Blocking α4β7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies

Abstract

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α₄β₇ with an anti-α₄β₇ monoclonal antibody (Rh-α₄β₇) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α₄β₇ integrin blockade, SHIV_SF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α₄β₇ or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α₄β₇ and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α₄β₇ integrin blockade may prolong virologic control by bNAbs in SHIV_SF162P3-infected macaques.

Fig. 1.. Anti–HIV bNAbs and Rh-α₄β₇ mAb combined treatment delays rebound viremia in SHIV-infected macaques.

(A) Shown is a schematic of the study design (LN bx, lymph node biopsy). (B) Log viral RNA (copies/ml) in plasma of macaques from the time of SHIV infection and after antibody treatment is shown (black lines, intravaginal infection; colored lines, intravenous infection). The time before the bNAb infusion is shaded in gray. The top graph shows the pVLs in the bNAbs-only group; the middle graph shows the VLs in the bNAbs–Rh-α₄β₇ combined treatment group, and the bottom graph shows the VLs in control, untreated animals. (C) Kaplan-Meier curves were generated for time to first detection of plasma viral RNA >200 copies/ml in the bNAbs-only group and bNAbs–Rh-α₄β₇ group. Curves are compared with the Gehan-Breslow-Wilcoxon test (P = 0.042). (D) Shown is the median ± range of log viral RNA (copies/ml) in plasma after week 14 p.i. (earliest time of pVL rebound) for the animals in the two treatment groups.

Fig. 2.. bNAb plasma concentrations do not differ between treatment groups before viral rebound.

(A) Shown are the plasma concentrations (means ± SD) of anti–HIV bNAbs VRC07–523LS and PGT128 6 days before the first detection of >200 copies/ml of SHIV RNA in the bNAbs-only and bNAbs–Rh-α₄β₇ treatment groups. (B) Shown are the medians ± range of CD4⁺ T cell counts in blood for different time points after SHIV infection in the three groups of animals. Differences between groups were tested at each time point by Kruskal-Wallis test, and differences between time points were tested with a mixed-effect analysis using Geisser-Greenhouse correction and Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.01.

Fig. 3.. bNAbs–Rh-α₄β₇ treatment reduces viral RNA and DNA in gut tissues of SHIV-infected macaques.

(A and B) Copies of SHIV RNA (A) and SHIV DNA (B) from the indicated tissues at the time of necropsy were quantified by RT-qPCR (normalized to RNA content) and gag-qPCR (normalized to albumin), respectively. Bars represent means ± SD. The dashed line indicates the lower limit of detection of the assay. Data from the treatment groups are compared to data from the control group by Kruskal-Wallis test and the results of the Dunn’s multiple comparisons post hoc test. **P < 0.01. MLN, mesenteric lymph nodes; ING, inguinal lymph nodes; ILIAC, iliac lymph nodes.

Fig. 4.. Effects of bNAbs–Rh-α₄β₇ treatment of SHIV-infected macaques on T cells, NK cells, and B cells in different tissues.

(A) Left: tSNE plot displaying kinetics of expression of α₄β₇ on live single-cell populations in blood from SHIV-infected macaques, including four animals from the bNAbs-only group, six from the bNAbs–Rh-α₄β₇ group, and three controls. Gating of α₄β₇-positive cell populations on the tSNE plot was based on antigen expression and discrete density clustering. P1 to P6 (red areas in black circles) on the tSNE plot indicate the different immune cell populations expressing α₄β₇. Right: Heatmap depicting the fold change in expression of the antigens CD3, CD8, CD95, CD28, CD4, and CD69 for each of the immune cell populations in P1 to P6 compared to the channel values for cell populations negative for these antigens. (B and C) Shown are the frequencies of cell subsets within the CD3⁺ CD4⁺ T cell population [CD4⁺ central memory T cells (CM), CCR6⁺] (B) and the CD3⁺ CD8⁺ T cell population [CD69⁺CD8⁺ effector memory T cells (EM)] (C) among live cells isolated from PBMCs at weeks 10 and 17 p.i. Data are compared by two-way ANOVA with Bonferroni multiple comparisons correction. (D and E) Shown is the frequency of CXCR5⁺ PD1^high T follicular helper cells within the CD3⁺ CD4⁺ T cell population (D) and NKp46⁺ cells within the NKG2A⁺ CD3⁻ cell population (E) among live cells isolated from LN bx at weeks 17 or 20 p.i. (F and G) Shown are frequencies of NKp46⁺ cells (F) within the NKG2A⁺ CD3⁻ cell population and CD20⁺ B cells (G) among live cells isolated from MLN at necropsy. (H and I) Shown are frequencies of total CD20⁺ B cells and CD38⁺ IgM⁺ B cells isolated from a small portion of macaque ileum at necropsy. Data from the bNAbs-only and bNAbs–Rh-α₄β₇ treatment groups were compared directly with a Mann-Whitney nonparametric unpaired test and were compared with the control group using the Kruskal-Wallis test and the results of the Dunn’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. Bars represent means ± SD.

Fig. 5.. bNAbs–Rh-α₄β₇ treatment reduces B cell follicles and aggregates compared to bNAbs-only treatment.

(A to D) Tissue sections (5 μm) were cut from formalin-fixed MLN and ileum tissue blocks (two blocks per animal, one section per block) and were stained with anti-CD20 antibody. Digitally scanned images were analyzed with QuPath version 0.2.3, and results are expressed as a percentage of tissue area covered by B cell follicles (A and B) or lymphoid aggregates (C and D). Representative full-scale images from one tissue per treatment group are shown, with higher magnification of a lymphoid aggregate detailed in the insets (red boxes). Full image scale bars, 2 mm; inset scale bars, 250 μm. Data from the bNAbs-only and bNAbs–Rh-α₄β₇ treatment groups are compared directly with Mann-Whitney test (nonadjusted P value shown) and with the Kruskal-Wallis test and Dunn’s multiple comparisons post hoc test (shown for comparisons with the control group). *P < 0.05. Bars represent means ± SD.

Fig. 6.. bNAbs–Rh-α₄β₇ treatment increases T cell responses with no change in total anti–HIV Env antibodies.

(A) Antibody titers against the SHIV_SF162P3 gp140 trimer were measured before week 10 p.i. and after week 17 p.i. in the bNAbs-only and bNAbs–Rh-α₄β₇ treatment groups. Results are compared by two-way ANOVA with Sidak’s multiple comparisons test. (B) Shown is the frequency of CD4⁺ T cells (left) and CD8⁺ T cells (right) secreting IFN-γ, TNF-α, or both, after PBMC stimulation for 6 hours with CD28/CD49d and a HIV consensus B envelope peptide pool, a gag pool, or a pool of 15-mer overlapping peptides from the V2 loop of the SHIV_SF162P3 Env. Data from the two treatment groups are compared by Kruskal-Wallis test and the results of the Dunn’s multiple comparisons post hoc test. *P < 0.05. Bars represent means ± SD.