Boosting with Subtype C CN54rgp140 Protein Adjuvanted with Glucopyranosyl Lipid Adjuvant after Priming with HIV-DNA and HIV-MVA Is Safe and Enhances Immune Responses: A Phase I Trial

Background A vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF) in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA). Methods Forty volunteers (35 vaccinees and five placebo recipients) were given two CN54rgp140/GLA-AF immunizations 30-71 weeks after the last HIV-MVA vaccination. These immunizations were delivered intramuscularly four weeks apart. Results The vaccine was safe and well tolerated except for one episode of asymptomatic hypoglycaemia that was classified as severe adverse event. Two weeks after the second HIV-MVA vaccination 34 (97%) of the 35 previously vaccinated developed Env-specific binding antibodies, and 79% and 84% displayed IFN-gamma ELISpot responses to Gag and Env, respectively. Binding antibodies to subtype C Env (included in HIV-DNA and protein boost), subtype B Env (included only in HIV-DNA) and CRF01_AE Env (included only in HIV-MVA) were significantly boosted by the CN54rgp140/GLA-AF immunizations. Functional antibodies detected using an infectious molecular clone virus/peripheral blood mononuclear cell neutralization assay, a pseudovirus/TZM-bl neutralization assay or by assays for antibody-dependent cellular cytotoxicity (ADCC) were not significantly boosted. In contrast, T-cell proliferative responses to subtype B MN antigen and IFN-gamma ELISpot responses to Env peptides were significantly enhanced. Four volunteers not primed with HIV-DNA and HIV-MVA before the CN54rgp140/ GLA-AF immunizations mounted an antibody response, while cell-mediated responses were rare. After the two Env subtype C protein immunizations, a trend towards higher median subtype C Env binding antibody titers was found in vaccinees who had received HIV-DNA and HIV-MVA prior to the two Env protein immunizations as compared to unprimed vaccinees (p = 0.07). Conclusion We report excellent tolerability, enhanced binding antibody responses and Env-specific cell-mediated immune responses but no ADCC antibody increase after two immunizations with a subtype C rgp140 protein adjuvanted in GLA-AF in healthy volunteers previously immunized with HIV-DNA and HIV-MVA

A comparative phase I study of combination, homologous subtype-C DNA, MVA, and Env gp140 protein/adjuvant HIV vaccines in two immunization regimes

There remains an urgent need for a prophylactic HIV vaccine. We compared combined MVA and adjuvanted gp140 to sequential MVA/gp140 after DNA priming. We expected Env-specific CD4+ T-cells after DNA and MVA priming, and Env-binding antibodies in 100% individuals after boosting with gp140 and that combined vaccines would not compromise safety and might augment immunogenicity. Forty volunteers were primed three times with DNA plasmids encoding (CN54) env and (ZM96) gag-pol-nef at 0, 4 and 8 weeks then boosted with MVA-C (CN54 env and gag-pol-nef) and glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) adjuvanted CN54gp140. They were randomised to receive them in combination at the same visit at 16 and 20 weeks (accelerated) or sequentially with MVA-C at 16, 20, and GLA-AF/gp140 at 24 and 28 weeks (standard). All vaccinations were intramuscular. Primary outcomes included ≥grade 3 safety events and the titer of CN54gp140-specific binding IgG. Other outcomes included neutralization, binding antibody specificity and T-cell responses. Two participants experienced asymptomatic ≥grade 3 transaminitis leading to discontinuation of vaccinations, and three had grade 3 solicited local or systemic reactions. A total of 100% made anti-CN54gp140 IgG and combining vaccines did not significantly alter the response; geometric mean titer 6424 (accelerated) and 6578 (standard); neutralization of MW965.2 Tier 1 pseudovirus was superior in the standard group (82 versus 45% responders,  = 0.04). T-cell ELISpot responses were CD4+ and Env-dominant; 85 and 82% responding in the accelerated and standard groups, respectively. Vaccine-induced IgG responses targeted multiple regions within gp120 with the V3 region most immunodominant and no differences between groups detected. Combining MVA and gp140 vaccines did not result in increased adverse events and did not significantly impact upon the titer of Env-specific binding antibodies, which were seen in 100% individuals. The approach did however affect other immune responses; neutralizing antibody responses, seen only to Tier 1 pseudoviruses, were poorer when the vaccines were combined and while T-cell responses were seen in >80% individuals in both groups and similarly CD4 and Env dominant, their breadth/polyfunctionality tended to be lower when the vaccines were combined, suggesting attenuation of immunogenicity and cautioning against this accelerated regimen

Envelope-Specific Recognition Patterns of HIV Vaccine-Induced IgG Antibodies Are Linked to Immunogen Structure and Sequence

Background: A better understanding of the parameters influencing vaccine-induced IgG recognition of individual antigenic regions and their variants within the HIV Envelope protein (Env) can help to improve design of preventive HIV vaccines. Methods: Env-specific IgG responses were mapped in samples of the UKHVC003 Standard Group (UK003SG, n = 11 from UK) and TaMoVac01 (TMV01, n = 17 from Tanzania) HIV vaccine trials. Both trials consisted of three immunizations with DNA, followed by two boosts with recombinant Modified Vaccinia Virus Ankara (MVA), either mediating secretion of gp120 (UK003SG) or the presentation of cell membrane bound gp150 envelopes (TMV01) from infected cells, and an additional two boosts with 5 μg of CN54gp140 protein adjuvanted with glucopyranosyl lipid adjuvant (GLA). Env immunogen sequences in UK003SG were solely based on the clade C isolate CN54, whereas in TMV01 these were based on clades A, C, B, and CRF01AE. The peptide microarray included 8 globally representative Env sequences, CN54gp140 and the MVA-encoded Env immunogens from both trials, as well as additional peptide variants for hot spots of immune recognition. Results: After the second MVA boost, UK003SG vaccinees almost exclusively targeted linear, non-glycosylated antigenic regions located in the inter-gp120 interface. In contrast, TMV01 recipients most strongly targeted the V2 region and an immunodominant region in gp41. The V3 region was frequently targeted in both trials, with a higher recognition magnitude for diverse antigenic variants observed in the UK003SG (p < 0.0001). After boosting with CN54gp140/GLA, the overall response magnitude increased with a more comparable recognition pattern of antigenic regions and variants between the two trials. Recognition of most immunodominant regions within gp120 remained significantly stronger in UK003SG, whereas V2-region recognition was not boosted in either group. Conclusions: IgG recognition of linear antigenic Env regions differed between the two trials particularly after the second MVA boost. Structural features of the MVA-encoded immunogens, such as secreted, monomeric gp120 vs. membrane-anchored, functional gp150, and differences in prime-boost immunogen sequence variability most probably contributed to these differences. Prime-boosting with multivalent Env immunogens during TMV01 did not improve variant cross-recognition of immunodominant peptide variants in the V3 region

Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

<div><p>Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.</p></div

T cell responses induced by tested vaccine candidates.

<p>(A) Mice (<i>n</i> = 3 per group) were immunized once with 5 μg of DREP-C-ENV or 5 μg of DREP-C-GPN alone, or both constructs either mixed or given at separate sites. DREP constructs were given i.d. followed by EP. Splenocytes were assayed 10 days post-immunization with IFN-γ Elispot using peptides PADPNQEM (Env), VGPTPVNI (Pol1) and YYDPSKDLI (Pol2). (B-D) DREP-C-ENV and DREP-C-GPN induce T cells and antibodies that are boosted by multiple administrations. Mice (<i>n</i> = 5 per group) were immunized by i.d. EP one to three times with 5 or 0.1 μg of each DREP-C-ENV and DREP-C-GPN given at separate sites. One group was given DNA-C (5 μg of each construct) 3 times i.m. Boost immunizations were given with a 3 week interval between each administration. (B) Serum was assayed with ELISA for anti-gp140 IgG antibodies 3 weeks after the last immunization. Responses are shown as box plots, with whiskers representing 5–95 percentiles. (C) Splenocytes were assayed with IFN-γ Elispot 10 days and 3 weeks after last immunization using the peptides described above. (D) ICS for CD107a, IFN-γ, IL-2 and TNF-α was performed on splenocytes 10 days after the last immunization. The percentage of CD8+ T cells expressing a certain number of cytokines is shown. Specific markers expressed are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.s002" target="_blank">S2 Fig.</a> Statistical analyses were performed to compare mice given a different number of administrations of the same dose of DREP-C. In addition, mice given the same number of administrations but with different doses/regimens were compared in separate statistical comparisons. Abbreviations: D-ENV, DREP-C-CN54ENV; D-GPN, DREP-C-ZM96GPN. * <i>P</i> < 0.05; ** <i>P</i> < 0.01</p

T cell responses following DREP-C prime and boost with MVA-C and/or gp140/GLA.

<p>Mice (<i>n</i> = 5 per group) were primed one or three times with DREP-C, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.g001" target="_blank">Fig. 1B-D</a>. Six weeks after the last administration of DREP-C, mice were boosted with one of the following: (A) MVA in one side, (B) CN54gp140/GLA in one side, (C) MVA-C in one side followed by gp140/GLA in the other side three weeks after MVA-C administration, or (D) MVA-C in one side and gp140/GLA in the other side simultaneously. The DREP-C doses are stated in the figure. The booster doses given were 5×10⁶ TCID50 of MVA, 10 μg of gp140 mixed with 0.7 μg of GLA. Three weeks after the last immunization, splenocytes were assayed with IFN-γ Elispot. For peptide designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.g001" target="_blank">Fig. 1A</a>. Groups that were given the same booster following a DREP-C prime were compared statistically. Please note that the Y-axis scale of (B) differs from the others. * <i>P</i> < 0.05.</p

Antibody responses following DREP-C prime and boost with MVA-C and/or gp140/GLA.

<p>Mice (<i>n</i> = 5 per group) were primed 1 or 3 times with DREP-C, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117042#pone.0117042.g001" target="_blank">Fig. 1B-D</a>. Six weeks after the last administration of DREP-C, mice were boosted with one of the following: (A, E) MVA in one side, (B, F) CN54gp140/GLA in one side, (C, G) MVA-C in one side followed by CN54gp140/GLA in the other side three weeks after MVA-C administration, or (D, H) MVA-C in one side and gp140/GLA in the other side simultaneously. The DREP-C doses are stated in the figure. The booster doses given were 5×10⁶ TCID50 of MVA, 10 μg of gp140 mixed with 0.7 μg of GLA. Three weeks after the last immunization, serum was assayed with ELISA for anti-gp140 (A-D) IgG, (E-H) IgG1 (red) and IgG2a (blue). Responses are shown as box plots, with whiskers representing 5–95 percentiles. (I-L) IgG2a:IgG1 ratio means, with error bars representing standard error of the mean. Groups that were given the same booster following a DREP-C prime were compared statistically. * <i>P</i> < 0.05.</p

Glucopyranosyl lipid A adjuvant significantly enhances HIV specific T and B cell responses elicited by a DNA-MVA-protein vaccine regimen

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