Immunogenicity and lower dose requirement of polymer entrapped tetanus toxoid co-administered with alum

Low adjuvanticity of microparticles based vaccine formulation necessitates the use of alum along with particles to elicit improved antibody titers from single point immunization. It was observed that antibody response from immunization with admixture of alum and polymer entrapped antigen was dependent on particle size, amount of antigen released during burst phase and dose of microencapsulated antigen. In the animals immunized with polymer entrapped tetanus toxoid (TT) very large particles (50-150 microm) did not elicited high antibody titers where as microparticles in the range 2-8 microm exhibited remarkable improvement in the antibody response. Very small size particles (<2 microm) were also not as effective as 2-8 microm size particles for generation of antibody response. Presence of alum improved the immune response by adsorbing the burst released antigen from the particle surfaces. Role of alum in potentiation of immune response from polymer entrapped TT was highly significant at lower dose regimes. Polymer entrapped TT as little as 0.1 Lf when immunized along with alum generated antibody responses superior to those elicited by 10 Lf soluble antigen. Immunization with admixture of particles and alum generated two to three times higher antibody titer than that observed from immunization with similar single doses of alum adsorbed TT. Single point immunization of admixture of particles entrapped TT and alum generated sustained long lasting antibody responses comparable to two divided doses of alum-adsorbed antigen. Superiority of single dose polymeric formulation in comparison to two divided doses of alum adsorbed TT was more evident at lower doses of TT immunization. This reflected the profound synergistic effect of both the adjuvants at lower doses. Affinity of antibodies generated from single point immunization was comparable to that achieved with two doses of alum adsorbed TT immunization. Particle alone elicited more of IgG2a type antibody where as immunization with admixture of alum and particles improved the overall antibody response and more of Th2 type response.     

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.     

Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles

Virus-like particles (VLPs) composed of rotavirus VP2, VP6, and VP7 of G1 or G3 serotype specificity were produced in insect cells coinfected with recombinant baculoviruses expressing single rotavirus genes. The VLPs were purified and subsequently evaluated for immunogenicity and protection in the adult mouse model of rotavirus infection. Mice were vaccinated twice intramuscularly with G1 VLPs formulated with Quillaja saponaria (QS-21) or adsorbed to aluminium hydroxide (AlOH), or with G1 VLPs alone. G3 VLPs, G1 plus G3 VLPs, inactivated SA11 virions formulated with QS-21, or adjuvants were similarly inoculated as controls. Mice were examined for serum and fecal antibody responses by ELISA or microneutralization assays. Protective efficacy of the VLP vaccine formulations against oral challenge with the G3 murine ECwt rotavirus was assessed by comparing the antigen shed in stool of the VLP-vaccinated mice to that of the adjuvant-immunized mice. G1 VLPs in QS-21 induced significantly higher serum and intestinal antibody titers than G1 VLPs in AlOH or G1 VLPs alone. QS-21 also heightened serum and fecal antibody responses to G3 VLPs. These QS-21-augmented antibody responses were further characterized by equivalent IgG1 and IgG2a titers in sera, suggesting that G1 or G3 VLPs in QS-21 induced a balanced Th1/Th2 response. G1 VLPs in QS-21 induced partial protection (88%) against oral challenge with the heterotypic ECwt virus, whereas G3 VLPs in QS-21 induced complete protection (100%). In contrast, G1 VLPs when formulated with AlOH induced a predominant Th2 response and did not protect (1%) mice from virus challenge. Our results indicate that the type of adjuvant used clearly influences both antibody responses to rotavirus VLPs and the protective efficacy against rotavirus infections. These data have important implications for the development of parenteral vaccines to ameliorate rotavirus disease.     

Contribution of TLR4 and MyD88 for adjuvant monophosphoryl lipid A (MPLA) activity in a DNA prime–protein boost HIV-1 vaccine

Recombinant protein vaccines are commonly formulated with an immune-stimulatory compound, or adjuvant, to boost immune responses to a particular antigen. Recent studies have shown that, through recognition of molecular motifs, receptors of the innate immune system are involved in the functions of adjuvants to generate and direct adaptive immune responses. However, it is not clear to which degree those receptors are also important when the adjuvant is used as part of a novel heterologous prime-boost immunization process in which the priming and boosting components are not the same type of vaccines. In the current study, we compared the immune responses elicited by a pentavalent HIV-1 DNA prime–protein boost vaccine in mice deficient in either Toll-like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88) to wildtype mice. HIV gp120 protein administered in the boost phase was formulated with either monophosphoryl lipid A (MPLA), QS-21, or Al(OH)₃. Endpoint antibody titer, serum cytokine response and T-cell memory response were assessed. Neither TLR4 nor MyD88 deficiency had a significant effect on the immune response of mice given vaccine formulated with QS-21 or Al(OH)₃. However, TLR4- and MyD88-deficiency decreased both the antibody and T-cell responses in mice administered HIV gp120 formulated with MPLA. These results further our understanding of the activation of TLR4 and MyD88 by MPLA in the context of a DNA prime/protein boost immunization strategy.     

Increased immunogenicity of HIV envelope subunit complexed with alpha2-macroglobulin when combined with monophosphoryl lipid A and GM-CSF

Critical to the success of HIV-1 subunit vaccines is the development of strategies to augment vaccine immunogenicity. Successful adjuvants must not only improve immunogenicity above current adjuvant levels, but must also decrease the dose of immunogen required for optimal immunogenicity. We have evaluated activated alpha2-macroglobulin (alpha2M*) and a squalene-based stable emulsion containing monophosphoryl lipid A (MPL-SE) with granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvants to enhance the immunogencity of candidate HIV immunogens. Balb/c mice were subcutaneously immunized on days 0, 14 and 28 with 100-0.1 microg of HIV-1 envelope gp120 C4-V3 immunogens from either HIV IIIB (C4-V3(IIIB)) or SHIV 89.6P (C4-V3(89.6P)). Immunogens were tested covalently coupled to alpha2M*, formulated with MPL-SE/GM-CSF, or as a combination of both. Using CFA/IFA, only 50 and 100 microg, but not lower doses of C4-V3(IIIB) peptides, induced antibody responses. In contrast, peak antibody responses were detected in mice immunized with 10 microg of C4-V3 peptide coupled to alpha2M* (alpha2M*-peptide). Similar to CFA/IFA, MPL-SE/GM-CSF induced optimal antibody responses at 50 and 100 microg of C4-V3 immunogen. However, the combination of MPL-SE/GM-CSF with alpha2M*-C4-V3 peptide decreased the dose of C4-V3 required for optimal response to 5 microg for C4-V3(IIIB), and to 0.1 microg for C4-V3(89.6P). Taken together, HIV envelope gp120 C4-V3 peptides covalently complexed with alpha2M* and formulated with MPL-SE/GM-CSF resulted in a subunit HIV immunogen capable of inducing anti-HIV envelope antibody responses at doses up to 100-fold less than those needed with CFA/IFA or MPL-SE/GM-CSF alone.     

An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model

The safety, immunogenicity, and protective efficacy of two non-replicating antigen-based vaccines and one live-attenuated virus (LAV) vaccine for dengue type-2 (dengue-2) virus were evaluated in the rhesus macaque model. The non-replicating vaccines consisted of whole, purified inactivated virus (PIV) and a recombinant subunit protein containing the amino-(N)-terminal 80% of envelope protein (r80E), each formulated with one of five different adjuvants. Each formulation was administered to three animals on a 0, 3-month schedule. Following the primary immunizations, 37 of 39 animals demonstrated dengue-2 virus neutralizing antibodies. After the booster immunizations all animals had dengue neutralizing antibodies with peak titers ranging from 1:100 to 1:9700. The highest neutralizing antibody titers were observed in the groups that received r80E antigen formulated with AS04, AS05, or AS08 adjuvant, and PIV formulated with AS05 or AS08 adjuvant. These newer adjuvants are based on alum, fraction QS-21 of saponin, and monophosphoryl lipid A (MPL). Protection was evaluated by dengue-2 virus challenge 2 months after the booster by the measurement of circulating virus (viremia) and post-challenge immune responses. Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays. None of the vaccines conferred sterile immunity.     

QS-21 structure/function studies: effect of acylation on adjuvant activity

QS-21 is a natural saponin adjuvant derived from the tree Quillaja saponaria Molina. Previous studies over a limited dose range suggested the acylation is critical to adjuvant activity. In this study, we prepared DS-1 (deacylated QS-21) and RDS-1 (reacylated DS-1 with dodecylamine at a different site than QS-21) to determine the effect on a dose-response curve over a wider range in mice. DS-1 and RDS-1 induced IgG1 responses at higher doses compared to that induced by QS-21. DS-1 was inactive for inducing IgG2a or CTL responses at any doses. RDS-1 showed moderate IgG2a response at 240 microg, but did not show CTL response at any dose evaluated.     

Adjuvant-enhanced antibody responses to recombinant proteins correlates with protection of mice and monkeys to orthopoxvirus challenges

Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum+CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations 3 weeks apart. In addition, monkeys immunized with recombinant vaccinia virus proteins and QS-21 developed neutralizing antibody to monkeypox virus and had reduced virus load, skin lesions, and morbidity compared to the non-immunized group following monkeypox virus challenge.     

Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production

The present 2-year study investigated the feasibility of using porcine zona pellucidae (pZP) as antigen for immunocontraception in American black bears. Sows, 3-6 years of age, were administered either two doses of 250 microg pZP with Freund's adjuvant (n = 10) or adjuvant alone (n = 5), one in April and one in May, and were kept away from the boars until June. Serum samples were collected before injections and before denning (November). The presence of sows with cubs at side was observed during premature emergence from denning. First-year results indicated that anti-pZP antibody titres in vaccinated sows were 2.5-9.0-fold (range) higher compared with non-vaccinated sows and that the vaccinated sows were threefold less likely to become pregnant (P = 0.167). Control and vaccinated bears produced 1.6 and 0.2 cubs per sow, respectively (P = 0.06). The second-year study investigated the feasibility of using pZP sequestered in a controlled-release pellet and a water-soluble adjuvant (QS-21) to avoid regulatory problems associated with Freund's adjuvant. Sows in the treatment group (n = 22) were administered a single dose of an emulsion of 250 microg pZP and 150 microg QS-21 plus a pellet containing 70-90 microg pZP for delayed release as booster dose. Control sows (n = 5) received the QS-21 adjuvant in pellet alone. Serum samples were collected before inoculations (April) and before denning (November). Seven cubs were born to the five control sows, but none was born to the 22 vaccinated sows (P < 0.001). Anti-pZP antibody mean absorbance ratios in control sows remained at background levels, whereas vaccinated sows had ratios fourfold higher than controls. Two-dimensional polyacrylamide gel electrophoresis and immunohistochemical localisation confirmed immunoreactivity of sera from inoculated bears. We conclude that cub production in the American black bear can be effectively limited with either two injections of 250 microg pZP or a single inoculation of partially purified pZP sequestered in controlled-release pellets.     

Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine.

A parenterally administered rotavirus vaccine composed of virus-like particles (VLPs) is being evaluated for human use. VLPs composed of bovine VP6 and simian VP7 (SA11, G3) proteins (6/7-VLPs) or of bovine VP2, bovine VP6, and simian VP7 (SA11, G3) proteins (2/6/7-VLPs) were synthesized and purified from Sf9 insect cells co-infected with recombinant baculoviruses. 6/7- and 2/6/7-VLP administered parenterally (i.m.) in mice had comparable immunogenicity, but the 2/6/7-VLPs were more homogeneous and stable. The inclusion of the VP2 capsid contributed to particle formation and stability. The adjuvant QS-21 significantly enhanced the immunogenicity of 2/6/7-VLPs over A10H or saline alone. Equivalent serum neutralizing antibody responses were induced over the range of 1-15 microg/dose of 2/6/7-VLPs administered with the range of 5-20 microg/dose of QS-21. The immunogenicity of 2/6/7-VLPs and inactivated SA11 virus were comparable. 2/6/7-VLPs are a promising candidate for a parenterally delivered rotavirus subunit vaccine.     

HIV-1 Tat-coated nanoparticles result in enhanced humoral immune responses and neutralizing antibodies compared to alum adjuvant

HIV-1 Tat has been identified as an attractive target for vaccine development and is currently under investigation in clinical trials as both a therapeutic and preventative vaccine for HIV-1. It is well known that protein based vaccines produce poor immune responses by themselves and therefore require adjuvants to enhance immune responses. We have previously reported on the use of anionic nanoparticles (NPs) for enhancing cellular and humoral immune responses to Tat (1-72). The purpose of this study was to further evaluate the immune response of HIV-1 Tat (1-72) coated on anionic nanoparticles compared to alum using various doses of Tat (1-72). Nanoparticles were effective at generating comparable antibody titers at both 1 and 5 microg doses of Tat (1-72), whereas the antibody titers significantly decreased at the lower dose of Tat (1-72) using alum. Anti-sera from Tat (1-72) immunized mice reacted greatest to the N-terminal and basic regions of Tat, with the NP groups showing stronger reactivity to these regions compared to alum. Moreover, the anti-sera from all Tat (1-72) immunized groups contained Tat-neutralizing antibodies and were able to significantly inhibit Tat-mediated long terminal repeat (LTR) transactivation.     

Lack of immune interference between inactivated polio vaccine and inactivated rotavirus vaccine co-administered by intramuscular injection in two animal species

A parenteral inactivated rotavirus vaccine (IRV) in development could address three problems with current live oral rotavirus vaccines (ORV): their lower efficacy in low and middle-income countries (LMICs), lingering concerns about their association with intussusception, and their requirement for a separate supply chain with large volume cold storage. Adding a new parenteral IRV to the current schedule of childhood immunizations would be more acceptable if it could be combined with another injectable vaccine such as inactivated polio vaccine (IPV). Current plans for polio eradication call for phasing out oral polio vaccine (OPV) and transitioning to IPV, initially in LMICs as a single dose booster after two doses of OPV and ultimately as a two dose schedule. Today in many LMICs, IPV is administered as a standalone vaccine, which involves a separate cold chain and is relatively costly. We therefore tested in two animal models formulations of IPV with IRV to determine whether co-administration might interfere with the immune response to each product and spare antigen dose for both vaccines. Our results demonstrate that IRV when adjuvanted with alum and administered alone or in combination with IPV did not impair the immune responses to either rotavirus or poliovirus serotypes 1, 2 and 3. Similarly, IPV when formulated and administered alone or together with IRV induced comparable levels of neutralizing antibody to poliovirus type 1, 2 and 3. Furthermore, comparable antibody titers were observed in animals vaccinated with low, middle or high dose of IPV or IRV in combination. This dose sparing and the lack of interference between IPV and IRV administered together represent another step to support the further development of this novel combination vaccine for children.     

Microparticles in MF59, a potent adjuvant combination for a recombinant protein vaccine against HIV-1

Novel adjuvant formulations involving PLG microparticles with entrapped recombinant protein antigens (env gp120 and p24 gag) from human immunodeficiency virus type-1 (HIV-1), dispersed in the emulsion adjuvant MF59 were evaluated as potential HIV-1 vaccine candidates in mice and baboons. In mice, the adjuvant combination induced significantly enhanced antibody responses in comparison to either adjuvant used alone. In addition, the polylactide co-glycolide polymer (PLG) microparticles and MF59 combination induced CTL activity against HIV-1 p24 gag. In baboons, the adjuvant combination induced significantly enhanced antibody titers after a single dose of gp120, but the responses were comparable to gp120 in MF59 alone after boosting. Both MF59+gp120 alone and PLG/gp120 in MF59 induced neutralizing antibodies against a T cell line-adapted (TCLA) strain and a primary isolate of HIV-1. In contrast to the observations with gp120, immunization in baboons with PLG/p24 in MF59 induced significantly enhanced antibody responses after boosting, in comparison to immunization with MF59 alone + p24.     

A recombinant baculovirus-expressed S glycoprotein vaccine elicits high titers of SARS-associated coronavirus (SARS-CoV) neutralizing antibodies in mice

A recombinant SARS-CoV spike (S) glycoprotein vaccine produced in insect cells in a pre-clinical development stage is described. A truncated version of S glycoprotein, containing only the ecto-domain, as well as a His-tagged full-length version were cloned and expressed in a serum-free insect cell line, ExpresSF+. The proteins, purified to apparent homogeneity by liquid column chromatography, were formulated without adjuvant at 3, 9, 27, and 50 microg per dose in phosphate saline and used to immunize mice. Both antigens in each formulation elicited a strong immune response after two or three vaccinations with the antigen. Neutralizing antibody titers correlated closely with standard ELISA reactivity against the S glycoprotein. The truncated S protein was also formulated with an adjuvant, aluminum hydroxide, at 1 microg per dose (+/-adjuvant), and 5 microg per dose (+/-adjuvant). Significantly enhanced immune responses, manifested by higher titers of serum ELISA and viral neutralizing antibodies, were achieved in adjuvanted groups with fewer doses and lower concentration of S glycoprotein. These findings indicate that the ecto-domain of SARS-CoV S glycoprotein vaccine, with or without adjuvant, is immunogenic and induces high titers of virus neutralizing antibodies to levels similar to those achieved with the full S glycoprotein vaccine.     

HIV-1 Envgp140 trimers elicit neutralizing antibodies without efficient induction of conformational antibodies

Currently, no vaccine for human immunodeficiency virus (HIV-1) provides protection from virus infection. One reason for these disappointing results has been the difficulty of current vaccine candidates to elicit high-titer, broadly reactive immunity to a large number of viral proteins. Recently, our laboratory demonstrated that the coupling of C3d to a soluble trimerized HIV-1 envelope (Env(gp140(FT))) elicited higher titers of neutralizing antibodies than monomers of Env(gp120) coupled to C3d [Bower JF, Yang X, Sodroski J, Ross TM. Elicitation of neutralizing antibodies with DNA vaccines expressing soluble stabilized human immunodeficiency virus type 1 envelope glycoprotein trimers conjugated to C3d. J Virol 2004;78(9):4710-9]. To determine if the induction of conformational antibodies correlated with neutralization, mice (BALB/c) were primed (2x) with DNA plasmids expressing monomeric Env(gp120) or trimeric Env(gp140) alone or fused to mC3d(3) at one of two doses (2.0microg or 0.2microg), followed by a boost of recombinant uncleaved, trimeric Env(gp140). Regardless of the priming dose of DNA, all mice had high-titer anti-Env IgG antibodies. Interestingly, Env(gp140) trimers did not elicit higher titers of antibodies that recognized conformational Env epitopes compared to monomers of Env(gp120). Therefore, additional parameters were examined for correlation with neutralization. For neutralization-resistant HIV-1 isolates, ADA and YU-2, neutralization correlated with high-titer, high avidity antibodies, with Env(gp140) eliciting slightly higher neutralization titers than Env(gp120). In contrast, none of the measured parameters correlated with neutralization for the more neutralization-sensitive isolates, MN or 89.6. Therefore, even though soluble, uncleaved Env(gp140) trimers may be marginally more effective at eliciting neutralizing antibodies than Env(gp120), neutralization does not appear to correlate with the elicitation of conformationally dependent antibodies.     

IL-33 enhances the kinetics and quality of the antibody response to a DNA and protein-based HIV-1 Env vaccine

Induction of a sustained and broad antibody (Ab) response is a major goal in developing a protective HIV-1 vaccine. DNA priming alone shows reduced levels of immunogenicity; however, when combined with protein boosting is an attractive vaccination strategy for induction of humoral responses. Using the VC10014 DNA and protein-based vaccine consisting of HIV-1 envelope (Env) gp160 plasmids and trimeric gp140 proteins derived from an HIV-1 clade B infected subject who developed broadly neutralizing serum Abs, and which has been previously demonstrated to induce Tier 2 heterologous neutralizing Abs in rhesus macaques, we evaluated whether MPLA and IL-33 when administered during the DNA priming phase enhances the humoral response in mice. The addition of IL-33 during the gp160 DNA priming phase resulted in high titer gp120-specific plasma IgG after the first immunization. The IL-33 treated mice had higher plasma IgG Ab avidity, breadth, and durability after DNA and protein co-immunization with alum adjuvant as compared to MPLA and alum only treated mice. IL-33 was also associated with a significant IgM Env-specific response and expansion of peritoneal and splenic B-1b B cells. These results indicate that DNA priming in the presence of exogenous IL-33 qualitatively alters the HIV-1 Env-specific humoral response, improving the kinetics and breadth of potentially protective Ab.     

A bivalent conjugate vaccine in the treatment of biochemically relapsed prostate cancer: a study of glycosylated MUC-2-KLH and Globo H-KLH conjugate vaccines given with the new semi-synthetic saponin immunological adjuvant GPI-0100 OR QS-21

GPI-0100 is a semi-synthetic saponin with modifications designed to augment stability and diminish toxicity. Two batches of GPI-0100 (the second with higher purity) were tested with doses ranging between 100 and 5000 microg in groups of five treated prostate cancer patients who had no evidence of disease except for rising PSA levels. GPI-0100 was mixed with a bivalent vaccine containing the glycolipid Globo H and the glycosylated mucin MUC2 conjugated to keyhole limpet hemocyanin (KLH). All doses were well tolerated and antibody titers against Globo H and MUC-2 escalated with the increasing dose levels. At the 5000 microg dose level in this patient population, toxicity remained minimal with only occasional grade II local toxicity at vaccination sites and occasional sporadic grade I elevations in ALT. Compared with a subsequent trial with the same bivalent vaccine plus QS-21 at the maximal tolerated dose of 100 microg, the 5000 microg dose of GPI-0100 produced comparable antibody titers.     

Effect of aluminum hydroxide adjuvant and formaldehyde in the formulation of rPA anthrax vaccine

The serological response and efficacy of Bacillus anthracis recombinant protective antigen (rPA) vaccines formulated with aluminum hydroxide adjuvant, either with or without formaldehyde, were evaluated in rabbits. Rabbits that had been injected with a single dose of 25 microg of rPA adsorbed to 500 microg of aluminum in aluminum hydroxide gel (Alhydrogel) had a significantly higher quantitative anti-rPA IgG ELISA titers (p<0.0001) and toxin neutralizing antibody (TNA) assay titers (p<0.0001) than rabbits tested at the next lowest concentration of aluminum (158 microg). Rabbits injected with two doses of 50 microg of rPA formulated with 500 microg of aluminum also had significantly higher serological responses, as measured by a quantitative anti-rPA IgG ELISA (p<0.0001) and TNA assay (p<0.0001), than sera from rabbits injected with a rPA vaccine formulated without adjuvant. Short-term protection against an aerosol spore challenge (448 LD(50)), however, was not significantly different between the two groups (12/12 and 11/12, respectively). Rabbits injected with a single dose of 50 microg of rPA formulated with 500 microg of aluminum and 0.2% formaldehyde had significantly higher ELISA (p<0.0001) and TNA assay (p<0.0001) titers than rabbits that had been injected with a rPA vaccine formulated with adjuvant but without formaldehyde. Short-term protection against a 125 LD(50) parenteral spore challenge, however, was not significantly different between the two groups (14/24 and 9/24, respectively; p=0.2476). Under the conditions tested in the rabbit animal model, significantly higher serological responses were observed in rabbits that had been injected with rPA formulated with aluminum hydroxide gel adjuvant and formaldehyde. However, differences in short-term efficacy were not observed.     

Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates

A year ago we described a comparison of 19 immunological adjuvants for their ability to augment antibody and T-cell responses against vaccines containing two cancer antigens, GD3 ganglioside and MUC1 peptide, covalently attached to keyhole limpet hemocyanin (KLH). As in our previous experience, the saponin fraction QS-21 was the most potent single adjuvant but several other adjuvants also had potent adjuvant activity. Induction of an immune response against cancer antigens is generally difficult because these antigens are autoantigens. To get maximal benefit from the adjuvant component of cancer vaccines we have now tested whether combinations of the optimal adjuvants induced an improved immune response compared to QS-21 alone. Since over the intervening year a new semi-synthetic saponin adjuvant (GPI-0100) containing the dodecylamide derivative of hydrolyzed naturally-occurring saponins had become available, this was tested as well. Twelve different adjuvant combinations and GPI-0100 were compared for their ability to augment (1) antibody responses against GD3 and MUC1 and (2) T-cell responses against GD3, MUC1 and KLH. GPI-0100 and five adjuvant combinations were superior to QS-21 alone for induction of IgM and IgG antibodies against MUC1 and/or GD3: QS-21 plus bacterial nucleotide CpG, QS-21 plus monophosphoryl lipid A (MPL), QS-21 plus non-ionic block copolymer CRL-1005, QS-21 plus Titermax and Titermax plus CpG. Antibody responses were documented both by ELISA against purified antigens and by FACS for cell surface reactivity. There was no evidence for T-cell immunity against GD3 or MUC1. The antibody responses against GD3 and MUC1 were, however, strongly correlated with IFN-gamma release and DTH against KLH. These results demonstrate that combinations of immunological adjuvants are able to augment antibody and T-cell responses to these conjugates beyond that attainable with QS-21 alone, and again confirm the absolute necessity of potent adjuvants or adjuvant combinations for optimal immunogenicity with conjugate vaccines.     

Durable HIV-1 antibody and T-cell responses elicited by an adjuvanted multi-protein recombinant vaccine in uninfected human volunteers

BACKGROUND: Use of the recombinant proteins NefTat and gp120(W61D) formulated with the AS02A adjuvant system was previously shown to protect against AIDS in a rhesus macaque SHIV animal model system. METHODS: Eighty-four HIV uninfected human participants were vaccinated intramuscularly at 0, 1, and 3 months and evaluated for safety. Immune responses were analyzed for the presence of vaccine-induced antibody and T lymphocyte responses. RESULTS: The vaccines were safe and well tolerated at all doses. Nef-, Tat-, and gp120-specific binding antibodies were induced in all individuals that received the respective antigen, lasting up to 9 months after the final immunization. Antibodies able to neutralize the T-cell laboratory-adapted strain of HIV-1(W61D) were detected in the majority of vacinees, but did not neutralize primary isolates. Envelope-specific antibody-dependent cell cytoxicity was detected in most of the individuals receiving gp120. Robust and persistent HIV-specific lymphoproliferative responses were detected against all subunit proteins in the majority of immunized participants. As expected, HIV-specific CD8 T-cell responses were not detected. CONCLUSIONS: Despite the lack of primary isolate neutralizing antibody induction, the observed high frequency and magnitude of other immune responses warrant further work with this vaccine or vaccine components.