or co-administration of immunopotentiating adjuvants can enhance the cellular responses. DNA- and RNA-based vaccines are generally believed to induce better cellular than humoral responses, although a number of animal studies have shown the potency of such vaccines to also induce significant humoral responses. An important question remains as to whether combinations of gene-based and protein-based vaccines will enhance both cellular and humoral responses resulting in protection against disease. Another important issue is whether any of the vaccine types discussed in this review can be applied mucosally, parenterally, or through a combination of mucosal and parenteral immunization. The latter mode has been shown to significantly enhance mucosal and systemic immune responses^{149, 150}.

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