Combination anti-HIV-1 antibody therapy is associated with increased virus-specific T cell immunity

Abstract

Combination antiretroviral therapy (ART) is highly effective in controlling human immunodeficiency virus (HIV)-1 but requires lifelong medication due to the existence of a latent viral reservoir1,2. Potent broadly neutralizing antibodies (bNAbs) represent a potential alternative or adjuvant to ART. In addition to suppressing viremia, bNAbs may have T cell immunomodulatory effects as seen for other forms of immunotherapy3. However, this has not been established in individuals who are infected with HIV-1. Here, we document increased HIV-1 Gag-specific CD8+ T cell responses in the peripheral blood of all nine study participants who were infected with HIV-1 with suppressed blood viremia, while receiving bNAb therapy during ART interruption4. Increased CD4+ T cell responses were detected in eight individuals. The increased T cell responses were due both to newly detectable reactivity to HIV-1 Gag epitopes and the expansion of pre-existing measurable responses. These data demonstrate that bNAb therapy during ART interruption is associated with enhanced HIV-1-specific T cell responses. Whether these augmented T cell responses can contribute to bNAb-mediated viral control remains to be determined.

Conflict of interest statement

There are patents on 3BNC117 (PTC/US2012/038400) and 10-1074 (PTC/US2013/065696) that list M.C.N. as an inventor. M.C.N. is a member of the Scientific Advisory Board of Frontier Bioscience. Gilead has the rights to develop the 3BNC117 and 10-1074 antibody combination for clinical use.

Figures

Fig. 1. Increased frequency of Gag-specific T cells during ATI in bNAb-treated individuals.

a, Study design. b,c, Net frequency of cytokine+ CD8+ (b) or CD4+ cells (c) after Gag stimulation at weeks −2, 6/7, 12 and 18. Total cytokine+ cells include cells that express at least one cytokine and effector function upon Gag stimulation (CD107A, IFN-γ, MIP1-β and/or TNF-α for CD8+; CD40L, IFN-γ, IL-2 and/or TNF-α for CD4+). Net value was calculated by subtracting the frequency of cytokine+ cells detected in a DMSO control. Bars show median values. Symbols represent biologically independent samples from n = 9 (weeks –2, 6/7 and 12) and n = 7 (week 18) bNAb-treated individuals with suppressed viral load during ATI (week 18 sample was not available for individual 9244 and individual 9242 reinitiated ART after viral rebound at week 15). Lines connect data from the same donor. P values comparing responses at week 6/7, 12 or 18 versus baseline (week –2) were calculated using a paired two-tailed Wilcoxon test.

Fig. 2. Polyfunctionality of Gag-specific T cells.

T cell cytokine coexpression after HIV-1 Gag peptide pool stimulation was evaluated in bNAb + ATI individuals by ICS and analyzed using combination gates. a, Coexpression of CD107A, IFN-γ, MIP1-β and TNF-α in CD8+ T cells. b, Coexpression of CD40L, IFN-γ, IL-2 and TNF-α in CD4+ T cells. Box-and-whisker plots show median values (line), 25th to 75th percentiles (box outline) and minimum and maximum values (whiskers); n = 9 (weeks −2, 6/7 and 12) and n = 7 (week 18) biologically independent samples from bNAb-treated individuals with suppressed viral load during ATI. P values comparing responses at week 6/7, 12 or 18 versus baseline (week −2) were calculated using a paired two-tailed Wilcoxon test.

Fig. 3. AIM assay evaluation of T cell responses to multiple HIV-1 antigens.

AIM+ T cells include cells that were PD-L1+CD69+ or 4-1BB+CD69+ or PD-L1+4-1BB+. a, Net frequency of HIV-1 Gag-specific AIM+CD8+ (left, red) and CD4+ T cells (right, blue) for each bNAb + ATI individual. Net frequency of the Gag-stimulated condition was calculated by subtracting the frequency detected in a DMSO control. b, Comparison of net frequency HIV-1 Gag-specific AIM+CD8+ (left, blue) and CD4+ T cells (right, red) at week −2 and week 12. Symbols represent biologically independent samples from n = 9 bNAb + ATI individuals. Lines connect data from the same donor. Bars show median values. c, Comparison of the frequency of Gag-specific CD8+ or CD4+ T cells identified by AIM assay or ICS. The or-gate strategy was used for both assays. Symbols represent biologically independent samples from n = 9 bNAb + ATI individuals (samples obtained at week −2 and 12 for each individual were included for comparison). Lines connect data from the same donor and time point. d, Relationship between the frequency of Gag-specific CD8+ and CD4+ T cell responses identified either by AIM assay or ICS. Symbols represent biologically independent samples from n = 9 bNAb + ATI individuals (samples obtained at week −2 and 12 for each individual were included for comparison). Association was determined by Spearman correlation. e, Net frequency of HIV-1 Pol, Nef, gp120 or gp41-specific CD8+ (upper graphs) or CD4+ T cells (lower graphs) identified by AIM assay in bNAb + ATI individuals at week −2 and week 12. Symbols represent biologically independent samples from n = 9 bNAb + ATI individuals. Lines connect data from the same donor. Median values are shown as bars. P values indicated in b,c,e were calculated by a paired two-tailed Wilcoxon test.

Fig. 4. Responses to HIV-1 Gag epitopes.

PBMCs obtained at week −2 and 12 were evaluated for IFN-γ ELISpot responses to 123 peptides spanning the entire HIV-1 Gag protein (Consensus Clade B sequence). a–d, Plots showing PBMC IFN-γ ELISpot response calculated as spot-forming units (s.f.u.) per 106 PBMCs for individuals with broadened IFN-γ ELISpot response at week 12 (dark gray bars) compared to week −2 (white bars): 9244, 9246, 9252 and 9255. e, PBMC IFN-γ ELISpot responses at week −2 were plotted against week 12 responses for all nine individuals in the bNAb + ATI group. White symbols represent responses that were below the limit of detection (LOD) for both time points. Responses were considered as new (red symbols) if responses were undetectable for week −2 and detectable for week 12. Responses were considered as unchanged (light gray symbols) if number of spots did not differ by more than twofold between both time points. Responses were considered as increased (blue symbols) if number of spots for week −2 were increased by more than twofold for week 12 versus week −2. f, Doughnut chart depicting proportion of new, increased or unchanged IFN-γ ELISpot responses within all detectable responses (n = 22) of the nine individuals at week 12. g, Summary of detectable IFN-γ ELISpot responses at week 12 for all nine bNAb study participants. The LOD of 50 s.f.u. per 106 PBMCs is indicated as a dashed line in a–e.

Extended Data Fig. 1. Study participant clinical characteristics.

(a) Study participant demographics and baseline clinical data. Amer Indian: American Indian; Hisp: Hispanic; cobi: cobicistat; DTG: dolutegravir; EFV: efavirenz; EVG: elvitegravir; FTC: emtricitabine; RPV: rilpivirine; TAF: tenofovir alafenamide fumarate; TDF: tenofovir disoproxil fumarate. NNRTI-based regimens were switched four weeks before ART interruption due to longer half-lives of NNRTIs. All participants harboured clade B viruses. Viral load <20D: plasma HIV-1 RNA detected but not quantifiable by clinical assays. d0: day 0; dx: diagnosis; Scr: screening. (b) Levels of plasma HIV-1 RNA (black; left y axis) and serum concentration of 3BNC117 (red) and 10-1074 (blue, right y axis) in the 9 participants enrolled in the bNAb+ATI trial.

Extended Data Fig. 2. Frequency of Gag-specific CD4+ and CD8+ unchanged in ART-treated individuals over time.

T cell cytokine coexpression after 6h HIV-1 Gag peptide pool stimulation was evaluated by intracellular cytokine staining (ICS) in individuals on continuous ART. (a) Demographics and clinical data of ART-treated individuals. 3TC: lamivudine; ABC: abacavir; cobi: cobicistat; DRV: darunavir; DTG: dolutegravir; EFV: efavirenz; EVG: elvitegravir; FTC: emtricitabine; RAL: raltegravir; rit: ritonavir; RPV: rilpivirine; SQV: saquinavir; TAF: tenofovir alafenamide fumarate; TDF: tenofovir disoproxil fumarate. Viral load <20D: plasma HIV-1 RNA detected but not quantifiable by clinical assays. n.d.: not determined. (b) Cytokine analysis of CD8+ and CD4+ after HIV-1 Gag peptide pool stimulation at week 0 and 12. Symbols represent biologically independent samples from n=13 individuals on continuous ART. Lines connect data from the same donor. Bars show median values. P values were calculated by paired two-tailed Wilcoxon test.

Extended Data Fig. 3. Individual Gag-specific T cell responses measured by ICS.

(ab) Net frequency of total cytokine+ CD8+ (a) or CD4+ cells (b) after Gag stimulation for each individual study participant. Total cytokine+ cells include cells that express at least one cytokine/effector function upon Gag stimulation (CD107A, IFNγ, MIP1β and/or TNFα for CD8+; CD40L, IFNγ, IL-2 and/or TNFα for CD4+). Net value was calculated by subtracting frequency of total cytokine+ cells detected in a DMSO control. ND: Week 18 sample was not available for individual 9244. *9242 week 18 on ART after viral rebound at week 15.

Extended Data Fig. 4. Unchanged frequency of CMV-specific effector T cells detected by intracellular cytokine staining.

PBMCs were stimulated with CMV pp65 peptide pools for 6h and cytokine production was evaluated by ICS in bNAb+ATI individuals at week -2 and week 12. (ab) Cytokine analysis of CD8+ (a) or CD4+ T cells (b) at week -2 and week 12 after CMV pp65 stimulation. Net frequency of stimulated condition was calculated by subtracting frequency detected in a DMSO control. Symbols represent biologically independent samples from n=9 bNAb+ATI individuals. Lines connect data from the same donor. Bars show median values. P values are indicated in graphs and were calculated by paired two-tailed Wilcoxon test.

Extended Data Fig. 5. HIV-1 Gag-specific T cell responses in early rebounders with bNAb-resistant reservoir.

(a) Study participant demographics and baseline clinical data. Hisp: Hispanic; cobi: cobicistat; EVG: elvitegravir; FTC: emtricitabine; TAF: tenofovir alafenamide fumarate; TDF: tenofovir disoproxil fumarate. All participants harboured clade B viruses. d0: day 0; dx: diagnosis; Scr: screening. (b) Levels of plasma HIV-1 RNA (black; left y axis) and serum concentration of 3BNC117 (red) and 10-1074 (blue, right y axis) in the 2 participants enrolled in the bNAb+ATI trial with early rebound due to bNAb-resistant reservoir. (c) Net frequency of total cytokine+ CD8+ or CD4+ T cells after HIV-1 Gag stimulation in both individuals at weeks -2, 7 and 11 (9245), or weeks -2, 6 and 12 (9251).

Extended Data Fig. 6. No change in HLA-DR/CD38 and PD-1 expression of HIV-1-specific T cell responses.

HIV-1-specific T cell responses identified by PD-L1/CD69/4-1BB AIM assay were analyzed for surface expression of HLA-DR/CD38 and PD-1. (a) Representative plot showing expression of HLA-DR/CD38 and PD-1 on AIM+ HIV-1-specific CD8+ (upper graphs) and CD4+ T cells (lower graphs). Flow panels are representative of n=6 (CD8+) or n=8 (CD4+) biologically independent bNAb+ATI individuals. (b) Frequency of HLA-DR+CD38+ or PD-1+ of HIV-1-specific CD8+ and CD4+ T cell responses at week -2 or week 12. Symbols represent biologically independent samples from n=6 (CD8+) and n=8 (CD4+) bNAb+ATI individuals. Only samples with AIM-responses that are at least 2-fold over DMSO-stimulated control condition were analyzed for phenotype to limit the contribution of background events. Lines connect data from the same donor. Bars show median values. P values are indicated in graphs and were calculated by paired two-tailed Wilcoxon test.

Extended Data Fig. 7. Dynamics of T cell responses to multiple HIV-1 antigens in bNAb+ATI individuals.

(ab) Frequency of CD8+ (a) and CD4+ T cells (b) specific to HIV-1 Pol, Nef, gp120 or gp41 were evaluated by CD69/PD-L1/4-1BB AIM assay at weeks -2 and 12. Individuals with viral suppression >30 weeks (9254, 9255) are marked with an asterisk (*). (c) Number of increased HIV-specific CD4+ and CD8+ T cell responses in n=7 biologically independent individuals with 15-26 weeks of viral control after ATI (15-26 weeks: 9241, 9242, 9243, 9244, 9246, 9247, 9252) versus n=2 biologically independent individuals with viral control beyond 30 weeks after ATI (>30 weeks: 9254 and 9255). Number of increased HIV-specific T cell responses was calculated as the sum of single HIV antigens (Gag, Pol, Nef, gp120, gp41) for which we observed an increase at week 12 versus week -2. Bars represent median values with IQR.

Extended Data Fig. 8. PBMC IFNγ ELISpot responses were undetectable/not changed in 5 individuals.

PBMCs were evaluated for IFNγ ELISpot responses to 123 HIV-1 Gag peptides spanning the entire Gag protein. Spot forming units (SFU) were calculated as number of spots in test wells subtracted by mean number of spots in media control wells and normalized to SFU/106 PBMCs. A response was considered positive if greater than 50 SFU/106 PBMCs (=limit of detection, LOD, dashed line). (a-e) Graph representing IFNγ ELISpot responses for individuals with either undetectable (9242 (b), 9243 (c), 9254 (e)) or unchanged response (9241 (a), 9247 (d)) at week -2 (left axis, white bars) and week 12 (right axis, gray bars). (f) Number of responses above LOD for n=9 biologically independent bNAb+ATI individuals at week -2 and week 12. Lines connect data from the same donors, bars represent median values. P value was calculated by paired two-tailed Wilcoxon test.

Extended Data Fig. 9. In vitro HIV-1 inhibition assay for individuals 9252 and 9246.

PHA-activated CD4+ T cells were infected in vitro with HIV-1BaL, cultured alone or in presence with autologous CD8+ T cells for 3, 5 and 7 days, and analyzed for infection using flow cytometry. (a) Example plot showing the frequency of infected CD4+ T cells (HIV-1 Gag+ cells with down-regulated surface CD4) for bNAb+ATI individual 9252. Flow panels are representative of n=3 technical replicates. (b) Fraction of residual HIV-1 Gag+CD4- T cells after 3, 5 or 7 days of co-culture with CD8+ T cells obtained at week -2 or week 12 for n=2 biologically independent individuals (9252 and 9246) normalized to infected CD4+ T cells cultured without CD8+ T cells. (c) Fraction of residual HIV-1 Gag+CD4- T cells after 3, 5 or 7 days of co-culture with autologous CD8+ T cells for one HIV-1-uninfected control individual. Each condition was done in technical duplicates or triplicates depending on cell availability and mean values are shown.

Extended Data Fig. 10. Comparison of the circulating latent reservoir and rebound viruses.

Maximum likelihood phylogenetic trees of full-length gag sequences isolated from CD4+ T cell genomic near-full length (NFL) HIV-1 sequencing and rebound plasma SGA from participants 9241, 9242, 9243, 9244, 9246, 9247 and 9252. Open and closed black rectangles indicate NFL-derived viruses from pre-infusion (week -2) and week 12, respectively. Viruses obtained at the time of rebound are indicated by red rectangles (plasma SGA). Asterisks indicate individuals where there is at least one identical match between a gag sequence from the latent reservoir and the rebound viruses.