The tissue origin(s) and the earliest viral dynamics of HIV rebound after antiretroviral therapy (ART) remain unclear. Here, using barcoded SIVmac239 in rhesus macaques (n = 24), we defined the distribution of barcode-specific viral RNA expression in tissues during ART (n = 6) and then assessed initial clonal rebound 5 and 7 days after ART cessation by identifying barcodes in individual tissues that exceeded the 99th percentile of the on-ART distribution ('outliers'). In 4 of 11 aviraemic and 6 of 7 viraemic animals, 32 such outlier barcodes were identified. Sixteen of these barcodes were also identified in rebound viraemia, confirming specific tissues as rebound origin and early amplification sites. Overall, 27 of the 32 outlier barcodes were determined to reflect rebound origins, of which 96% were in the gastrointestinal tract (26%) or gastrointestinal tract-associated lymphoid tissues (70%). These results indicate that distinct tissue sites differentially support post-ART viral rebound, with potential therapeutic implications for interventions designed to prevent or control these events.

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.

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.

Lack of access to antiretroviral therapy (ART) leads to the transmission of human immunodeficiency virus in ~120,000 children annually, emphasizing the need for new strategies to prevent lifelong infection. Here in an infant rhesus macaque model of peripartum oral infection, we show that broadly neutralizing antibodies directed to the viral envelope protein were insufficient to prevent latent reservoir establishment, independent of daily ART. Blockade of the human immunodeficiency virus co-receptor CCR5 via the antibody leronlimab also failed to prevent reservoir establishment, but it significantly reduced reservoir seeding in lymphoid and gastrointestinal tissues. Following the treatment of infants with viraemia at 72 h post-infection with the combination of broadly neutralizing antibodies, ART and leronlimab, no subsequent evidence of replicating or latent virus and antiviral immunity was observed 1 year after treatment interruption. These findings suggest a synergy between broadly neutralizing antibodies, ART and CCR5 blockade for preventing viral reservoir establishment and offer potential improvements over current therapies for newborns exposed to human immunodeficiency virus.

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.

A vaccine is widely regarded as necessary for the control of the HIV pandemic and eventual eradication of AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have both been shown capable of vaccine protection against the simian counterpart of HIV, SIV, in rhesus macaques. Here we provide preliminary evidence that combining these orthogonal antiviral mechanisms can provide increased protection against SIV challenge such that replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced in the presence of passively administered incompletely protective levels of neutralizing antibody. The report invites studies involving larger cohorts of macaques and alternate routes of providing neutralizing antibody.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of neonatal neurological impairment worldwide, but the viral factors enabling vertical spread across the placenta remain undetermined. The pentameric complex (PC), composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be important for entry into nonfibroblast cells in vitro. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development. To determine the relevance of the PC for congenital transmission in a translational nonhuman primate model, we engineered a rhesus CMV (RhCMV) mutant lacking the orthologs of UL128 and UL130, which demonstrated diminished infection of epithelial cells in vitro. However, intravenous inoculation of either CD4+ T cell-depleted or immunocompetent RhCMV-seronegative pregnant rhesus macaques (RMs) in the early second trimester with the PC-deficient mutant resulted in maternal RhCMV peak plasma viremia similar to inoculations with PC-intact RhCMV, although virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced IgG responses that neutralized RhCMV entry into epithelial cells in tissue culture. These responses were reduced, but not absent, from animals infected with the PC-deficient virus, which also induced IgG responses against gH. Moreover, congenital CMV transmission was confirmed in multiple animals infected with PC-deficient virus by detecting viral DNA in the amniotic fluid, indicating that transplacental transmission in RMs is not contingent on the PC.

Strain 68-1 rhesus CMV (RhCMV) vectors induce immune responses that mediate early, complete replication arrest of SIV infection in ∼60% of vaccinated rhesus macaques (RMs). This unique efficacy depends on the ability of these vectors to elicit effector memory (EM)-biased CD8+ T cells recognizing SIV peptides presented by MHC-E, rather than MHC-Ia. These efficacious responses still occurred when spread of the 68-1 vector was impaired by deletion of the viral anti-host intrinsic immunity factor phosphoprotein 71 (pp71), but efficacy was lost with a more stringent attenuation strategy based on destabilization of Rh108, the ortholog of the essential human CMV (HCMV) transcription factor UL79 that is required for late viral gene expression. Although unable to produce infectious progeny (ie single-cycle infection), Rh108-deficient vectors elicited durable, high frequency, EM-biased, SIV-specific CD8+ T-cell responses in RMs, but these responses were MHC-Ia–restricted and therefore non-efficacious. Here, we tested a different single-cycle attenuation strategy based on deletion (Δ) of the glycoprotein L (gL) that is essential for viral entry but allows for late gene expression and viral assembly. ΔgL 68-1 RhCMV/SIV vectors, grown on gL-complementing fibroblasts, were robustly immunogenic at doses above 105 PFU, generating high frequency, EM-biased, SIV-specific CD8+ T-cell responses that were also unconventionally restricted, including the MHC-E restriction associated with efficacy. Indeed, these single-cycle vectors manifested replication arrest efficacy in 70% of vaccinated RMs, further linking MHC-E restriction with efficacy, and demonstrating that 68-1 RhCMV/SIV efficacy does not require vector dissemination within the host.

Background: Leronlimab is a humanized κ-IgG4 monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). We investigated leronlimab as a treatment option for people living with multidrug-resistant HIV-1. Setting and Methods: In a phase 2b/3, multicenter, randomized, double-blind, placebo-controlled study conducted in 21 hospital centers in the United States, treatment-experienced people living with HIV (PLWH) with documented drug resistance were randomly assigned once weekly leronlimab (350 mg subcutaneously) or matching placebo for one week overlapping existing failing antiretroviral treatment (ART), followed by a 24-week single-arm extension with weekly leronlimab combined with a new optimized background treatment (OBT). The primary endpoint was achieving ≥0.5 log 10 reduction in plasma HIV-1 RNA from baseline at the end of the one-week double-blinded treatment period. Results: 52 participants were enrolled (25 leronlimab and 27 placebo). After the one-week randomized phase, by the intent-to-treat analysis 64.0% (16/25) receiving leronlimab achieved ≥0.5 log 10 reduction in plasma HIV-1 RNA versus 23.1% (6/26) receiving placebo (p=0.0032), while by per protocol analysis 72.7% (16/22) receiving leronlimab achieved ≥0.5 log 10 reduction in plasma HIV-1 RNA versus 24.0% (6/25) receiving placebo (p=0.0008). Leronlimab was generally well tolerated with no drug-related SAEs reported. Overall, 175 adverse events were reported by 34/52 participants, with 120 (68.6%) adverse events categorized as mild. Conclusions: Leronlimab resulted in significantly reduced plasma HIV-1 within one week after addition to failing ART. After 24 weeks combined with an OBT, most participants had plasma HIV-1 RNA levels <50 copies per mL plasma, suggesting utility of leronlimab as a component of salvage therapy.

Infection of rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) elicits MHC-I restricted CD8 T cell responses whereas RhCMV deleted for homologs of HCMV UL128, UL130 and UL146 induces MHC-II and MHC-E restricted CD8 T cells. MHC-E restricted T cells are required for RhCMV-based SIV vaccines to provide “control and clear” protection against highly virulent SIV. Vaccination of Mauritian cynomolgus macaques (MCM) with similarly modified Cynomolgus (Cy)CMV/SIV vectors elicited MHC-E-restricted T cells and protected against SIV challenge. In contrast, immunization of RM with CyCMV or MCM with RhCMV failed to elicit MHC-E-restricted T cells suggesting species specificity of CMV-mediated T cell programming. To determine the viral determinants of this species specificity we generated RhCMV/CyCMV chimeras using transformation-associated recombination cloning in yeast. RhCMV and CyCMV genomes were cloned as overlapping fragments that were assembled into quarter genomes by homologous recombination using yeast artificial chromosomes. Quarter genomes were then assembled as four chimeras consisting of three quarters of RhCMV and one quarter of CyCMV. The resulting recombinants were transferred into E. coli and recombinant vectors were recovered in primary rhesus fibroblasts. Analysis of the T cell responses to recombinant Cy/RhCMV chimeras in RM and MCM revealed that a single genomic region, and possibly a single RhCMV gene, is required for unconventional CD8 T cell induction. RO1 AI095113, R37 AI054292, U19 AI128741, P01 AI174856, R01 AI059457, R01 AI175459, R01 AI140888, R01 AI129703, P51OD011092 Viral Immunology (VIR)

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.

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies. The role of viral Fc-gamma receptors in rhesus cytomegalovirus (RhCMV) infection is unclear. Here, the authors characterized RhCMV vFcγRs and report that their deletion did not affect virus replication, tropism or superinfection in rhesus macaques but increased susceptibility of the virus to antibody control.

A vaccine is considered essential for controlling the HIV pandemic and ultimately eradicating AIDS. Neutralizing antibodies and MHC-E-restricted CD8+ T cells have shown the ability to protect against the simian counterpart of HIV, SIV, in rhesus macaques. In this study, we provide preliminary evidence that combining these orthogonal antiviral mechanisms can offer increased protection against SIV. Specifically, the replication arrest observed following vaccination with a rhesus cytomegalovirus (RhCMV/SIV)-based vaccine was enhanced by the presence of a passively administered neutralizing antibody at incompletely protective levels. This report encourages studies involving larger cohorts of macaques and alternative methods for administering neutralizing antibodies.

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.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a deadly infectious disease having a major impact on global health. Using the CMV vector for development of novel vaccines is a promising new strategy that elicits strong and durable, high frequency memory T cell responses against heterologous immunogens. We conducted functional transcriptomic analysis of whole blood samples collected from cohorts of rhesus (Rh) macaques that were administered RhCMV/TB vector using a prime-boost strategy. Two modified CMV vectors were used in this study, including 68-1 RhCMV/TB-6Ag (encoding 6 Mtb protein immunogens, including Ag85A, ESAT-6, Rv3407, Rv2626, Rpf A, and Rpf D) and its attenuated variant, 68-1 RhCMV/Δpp71-TB-6Ag (a cell-to-cell spread-deficient vaccine vector lacking the Rh110 gene encoding the pp71 tegument protein). Bulk mRNA sequencing, differential gene expression, and functional enrichment analyses showed that these RhCMV/TB vaccines induce the innate and adaptive immune responses with specific transcriptomic signatures, including the IL-15-induced protective gene signature previously defined to be linked with protection against simian immunodeficiency virus (SIV) by the 68-1 RhCMV/SIV vaccine. While both vectors exhibited a transcriptomic response of the IL-15 protective signature in whole blood, we show that lack of pp71 does not maintain induction of the protective signature for the full duration of the study compared to the parental non-attenuated vector. Our observations indicate that RhCMV vector vaccines induce a transcriptomic response in whole blood that include a conserved IL-15 signature of which vector-encoded pp71 is an important component of response durability that upon future Mtb challenge may define specific vaccine protection outcomes against Mtb infection.

AbstractAn influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E–restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E–restricted CD8 + T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E–restricted CD8 + T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E + prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8 + T cell–based immunotherapies.

Background:RhCMV/SIV vaccines protect ∼59% of vaccinated rhesus macaques against repeated limiting-dose intra-rectal exposure with highly pathogenic SIVmac239M, but the exact mechanism responsible for the vaccine efficacy is not known. It is becoming evident that complex interactions exist between gut microbiota and the host immune system. Here we aimed to investigate if the rhesus gut microbiome impacts RhCMV/SIV vaccine-induced protection. Methods:Three groups of 15 rhesus macaques naturally pre-exposed to RhCMV were vaccinated with RhCMV/SIV vaccines. Rectal swabs were collected longitudinally both before SIV challenge (after vaccination) and post challenge and were profiled using 16S rRNA based microbiome analysis. Results:We identified ∼2,400 16S rRNA amplicon sequence variants (ASVs), representing potential bacterial species/strains. Global gut microbial profiles were strongly associated with each of the three vaccination groups, and all animals tended to maintain consistent profiles throughout the pre-challenge phase. Despite vaccination group differences, using newly developed compositional data analysis techniques we identified a common gut microbial signature predictive of vaccine protection outcome across the three vaccination groups. Part of this microbial signature persisted even after SIV challenge. We also observed a strong correlation between this microbial signature and an early signature derived from whole blood transcriptomes in the same animals. Conclusions:Our findings indicate that changes in gut microbiomes are associated with RhCMV/SIV vaccine-induced protection and early host response to vaccination in rhesus macaques.

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.