Biodegradable PLGA microspheres as a delivery system for malaria synthetic peptide SPf66

SPf66 is the first chemically synthesised vaccine to elicit a partial protective immune response against malaria. The aluminium hydroxide (alum)-adsorbed SPf66 vaccine is weakly immunogenic and of poor to moderate efficacy in humans. To investigate the possibility of improving SPf66 vaccine immunogenicity, a delivery system based on poly-D,L-lactide-co-glycolide (PLGA) microspheres was developed and the immune response induced after its subcutaneous administration into mice was evaluated. Microspheres were prepared by a solvent extraction/double emulsion (w/o/w) method and characterised for morphology, size, peptide loading, release profile and peptide integrity. The in vitro and in vivo results obtained showed that there was no apparent effect of the encapsulation procedure on SPf66 integrity and immunogenicity. The subcutaneous administration of microspheres showed a significantly higher immune response (serum IgG levels) than that obtained with alum adsorbed SPf66 and it was comparable to that of SPf66 emulsified with Freund's adjuvant (FA). These observations illustrate the potential of PLGA microspheres as a delivery system for chemically synthesised antigens.     

Safety,tolerability and immunogenicity of new formulations of the Plasmodium falciparum malaria peptide vaccine SPf66 combined with the immunological adjuvant QS-21

SPf66 is a synthetic malaria peptide vaccine, which has been widely tested in combination with aluminium hydroxide (alum) as the adjuvant. Since this formulation is weakly immunogenic, we sought to improve its immunogenicity by using the saponin adjuvant QS-21. SPf66/QS-21 vaccines were evaluated for safety, tolerability and immunogenicity in healthy adults. The vaccines were found to be safe in 87/89 (97.8%) volunteers studied. However, two individuals developed severe vaccine allergy following the third dose of 1/3 SPf66/QS-21 formulations tested. Vaccine formulations containing QS-21 induced a 45- to over 200-fold increase in anti-SPf66 IgG titres over the alum formulation after the second and third doses, respectively. Anti-SPf66 antibody from some subjects reacted against asexual blood stage parasites, as demonstrated by immunofluorescence and immunoblotting. Antibody responses generated by the QS-21 formulations were of longer duration compared to those evoked by the alum formulation. While SPf66/alum has been found to induce only CD4+ T cell response, the QS-21 formulations exhibited the potential to also elicit SPf66-specific CD8+ responses. These observations demonstrate that the use of QS-21 can substantially enhance the immunogenicity of peptide vaccines, such as SPf66.     

Remarkably high antibody levels and protection against P. falciparum malaria in Aotus monkeys after a single immunisation of SPf66 encapsulated in PLGA microspheres

Single dose immunisation is a major goal in vaccine design. The purpose of this study was the development of a single dose delivery system for the SPf66 malaria vaccine, based on this antigen's microencapsulation in PLGA microspheres by double emulsion method.Results indicate that a single immunisation in mice and monkeys with the SPf66 malaria vaccine, encapsulated in a mixture of two formulations of PLGA microspheres, induced a remarkably high and long-lasting immune response as assessed by ELISA and Western Blott. This immune response was associated with a good protective capacity in Aotus monkeys, after experimental challenge, indicating that antigen integrity lasted following the microencapsulation process. PLGA biodegradable microspheres thus serve as an effective delivery system for the design of a single dose immunisation vaccine, such as the SPf66 synthetic malaria vaccine.     

A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component(s). Diphtheria-tetanus-pertussis (DPT) vaccine contains not only aluminum hydrate (alum) to enhance the immune response to the vaccine ingredients, but also, both for that purpose and as a principal ingredient, pertussis toxin (PT). However, both adjuvants strongly promote T helper (Th) 2 type immune responses. Th1 and Th2 type immune responses are counterbalanced in vivo, and a Th2-prone immune response is not effective against intracellular infections but promotes IgE production, which is related to allergic disease. In this study, we used the CpG motif contained in oligodeoxynucleotide (CpG-ODN), which has an adjuvant effect and also induces the Th1 response, as an adjuvant to this vaccine, and we investigated its adjuvanticity and its potential to modulate immune responses to DPT vaccine. Administration of DPT vaccine with CpG-ODN (DPT-alum/ODN) to mice significantly reduced the total IgE levels and increased the anti-PT specific IgG2a titer in serum, in comparison with ordinary DPT vaccine (DPT-alum). Moreover, we investigated the antibody response to orally administrated ovalbumin (OVA) after vaccine administration. In the DPT-alum/ODN-administered group, the OVA specific IgE production in serum greatly decreased in comparison with that in the DPT-alum-administered group. These data indicate that CpG-ODN was not useful only as an efficient vaccine adjuvant but also shifted the immune responses substantially toward Th1 and modulated the Th1/Th2 immune response in DPT vaccine. These data suggested new applications of CpG-ODN as adjuvants in DPT vaccine.     

Study of the safety and immunogenicity of the synthetic malaria SPf66 vaccine in children aged 1-14 years.

Safety and immunogenicity tests of the SPf66 malaria vaccine have been carried out on a population of children, aged 1 to 14 years, in the town of Tumaco, Colombia. Adverse reactions measured after each vaccination were local and minimal, and observed in only a small percentage of the vaccinated children. One year later, no delayed reaction was evident. The majority of the child population developed high antibody titres against SPf66 and the degree of response did not vary with age. These induced antibodies recognize the native parasite proteins, in particular the molecules from which the amino acid sequence of this vaccine was deduced. These studies demonstrate that the SPf66 vaccine is safe and highly immunogenic for use in children greater than 1 year old.     

The first field trials of the chemically synthesized malaria vaccine SPf66: safety, immunogenicity and protectivity.

This paper reports the results of the first field study performed to assess the safety, immunogenicity and protectivity of the synthetic malaria vaccine SPf66 directed against the asexual blood stages of Plasmodium falciparum. Clinical and laboratory tests were performed on all volunteers prior to and after each immunization, demonstrating that no detectable alteration was induced by the immunization process. The vaccines were grouped as high, intermediate or low responders according to their antibody titres directed against the SPf66 molecule. Two of the 185 (1.08%) SPf66-vaccinated and nine of the 214 (4.20%) placebo-vaccinated volunteers developed P. falciparum malaria. The efficacy of the vaccine was calculated as 82.3% against P. falciparum and 60.6% against Plasmodium vivax.     

Combination of immune stimulating adjuvants with poly(lactide-co-glycolide) microspheres enhances the immune response of vaccines

The development of vaccines that generate mixed humoral and cellular immune responses is a challenge in vaccinology. Poly(lactide-co-glycolide) microspheres are vaccine adjuvants which possess the advantage of allowing the coencapsulation of other adjuvants in addition to the antigen. Thus, we can stimulate the immune system from different ways and resemble the effects of a natural infection. In this study, we have coencapsulated BSA with monophosphoryl lipid A, polyinosinic-polycytidylic acid, α-galactosylceramide and alginate into PLGA microspheres. All the microspheres have developed a higher humoral immune response, in terms of release of total IgG, in comparison to the administration of soluble antigen. In addition, they triggered a more balanced IgG1/IgG2a response. The combination of MPLA and α-galactosylceramide within the microspheres developed the higher cellular response, confirming that combination of adjuvants with different action mechanisms is a good strategy to increase vaccines’ immunogenicity.     

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)₃. Clear differences in the IgG profiles induced by G3, Al(OH)₃ or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)₃ even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)₃ increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.     

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP1(19) in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSP1(19)-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.     

Novel synthetic LPS receptor agonists boost systemic and mucosal antibody responses in mice

Safe and cost-effective adjuvants are a critical requirement for subunit vaccine development. We report here the in vivo activity of a series of fully synthetic LPS receptor agonists that have been shown to activate NF-kappaB signaling through the Toll-like receptor 4 (TLR4). These compounds boost antibody responses to protein antigens when coadministered at microgram doses in mice. At these dosage levels no adverse effects are observed. Antibody responses are largely IgG1, with enhanced IgG2a, and down-regulated IgE as compared to alum adjuvanted immunization. Stimulation of Th1 is confirmed by enhanced gamma-interferon production after in vitro antigen restimulation of spleen cells from mice immunized with the synthetic adjuvants. The adjuvants are active by both subcutaneous and intranasal routes of vaccine administration, and in the latter case can amplify both serum IgG and serum and mucosal IgA responses. The compounds must be administered at the same site with antigen to boost anti-vaccine antibody. These fully synthetic ligands of the innate immune system offer the potential for use as effective, safe, and nonbiologically-derived adjuvants.     

12. Reduction in the mean number of Plasmodium falciparum genotypes in Gambian children immunized with the malaria vaccine SPf66

SPf66, a synthetic peptide Plasmodium falciparum vaccine, did not protect young Gambian children against clinical attacks of malaria. Nevertheless, Gambian children who had been vaccinated with SPf66 and who were parasitaemic at the end of the first malaria transmission season after vaccination had significantly fewer detectable P. falciparum genotypes than control children, as determined by polymerase chain reaction analysis of 3 polymorphic loci—the msp1 block 2 repeat region, the msp2 repeat region, and the R11 region of the glutamate-rich protein gene (glurp). Geometric mean numbers of genotypes were 1·66 vs. 1·87, 1·95 vs. 2·43, and 1·21 vs. 1·50 for msp1,msp2 and glurp, respectively (P = 0·31, P = 0·04 and P < 0·01). Differences between groups became a little more marked for msp1 and msp2 when children with symptomatic malaria were excluded. No significant difference was found between parasites obtained from SPf66-vaccinated or control children in the prevalences of amino acid alleles at positions 44 and 47 in the 11 amino acid sequence of the merozoite surface protein 1 molecule, which is present in SPf66. The reduction in the number of genotypes observed could not be explained by a difference in parasite densities between SPf66-vaccinated and control children, as geometric mean parasite densities were almost identical in the 2 groups. These observations suggest that SPf66 vaccine may have induced an immune response which reduced the incidence of new infections in immunized children or accelerated the rate of clearance of parasites of individual genotypes. However, no reduction in the prevalence or density of parasitaemia was recorded in SPf66-vaccinated children, suggesting the existence of some kind of density-dependent mechanism for controlling low levels of malaria parasitaemia.     

Starch microparticles as an adjuvant in immunisation: effect of route of administration on the immune response in mice

This paper describes the effects on the development of an immune response by changing the route of administration of a new vaccine adjuvant, starch microparticles with human serum albumin (HSA) as a model antigen. The model vaccine was administered to mice by oral, subcutaneous and intramuscular routes in various combinations and both the local secretory immunoglobulin antibody (s-IgA) and systemic humoral and cellular (delayed-type hypersensitivity assay (DTH)) responses were followed. The only immunisation regimens inducing a significant s-IgA response were those incorporating oral booster doses. Oral and subcutaneous immunisations had similar effects on the Th1/Th2 balance, as indicated by the IgG subclass ratios and cytokine analyses. However, significant differences between oral and intramuscular immunisations were seen in the IgG subclass ratios. The Th2 influence was stronger after oral primary immunisation than after intramuscular primary immunisation, while oral boosters elicited a comparatively stronger Th1 response than intramuscular boosters. This result was also supported by the DTH analyses. Subcutaneous immunisation induced a stronger Th2 response than intramuscular immunisation, as indicated by subclass ratio and the IgE response. In conclusion, our results show that the profile of an immune response depends on the route of administration, which should be considered when developing new vaccines or new routes of administration.     

Immunogenicity of the Plasmodium falciparum Pf332-DBL domain in combination with different adjuvants

The Plasmodium falciparum antigen 332 (Pf332) is a conserved blood-stage antigen, which has been suggested to play a role in parasite invasion. In the present study, we have investigated the immunogenicity of the Duffy-binding like (DBL)-domain of the Pf332 molecule in combination with different adjuvants in four animal species. Three of the adjuvants are applicable for human use (Montanide ISA 720, alum and levamisole), whilst Freund's adjuvant served as a positive control adjuvant. Montanide ISA 720 was able to generate a significant and Th2-biased IgG response in BALB/c and C57BL/6 mice. Alum was a strong inducer of a Th2-type immune response only in BALB/c mice, whereas it was a poor adjuvant together with Pf332-DBL in C57BL/6 mice, rabbits and rats. Levamisole did not show any obvious adjuvant effect in any of the immunized animals. Thus in the case with Pf332-DBL, Montanide ISA 720 may be an adjuvant to further explore in the development of a vaccine against malaria.     

Cationic lipid DC-Chol induces an improved and balanced immunity able to overcome the unresponsiveness to the hepatitis B vaccine.

Th1 and Th2 immune responses against antigens can be modulated by the use of adjuvants. Since antibody isotypes (IgG1 and IgG2a) and cytokines induced may reflect the Th differentiation taking place during the immune response, the humoral and cellular immune responses induced in mice against hepatitis B virus surface antigen (HBsAg) were examined when the antigen was either adsorbed to aluminum hydroxyde or administered with a new adjuvant the cationic lipid 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). The use of DC-Chol increased antibody responses in responding BALB/c mice, induced more consistent IgG1 and IgG2a antibody responses in OF1 mice and overcame the nonresponse to HBsAg in B10.M mice. Furthermore, DC-Chol was able to induce cellular immune responses to HBsAg. The DC-Chol induced a balanced Th1/Th2 response, which enabled mice to overcome the inherited unresponsiveness to HBsAg encountered with aluminum-adjuvanted vaccine. Thus, the DC-Chol provides a signal to switch on both Th1 and Th2 responses, which may have important implications for vaccination against hepatitis B virus, as well as for enhancing weak immunogenicity of other recombinant purified antigens in a nonresponder population.     

Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch

Beta-amyloid (Abeta) peptide has been proposed to be a causal factor in Alzheimer's disease (AD). Currently being investigated, active and passive Abeta-immunotherapy significantly reduce Abeta plaque deposition, neuritic dystrophy, and astrogliosis in the brains of APP transgenic (APP/Tg) mice. Immunization with Abeta42 formulated in the Th1-type adjuvant QS21 was beneficial for AD patients with significant titers of anti-Abeta antibodies, however, 6% of participants developed meningoencephalitis, likely due to anti-Abeta-specific autoimmune Th1 cells. Thus, successful Abeta vaccination requires the development of strong antibody responses without Th1-type cellular immunity. In this study, we compared the induction of humoral immune responses with Th1-type (Quil A) and Th2-type (Alum) adjuvants singly and in combination, using our novel epitope vaccine composed of self B cell epitope Abeta(1-15) and foreign T cell epitope PADRE (PADRE-Abeta(1-15)-MAP). Formulated in Quil A, this vaccine resulted in significantly higher anti-Abeta antibody responses in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, compared with Alum. Anti-Abeta antibodies induced by Alum were predominantly IgG1 type accompanied by lower levels of IgG2a and IgG2b. Quil A induced robust and almost equal titers of anti-Abeta antibodies of IgG1 and IgG2a isotypes and slightly lower levels of IgG2b. Switching adjuvants from Alum to Quil A induced higher concentrations of antibodies than injections with Alum only, however slightly lower than Quil A only. Switching both adjuvants did not change the profile of antibody responses generated by the initial adjuvant injected. These results suggest that switching from Alum to Quil A would be beneficial for AD patients because anti-Abeta antibody production was enhanced without changing the initially generated and likely beneficial Th2-type humoral response.     

Novel Th1-biased adjuvant, SPO1, enhances mucosal and systemic immunogenicity of vaccines administered intranasally in mice

Oil-in-water emulsions are potent human adjuvants commonly used in effective pandemic influenza vaccines; however, such emulsions that can induce both Th1-biased systemic immune responses and strong mucosal immune responses via an easy method of administration are lacking. To address this need for new adjuvants, we developed a novel oil/water emulsion, SPO1, which allows convenient mucosal immunization via an intranasal spray as well as by parenteral routes. Our report shows that SPO1 was able to boost up immunological resistance by inducing effective mucosal and serum antibodies, and the immune response was polarized to a Th1 pattern, as demonstrated by high IgG2α antibody levels and interferon-gamma production by splenocytes from intranasally (i.n.) immunized mice. Up-regulation of co-stimulatory and antigen-presenting molecules on dendritic cells was also observed in vivo after i.n. immunization, suggesting a possible mechanism for the adjuvant effects of SPO1. Another explanation may simply be a depot of antigen at the immunization site, as evidenced by in vivo imaging of i.n. immunized mice. In conclusion, our results demonstrate that a novel oil/water emulsion, SPO1, is a potent Th1 adjuvant for use in influenza and other vaccines, as it induces strong mucosal and systemic immune responses.     

The increase in intra-macrophage thiols induced by new pro-GSH molecules directs the Th1 skewing in ovalbumin immunized mice

In the present work, the capacity of new pro-GSH molecules to increase the intra-macrophage thiol content in vitro and in vivo as well as to shift the immune response to Th1 in ovalbumin (Ova)-sensitized mice were examined. The molecules were the N-butanoyl GSH derivative, GSH-C4, and a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA), I-152. In vitro, 2 h-incubation with both molecules was found to increase intra-macrophage thiol content; in vivo, Ova-sensitized mice pre-treated by intraperitoneal administration of the pro-GSH molecules showed an increase in plasma anti-Ova IgG2a and IgG2b, characterizing Th1 immune response, and a decrease in IgG1, typical of the Th2 response. Such findings were connected to a shift to a Th1 response also involving splenocyte IFN-γ production as revealed by ELISPOT assay and higher levels of IL-12 in circulation. Although immune responses are in vivo mediated both by dendritic cells and macrophages, the data reported in this paper corroborate the suggestion that the pro-GSH molecules, increasing the intra-cellular thiol pool, modulate the Th1/Th2 balance favouring Th1-type responses and may be employed as Th1-directing adjuvants in new vaccination protocols and as immunomodulators in those diseases where Th1 response patterns are compromised in favour of Th2.     

Development of adjuvant nanocarrier systems for seasonal influenza A (H3N2) vaccine based on Astragaloside VII and gum tragacanth (APS)

Adjuvants are chemical/biological substances that are used in vaccines to increase the immunogenicity of antigens. A few adjuvants have been developed for use in human vaccines because of their limitations including lack of efficacy, unacceptable local or systemic toxicity, the difficulty of manufacturing, poor stability, and high cost. For that reasons, novel adjuvants/adjuvant systems are under search. Astragaloside VII (AST-VII), isolated from Astragalus trojanus, exhibited significant cellular and humoral immune responses. The polysaccharides (APS) obtained from the roots of Astragalus species have been used in traditional Chinese medicine and possess strong immunomodulatory properties. In the present study, the immunomodulatory effects of a newly developed nanocarrier system (APNS: APS containing carrier) and its AST-VII containing formulation (ANS: AST-VII + APNS), on seasonal influenza A (H3N2) vaccine were investigated. Inactivated H3N2 alone or its combinations with test compounds/formulations were intramuscularly injected into Swiss albino mice. Four weeks after immunization, the immune responses were evaluated in terms of antibody and cytokine responses as well as splenocyte proliferation. APNS demonstrated Th2 mediated response by increasing IgG1 antibody titers, whereas ANS showed response towards Th1/Th2 balance and Th17 by producing of IFN-γ, IL-17A and IgG2a. Based on these results, we propose that APNS and ANS are good candidates to be utilized in seasonal influenza A vaccines as adjuvants/carrier systems.     

Post-fusion influenza vaccine adjuvanted with SA-2 confers heterologous protection via Th1-polarized,non-neutralizing antibody responses

Development of a universal influenza vaccine that can provide robust and long-lasting protection against heterologous infections is a global public health priority. A variety of vaccine antigens are designed to increase the antigenicity of conserved epitopes to elicit cross-protective antibodies that often lack virus-neutralizing activity. Given the contribution of antibody effector functions to cross-protection, adjuvants need to be added to modulate antibody effector functions as well as to enhance antibody quantity. We previously showed that post-fusion influenza vaccine antigens elicit non-neutralizing but cross-protective antibodies against conserved epitopes. Here, using a murine model, we comparably assessed the adjuvanticity of the newly developed SA-2 adjuvant containing a synthetic TLR7 agonist DSP-0546 and squalene-based MF59 analog as representative Th1- or Th2-type adjuvants, respectively. Both types of adjuvants in the post-fusion vaccine comparably enhanced cross-reactive IgG titers against heterologous strains. However, only SA-2 skewed the IgG subclass into the IgG2c subclass in association to its Th1-polarizing nature. SA-2-enhanced IgG2c responses exhibited antibody-dependent cellular cytotoxicity against heterologous virus strains, without cross-neutralizing activity. Eventually, the SA-2-adjuvanted vaccination provided protection against lethal infection by heterologous H3N2 and H1N1 viruses. Together, we conclude that the combination with a SA-2 is advantageous for enhancing the cross-protective capability of post-fusion HA vaccines that elicit non-neutralizing IgG antibodies.     

Interleukin-12 redirects murine immune responses to soluble or aluminum phosphate adsorbed HSV-2 glycoprotein D towards Th1 and CD4+ CTL responses

The type of immune response elicited against HSV-2 infection may be a factor in the frequency and severity of recurrent disease, with non-recurrent status being associated with a Th1-like response. As administration of glycoprotein D subunit formulated with an aluminum-based adjuvant induces predominantly Th2-like immune responses, we sought to assess the ability of IL-12 to redirect anti-HSV immunity towards a Th1 response. Co-administration of gD with IL-12 resulted in gD-specific antibody subclass switching from predominantly IgG1 observed in mice immunized with either gD or gD/AlPO4 to a more balanced combination of IgG1 and IgG2a, and enhanced virus neutralizing activity. Spleen cells from mice immunized with gD and IL-12, and restimulated in vitro with HSV-2, developed into effector cells capable of secreting IFN-gamma and lysing HSV-2 infected targets, while those obtained from gD or gD/ALPO4 immunized mice did not express lytic activity. In vitro studies determined that these CTLs were CD4+ and that the cytotoxicity was primarily perforin dependent. Vaginal challenge with HSV-2 demonstrated that IL-12 co-administration with gD resulted in increased efficacy of this vaccine as compared to administration of gD antigen alone. This acquired protection persisted up to 1 year. Finally, adsorbing gD and IL-12 to AlPO4 decreased the optimal dose of IL-12 required to enhance gD immunogenicity and shift responses towards a Th1-like profile.