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, 9 (1), e84707
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Glucopyranosyl Lipid A Adjuvant Significantly Enhances HIV Specific T and B Cell Responses Elicited by a DNA-MVA-protein Vaccine Regimen

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Glucopyranosyl Lipid A Adjuvant Significantly Enhances HIV Specific T and B Cell Responses Elicited by a DNA-MVA-protein Vaccine Regimen

Paul F McKay et al. PLoS One.

Abstract

Using a unique vaccine antigen matched and single HIV Clade C approach we have assessed the immunogenicity of a DNA-poxvirus-protein strategy in mice and rabbits, administering MVA and protein immunizations either sequentially or simultaneously and in the presence of a novel TLR4 adjuvant, GLA-AF. Mice were vaccinated with combinations of HIV env/gag-pol-nef plasmid DNA followed by MVA-C (HIV env/gag-pol-nef) with HIV CN54gp140 protein (+/-GLA-AF adjuvant) and either co-administered in different muscles of the same animal with MVA-C or given sequentially at 3-week intervals. The DNA prime established a population of B cells that were able to mount a statistically significant anamnestic response to the boost vaccines. The greatest antigen-specific antibody response was observed in animals that received all vaccine components. Moreover, a high proportion of the total mucosal IgG (20 - 50%) present in the vaginal vault of these vaccinated animals was vaccine antigen-specific. The potent elicitation of antigen-specific immune responses to this vaccine modality was also confirmed in rabbits. Importantly, co-administration of MVA-C with the GLA-AF adjuvanted HIV CN54gp140 protein significantly augmented the antigen-specific T cell responses to the Gag antigen, a transgene product expressed by the MVA-C vector in a separate quadriceps muscle. We have demonstrated that co-administration of MVA and GLA-AF adjuvanted HIV CN54gp140 protein was equally effective in the generation of humoral responses as a sequential vaccination modality thus shortening and simplifying the immunization schedule. In addition, a significant further benefit of the condensed vaccination regime was that T cell responses to proteins expressed by the MVA-C were potently enhanced, an effect that was likely due to enhanced immunostimulation in the presence of systemic GLA-AF.

Conflict of interest statement

Competing Interests: The authors received funding for equipment from Dormeur Investment Service Ltd and can declare that charitable funding from this commercial funder does not alter adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Gp140-Specific Serum IgG responses in mice primed with DNA.
(A) Animals (n = 10) were inoculated 3 times with 100 µg plasmid DNA (IM at week 0, 3 and 6) then boosted with variations of MVA-C (107 PFU) and/or recombinant protein with/without GLA-AF adjuvant (IM inoculation at week 12 and 15). CN54gp140 recombinant protein (10 µg) and GLA-AF adjuvant (20 µg) were admixed and injected into one quadricep muscle. If MVA-C was administered at the same time it was inoculated into the other quadricep muscle. Serum was taken one day prior to each vaccination and the antigen-specific IgG antibody was assessed by an immunoglobulin ELISA with a murine purified IgG standard curve. (B) Comparison of the serum IgG or IgA levels in each DNA primed group at peak response (Week 15). Antigen-specific IgG concentrations are shown in µg/ml (+/− SEM). Statistical comparisons were performed using a Mann-Whitney test (***p<0.0001, comparison to DNA – MVA-C group).
Figure 2
Figure 2. Gp140-Specific Mucosal IgG or IgA responses and serum IgG Th1/Th2 bias in DNA primed mice boosted with various combinations of MVA-C and/or CN54gp140 and GLA-AF.
Female BALB/c mice (n = 10) inoculated with a total of 100 ug plasmid DNA (50 ug per leg; IM at week 0, 3 and 6) then boosted with variations of 107 PFU MVA-C and/or 10 µg recombinant protein with/without 20 µg GLA-AF adjuvant (IM inoculation at week 12) were assessed at week 15, three weeks after the first boost event. (A) Mucosal vaginal wash samples contained moderate levels of vaccine antigen-specific IgG but very low levels of specific IgA (***p<0.0001; **p = 0.0015). (B) The effect of the boost inoculation upon the Th1:Th2 bias was assessed with the ratio of the IgG2a∶IgG1 immunoglobulin isotype. Antigen-specific IgG, IgG1, IgG2a or IgA antibody was assessed by an immunoglobulin ELISA with a murine purified IgG, IgG1, IgG2a or IgA standard curve. Antigen-specific Ig concentrations are shown in µg/ml (+/− SEM). Statistical comparisons were performed using a Mann-Whitney test.
Figure 3
Figure 3. Anamnestic humoral responses after plasmid DNA vaccination.
(A) Mice that had been previously inoculated three times with plasmid DNA expressing the CN54gp140 Env transgene elicited a memory B cell population that differentiated into antigen-specific antibody secreting cells after a single MVA/gp140+GLA vaccination. (B) MVA alone was also able to re-stimulate this memory population of B cells and this was again further boosted by subsequent inoculations of the recombinant adjuvanted antigen CN54gp140. Antigen-specific IgG antibody was assessed by an immunoglobulin ELISA and concentrations are shown in µg/ml (+/− SEM).
Figure 4
Figure 4. Splenocyte IFN-γ ELISpot responses to specific vaccine antigen derived peptide pools.
Cells isolated from mouse spleens at termination were stimulated with peptide pools (15 mers overlapping by 11 aa) that covered the entire coding sequence of the Gag and Env protein (two pools for each peptide) that were expressed from the transgenes present in the two plasmid DNAs and the MVA-C vector. (A) Specific peptide pool responses of splenocytes from all study groups. (B) Comparison of splenocyte IFN-γ ELISpot responses to the Env and the Gag peptide pools in animals that had been DNA primed then inoculated with either MVA-C and CN54gp140+GLA sequentially or with the MVA and protein or with all three boost vaccine components concurrently. Responses from each group of animals are shown as group mean spot forming units (SFU)/million splenocytes +/− SEM. Groups were statistically compared using a one-way Anova (*p<0.05; **p<0.01; ***p<0.005; **** p<0.001).
Figure 5
Figure 5. Polyfunctional cytokine responses of peptide pool stimulated splenocytes.
Cells from each animal were stimulated with each different peptide pool for 6°C in the presence of a golgi secretion inhibitor. CD4+ or CD8+ T cell populations were gated and single, double or triple cytokine expressing cell percentage enumerated. The percentage cytokine expression of each animal was averaged within the group and is expressed as a pie-chart of relative proportions of the total cytokine expressing cell population. Pie slice colour key indicates the particular cytokine(s) expressed.
Figure 6
Figure 6. Vaccine elicited CN54gp140-specific serum and mucosal IgG responses in rabbits.
Male and female NZW rabbits were primed with three DNA inoculations (8 mg total; 4 mg each leg) into their quadricep muscles then boosted with either MVA – gp140+GLA given sequentially or MVA/gp140+GLA administered concurrently in separate leg muscles (MVA-C at 1.3×108 PFU in 500 µl; CN54gp140 at 100 µg in 400 µl; GLA-AF at 5 µg in 400 µl – co-formulated with CN54gp140 recombinant protein). The antigen-specific serum IgG response over the course of the vaccine regimen (A) and the mucosal IgG response at termination (B). Antigen-specific rabbit IgG antibody was assessed by an immunoglobulin ELISA with a rabbit purified IgG standard curve. Antigen-specific IgG concentrations are shown in µg/ml (+/− SEM).

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