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CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption

Abstract

The primary human immunodeficiency virus (HIV) reservoir is composed of resting memory CD4+ T cells, which often express the immune checkpoint receptors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which limit T cell activation via synergistic mechanisms. Using simian immunodeficiency virus (SIV)-infected, long-term antiretroviral therapy (ART)-treated rhesus macaques, we demonstrate that PD-1, CTLA-4 and dual CTLA-4/PD-1 immune checkpoint blockade using monoclonal antibodies is well tolerated, with evidence of bioactivity in blood and lymph nodes. Dual blockade was remarkably more effective than PD-1 blockade alone in enhancing T cell cycling and differentiation, expanding effector-memory T cells and inducing robust viral reactivation in plasma and peripheral blood mononuclear cells. In lymph nodes, dual CTLA-4/PD-1 blockade, but not PD-1 alone, decreased the total and intact SIV-DNA in CD4+ T cells, and SIV-DNA and SIV-RNA in B cell follicles, a major site of viral persistence during ART. None of the tested interventions enhanced SIV-specific CD8+ T cell responses during ART or viral control after ART interruption. Thus, despite CTLA-4/PD-1 blockade inducing robust latency reversal and reducing total levels of integrated virus, the degree of reservoir clearance was still insufficient to achieve viral control. These results suggest that immune checkpoint blockade regimens targeting PD-1 and/or CTLA-4, if performed in people living with HIV with sustained aviremia, are unlikely to induce HIV remission in the absence of additional interventions.

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Fig. 1: CTLA-4 and PD-1 blockade is biologically active in SIV-infected, ART-treated RMs.
Fig. 2: T cell proliferative and effector responses are synergistically improved by dual CTLA-4/PD-1 blockade.
Fig. 3: CTLA-4 and PD-1 combined blockade induces robust viral reactivation in ART-treated, SIV-infected RMs.
Fig. 4: CTLA-4 and dual blockade, but not PD-1 blockade, reduces the total and intact SIV-DNA in LN.

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request. Sequences of gp160 env are available in GenBank (accession nos. MN856887MN857133).

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Acknowledgements

We thank S. Ehnert, C. Souder and E. Strobert (Research Resources and Veterinary Medicine) at YNPRC for providing animal and veterinary care, as well as the Emory Flow Cytometry Core. We thank C. Ashman, G. Jones, L. Anderson and A. Barnard from GSK for assisting with the preparation and quality control of the therapeutic antibodies, as well as K. Fraley, A. Mayer and G. Page from GSK for measuring antibody and anti-drug antibody levels. We also thank R. Shoemaker, K. Oswald and W. Bosche at Leidos Biomedical Research for technical assistance. The SIVmac239 strain used to infect the RMs was provided by K. Van Rompay of UC-Davis, anti-Gag tetramers were provided by D. Long at the NIH Tetramer Core Facility at Emory, and ART was supplied through ViiV Healthcare and GSK. This work was supported by the NIAID, NIH, under awards R01AI116379 and R21/R33AI116171 to M.P. and award UM1AI124436 (Emory CIAR). Support for this work was also provided by GlaxoSmithKline and Qura Therapeutics under subcontract 5105399 from the Collaboratory of AIDS Researchers for Eradication (CARE, 1UM1AI126619-01) to D.M., the Virology & Drug Discovery Core of Emory CFAR (P30AI050409), ORIP/OD awards P51OD011132 (YNPRC) and P51OD011092 (ONPRC), and in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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Authors

Contributions

J.H. contributed to conceptualization, methodology, formal analysis, investigation, writing (original draft, review and editing) and visualization. S.G. contributed to conceptualization, formal analysis, investigation and writing the original draft. C.N.C. contributed to formal analysis, investigation and visualization. H.W., C.G., S.L.M.R., J.L.R., K.N., M.N., K.B.-S., C.K., M.P., L.M., B.C., S.J., A.S. and A.A.K. contributed to investigation. E.L. and J.S. contributed to formal analysis and investigation. S.D.F. contributed to methodology and investigation. C.S.M. contributed to conceptualization and investigation. B.J. contributed to resources. H.A.-M., J.L. and J.D.E. contributed to methodology and writing (review and editing). D.M.M. contributed to methodology, resources, writing (review and editing) and funding acquisition. G.S. contributed to writing (review and editing) and funding acquisition. K.J.B. contributed to methodology, formal analysis, and writing (review and editing). D.F. contributed to conceptualization, methodology, resources, writing (review and editing), supervision and funding acquisition. M.P. contributed to conceptualization, methodology, resources, writing (original draft, review and editing), supervision and funding acquisition.

Corresponding author

Correspondence to Mirko Paiardini.

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Competing interests

S.G., C.G., J.S., A.S., B.J., A.A.K., H.A.-M. and D.F. are employed by and/or have financial interests in GlaxoSmithKline or ViiV Healthcare.

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Peer review information Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Plasma viral loads during acute SIV infection and subsequent decay during ART.

Viral loads (SIVmac239 RNA copies/mL) were quantified in plasma utilizing RT-qPCR and represented on a logarithmic scale. The terminal measurement (indicated in light red) for each animal indicates the start of ICB therapy (d0 p.t.). Initiation of ICB therapy was dependent on duration of undetectable viremia in plasma. Data sets are grouped based on ICB treatment as follows: (a) VHDUM (control; n=6), black; (b) anti-CTLA-4 mAb (αCTLA-4; n=6), blue; (c) anti-PD-1 mAb (αPD-1; n=6), pink; (d) anti-CTLA-4 plus anti-PD-1 mAb (combined blockade; n=7), red; and (e) anti-CTLA-4/PD-1 mAbdAb (BsAb; n=8), purple. Individual RMs are indicated by shape with closed data points indicating viral reactivation in plasma during subsequent ICB. The vertical dotted line indicates ART initiation (d60 p.i.) and the shaded gray area represents ongoing ART administration. The horizontal dotted line indicates the assay’s limit of detection (≤60 copies/mL) with undetectable events represented as 30 copies/mL.

Extended Data Fig. 2 Macaque characteristics and ICB antibodies.

aAge in months at SIV infection (d0 p.i.; post-infection) and at ICB (d0 p.t.; post-treatment). bIndicates the number of CD4+ T cells per μL of PB at chronic infection prior to ART initiation (d52 p.i.) as determined by complete blood counts combined with flow cytometry. cSIV-RNA viral loads per mL of plasma was measured by quantitative RT-qPCR with a limit of detection of ≤60 copies/mL at acute (d14 p.i) and chronic infection (d52 p.i.). dDays of undetectable (UD) viral load prior to ICB treatment were calculated from the first UD observation following ART initiation and does not account for subsequent blips. eIndicates whether Fc gamma receptor has been disabled by a LAGA mutation (L235A and G237A) and is capable of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), ect. fIndicates the pharmacological generic equivalent of the variable domain and epitope specificity.

Extended Data Fig. 3 Blockade pharmacokinetics and emergence of anti-drug antibodies.

(a) Plasma concentrations (ng/mL) of Co-IR blocking monoclonal antibodies (mAb) were quantified with an antibody capture assay; stratified by treatment group; and represented on a logarithmic scale. Blood was drawn immediately prior to- and 5 mins following ICB administration. (b) Using only blood drawn prior to ICB infusion, the levels of anti-drug antibodies (ECL, electrochemiluminescence) were quantified by MSD assay, which were utilized to calculate the percentage of inhibition. Vertical arrows and dotted lines represent either an ICB infusion or ART interruption, as indicated, with the gray shaded area representing ongoing ART. Individual RMs are indicated by shape with closed data points indicating animals with viral reactivation in plasma.

Extended Data Fig. 4 Combined blockade leads to a limited restoration of cytolytic functionality in ICR-expressing CD8+ T cell subsets.

By flow cytometry, the frequencies of granzyme B+ cells were determined within (a) CTLA-4+PD-1+ and (b) CTLA-4+PD-1- memory CD8+ T cells in LN following the fourth ICB infusion (d28 p.t.). All RMs are color-coded and grouped based on ICB therapy with population sizes as indicated for all analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. Averaged data are presented as the mean ± s.e.m. and were analyzed with a two-sided Kruskal-Wallis test with Dunn’s correction for multiple comparisons relative to controls.

Extended Data Fig. 5 SIV-specific CD8+ T cells display an exhausted phenotype that is not rescued by ICB during suppressive ART, but by viral rebound following ATI.

Within memory CD8+ T cells at the on-ART, pretreatment baseline (d-29 p.t.; n=5 ICB-treated, Mamu-A*01+ RMs) the frequencies of exhaustion, proliferative and activation biomarkers (as indicated above) were quantified via flow cytometry in SIV-specific and non-specific subsets, as assessed by anti-Gag-CM9 staining (as indicated below) in (a) LN and (b) PBMCs. Likewise, within LN SIV-specific memory CD8+ T cells the frequencies of (c) Ki-67+, (d) HLA-DR+CD38+, (e) T-bet+, and (f) Granzyme B+ cells were measured at the pre-ICB baseline and following the fourth ICB infusion (d28 p.t.). Likewise, the frequencies of (g) Ki-67+, (h) HLA-DR+CD38+, (i) T-bet+, and (j) Granzyme B+ cells within SIV-specific memory CD8+ T cells in PBMCs were quantified at the pre-ICB baseline, following the third ICB treatment (d21 p.t.), and 15 days following ART interruption (d50 p.t.). Analyses were conducted in up to 6 Mamu-A*01+ RMs (g,h,i,j), of which 5 were ICB-treated (a,b,c,d,e,f). Each data point represents an individual animal, as indicated by shape, and those with ICB-related viral reactivation in plasma are represented as closed data points. Averaged data are presented as the mean ± s.e.m. and were analyzed with (a,b) a one-way, pair-wise ANOVA using a Bonferroni correction for multiple corrections.

Extended Data Fig. 6 SIV-specific CD8+ T cell responses are not enhanced by blockade of PD-1 and/or CTLA-4 during long-term ART.

In 5 Mamu-A*01+, ICB-treated RMs, the frequency of SIV-specific cells, as determined via anti-Gag-CM9 tetramer staining with flow cytometry, were measured in (a) LN and (b) PBMC memory CD8+ T cells at treatment baseline (d-29 p.t.) and following ICB (d21 or 28 p.t.). The number of interferon gamma (IFN-γ) spot forming units (SFU) per 106 mononuclear cells were quantified by ELISpot upon SIV-Gag stimulation in (c) PBMCs as a fold-change (d21 p.t. relative to d-29 p.t.) and (d) cross-sectionally in LN (d21 p.t). Points represent unique animals, as indicated by shape and fill, as an average of three replicates with DMSO background subtracted. All RMs are color-coded and grouped based on ICB therapy as follows with population sizes as indicated for all ELISpot analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=5), pink; combined blockade (n=7), red; and BsAb (n=8), purple. From ex vivo SIV-Gag-stimulated PBMCs with unstimulated background subtracted, the distribution of cytokine co-expression was determined via flow cytometry in memory (e) CD4+ and (f) CD8+ T cells before (d-29 p.t.) and during (d21 p.t.; indicated at left) ICB administration (indicated above). Rainbow-colored inner pie wedges represent each Boolean combination of cytokine co-expression (annotated at far right); whereas, the stacked, outer concentric rings overlap with pie wedges that are positive for that cytokine (indicated at middle right): IFN-γ+, red; IL-2+, orange; TNFα+, green; and CD107a+, purple. Population sizes for all cytokine analyses are as follows: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. Averaged data are presented as the mean ± s.e.m., and were analyzed with a two-sided (c,d) Mann-Whitney U test or (e,f) a Permutation test.

Extended Data Fig. 7 Combined blockade increases the ratio of cell-associated SIV-RNA relative to SIV-DNA content in PBMCs and limit intact provirus in LN CD4+ T cells.

(a) The number of log-transformed cell-associated SIV-DNA copies per 106 PBMCs were quantified by 12 replicate reaction RT-qPCR prior to the first infusion (d0 p.t.), and 1 week following the first (d7 p.t.) and fourth infusions (d28 p.t.); from which (b) the ratio of SIV-RNA to -DNA copies was computed. All RMs are color-coded and grouped based on ICB therapy as follows with population sizes as indicated for all RT-qPCR analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. By IPDA with ddPCR, (c) the DNA shearing index pre- and post-ICB, and (d) the fold change in intact provirus per 106 cells relative to pre-treatment baseline were quantified in LN CD4+ cells. For all IPDA analyses, population sizes are as follows: control (n=3), αCTLA-4 (n=6), αPD-1 (n=5), combined blockade (n=7), and BsAb (n=7). (e) An example IPDA plot is given for the amplification of the env relative response element (RRE) on VIC dye against pol on FAM dye in relative fluorescence units (RFU). A positivity cutoff of 1250 is used for env and 2000 for pol. Sample plots shown (as indicated in red) are as follows: negative control, merged analysis from no-template control and PBMCs at pre-infection from RLt16; env positive control, env gBlock (IDT); pol positive control, pol gBlock (IDT); and IPDA (shown are LN CD4+ cells at d-29 p.t. from RWs16; 1 of 58 image sets of 8 replicate reactions). Averaged data are presented as the mean ± s.e.m. Each data point represents an individual animal, as indicated by shape, and those with ICB-related viral reactivation in plasma are represented as closed data points. Data were analyzed with a two-sided (a,b) Wilcoxon matched-pairs signed rank test, (d) a Mann-Whitney U test, or (c) a two-way ANOVA with Bonferroni’s correction for multiple comparisons relative to baseline and controls.

Extended Data Fig. 8 ICB does not differentially impact viral reservoir content between lymphoid and myeloid lineages, and vDNA content correlates with intact proviral genomes.

(a) The number of LN vRNA+ cells per 105 cells were quantified by RNAscope before (d-29) and after ICB therapy (d28 p.t.). All RMs are color-coded and grouped based on ICB therapy as follows with population sizes as indicated for RNAscope analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. In the LN B cell follicle (BCF) and T cell zone (TCZ), the number of (b) vDNA or (c) vRNA cells per 105 cells were measured based on their co-expression of CD3 (lymphoid) or CD68/CD163 (MØ; myeloid) in a subset of ICB-responsive RMs (n=19). (d) In situ immunofluorescence staining for CD3 (green), CD68/CD163 (blue), and DAPI (grey) combined with hybridization for SIV vDNA (red) in LN TCZ or BCF (annotated at upper right) prior to or following ICB in representative RMs (3 of 38 unique samples; up to two tissue sections were analyzed). vDNA positive cells are annotated with a white arrow and successive magnification within the indicated boxed area (white) are shown below. (e) From LN at d28 p.t., the log-transformed frequency of vDNA+ cells per 106 cells were correlated against the contemporaneous frequency of log-transformed intact proviral genomes per 106 CD4+ cells (n=29). Averaged data are presented as the mean ± s.e.m. and were analyzed with (a,b,c) a two-sided Wilcoxon matched-pairs signed rank test relative to baseline or (e) a Pearson correlation coefficient.

Extended Data Fig. 9 Dual CTLA-4/PD-1 blockade does not control or delay viral rebound after ATI.

The delay in viral rebound by ICB was analyzed as the average number of days until detectable viral loads were observed in plasma with RT-qPCR. Plasma viral loads following ATI are shown for individual RMs by ICB: (b) control, (c) αCTLA-4, (d) αPD-1, (e) combined blockade, and (f) BsAb. Mamu-A*01+ RMs are shown with a bold connecting line and animals with prior ICB-related viral reactivation in plasma are represented as closed data points. The dotted horizontal line indicates the PCR assay limit of detection, with undetectable events plotted as 30 copies/mL. (g) Plasma viral loads following ATI were averaged by ICB and are shown with the mean as the solid line in bold and the s.e.m. represented by the color-matched shaded region. (h) The attenuation in set-point plasma viremia was analyzed between chronic infection (d52 p.i.) and following rebound (d170 post-ATI, p.A.). Population sizes are as follows for all viral load and rebound delay analyses: controls (n=6), black; αCTLA-4 (n=6), blue; αPD-1 (n=6), pink; combined blockade (n=7), red; and BsAb (n=8), purple. (i) By 12 replicate reaction RT-qPCR, the number of cell-associated SIV-RNA copies per 106 cells was quantified in axillary LN at necropsy: controls (n=6), αCTLA-4 (n=6), αPD-1 (n=5), combined blockade (n=6), and BsAb (n=7). Averaged data are presented as the mean ± s.e.m., and were analyzed with a two-sided (g) two-way ANOVA with Dunnett’s correction for multiple comparisons or (a,h,i) a Mann-Whitney U test and/or Wilcoxon ranked sign test. Color-coded statistical asterisks indicate significance between control animals and the treatment group of that color. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Extended Data Fig. 10 Phylogenetic trees of gp160 env sequences from plasma.

Single genome sequencing-derived gp160 env sequences from the plasma of 9 RMs are displayed in maximum-likelihood phylogenetic trees. Virus obtained from different experimental phases are as follows: blue, SIVmac239 inoculum; black, chronic infection prior to ART initiation (d52 p.i.); red, during on-ART ICB-mediated reactivation (d7-28 p.t.); and green, during acute rebound post-ATI. A scale bar indicating diversity for each phylogenetic tree is indicated below and lineage determinations are annotated for RUm16 (at right). Lineages were deemed distinct if they differed at 3 or more nucleotide positions over env.

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Harper, J., Gordon, S., Chan, C.N. et al. CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption. Nat Med 26, 519–528 (2020). https://doi.org/10.1038/s41591-020-0782-y

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