Identifying a minor histocompatibility antigen in mauritian cynomolgus macaques encoded by APOBEC3C

Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark malignant and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans

Antibodies used for ICS assay.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.</div

Frequency of Gag GW9-specific CD8+ T cells from post-ART SIV PTCs, blippers, and rebounders expressing exhaustion markers on ART and after ART withdrawal.

(A) Frequency of Gag GW9-specific CD8+ T cells, and (B-E) frequency of Gag GW9-specific CD8+ T cells expressing LAG3 (B), CTLA4 (C), PD1 (D), and TIGIT (E) in the PTCs (orange), blippers (teal) and rebounders (pink) during ART treatment (on ART) and 12 weeks post ART withdrawal (post ART). Results are displayed for each animal individually with the lines at the median. * P≤0.05, ** P≤0.01. P values were calculated using two-way ANOVA tests with Šídák correction for multiple comparisons. (EPS)</p

T cell gating schematic.

Representative gating strategy used to evaluate frequency, memory phenotype, and exhaustion marker expression of bulk CD4+, CD8+, and CD4+CD8+ T cells, and antigen-specific CD8+ T cells. (EPS)</p

Differential gene expression of CD8+ T cells from PTCs and rebounders on ART and after ART withdrawal.

(A) Heat map showing the mean-centered value of statistically significant differentially expressed genes between four PTCs (gray) and four rebounders (pink) on ART (time point 1, dark gray) and after ART withdrawal (time point 2, white). (B) Box plots showing the values for differentially expressed genes in (A) that are related to immune function. Genes were considered significant if they had a false discovery rate of (EPS)</p

Antibodies used for T cell phenotyping.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.</div

The frequency of CD8+ T cell subsets from post-ART SIV PTCs, blippers, and rebounders expressing CTLA4 on ART and after ART withdrawal.

Frequency of CTLA4+ (A) bulk, (B) CD8+ TCM, (C) CD8+ TTM, and (D) CD8+ TEM cells from the post-ART SIV PTCs (orange), blippers (teal), and rebounders (pink) during ART treatment (on ART) and 12 weeks post ART withdrawal (post ART). * P≤0.05, ** P≤0.01. P values were calculated using two-way ANOVA tests with Šídák correction for multiple comparisons. (EPS)</p

Experimental design, ART drug concentrations, and immunological and virological analyses.

(A) Study design depicting the timeline of SIVmac239M infection, ART initiation, ART withdrawal, SIVmac239 rechallenge, administration of the CD8a depleting antibody, and necropsy. Animals that previously received therapeutic interventions [32] are displayed as open symbols. (B) Individual plasma viral loads for the entire study. Viral loads are displayed as log10ceq/mL. (C) Plasma concentrations of DTG (left), FTC (center), and TFV (right) during ART treatment (the -4 week time point) and after ART withdrawal (4 and 12 week time points). Amounts BLQ are displayed as zero ng/mL. (D) IUPM for each animal ~19–21 weeks after ART withdrawal determined by QVOA. Square symbols denote animals where zero positive replicates were observed; posterior median estimate is displayed. (E-F) The frequency of CD8+ T cells (E) and NK cells (F) before and 10 days after administration of the CD8α depleting antibody. Results are displayed for each animal individually with the lines at the median. ** P = 0.0078. P values were calculated using Wilcoxon signed-rank tests. (G) Representative gating strategy used to evaluate CD8+ T cells (top images) and NK cells (bottom images) before (left) and 10 days after CD8α depletion (right). Animal shape graphics were created with BioRender.com.</p

Immunological changes before and after SIVmac239 rechallenge.

(A) The frequency of CD4+ T cells (left), CD8+ T cells (center), and CD4+CD8+ T cells (right) before, one week after, and six weeks after SIVmac239 rechallenge in the animals that had undetectable (circles) or detectable (triangles) viremia after rechallenge. Results are displayed for each animal individually with the lines at the median. (B) IFN-γ ELISPOT assays were performed before and one week after SIVmac239 rechallenge to assess responses to Gag386-394GW9, Nef103-111RM9, and a Gag peptide pool in the animals that had undetectable (circles) or detectable (triangles) viremia after rechallenge. Results are displayed for each animal individually with the lines at the median. Animals that previously received therapeutic interventions are displayed as open symbols. * P = 0.0286. P values were calculated using Mann-Whitney U tests. (EPS)</p