Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Cytomegalovirus-vaccine-induced unconventional T cell priming and control of SIV replication is conserved between primate species.

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine.

Suppression of human and simian immunodeficiency virus replication with the CCR5-specific antibody Leronlimab in two species.

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.

CCR5 Receptor Occupancy Analysis Reveals Increased Peripheral Blood CCR5+CD4+ T Cells Following Treatment With the Anti-CCR5 Antibody Leronlimab.

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes.

Identification and Characterization of Antigen-Specific CD8+ T Cells Using Surface-Trapped TNF-α and Single-Cell Sequencing.

CD8+ T cells are key mediators of antiviral and antitumor immunity. The isolation and study of Ag-specific CD8+ T cells, as well as mapping of their MHC restriction, has practical importance to the study of disease and the development of therapeutics. Unfortunately, most experimental approaches are cumbersome, owing to the highly variable and donor-specific nature of MHC-bound peptide/TCR interactions. Here we present a novel system for rapid identification and characterization of Ag-specific CD8+ T cells, particularly well suited for samples with limited primary cells. Cells are stimulated ex vivo with Ag of interest, followed by live cell sorting based on surface-trapped TNF-α. We take advantage of major advances in single-cell sequencing to generate full-length sequence data from the paired TCR α- and ß-chains from these Ag-specific cells. The paired TCR chains are cloned into retroviral vectors and used to transduce donor CD8+ T cells. These TCR transductants provide a virtually unlimited experimental reagent, which can be used for further characterization, such as minimal epitope mapping or identification of MHC restriction, without depleting primary cells. We validated this system using CMV-specific CD8+ T cells from rhesus macaques, characterizing an immunodominant Mamu-A1*002:01-restricted epitope. We further demonstrated the utility of this system by mapping a novel HLA-A*68:02-restricted HIV Gag epitope from an HIV-infected donor. Collectively, these data validate a new strategy to rapidly identify novel Ags and characterize Ag-specific CD8+ T cells, with applications ranging from the study of infectious disease to immunotherapeutics and precision medicine.

Antibody-based CCR5 blockade protects Macaques from mucosal SHIV transmission.

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials.

Rhesus Cytomegalovirus-Specific CD8+ Cytotoxic T Lymphocytes Do Not Become Functionally Exhausted in Chronic SIVmac239 Infection.

CD8+ cytotoxic T lymphocytes (CTLs) exert potent antiviral activity after HIV/SIV infection. However, efforts to harness the antiviral efficacy of CTLs for HIV/SIV prophylaxis and therapy have been severely hindered by two major problems: viral escape and exhaustion. By contrast, CTLs directed against human cytomegalovirus (HCMV), a ubiquitous chronic herpesvirus, seldom select for escape mutations and remain functional and refractory to exhaustion during chronic HCMV and HIV infection. Recently, attempts have been made to retarget HCMV-specific CTLs for cancer immunotherapy. We speculate that such a strategy may also be beneficial in the context of HIV/SIV infection, facilitating CTL-mediated control of HIV/SIV replication. As a preliminary assessment of the validity of this approach, we investigated the phenotypes and functionality of rhesus CMV (RhCMV)-specific CTLs in SIVmac239-infected Indian rhesus macaques (RMs), a crucial HIV animal model system. We recently identified two immunodominant, Mamu-A∗02-restricted CTL epitopes derived from RhCMV proteins and sought to evaluate the phenotypic and functional characteristics of these CTL populations in chronic SIVmac239 infection. We analyzed and directly compared RhCMV- and SIVmac239-specific CTLs during SIVmac239 infection in a cohort of Mamu-A∗01+ and Mamu-A∗02+ RMs. CTL populations specific for at least one of the RhCMV-derived CTL epitopes were detected in ten of eleven Mamu-A∗02+ animals tested, and both populations were detected in five of these animals. Neither RhCMV-specific CTL population exhibited significant changes in frequency, memory phenotype, granzyme B expression, exhaustion marker (PD-1 and CTLA-4) expression, or polyfunctionality between pre- and chronic SIVmac239 infection timepoints. In chronic SIVmac239 infection, RhCMV-specific CTLs exhibited higher levels of granzyme B expression and polyfunctionality, and lower levels of exhaustion marker expression, than SIVmac239-specific CTLs. Additionally, compared to SIVmac239-specific CTLs, greater proportions of RhCMV-specific CTLs were of the terminally differentiated effector memory phenotype (CD28- CCR7-) during chronic SIVmac239 infection. These results suggest that, in contrast to SIVmac239-specific CTLs, RhCMV-specific CTLs maintain their phenotypes and cytolytic effector functions during chronic SIVmac239 infection, and that retargeting RhCMV-specific CTLs might be a promising SIV immunotherapeutic strategy.

MHC-E-Restricted CD8+ T Cells Target Hepatitis B Virus-Infected Human Hepatocytes.

Currently 247 million people are living with chronic hepatitis B virus infection (CHB), and the development of novel curative treatments is urgently needed. Immunotherapy is an attractive approach to treat CHB, yet therapeutic approaches to augment the endogenous hepatitis B virus (HBV)-specific T cell response in CHB patients have demonstrated little success. In this study, we show that strain 68-1 rhesus macaque (RM) CMV vaccine vectors expressing HBV Ags engender HBV-specific CD8+ T cells unconventionally restricted by MHC class II and the nonclassical MHC-E molecule in RM. Surface staining of human donor and RM primary hepatocytes (PH) ex vivo revealed the majority of PH expressed MHC-E but not MHC class II. HBV-specific, MHC-E-restricted CD8+ T cells from RM vaccinated with RM CMV vaccine vectors expressing HBV Ags recognized HBV-infected PH from both human donor and RM. These results provide proof-of-concept that MHC-E-restricted CD8+ T cells could be harnessed for the treatment of CHB, either through therapeutic vaccination or adoptive immunotherapy.

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

Viral opportunistic infections in Mauritian cynomolgus macaques undergoing allogeneic stem cell transplantation mirror human transplant infectious disease complications.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

Vaccine-Mediated Inhibition of the Transporter Associated with Antigen Processing Is Insufficient To Induce Major Histocompatibility Complex E-Restricted CD8+ T Cells in Nonhuman Primates.

Major histocompatibility complex E (MHC-E) is a highly conserved nonclassical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs coexpressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus 2 ICP47, bovine herpes virus 1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia but not MHC-E. These data demonstrate that, in contrast to results in mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.IMPORTANCE Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.

A live-attenuated RhCMV/SIV vaccine shows long-term efficacy against heterologous SIV challenge.

Previous studies have established that strain 68-1-derived rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) proteins (RhCMV/SIV) are able to elicit and maintain cellular immune responses that provide protection against mucosal challenge of highly pathogenic SIV in rhesus monkeys (RMs). However, these efficacious RhCMV/SIV vectors were replication and spread competent and therefore have the potential to cause disease in immunocompromised subjects. To develop a safer CMV-based vaccine for clinical use, we attenuated 68-1 RhCMV/SIV vectors by deletion of the Rh110 gene encoding the pp71 tegument protein (ΔRh110), allowing for suppression of lytic gene expression. ΔRh110 RhCMV/SIV vectors are highly spread deficient in vivo (~1000-fold compared to the parent vector) yet are still able to superinfect RhCMV+ RMs and generate high-frequency effector-memory-biased T cell responses. Here, we demonstrate that ΔRh110 68-1 RhCMV/SIV-expressing homologous or heterologous SIV antigens are highly efficacious against intravaginal (IVag) SIVmac239 challenge, providing control and progressive clearance of SIV infection in 59% of vaccinated RMs. Moreover, among 12 ΔRh110 RhCMV/SIV-vaccinated RMs that controlled and progressively cleared an initial SIV challenge, 9 were able to stringently control a second SIV challenge ~3 years after last vaccination, demonstrating the durability of this vaccine. Thus, ΔRh110 RhCMV/SIV vectors have a safety and efficacy profile that warrants adaptation and clinical evaluation of corresponding HCMV vectors as a prophylactic HIV/AIDS vaccine.

The human IL-15 superagonist ALT-803 directs SIV-specific CD8+ T cells into B-cell follicles.

Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques.

MHC-E is a highly conserved nonclassical MHC class Ib molecule that predominantly binds and presents MHC class Ia leader sequence-derived peptides for NK cell regulation. However, MHC-E also binds pathogen-derived peptide Ags for presentation to CD8+ T cells. Given this role in adaptive immunity and its highly monomorphic nature in the human population, HLA-E is an attractive target for novel vaccine and immunotherapeutic modalities. Development of HLA-E-targeted therapies will require a physiologically relevant animal model that recapitulates HLA-E-restricted T cell biology. In this study, we investigated MHC-E immunobiology in two common nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM). Compared to humans and MCM, RM expressed a greater number of MHC-E alleles at both the population and individual level. Despite this difference, human, RM, and MCM MHC-E molecules were expressed at similar levels across immune cell subsets, equivalently upregulated by viral pathogens, and bound and presented identical peptides to CD8+ T cells. Indeed, SIV-specific, Mamu-E-restricted CD8+ T cells from RM recognized antigenic peptides presented by all MHC-E molecules tested, including cross-species recognition of human and MCM SIV-infected CD4+ T cells. Thus, MHC-E is functionally conserved among humans, RM, and MCM, and both RM and MCM represent physiologically relevant animal models of HLA-E-restricted T cell immunobiology.

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques.

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.

Allogeneic stem cell transplantation in fully MHC-matched Mauritian cynomolgus macaques recapitulates diverse human clinical outcomes.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.

Cross-Species Rhesus Cytomegalovirus Infection of Cynomolgus Macaques.

Cytomegaloviruses (CMV) are highly species-specific due to millennia of co-evolution and adaptation to their host, with no successful experimental cross-species infection in primates reported to date. Accordingly, full genome phylogenetic analysis of multiple new CMV field isolates derived from two closely related nonhuman primate species, Indian-origin rhesus macaques (RM) and Mauritian-origin cynomolgus macaques (MCM), revealed distinct and tight lineage clustering according to the species of origin, with MCM CMV isolates mirroring the limited genetic diversity of their primate host that underwent a population bottleneck 400 years ago. Despite the ability of Rhesus CMV (RhCMV) laboratory strain 68-1 to replicate efficiently in MCM fibroblasts and potently inhibit antigen presentation to MCM T cells in vitro, RhCMV 68-1 failed to productively infect MCM in vivo, even in the absence of host CD8+ T and NK cells. In contrast, RhCMV clone 68-1.2, genetically repaired to express the homologues of the HCMV anti-apoptosis gene UL36 and epithelial cell tropism genes UL128 and UL130 absent in 68-1, efficiently infected MCM as evidenced by the induction of transgene-specific T cells and virus shedding. Recombinant variants of RhCMV 68-1 and 68-1.2 revealed that expression of either UL36 or UL128 together with UL130 enabled productive MCM infection, indicating that multiple layers of cross-species restriction operate even between closely related hosts. Cumulatively, these results implicate cell tropism and evasion of apoptosis as critical determinants of CMV transmission across primate species barriers, and extend the macaque model of human CMV infection and immunology to MCM, a nonhuman primate species with uniquely simplified host immunogenetics.

TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection.

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

Broadly targeted CD8⁺ T cell responses restricted by major histocompatibility complex E.

Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8αß(+) T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E-restricted CD8(+) T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells.

Nef-specific CD8(+) T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165-173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication.