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.

Characterization of Mauritian Cynomolgus Macaque FcγR Alleles Using Long-Read Sequencing.

The FcγRs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. FcγRs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize FcγR diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM FcγR genes. We also developed a high-throughput FcγR genotyping assay, which we used to determine allele frequencies and identify FcγR haplotypes in more than 500 additional MCMs. We found three alleles for FcγR1A, seven each for FcγR2A and FcγR2B, and four for FcγR3A; these segregate into eight haplotypes. We also assessed whether different FcγR alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of FcγR responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies.

Pegivirus avoids immune recognition but does not attenuate acute-phase disease in a macaque model of HIV infection.

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis-as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses-suggesting that HPgV's protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses-an understudied group of viruses with a high prevalence in the global human population-and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.

Improved full-length killer cell immunoglobulin-like receptor transcript discovery in Mauritian cynomolgus macaques.

Killer cell immunoglobulin-like receptors (KIRs) modulate disease progression of pathogens including HIV, malaria, and hepatitis C. Cynomolgus and rhesus macaques are widely used as nonhuman primate models to study human pathogens, and so, considerable effort has been put into characterizing their KIR genetics. However, previous studies have relied on cDNA cloning and Sanger sequencing that lack the throughput of current sequencing platforms. In this study, we present a high throughput, full-length allele discovery method utilizing Pacific Biosciences circular consensus sequencing (CCS). We also describe a new approach to Macaque Exome Sequencing (MES) and the development of the Rhexome1.0, an adapted target capture reagent that includes macaque-specific capture probe sets. By using sequence reads generated by whole genome sequencing (WGS) and MES to inform primer design, we were able to increase the sensitivity of KIR allele discovery. We demonstrate this increased sensitivity by defining nine novel alleles within a cohort of Mauritian cynomolgus macaques (MCM), a geographically isolated population with restricted KIR genetics that was thought to be completely characterized. Finally, we describe an approach to genotyping KIRs directly from sequence reads generated using WGS/MES reads. The findings presented here expand our understanding of KIR genetics in MCM by associating new genes with all eight KIR haplotypes and demonstrating the existence of at least one KIR3DS gene associated with every haplotype.

Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication.

Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute "pre-peak" phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque 'elite controllers' with a short-course of dextran sulfate sodium (DSS)-stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host's capacity to contain infection.

Conditional Immune Escape during Chronic Simian Immunodeficiency Virus Infection.

UNLABELLED: Anti-HIV CD8 T cells included in therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Identifying the epitopes that do not accumulate variants but retain immunogenicity depends on both host major histocompatibility complex (MHC) genetics and the likelihood for an epitope to tolerate variation. We previously found that immune escape during acute SIV infection is conditional; the accumulation of mutations in T cell epitopes is limited, and the rate of accumulation depends on the number of epitopes being targeted. We have now tested the hypothesis that conditional immune escape extends into chronic SIV infection and that epitopes with a preserved wild-type sequence have the potential to elicit epitope-specific CD8 T cells. We deep sequenced simian immunodeficiency virus (SIV) from Mauritian cynomolgus macaques (MCMs) that were homozygous and heterozygous for the M3 MHC haplotype and had been infected with SIV for about 1 year. When interrogating variation within individual epitopes restricted by M3 MHC alleles, we found three categories of epitopes, which we called categories A, B, and C. Category B epitopes readily accumulated variants in M3-homozygous MCMs, but this was less common in M3-heterozygous MCMs. We then determined that chronic CD8 T cells specific for these epitopes were more likely preserved in the M3-heterozygous MCMs than M3-homozygous MCMs. We provide evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are homozygous for a set of MHC alleles are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. IMPORTANCE: Anti-HIV CD8 T cells that are part of therapeutic treatments will need to target epitopes that do not accumulate escape mutations. Defining these epitope sequences is a necessary precursor to designing approaches that enhance the functionality of CD8 T cells with the potential to control virus replication during chronic infection or after reactivation of latent virus. Using MHC-homozygous and -heterozygous Mauritian cynomolgus macaques, we have now obtained evidence that epitopes known to escape from chronic CD8 T cell responses in animals that are MHC homozygous are preserved and retain immunogenicity in a host that is heterozygous for the same MHC alleles. Importantly, our findings support the conditional immune escape hypothesis, such that the potential to present a greater number of CD8 T cell epitopes within a single animal can delay immune escape in targeted epitopes. As a result, certain epitope sequences can retain immunogenicity into chronic infection.

Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection.

Human pegivirus (HPgV)-formerly known as GB virus C and hepatitis G virus-is a poorly characterized RNA virus that infects about one-sixth of the global human population and is transmitted frequently in the blood supply. We create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly variable acute phase of SPgV infection, showing that the viral load trajectory early in infection is dependent on the infecting dose, whereas the chronic-phase viremic set point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection-days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing most of the virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.

Whole genome sequencing of SIV-infected macaques identifies candidate loci that may contribute to host control of virus replication.

BACKGROUND: A small percentage of human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected macaques control virus replication without antiretroviral treatment. The major determinant of this control is host expression of certain major histocompatibility complex alleles. However, this association is incompletely penetrant, suggesting that additional loci modify the major histocompatibility complex's protective effect. Here, to identify candidate control-modifying loci, we sequence the genomes of 12 SIV-infected Mauritian cynomolgus macaques that experienced divergent viral load set points despite sharing the protective M1 major histocompatibility complex haplotype. RESULTS: Our genome-wide analysis of haplotype-level variation identifies seven candidate control-modifying loci on chromosomes 2, 3, 7, 8, 9, 10, and 14. The highest variant density marks the candidate on chromosome 7, which is the only control-modifying locus to comprise genes with known immunological function. Upon closer inspection, we found an allele for one of these genes, granzyme B, to be enriched in M1(+) controllers. Given its established role as a cytotoxic effector molecule that participates in CD8-mediated killing of virus-infected cells, we test the role of variation within gzmb in modifying SIV control by prospectively challenging M1(+) granzyme B-defined macaques. CONCLUSIONS: Our study establishes a framework for using whole genome sequencing to identify haplotypes that may contribute to complex clinical phenotypes. Further investigation into the immunogenetics underlying spontaneous HIV control may contribute to the rational design of a vaccine that prevents acquired immune deficiency syndrome.

Rapid, repeated, low-dose challenges with SIVmac239 infect animals in a condensed challenge window.

BACKGROUND: Simian immunodeficiency virus (SIV) infection of nonhuman primates is the predominant model for preclinical evaluation of human immunodeficiency virus (HIV) vaccines. These studies frequently utilize high-doses of SIV that ensure infection after a single challenge but do not recapitulate critical facets of sexual HIV transmission. Investigators are increasingly using low-dose challenges in which animals are challenged once every week or every two weeks in order to better replicate sexual HIV transmission. Using this protocol, some animals require over ten challenges before SIV infection is detectable, potentially inducing localized immunity. Moreover, the lack of certainty over which challenge will lead to productive infection prevents tissue sampling immediately surrounding the time of infection. FINDINGS: Here we challenged Mauritian cynomolgus macaques with 100 50% tissue culture infectious doses (TCID50) of SIVmac239 intrarectally three times a day for three consecutive days. Ten of twelve animals had positive plasma viral loads after this challenge regimen. CONCLUSIONS: This approach represents a straightforward advance in SIV challenge protocols that may avoid induction of local immunity, avoid inconsistent timing between last immunization and infection, and allow sampling immediately after infection using low-dose challenge protocols.

Discovery and full genome characterization of a new SIV lineage infecting red-tailed guenons (Cercopithecus ascanius schmidti) in Kibale National Park, Uganda.

BACKGROUND: Human immunodeficiency virus (HIV) type 1 and 2, the causative agents of acquired immunodeficiency syndrome (AIDS), emerged from African non-human primates (NHPs) through zoonotic transmission of simian immunodeficiency viruses (SIV). Among African NHPs, the Cercopithecus genus contains the largest number of species known to harbor SIV. However, our understanding of the diversity and evolution of SIVs infecting this genus is limited by incomplete taxonomic and geographic sampling, particularly in East Africa. In this study, we screened blood specimens from red-tailed guenons (Cercopithecus ascanius schmidti) from Kibale National Park, Uganda, for the presence of novel SIVs using unbiased deep-sequencing. FINDINGS: We describe and characterize the first full-length SIV genomes from wild red-tailed guenons in Kibale National Park, Uganda. This new virus, tentatively named SIVrtg_Kib, was detected in five out of twelve animals and is highly divergent from other Cercopithecus SIVs as well as from previously identified SIVs infecting red-tailed guenons, thus forming a new SIV lineage. CONCLUSIONS: Our results show that the genetic diversity of SIVs infecting red-tailed guenons is greater than previously appreciated. This diversity could be the result of cross-species transmission between different guenon species or limited gene flow due to geographic separation among guenon populations.

Technical advance: liposomal alendronate depletes monocytes and macrophages in the nonhuman primate model of human disease.

Nonhuman primates are critical animal models for the study of human disorders and disease and offer a platform to assess the role of immune cells in pathogenesis via depletion of specific cellular subsets. However, this model is currently hindered by the lack of reagents that safely and specifically ablate myeloid cells of the monocyte/macrophage Lin. Given the central importance of macrophages in homeostasis and host immunity, development of a macrophage-depletion technique in nonhuman primates would open new avenues of research. Here, using LA at i.v. doses as low as 0.1 mg/kg, we show a >50% transient depletion of circulating monocytes and tissue-resident macrophages in RMs by an 11-color flow cytometric analysis. Diminution of monocytes was followed rapidly by emigration of monocytes from the bone marrow, leading to a rebound of monocytes to baseline levels. Importantly, LA was well-tolerated, as no adverse effects or changes in gross organ function were observed during depletion. These results advance the ex vivo study of myeloid cells by flow cytometry and pave the way for in vivo studies of monocyte/macrophage biology in nonhuman primate models of human disease.

Acute phase CD8+ T lymphocytes against alternate reading frame epitopes select for rapid viral escape during SIV infection.

CD8+ T Lymphocytes (CTL) can control AIDS virus replication. However, natural selection favoring viral variants that escape CTL recognition is a common feature of both simian immunodeficiency virus (SIV) infection of macaques and HIV infection of humans. Emerging data indicate that CTL directed against alternate reading frame (ARF)-derived epitopes (a.k.a. cryptic epitopes) are important components of the total virus-specific response in SIV and HIV infection but the contributions of these responses during the critical first several weeks of infection have not been determined. We used a focused deep sequencing approach to examine acute phase viral evolution in response to CTL targeting two polypeptides encoded by ARFs of SIVmac239 in SIV-infected rhesus macaques. We report high magnitude CTL responses as early as three weeks post-infection against epitopes within both ARFs, which both overlap the 5' end of the env gene. Further, mutations accumulated in the epitopes by three to four weeks post infection consistent with viral escape. Interestingly, these mutations largely maintained the primary amino acid sequence of the overlapping Envelope protein. Our data show that high frequency CTL target cryptic epitopes and exert selective pressure on SIV during the acute phase, underscoring the importance of these unique immune responses.

Vaccine-induced CD8+ T cells control AIDS virus replication.

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.

Specific CD8+ T cell responses correlate with control of simian immunodeficiency virus replication in Mauritian cynomolgus macaques.

Specific major histocompatibility complex (MHC) class I alleles are associated with an increased frequency of spontaneous control of human and simian immunodeficiency viruses (HIV and SIV). The mechanism of control is thought to involve MHC class I-restricted CD8(+) T cells, but it is not clear whether particular CD8(+) T cell responses or a broad repertoire of epitope-specific CD8(+) T cell populations (termed T cell breadth) are principally responsible for mediating immunologic control. To test the hypothesis that heterozygous macaques control SIV replication as a function of superior T cell breadth, we infected MHC-homozygous and MHC-heterozygous cynomolgus macaques with the pathogenic virus SIVmac239. As measured by a gamma interferon enzyme-linked immunosorbent spot assay (IFN-γ ELISPOT) using blood, T cell breadth did not differ significantly between homozygotes and heterozygotes. Surprisingly, macaques that controlled SIV replication, regardless of their MHC zygosity, shared durable T cell responses against similar regions of Nef. While the limited genetic variability in these animals prevents us from making generalizations about the importance of Nef-specific T cell responses in controlling HIV, these results suggest that the T cell-mediated control of virus replication that we observed is more likely the consequence of targeting specificity rather than T cell breadth.

Escape from CD8(+) T cell responses in Mamu-B*00801(+) macaques differentiates progressors from elite controllers.

A small number of HIV-infected individuals known as elite controllers experience low levels of chronic phase viral replication and delayed progression to AIDS. Specific HLA class I alleles are associated with elite control, implicating CD8(+) T lymphocytes in the establishment of these low levels of viral replication. Most HIV-infected individuals that express protective HLA class I alleles, however, do not control viral replication. Approximately 50% of Mamu-B*00801(+) Indian rhesus macaques control SIVmac239 replication in the chronic phase in a manner that resembles elite control in humans. We followed both the immune response and viral evolution in SIV-infected Mamu-B*00801(+) animals to better understand the role of CD8(+) T lymphocytes during the acute phase of viral infection, when viral control status is determined. The virus escaped from immunodominant Vif and Nef Mamu-B*00801-restricted CD8(+) T lymphocyte responses during the critical early weeks of acute infection only in progressor animals that did not control viral replication. Thus, early CD8(+) T lymphocyte escape is a hallmark of Mamu-B*00801(+) macaques who do not control viral replication. By contrast, virus in elite controller macaques showed little evidence of variation in epitopes recognized by immunodominant CD8(+) T lymphocytes, implying that these cells play a role in viral control.

CD8+ T cell escape mutations in simian immunodeficiency virus SIVmac239 cause fitness defects in vivo, and many revert after transmission.

Virus-specific CD8(+) T lymphocytes select for escape mutations in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). To assess the effects of these mutations on viral fitness, we introduced escape mutations into 30 epitopes (bound by five major histocompatibility complex class I [MHC-I] molecules) in three different viruses. Two of these MHC-I alleles are associated with elite control. Two of the three viruses demonstrated reduced fitness in vivo, and 27% of the introduced mutations reverted. These findings suggest that T cell epitope diversity may not be such a daunting problem for the development of an HIV vaccine.

Reduction of CD4+ T cells in vivo does not affect virus load in macaque elite controllers.

A small percentage of human immunodeficiency virus (HIV)- and simian immunodeficiency virus (SIV)-infected individuals spontaneously control virus replication. The majority of these elite controllers mount high-frequency virus-specific CD4(+) T cell responses. To evaluate the role these responses might play in viral control, we depleted two elite controller macaques of CD4(+) cells. SIV-specific CD4(+) T cell responses did not return to baseline levels until 8 weeks postdepletion. Viral loads remained stable throughout the experiment, suggesting that SIV-specific CD4(+) T cell responses may not play a direct role in controlling chronic viral replication in these elite controllers.