Mucosal-associated invariant T (MAIT) cells are donor unrestricted T cells capable of both antigen-specific adaptive responses and cytokine driven innate-like functions. Although human MAIT cells uniformly express RORγt and IL23R, they generally produce IFN-γ, and only a small fraction produces IL-17. Recent studies show that combined TCR and cytokine stimulation can elicit functional heterogeneity in blood-derived MAIT cells. Here, we investigate the role of IL-23/IL-23R signaling in mediating the function and transcriptional profiles of lung MAIT cell clones. We demonstrate that BAL-derived lung MAIT cell clones exhibit distinct cytokine profiles and variable IL23R expression. Short-term IL-23 stimulation triggers clone-specific transcriptional programs and IL23R-dependent upregulation of type 17-associated genes. Prolonged conditioning of lung MAIT cell clones with TCR (5-OP-RU) and cytokine (IL-23) stimulation induces stable IL-17A production along with unique transcriptional changes. TCR + IL-23 conditioning alone upregulates clone-specific and shared cytoskeletal/structural gene programs, whereas subsequent PMA/Ionomycin stimulation further induces IL-12 family signaling and metabolic genes. Together, these findings demonstrate that IL23R expression and TCR signaling are required for IL-17A production, highlighting that these conditions may be met in tissue environments where MR1-specific antigens and proinflammatory cytokines coexist.

Abstract Tuberculosis (TB) remains the leading infectious cause of death worldwide, yet immunological protection against Mycobacterium tuberculosis (Mtb) remain poorly understood. We performed integrated single-cell transcriptomics and functional T-cell cloning on paired bronchoalveolar lavage (BAL) and peripheral blood samples from those exposed to TB, using IGRA and PET-CT to study potentially protective responses across the spectrum of TB infection/disease. Recent Mtb exposure alone was sufficient to remodel the pulmonary T-cell compartment. Longitudinal follow-up demonstrated that all participants who progressed to active TB had baseline PET-CT abnormalities, showing that PET-CT scan can be used to find those at high risk of progression. Furthermore, those who were PET-positive, but did not progress to active TB provide a natural model for protection, and exhibited enrichment of pulmonary cytotoxic CD8-associated T cells, identifying a candidate protective immune program. TAR-seq identified a discrete population of clonally expanded Mtb-responsive T cells and enabled direct linkage of antigen-responsive TCRs to transcriptional state through T cell cloning. Functional T-cell cloning further established a framework connecting antigen specificity, TCR sequence, and pulmonary cell state. Together, these findings provide a comprehensive atlas of human pulmonary T-cell immunity across the spectrum of TB, identify pulmonary immune features associated with durable control of infection, and establish an integrated platform for defining protective T-cell responses to inform next-generation TB vaccine development.

People living with HIV (PLWH) on suppressive antiretroviral therapy (ART) can face non-AIDS complications, partially driven by chronic immune activation. To define immune perturbations during ART-suppressed viral infection, we performed longitudinal single-cell transcriptomic and plasma proteomic analysis of rhesus macaques infected with SIVmac239M and ART-treated for 70 weeks. We identified broad, bi-phasic immune changes. Acute infection involves an interferon-driven signature, correlated with viral replication, that largely resolves with viral control. Cell-associated virus correlated with interferon-stimulated genes in most tissues; however, this was blunted in gut-associated lymph nodes, a feature that may contribute to reservoir persistence. Separate alterations manifest 54-66 weeks-post-infection, after 40 weeks of viral suppression, including broad TGF-β and NF-kB signaling and discrete bursts of inflammatory monocytes, largely restricted to bone marrow. These data highlight the biphasic remodeling of long-term ART-suppressed HIV, identifying specific tissues and cell populations with dysregulation, with implications for the treatment of PLWH.

Somatic gene editing therapies offer the potential for lasting treatments for a range of genetic disorders, with a host of therapies at various stages of clinical testing. The development of safe, effective therapies required relevant pre-clinical animal models capable of measuring on-target delivery and detecting off-target editing. Here we present the creation of a transgenic rhesus macaque encoding the Bivalent Enhanced Traffic Light Editing (BETLE) system, a multi-color fluorescent reporter that provides a simple readout for multiple modes of gene editing, including off-target effects. We used zygote microinjection and piggyBac transposase to generate preimplantation embryos that ultimately led to a healthy transgenic macaque. Extensive molecular analyses demonstrated single-copy integration of the intact reporter; however, the animal was mosaic, with only 5-8% of cells encoding the reporter, although all germ layers contained positive cells. The creation of this macaque provides important information to improve future transgenics, and progeny from this macaque could provide a powerful tool to evaluate the delivery and accuracy of gene therapies.

IntroductionThe functional responses of antigen (Ag) specific T cells are complex, clonotype-specific, context-dependent, and incompletely captured by dominant analytic approaches. Here we present T Cell Antigenic Recognition Sequencing (TAR-seq), a two-part process that uses single-cell RNA + T cell receptor sequencing (scRNA/TCR-seq) to provide a precise and comprehensive characterization of the differentiation state and functional response of T cells specific to a given antigen, ex vivo and in vivo.MethodsMany approaches use stimulation and activation-induced markers to identify Ag-specific T cells. We extend this by adding an scRNA/TCR-seq readout to identify the responding TCRs for each subject. We performed rigorous validation using tetramer-sorted T cells and developed a reusable probabilistic model to differentiate TCR-stimulated cells from bystander activation. These TCRs provide molecular barcodes to identify Ag-specific cells from unsorted scRNA/TCR-seq data, irrespective of stimulation, enabling a comprehensive view of their activity.ResultsWe applied this method to a cohort of SIV-vaccinated rhesus macaques (RMs). We performed SIV-infected cell recognition assays to identify the SIV-specific TCRs for each RM. We used these TCRs to subset SIV-specific cells and perform precise contrasts. Despite identical vaccination, we identified clone- and subject-level variability in their cytotoxic differentiation and cytokine production after incubation with SIV-infected targets. Per clonotype, the percentage of cells that responded after antigen exposure varied widely. Finally, we used TCRs as barcodes to precisely map the in vivo activity of CD8+ T cells 48-120H after SIV challenge. Precise TCR-based identification separated Ag-specific and bystander effects, measuring tissue-specific responses of SIV-specific T cells within 48 hours of infection.DiscussionTogether, our data validate a powerful method to comprehensively understand T cell activity, with relevance to infectious disease, cancer, and autoimmunity, across species.

The common marmoset (Callithrix jacchus), a small monkey native to Brazil, has been used as a biomedical model in the United States (US) since the 1950s, yet the origins, genomic diversity, and population structure of current colonies remain poorly defined. Through the NIH Marmoset Coordinating Center, we registered and sampled most US research marmosets (~2,300 living animals) and assembled pedigrees and historical records for >10,000 individuals. We present a resource of >800 whole-genome sequences, largely from US colonies. These data reveal an unexpected population structure that predates the establishment of research colonies. Indeed, this population structure mirrors variation found in marmosets across Brazil. Leveraging sequenced families, we generate the first pedigree-based recombination map and improved estimates of de novo mutation processes for this species. Our insights into genetic diversity, structure, and inbreeding will guide colony management, inform disease modelling and strengthen the marmoseťs standing as a biomedical model. Further, this work demonstrates how coordinated efforts across colonies can enable a self-sustaining "living laboratory", supporting data sharing and well-powered studies beyond the reach of single institutions.

Abstract Bacterial sepsis is a leading cause of neonatal mortality. Pro-inflammatory MR1-restricted T (MR1T) cells may help protect from sepsis by recognizing bacterial pathogens producing the canonical MR1 antigen 5-OP-RU. Most adult MR1T cells are mucosal-associated invariant T (MAIT) cells expressing a semi-invariant TCRα, while neonatal MR1T cells express diverse TCRα chains. Here, we perform combined single-cell RNA-sequencing and TCR repertoire analyses on MR1/5-OP-RU tetramer-positive cells from neonatal cord blood (CB) and adult blood. Compared to adult MR1T cells, CB MR1T cells exhibit greater TCR diversity, reduced cytotoxic and proinflammatory gene expression, diminished bacterial recognition and reduced binding to MR1/5-OP-RU. Structural analysis of a CB MAIT TCR reveals decreased β chain contribution to the TCR–MR1 interface relative to an adult MAIT TCR. These findings demonstrate developmental stage-specific differences in MR1T cell repertoire, function and MAIT TCR structure with implications for neonatal sepsis.

BACKGROUND:Due to their close evolutionary relationship with humans, rhesus macaques are an important pre-clinical model. While genetic diversity driven by short nucleotide variation has long been studied in rhesus macaques, there is comparatively little known about structural variation, with most published studies focused on cross-species comparative analyses. Understanding the degree and implications of intraspecies structural variation is essential to all biomedical research using rhesus macaques as a model. RESULTS:Here we present long-read sequencing of 59 rhesus macaques, identifying a catalog of 339,334 structural variants (SVs), which we subsequently genotype in a cohort of 2,645 individuals with short read whole genome sequencing data to create the largest public dataset of rhesus macaque SVs. These data reveal population structure within rhesus macaque SVs based on both geographic ancestry and to a lesser degree, breeding center. While there is evidence of strong purifying selection against SVs within exons, 0.7% of SVs overlap exons, with an average of 16.9 rare SVs per subject predicted to have a high impact on protein coding sequences. Notably, rhesus macaque SVs are dominated by Alu retrotransposition events, which comprise 55.7% of SVs and suggest significantly different modes of SV formation relative to humans and great apes. CONCLUSIONS:This dataset represents the largest study of structural variation in rhesus macaques to date and demonstrates use of both long and short-read datasets to generate SV genotype data. These data enable the consideration of structural variation impact in rhesus macaque-based research and will also aid the development of primate pangenomes.

Neonatal sepsis is a leading cause of childhood mortality. Understanding immune cell development can inform strategies to combat this. MR1-restricted T (MR1T) cells can be defined by their recognition of small molecules derived from microbes, self, and drug and drug-like molecules, presented by the MHC class 1-related molecule (MR1). In healthy adults, the majority of MR1T cells express an invariant α-chain; TRAV1-2/TRAJ33/12/20 and are referred to as mucosal-associated invariant T (MAIT) cells. Neonatal MR1T cells isolated from cord blood (CB) demonstrate more diversity in MR1T TCR usage, with the majority of MR1-5-OP-RU-tetramer(+) cells being TRAV1-2(-). To better understand this diversity, we performed single-cell-RNA-seq/TCR-seq (scRNA-seq/scTCR-seq) on MR1-5-OP-RU-tetramer(+) cells from CB (n=5) and adult participants (n=5). CB-derived MR1T cells demonstrate a less cytotoxic/pro-inflammatory phenotype, and a more diverse TCR repertoire. A panel of CB and adult MAIT and TRAV1-2(-) MR1T cell clones were generated, and CB-derived clones were unable to recognize several common riboflavin-producing childhood pathogens ( S. aureus, S. pneumoniae, M. tuberculosis ). Biochemical and structural investigation of one CB MAIT TCR (CB964 A2; TRAV1-2/TRBV6-2) showed a reduction in binding affinity toward the canonical MR1-antigen, 5-OP-RU, compared to adult MAIT TCRs that correlated with differences in β-chain contribution in the TCR-MR1 interface. Overall, this data shows that CB MAIT and TRAV1-2(-) MR1T cells, express a diverse TCR repertoire, a more restricted childhood pathogen recognition profile and diminished cytotoxic and pro-inflammatory capacity. Understanding this diversity, along with the functional ability of TRAV1-2(-) MR1T cells, could provide insight into increased neonatal susceptibility to infections.

Tuberculosis (TB) is the leading infectious disease killer worldwide and children suffer disproportionately. The ongoing burden of disease, despite widespread vaccination with BCG, highlights the need for novel vaccines. MR1-restricted T (MR1T) cells recognize small molecules, including microbial-derived molecules, presented by the monomorphic MHC class 1- related molecule (MR1). They have both "innate" effector capacity, allowing them to quickly respond to pathogens including Mycobacterium tuberculosis (Mtb), while also having adaptive features (effector memory cell surface phenotype and selective TCR usage). Feasibility of an MR1T cell-based vaccination remains unexplored and critical to this is whether or not MR1T cells possess the capacity for immunological memory. To begin to address this question, peripheral blood mononuclear cells (PBMC) were collected at 9-weeks of age from healthy term- infants in South Africa, who had either received BCG vaccination at birth (n=10) or who had BCG vaccination delayed (n=10). MR1/5-OP-RU tetramer positive cells were sorted using flow cytometry and single-cell RNA and TCR-seq was performed. Ex-vivo MR1T cells from vaccinated infants demonstrated increased expression of type I interferon response genes consistent with a cytokine mediated response to BCG vaccination. Using the TCR clustering algorithm TCRdist3, similar TCRs were grouped together, revealing a cluster significantly enriched in BCG-vaccinated infants. This cluster exhibited elevated expression of pro- inflammatory and cytotoxic genes, consistent with a recall response to prior vaccination and evidence of possible recognition of Mtb. This work provides the first step in addressing if MR1T cells demonstrate immunological memory, however, further work is needed to understand if these clonal expansions persist and possess the capacity for antigenic recall.

CD8+ resident memory T (Trm) cells comprise a small population of frontline sentinels compared with the large tissues they surveil, making outsized contributions to immune protection from infection. Here, we interrogated mechanisms of Trm cell function in primates. Intravenous immunization of macaques with a simian immunodeficiency virus (SIV)-gag-containing heterologous prime-boost-boost vaccine established memory T cells in >30 tissues, including visceral and mucosal compartments. Upon in vivo reactivation in the reproductive tract, antigen-sensing CD8+ Trm activated local stromal, parenchymal, and innate and adaptive immune cells. Stromal and parenchymal cells accentuated leukocyte migration and antiviral defenses. B and plasma cells mobilized into the vaginal mucosa, and bloodborne CD4+ T cells were recruited and adopted a host-defense program. Our findings demonstrate that systemic vaccination promotes a Trm cell response in barrier compartments and that Trm cells repurpose abundant neighboring stromal, parenchymal, and immune cells to amplify alarm signals and activate diverse host defenses.

Yellow fever virus (YFV) infection is fatal in 5%-10% of the 200,000 yearly cases. There is currently no available antiviral treatment. We showed previously that administration of 50 mg/kg of a YFV-specific neutralizing monoclonal antibody (nmAb) at 2 days postinfection (dpi), prior to the onset of severe disease, protected YFV-infected rhesus macaques from death. To further explore the clinical applicability of our nmAb MBL-YFV-01, we treated rhesus macaques with a lower dose (10 mg/kg) of this nmAb prophylactically or therapeutically at 3.5 dpi. We show that a single prophylactic or therapeutic i.v. dose of our nmAb protects rhesus macaques from death following challenge. A comprehensive analysis of 167 inflammatory cytokine and chemokines revealed that protection was associated with significantly reduced expression of 125 of these markers, including type I IFN, IL-6, and CCL2. This study further expands the potential clinical use of our YFV-specific nmAb, which could be used during an outbreak for immediate prophylactic immunity or for patients with measurable serum viremia.

IntroductionRNA sequencing (RNA-seq) can measure whole transcriptome gene expression from tissues or even individual cells, providing a powerful tool to study the immune response. Analysis of RNA-seq data involves mapping relatively short sequence reads to a reference genome, and quantifying genes based on the position of alignments relative to annotated genes. While this is usually robust, genetic polymorphism or genome/annotation inaccuracies result in genes with systematically missing or inaccurate data. These issues are frequently hidden or ignored, yet are highly relevant to immunologic data, where balancing selection has generated many polygenic gene families not accurately represented in a ‘one-size-fits-all’ reference genome.MethodsHere we present nimble, a tool to supplement standard RNA-seq pipelines. Nimble uses a previously developed pseudoaligner to process either bulk- or single-cell RNA-seq data using custom gene spaces. Importantly, nimble can apply customizable scoring criteria to each gene set, tailored to the biology of those genes.ResultsWe demonstrate that nimble recovers data in diverse contexts, ranging from simple cases (e.g., incorrect gene annotation or viral RNA), to complex immune genotyping (e.g., major histocompatibility or killer-immunoglobulin-like receptors). We use this enhanced capability to identify killer-immunoglobulin-like receptor expression specific to tissue-resident memory T cells and demonstrate allele-specific regulation of MHC alleles after Mycobacterium tuberculosis stimulation.DiscussionCombining nimble data with standard pipelines enhances the fidelity and accuracy of experiments, maximizing the value of expensive datasets, and identifying cellular subsets not possible with standard tools alone.

Hepatitis B virus (HBV) poses a significant global health challenge, necessitating the urgent development of curative therapeutics. However, this progress is impeded by the lack of robust, immunocompetent preclinical animal models due to HBV's strict species specificity. We previously showed that vector-mediated expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), renders macaques fully susceptible to HBV infection. In this study, we have generated transgenic macaques expressing hNTCP, marking the creation of the first transgenic nonhuman primate model for infectious disease research. We used PiggyBac (PB) transposon technology to insert a liver-specific hNTCP expression cassette into rhesus macaque zygotes and transferred the resulting embryos into surrogate females, resulting in two healthy transgenic offspring. In both animals, hNTCP is highly and selectively expressed in the liver. Most importantly, we show that isolated hepatocytes from these monkeys are susceptible to HBV infection. These findings lay the foundation for the development of a nonhuman primate HBV model, facilitating the advancement and validation of curative HBV therapies.

Single-cell RNA sequencing (scRNA-seq) allows cell classification using genome-wide transcriptional state; however, high-dimensional transcriptomic profiles, and the unsupervised analyses employed to interpret them, provide a systematically different view of biology than well-established functional/lineage definitions of immunocytes. Understanding these differences and limits is essential for accurate interpretation of these rich data. We present the Rhesus Immune Reference Atlas (RIRA), the first immune-focused macaque single-cell multi-tissue atlas. We contrasted transcriptional profiles against immune lineages, using surface protein and marker genes as ground truth. While the pattern of clustering can align with cell type, this is not always true. Especially within T and natural killer (NK) cells, many functionally distinct subsets lack defining markers, and strong shared expression programs, such as cytotoxicity, result in systematic intermingling by unsupervised clustering. We identified gene programs with high discriminatory/diagnostic value, including multi-gene signatures that model T/NK cell maturation. Directly measuring these diagnostic programs complements unsupervised analyses.

Abstract Malaria is still a global health issue that afects more than half of the worlds population. While a number of malaria vaccines are currently under development. The most potent and long-lived vaccine have been whole sporozoite vaccines that use live-attenuated sporozoites to generate both antibodies and liver resident memory T cells capable of blocking infection. This vaccine has a higher efficacy in a malaria naïve population (up to 100%), but this efficacy drops in a malaria endemic population (0-30%). It has been hypothesized that the immune profile of people living in malaria endemic regions is altered in a way that negatively affects vaccine induced immunity, but a better understanding of how this occurs is required to generate optimal vaccine strategies. Yet to understand how the parasite alters the immune system, thus causing vaccine hyporesponsive in endemic regions, requires detailed information on previous parasite exposure and access to tissue resident cells that reside in the liver and other lymphoid organs—both limiting our ability to study this phenomenon in humans. To overcome this, we used a non-human primate model of malaria that enables us to access tissues before and after infection and vaccination via minimally invasive biopsies. Here, we present ongoing analysis that shows the longitudinal changes in the immune landscape after infection, vaccination and infection after vaccination using flow cytometry and single cell transcriptomics.

Abstract Background Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. Methods We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. Results Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. Conclusions These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC. Graphical abstract

Transmission of HIV-1 to newborns and infants remains high, with 130,000 new infections in 2022 in resource-limited settings. Half of HIV-infected newborns, if untreated, progress to disease and death within 2 years. While immunologic immaturity likely promotes pathogenesis and poor viral control, little is known about immune damage in newborns and infants. Here we examined pathologic, virologic, and immunologic outcomes in rhesus macaques exposed to pathogenic simian-human immunodeficiency virus (SHIV) at 1-2 weeks, defined as newborns, or at 4 months of age, considered infants. Kinetics of plasma viremia and lymph node seeding DNA were indistinguishable in newborns and infants, but levels of viral DNA in gut and lymphoid tissues 6-10 weeks after infection were significantly higher in newborns versus either infant or adult macaques. Two of 6 newborns with the highest viral seeding required euthanasia at 25 days. We observed age-dependent alterations in leukocyte subsets and gene expression. Compared with infants, newborns had stronger skewing of monocytes and CD8+ T cells toward differentiated subsets and little evidence of type I interferon responses by transcriptomic analyses. Thus, SHIV infection reveals distinct immunological alterations in newborn and infant macaques. These studies lay the groundwork for understanding how immune maturation affects pathogenesis in pediatric HIV-1 infection.

Rhesus cytomegalovirus (RhCMV) vectors elicit major histocompatibility complex (MHC)-E-restricted CD8+ T cells that stringently control simian immunodeficiency virus (SIV) in rhesus macaques. These responses require deletion of eight RhCMV chemokine-like open reading frames (ORFs) that are conserved in human cytomegalovirus (HCMV). To determine whether HCMV encodes additional, nonconserved inhibitors of unconventional T cell priming, we inserted 41 HCMV-specific ORFs into a chemokine-deficient strain (68-1 RhCMV). Monitoring of epitope recognition revealed that HCMV UL18 prevented unconventional T cell priming, resulting in MHC-Ia-targeted responses. UL18 is homologous to MHC-I but does not engage T cell receptors and, instead, binds with high affinity to inhibitory leukocyte immunoglobulin-like receptor-1 (LIR-1). UL18 lacking LIR-1 binding no longer interfered with MHC-E-restricted T cell stimulation by RhCMV-infected cells or the induction of unconventionally restricted T cells. Thus, LIR-1 binding needs to be deleted from UL18 of HCMV/HIV vaccines to allow for the induction of protective MHC-E-restricted T cells.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.