Fig 1.
Experimental design and response to ART in SIV-infected infant RMs.
(A) Schematic of the study design. Sixteen infant RMs were infected orally with 105 TCID50 SIVmac251 (day 0), and starting on 4 weeks post infection (p.i.) treated with combination ART (TDF, FTC, DTG) for 15 months. Eight animals received 2 doses of Ad48-SIVsmE543 gag-pol-env (3x1010 viral particles, i.m.), 2 doses of MVA-SIVsmE543 gag-pol-env (1x108 PFU, i.m.), and 10 doses of GS-986 (0.3 mg/kg, o.g.), at the timepoints indicated (TV+TLR7). The remaining 8 animals served as ART-treated controls. At 64–71 weeks p.i. RMs underwent analytical treatment interruption (ATI) and all the animals were monitored for 4 to 6 months. PB, RB, and LN biopsies were collected at the indicated timepoints. Longitudinal analysis of plasma SIV RNA levels in (B) TV+TLR7 and (C) control RMs. The shaded area represents the period of ART treatment. The dashed line represents the limit of detection of the assay. Longitudinal analysis of peripheral CD4+ T cell frequency in (D) TV+TLR7 and (E) control RMs. The shaded area represents the period of ART treatment.
Table 1.
Experimental division of SIV-infected, ART-treated infant macaques.
Fig 2.
Cellular response measured by ELISPOT to therapeutic vaccination in SIV-infected, ART-treated infant RMs.
(A) Representative images of spot forming cells (SFC) in response to Gag, Pol, and Env at timepoints indicated above. Wpi, weeks post infection. (B) IFN-γ ELISPOT responses to Gag, Pol, and Env peptide pools from SIVmac239 were measured at week 22 prior to vaccination, week 34 after priming with two doses of Ad48, week 52 after boosting with two doses of MVA, and week 62 prior to analytical treatment interruption in control RMs (n = 4) and TV+TLR7 (n = 8) RMs. Bars represent mean ± SEM. Statistical analysis was performed to compare with week 22 using the non-parametric Friedman test with Dunn's multiple comparison test to correct for multiple comparisons. (C) Individual IFN-γ ELISPOT responses of TV+TLR7 RMs.
Fig 3.
Immunological response measured by ICS to therapeutic vaccination in SIV-infected, ART-treated infant RMs.
Pie charts depicting the ability of (A) memory CD8+ and (B) memory CD4+ T cells isolated from TV+TLR7 RMs to produce IFN-γ, IL-2 and/or TNF-α in response to stimulation with SIVmac239 Gag peptide pool at 22 (n = 5), 34 (n = 6), 52 (n = 4), and 62 (n = 7) weeks post infection (wpi). Total cytokine positive cells were compared to week 22 through permutation test. Cytokine positive subsets were compared to week 22 using a Wilcoxon Rank Sum Test, table representing P values is shown. (C) Representative flow plot of IFN-γ, TNF-α, and IL-2 expression in CD95+CD8+ T cells.
Fig 4.
Cellular breadth of immunological response following analytical treatment interruption (ATI) measured by ELISPOT to therapeutic vaccination in SIV-infected, ART-treated infant RMs.
(A) IFN-γ ELISPOT responses to 10-mer peptide subpools spanning the Gag, Pol, and Env proteins from SIVmac239 following ATI in TV+TLR7 RMs and control RMs. Bars represent median ± quartiles and the gray shading bordered by the horizontal dashed line represents the limit of detection. Statistical analysis was performed using Wilcoxon matched-pairs signed rank tests. SFC, spot forming cells. (B) Cellular immune breadth in TV+TLR7 RMs and control RMs as measured by total positive subpools of 10 peptides spanning the SIVmac239 Gag, Pol, and Env proteins following ATI. Black bar represents median. Groups were compared using a two-sided Mann-Whitney test (P < 0.05 was considered significant).
Fig 5.
Humoral response measured by ELISA and BAMA to therapeutic vaccination in SIV-infected, ART-treated infant RMs.
SIV-specific antibodies directed against gp120 from (A) vaccine strain, SIVsmE543, and (B) challenge strain, SIVmac251, were quantified by binding ELISA area under the curve (AUC) at week 4 prior to ART initiation, week 22 prior to vaccination, week 34 after priming with two doses of Ad48, week 52 after boosting with two doses of MVA, and week 62 prior to analytical treatment interruption (ATI) in TV+TLR7 RMs and equivalent time points for controls. (C) SIV-specific antibodies directed against SIVmac239 gp120, p27, and Nef were measured by Binding Antibody Multiplex Assay (BAMA) at same time points described above. Experimental groups were compared using a two-sided Mann-Whitney test and timepoints within groups were compared using the non-parametric Friedman test with Dunn's multiple comparison test to correct for multiple comparisons (P < 0.05 was considered significant).
Fig 6.
Immunological response to repeated oral GS-986 administration in SIV-infected, ART-treated infant RMs.
Frequency of (A) CD38+ and (B) CD69+ peripheral CD4+ and CD8+ T cells immediately prior to, 1 day post, and 14 days post oral GS-986 dose 1, 5, and 10 in TV+TLR7 or equivalent sample day in control RMs. (C) Frequency of CD169+ classical, intermediate, and nonclassical monocytes in TV+TLR7 and control RMs. Dose number is indicated by color. Bars represent mean ± SEM. Statistical analysis was performed using a Wilcoxon matched-pairs signed rank test.
Fig 7.
Impact of therapeutic vaccination and oral TLR-7 stimulation on SIV DNA persistence in CD4+ T cells of SIV-infected, ART-treated infant RMs.
Comparison of frequency of estimated SIV DNA levels in (A) peripheral, (B) LN, and (C) rectal CD4+ T cells before (12 weeks post infection, wpi) and after (62 wpi) therapeutic vaccine regimen in TV+TLR7 and control RMs as determined by PCR. Dashed line represents the limit of detection and open symbols represent values below the limit of detection. Statistical analysis was performed using a two-sided Wilcoxon rank-sum tests (P < 0.05 was considered significant). (D) Association between log reduction of SIV DNA in peripheral CD4+ and LN CD4+ T cells from pre-vaccination to post-vaccination and magnitude of SIV-specific T cells at week 62 prior to ART interruption measured by IFN-γ ELISPOT in TV+TLR7 RMs. Two-sided Spearman rank correlation test was used to determine statistical significance. R value indicates correlation coefficient.
Fig 8.
Influence of TV+TLR7 on time to rebound and post-rebound viremia following analytical treatment interruption (ATI) in SIV-infected, ART-treated infant RMs.
(A) Comparison of time to viral rebound in TV+TLR7 and control RMs depicted by Kaplan-Meier curves. Survival curves for groups were compared through Log-rank (Mantel-Cox) test. (B) Median longitudinal plasma SIV RNA levels in TV+TLR7 and control RMs following ATI. The solid line represents the median, the shaded area represents interquartile range, and the horizontal dashed line represents the limit of detection of the assay. (C) Set point viremia following 16 weeks of ATI of TV+TLR7 and control RMs determined by the mean of the final three viral load measurements. Dashed bars represent median. Groups were compared using a two-sided Mann-Whitney test (P < 0.05 was considered significant).
Fig 9.
Comparison of strong and weak cellular vaccine response within TV+TLR7 infant RMs.
(A) Magnitude of anti-SIV cellular immunity measured by IFN-γ ELISPOT prior to ATI at 62 weeks post infection of strong and weak responders. Dashed bars represent median. Groups were compared using a two-sided Mann-Whitney test (P < 0.05 was considered significant). (B) Pie charts depicting the ability of memory CD8+ and CD4+ T cells isolated from strong and weak TV+TLR7 responders to produce IFN-γ, IL-2 and/or TNF-α in response to stimulation with SIVmac239 Gag peptide pool at 52 weeks post infection (n = 4). (C) Longitudinal analysis of HLA-DR and PD-1 expression on effector memory (EM) and central memory (CM) CD8+ T cells in strong and weak responders within TV+TLR7 RMs (n = 8). The shaded area represents the period of ART treatment. AUC on ART was compared between groups using a two-sided Mann-Whitney test. (P < 0.05 was considered significant) Association between (D) HLA-DR expression on EM, (E) PD-1 expression on CM, and (F) PD-1 expression on EM CD8+ T cells during ART represented by AUC and magnitude of SIV-specific T cells following end of vaccine regimen prior to ATI measured by ELISPOT in TV+TLR7 RMs. Two-sided Spearman rank correlation test was used to determine statistical significance. R value indicates correlation coefficient. (G) Comparison of time to viral rebound in strong and weak TV+TLR7 responder RMs depicted by Kaplan-Meier curves. (H) Median longitudinal plasma SIV RNA levels between strong and weak responders following ATI. The solid line represents the median, the shaded area represents interquartile range, and the horizontal dashed line represents the limit of detection of the assay. (I) Set point viremia following 16 weeks of ATI of strong and weak TV+TLR7 responders determined by the mean of the final three viral load measurements. Dashed bars represent median. Groups were compared using a two-sided Mann-Whitney test (P < 0.05 was considered significant).