Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys

Abstract

The latent viral reservoir is the critical barrier for the development of a cure for HIV-1 infection. Previous studies have shown direct antiviral activity of potent HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) administered when antiretroviral therapy (ART) was discontinued, but it remains unclear whether bNAbs can target the viral reservoir during ART. Here we show that administration of the V3 glycan-dependent bNAb PGT121 together with the Toll-like receptor 7 (TLR7) agonist vesatolimod (GS-9620) during ART delayed viral rebound following discontinuation of ART in simian-human immunodeficiency virus (SHIV)-SF162P3-infected rhesus monkeys in which ART was initiated during early acute infection. Moreover, in the subset of monkeys that were treated with both PGT121 and GS-9620 and that did not show viral rebound after discontinuation of ART, adoptive transfer studies and CD8-depletion studies also did not reveal virus. These data demonstrate the potential of bNAb administration together with innate immune stimulation as a possible strategy for targeting the viral reservoir.

Extended Data Figure 1.. Study design.

44 rhesus monkeys (N=11 animals/group) were infected with SHIV-SF162P3 at week 0 and initiated ART at week 1 (day 7). GS-9620 administrations and PGT121 infusions are shown from weeks 96–114. ART was discontinued at week 130.

Extended Data Figure 2.. Immune activation following GS-9620 administration and prior to ART discontinuation.

(a) Activation of CD4+ T cells was assessed by CD38 expression on days 0 and 1 following GS-9620 administration, supplementing the data shown in Fig. 2a (N=11 animals/group). Representative data are shown following the fifth GS-9620 dose, which was comparable to the other doses. Red horizontal bars indicate median values. P-values reflect 2-sided Mann-Whitney tests. (b) Plasma levels of IFN-α, IL-1RA, I-TAC, Eotaxin, MIG, MCP-1, IL-1β, IL-6, IP-10 are shown on day 1 following GS-9620 administration (N=11 animals/group). Red bars represent mean values. Combined data from all GS-9620 administrations with pre-dose levels subtracted are shown.

Extended Data Figure 3.. Anti-drug antibody (ADA) assay prior to ART discontinuation.

ADA responses were assessed in the PGT121+GS-9620 and PGT121 alone groups every 2 weeks from weeks 106–124 an electrochemoluminescence (ECL) assay with an anti-PGT121 idiotypic mAb (N=11 animals/group). No ADA was detected. One animal in the PGT121+GS-9620 group had background reactivity in this assay at week 106 prior to PGT121 exposure (green bars).

Extended Data Figure 4.. PGT121 pharmacokinetics in serum and tissues prior to ART discontinuation.

(a) Peak serum PGT121 levels are shown (limit of detection 0.5 μg/ml) 1 hour following each of 5 infusions of PGT121 (weeks 106–114) and during the washout period (weeks 114–130) (N=11 animals/group). (b) PGT121 levels (limit of detection 0.5 μg/ml) were assessed in cell lysates (L) and initial wash supernatants (S) from 10⁶ lymph node (LN) and colorectal (CR) biopsies from week 120 (N=11 animals/group). Positive controls (Ctrl) included lymph node samples from naïve monkeys spiked with PGT121. Red bars represent median values.

Extended Data Figure 5.. IFN-γ ELISPOT responses prior to ART discontinuation.

Gag-, Env-, and Pol-specific IFN-γ ELISPOT responses in PBMC are shown at (a) week 4, (b) week 96, and (c) week 120 (N=11 animals/group). Spot-forming cells (SFC) per million PBMC are shown. Red horizontal bars indicate median values. The dotted line represents the assay limit of quantitation (55 SFC/million PBMC).

Extended Data Figure 6.. IFN-γ intracellular cytokine staining (ICS) responses prior to ART discontinuation.

Gag-, Env-, and Pol-specific IFN-γ ICS responses (a) in peripheral blood mononuclear cells (PBMC) and (b) in lymph node mononuclear cells are shown at week 120 (N=11 animals/group). Percent IFN-γ producing CD8+CD3+ T cells in PBMC and percent IFN-γ producing total CD8+CD3+ T cells (CD8) and follicular CXCR5+CD8+CD3+ T cells (fCD8) in lymph node mononuclear cells are shown. Red horizontal bars indicate median values. The dotted line represents the assay limit of quantitation (0.05% CD8+CD3+ T cells).

Extended Data Figure 7.. IFN-γ ELISPOT responses following ART discontinuation and trends for viral rebound.

(a) Gag-, Env-, and Pol-specific IFN-γ ELISPOT responses in PBMC are shown at day 140 following ART discontinuation (N=11 animals/group). Spot-forming cells (SFC) per million PBMC are shown. Monkeys in each group that demonstrated viral rebound vs. no rebound are shown separately. Red horizontal bars indicate median values. The dotted line represents the assay limit of quantitation (55 SFC/million PBMC). (b) Trends for viral rebound are shown in the GS-9620 alone and the PGT121 alone groups in relation to pre-ART week 1 viral loads, supplementing the data shown in Fig. 5c. Red horizontal bars indicate median values.

Extended Data Figure 8.. CD8 depletion efficiency.

CD8+ T cells per μl peripheral blood are shown in PGT121+GS-9620 treated monkeys before and after CD8 depletion in animals that exhibited viral rebound and post-rebound virologic control (N=5, left, red lines) and in animals that exhibited no viral rebound following ART discontinuation (N=5, right, black lines).

Extended Data Figure 9.. Computational model.

(a) LASSO and PLSR model identifies the parameters that correlate with delayed viral rebound (N=11 animals/group). Left, Individual monkeys are showed distributed by latent variables 1 and 3 of the model. Timing of viral rebound is indicated by the color gradient. R² = 0.176, root mean square error (RMSE) = 0.917, P < 0.001 in 2-sided permutation tests. Middle, The contribution of the selected features to model separation is displayed in variable importance in projection (VIP) scores, related to early (blue) or late (red) viral rebound. Right, Correlation between viral rebound and latent variable 1. P-value reflects a 2-sided Spearman rank-correlation test. (b) LASSO and PLSR model identifies the parameters that correlate with reduced total viral loads (N=11 animals/group). Left, Individual monkeys are showed distributed by latent variables 1 and 2. Total viral loads are indicated by the color gradient. R² = 0.282, root mean square error (RMSE) = 0.857, P < 0.001 in in 2-sided permutation tests. Middle, The contribution of the selected features to model separation is displayed in VIP scores, related to high (blue) or low (red) total viral loads. Right, Correlation between total viral loads and latent variable 1. P-value reflects a 2-sided Spearman rank-correlation test.

Extended Data Figure 10.. In vitro killing studies.

(a) GS-9620 treatment led to CD69 upregulation of CD56+ NK cells and CD3+ T cells in vitro following incubation of human PBMC with 1,000 nM GS-9620 for 5 days (N=9). (b) GS-9620 treatment augmented PGT121-mediated killing of PGT121 in vitro. Percent p24 reduction in CD4+ T cells (N=6) using an antibody-mediated killing assay (see Methods). Percent killing was calculated as the percent reduction of p24 in CD4+ T cells with PGT121 compared with no PGT121. P-values reflect 2-sided paired Student’s t tests.

Figure 1.. Plasma viral loads following SHIV-SF162P3 infection and prior to ART discontinuation.

44 rhesus monkeys were infected with SHIV-SF162P3 at week 0 and initiated ART at week 1 (day 7) (N=11 animals/group). GS-9620 administrations and PGT121 infusions were administered from weeks 96–114. ART was discontinued at week 130. Plasma viral loads are shown for (a) weeks 0–5 and (b) weeks 0–130. Log SIV RNA copies/ml are shown (limit of detection 2.3 log RNA copies/ml). Red lines indicate median values.

Figure 2.. Cellular immune activation following GS-9620 administration and prior to ART discontinuation.

Activation of (a) CD4+ T cells and (b) NK cells in peripheral blood was assessed by CD69 expression on days 0 and 1 following GS-9620 administration (N=11 animals/group). Representative data are shown following the fifth GS-9620 dose, which was comparable to the other doses. Red horizontal bars indicate median values. P-values reflect 2-sided Mann-Whitney tests.

Figure 3.. Viral DNA prior to ART discontinuation.

Log viral DNA copies/10⁶ CD4+ T cells are shown (limit of detection 3 DNA copies/10⁶ cells) in PBMC and lymph nodes prior to the interventions (a) at week 96 prior to the interventions and (b) at week 120 following the interventions (N=11 animals/group). Red horizontal bars indicate median values. P-values reflect 2-sided Mann-Whitney tests.

Figure 4.. Viral loads following ART discontinuation.

(a) Plasma viral loads are shown for 196 days following ART discontinuation (N=11 animals/group). Log SIV RNA copies/ml are shown (limit of detection 2.3 log RNA copies/ml). Numbers and percentages of animals that show viral rebound by day 196, defined as any confirmed detectable viral load, are depicted. (b) Summary of peak and setpoint viral loads following ART discontinuation (N=11 animals/group). Red lines and horizontal bars indicate median values. P-values reflect chi-square tests of area under the curve (AUC) total viral RNA following ART discontinuation.

Figure 5.. Analysis and correlations of viral rebound.

Comparison of time to viral rebound among groups as (a) dot plots depicting raw data and (b) Kaplan-Meier curves (N=11 animals/group). (c) Correlations of viral rebound in PGT121+GS-9620 treated animals with pre-ART week 1 log viral RNA copies/ml (N=11). Red horizontal bars indicate median values. P-values reflect (a) a censored Poisson regression model of incidence rate ratios, (b) a Kruskall-Wallis test, and (c) a 2-sided Spearman rank-correlation test.

Figure 6.. Adoptive transfer and CD8 depletion studies.

(a) Plasma viral loads are shown in recipient monkeys following adoptive transfer of 30 million PBMC and LNMC from PGT121+GS-9620 treated monkeys that exhibited viral rebound and post-rebound virologic control (N=2, left, red lines) and PGT121+GS-9620 and PGT121 treated monkeys that exhibited no viral rebound following ART discontinuation (N=5, 4 from the PGT121+GS-9620 group and 1 from the PGT121 alone group, right, black lines). (b) Plasma viral loads are shown in PGT121+GS-9620 treated monkeys before and after CD8 depletion (see Methods) in animals that exhibited viral rebound and post-rebound virologic control (N=5, left, red lines) and in animals that exhibited no viral rebound following ART discontinuation (N=5, right, black lines). CD8 depletion was performed on day 196 (arrows).