RhCMV serostatus and vaccine adjuvant impact immunogenicity of RhCMV/SIV vaccines

Abstract

Rhesus cytomegalovirus (RhCMV) strain 68-1-vectored simian immunodeficiency virus (RhCMV/SIV) vaccines are associated with complete clearance of pathogenic SIV challenge virus, non-canonical major histocompatibility complex restriction, and absent antibody responses in recipients previously infected with wild-type RhCMV. This report presents the first investigation of RhCMV/SIV vaccines in RhCMV-seronegative macaques lacking anti-vector immunity. Fifty percent of rhesus macaques (RM) vaccinated with a combined RhCMV-Gag, -Env, and -Retanef (RTN) vaccine controlled pathogenic SIV challenge despite high peak viremia. However, kinetics of viral load control by vaccinated RM were considerably delayed compared to previous reports. Impact of a TLR5 agonist (flagellin; FliC) on vaccine efficacy and immunogenicity was also examined. An altered vaccine regimen containing an SIV Gag-FliC fusion antigen instead of Gag was significantly less immunogenic and resulted in reduced protection. Notably, RhCMV-Gag and RhCMV-Env vaccines elicited anti-Gag and anti-Env antibodies in RhCMV-seronegative RM, an unexpected contrast to vaccination of RhCMV-seropositive RM. These findings confirm that RhCMV-vectored SIV vaccines significantly protect against SIV pathogenesis. However, pre-existing vector immunity and a pro-inflammatory vaccine adjuvant may influence RhCMV/SIV vaccine immunogenicity and efficacy. Future investigation of the impact of pre-existing anti-vector immune responses on protective immunity conferred by this vaccine platform is warranted.

Figure 1

Vaccination protocol. Schematic representation of the study protocol showing RhCMV/SIV or control RhCMV vaccination at 0, 12, and 24 weeks and weekly SIVmac251 challenges (up to 12) starting at 36 weeks after priming immunization.

Figure 2

Vaccine induction of antiviral T-cell responses. T-cell responses specific to (a) SIV Gag peptide pool and (b) Gag69 supertope elicited by RhCVM/SIV vaccines. Responses were evaluated by flow cytometric ICS for TNF and/or IFN-γ expression in gated CD4 or CD8 T-cells after stimulating cryopreserved PBMC thawed the same day, with overlapping peptides for SIV Gag protein or a 15-mer Gag_273-287(69) peptide. Shown in stacked bar charts are the frequencies of single TNF⁺ (labeled in red), single IFN-γ⁺ (labeled in blue) and TNF⁺IFN-γ⁺ (labeled in purple) responding T-cells. Mamu-B*17-positive RM were labeled in blue in box and whisker plots. n = 8 RM per group. Differences between frequencies of total responding T-cells of groups B and C were analyzed by the nonparametric Mann–Whitney tests (**, P < 0.01; NS, not significant).

Figure 3

Vaccine induction of antiviral antibodies. Antibody responses elicited by RhCVM/SIV vaccines. Longitudinal binding antibody concentrations specific for (a) RhCMV and (b) SIV Gag (SIVmac251 p27) in plasma of vaccinated RM. Black lines indicate median values. The second RhCMV SIV vaccine immunization boost significantly enhanced (c) anti-SIV p27 antibody concentrations in Group B RM and (d) anti-Env (SIVmac251 gp130) antibodies in the majority of Groups B and C animals. Shown are the comparison of IgG concentrations between baseline (week 0) and 1 week (week 25) or 2 weeks (week 26) after the second boost. n = 8 RM per group. The Friedman test was used to determine the significance of matched samples (P values shown) and when significant, the Dunn’s multiple comparison test was used to determine the significance of pairwise differences (**, P < 0.01; ***, P < 0.001).

Figure 4

Viral loads after IVAG challenge with SIVmac251. Acquisition of SIV infection and plasma viral load profiles in RhCMV/SIV-vaccinated and control RM. (a) Kaplan–Meier curves showing the numbers of challenges required to achieve SIV infection. The onset of infection was defined as positive plasma viral loads (PVL) (10³ copies per mL) for two weeks. A nonparametric logrank test indicated that acquisition curves were not significantly different. (b) Kinetics of PVL after SIV infection. RM that met controller criteria (PVL set points below 10³ copies/mL) were labeled in black and Mamu-B*17-positive SIV-infected RM were labeled in blue. (c) Peak and set-point pvls in groups A-C. Analysis represents RM demonstrating detectable viremia; n = 6–8 animals per group. Peak pvls (log values) were significantly lower in Group B RM versus Group A control RM when assessed by ANOVA and post-hoc Dunnett’s tests. Set-point pvls (log of mean values at or after week 6) significantly differed between groups by ANOVA but pairwise comparisons were not significant. Peak and set-point pvls were correlated.

Figure 5

Anti-viral antibody responses after IVAG challenge with SIVmac251. Kinetics of antibody responses to (a) Gag (p27) and (b) Env (gp130) following SIV infection in control and RhCMV/SIV-vaccinated RM. RM that met controller criteria were labeled in black and Mamu-B*17-positive RM were labeled in blue. Analysis represents RM demonstrating detectable viremia; n = 6–8 animals per group. Comparative analyses of anti-p27 antibody concentrations and anti-gp130-binding activity between all groups at 1 and 2 WPI were performed by Kruskal–Wallis test (P values shown) followed by Dunn’s multiple comparison test for pairwise comparison (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 6

Antiviral T-cell responses after IVAG challenge with SIVmac251. Cellular immune responses following SIV infection. Kinetics of SIV-Gag- and SIV-RTN-specific (a) CD4 and (b) CD8 T-cell responses in freshly isolated PBMC after SIV infection. Responses were determined by ICS for both TNF and IFN-γ expression after stimulating with overlapping peptide pools for each SIV protein. RM that met controller criteria were labeled in black and Mamu-B*17-positive RM were labeled in blue. Frequencies represent T cells stining positive for both TNF and IFN-γ expression. (c) Comparison of the SIV-specific CD4 and CD8 T-cell populations of PBMC and MNC freshly isolated from indicated tissues assessed at necropsy of SIV-infected RM. Time points for necropsy varied from 17–41 weeks (median 28 weeks) after SIV infection. Analysis represents RM that demonstrated detectable viremia; n = 6–8 animals per group. Shown in stacked bar charts are the mean frequencies (+ SEM) of responses to SIV-Gag (red) and SIV-RTN (blue). The Kruskal–Wallis test was used to determine the significance of overall differences of Gag- and RTN-specific T-cell responses, with P values shown in red and blue, respectively. If significant, the Dunn’s post test was used to determine the significance of pairwise differences (*, P < 0.05; NS, not significant).