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.

HIV/SIV-specific CD8+T cell responses are typically unable to control viral rebound following antiretroviral therapy (ART) interruption (ATI). To investigate whether enhancing the magnitude and activation of SIV-specific CD8+ T cells at the time of ATI can improve the immune interception of reactivating SIV infections, we vaccinated SIVmac239-infected rhesus macaques (RMs) on ART, boosting immediately prior to ATI, with a nucleoside-unmodified mRNA vaccine expressing SIVmac239 Gag (mRNA/SIVgag) alone or in combination with Nef (mRNA/SIVnef) and Pol (mRNA/SIVpol). The mRNA/SIVgag vaccine was effective in boosting Gag-specific CD8+ T cells in blood and lymphoid tissues. Following ATI, the mRNA/SIVgag vaccine group showed a significant delay in time to measurable viral rebound compared with controls and manifested lower plasma viral loads (PVLs) for up to 6 weeks after rebound. Similarly, RMs that received mRNA/SIVgag, mRNA/SIVnef, and mRNA/SIVpol also manifested a delay in SIV rebound compared with controls, suggesting that boosting SIV-specific CD8+ T cells during ATI can enhance early immune targeting of reactivating SIV infections. However, viral control was not sustained long term as PVLs were similar across vaccinees and controls by 24 weeks after rebound, highlighting the need for adjunctive therapies to improve the durability of virologic control elicited by CD8+ T cell-targeting vaccines.

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.

Adeno-associated virus (AAV) vectors are promising vehicles for the delivery and long-term expression of antibodies for treatment of chronic diseases. Given the strong link between the C-C chemokine receptor 5 (CCR5)-Δ32/Δ32 genotype and protection from HIV, we explored the ability of AAV vectors expressing the CCR5-blocking antibody leronlimab to mediate a functional cure in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques by interrupting viral access to the viral entry co-receptor CCR5. Delivery of AAV-leronlimab to rhesus macaques elicited antidrug antibodies (ADA) and rapid plasma leronlimab clearance in approximately half of the treated macaques. However, in these same animals, we observed spontaneous leronlimab transgene reemergence and detectable CCR5 receptor occupancy approximately 1 year later that subsequently persisted indefinitely without rebound of ADA. In macaques that did not mount robust ADA responses, detectable plasma leronlimab concentrations and CCR5 receptor occupancy were maintained for more than 1 year. Of the nine macaques producing sufficient leronlimab to achieve full CCR5 receptor occupancy on blood CD4+ T cells, AAV-leronlimab drove stringent or partial control of SHIV viremia in six macaques long term. In the three macaques with uncontrolled SHIV viremia, short-term administration of exogenous leronlimab induced complete SHIV suppression in two and a 100-fold reduction in the third, indicating that a threshold of leronlimab expression is necessary to effectively halt SHIV replication. These results demonstrate the potential of gene therapy-mediated long-term antibody-based CCR5 blockade for HIV functional cure but highlight challenges in achieving sufficient antibody expression when targeting an abundant self-antigen.

O'nyong-nyong virus (ONNV) is a mosquito-borne alphavirus first isolated in Uganda in 1959. Since its discovery, ONNV has caused several outbreaks in Africa, manifesting clinically as fever, rash, and joint/muscle pain lasting months. Currently, we have a limited understanding of ONNV infection and disease in relevant animal models, which restricts the evaluation of vaccines and therapeutics. In 1967, Binn et al. reported that infection of rhesus macaques (RMs) with ONNV failed to induce viremia in two animals. This may be attributed to the potential attenuation of the virus through extensive passaging. To mitigate this issue, we constructed an infectious clone from the sequence of ONNV-UVRI0804 (ONNV0804), a 2017 clinical isolate from a febrile patient in Uganda. This strain demonstrated high pathogenicity in immunocompetent mice, resulting in an earlier and more severe onset of disease and significantly higher viremia compared to a highly passaged control strain ONNVUgMP30. In the current study, three male and three female rhesus macaques were subcutaneously inoculated with ONNV0804. All animals became viremic at 2 days post inoculation (dpi). Both classical and nonclassical monocytes were activated (CD169+), peaking at 3 dpi, which corresponded with the peak of plasma viral RNA. Additionally, CD4+ and CD8 + effector memory T cells and memory B cells began proliferating in peripheral blood by day 7, peaking at day 10, which also corresponded to the timing of neutralizing antibody development, indicating a robust adaptive immune response to ONNV0804. Finally, key clinical disease manifestations were recapitulated, including lymphadenopathy and histological features of early-stage arthritis. Taken together, rhesus macaque infection with ONNV0804 clinical isolate is a promising model for investigating immune responses to alphaviruses and evaluating vaccines to protect against future epidemics.

Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in Central and South America in 2015-16. During that outbreak, ZIKV infection during pregnancy was associated with high rates of adverse outcomes, including miscarriage and a spectrum of neurological birth defects collectively termed congenital Zika syndrome (CZS). Ten years later, there are still no licensed ZIKV vaccines, underscoring the need for robust experimental models to evaluate vaccine efficacy against congenital disease. Here, we evaluated a highly pathogenic nonhuman primate pregnancy model for its ability to assess vaccine-mediated protection using a Zika virus-like particle (VLP) vaccine as a test platform. First, non-pregnant rhesus macaques (RM) were vaccinated with a prime-boost method to compare control, VLP alone, and VLP plus Alhydrogel (alum) adjuvant. Vaccination plus alum elicited strong neutralizing antibody responses and reduced viral dissemination compared to controls. Next, we evaluated the vaccine's protective efficacy against adverse pregnancy complications. Two cohorts of female RM were vaccinated with ZIKV-VLP adjuvanted with alum or alum alone prior to time-mated breeding. At gestational day (GD) 30 (mid first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain previously shown to induce frequent first-trimester pregnancy loss. Within the vaccinated cohort, two of three animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. One vaccinated animal experienced early pregnancy loss despite the absence of detectable virus in fetal or placental tissues. In the sham vaccine control pregnancy cohort, severe adverse outcomes included miscarriage and hydrops fetalis with widespread viral RNA and placental pathology in two animals. These results support a high risk of early pregnancy loss following African-lineage ZIKV-DAK 41524 infection in RM. This model can be further used to understand the complexities of placental immunological features underlying miscarriage following ZIKV infection.

Zika virus (ZIKV) is an arthropod-borne Orthoflavivirus that caused a major outbreak in the Americas in 2015-16. In Brazil, up to 46% of ZIKV positive pregnancies resulted in congenital Zika syndrome (CZS). CZS is characterized by a wide range of neurologic birth defects and miscarriage in up to 7.6% of affected pregnancies. With no current licensed ZIKV vaccines, we sought to evaluate a Zika virus-like particle (VLP) vaccine candidate in a rhesus macaque (RM) pregnancy model. VLPs were produced in mammalian cells expressing the pre-membrane-envelope region of the Asian-lineage ZIKV strain PRVABC59, which belongs to the Asian ZIKV lineage that is associated with outbreaks of congenital disease. To evaluate vaccine protection against adverse pregnancy complications, two cohorts of female RM were vaccinated with ZIKV-VLP with adjuvant Alhydrogel (alum) or adjuvant alone prior to mating. At gestational day (GD) 30 (early first trimester), pregnant animals were challenged with ZIKV-DAK 41524, an African-lineage strain shown to induce 1st-trimester fetal demise in 78% (n=11/14 animals) of RM, making it an ideal and stringent model for evaluating ZIKV vaccines. Within the vaccinated cohort, 2 of 3 animals reached the study endpoint of GD 90 with no observed adverse pregnancy outcomes. The third animal experienced pregnancy loss at GD 49 (18 d post infection), although no infectious virus was detected in placental or fetal tissues. In the unvaccinated cohort, two animals had severe adverse events. One animal experienced preterm labor, and another developed early-onset hydrops fetalis with widespread ZIKV-RNA detected via RNAscope and extensive placental damage. These results confirm a significant risk for early pregnancy loss in RM infected with ZIKV-DAK 41524. This model can be further used to understand the complexities of placental immunological features underlying stillbirth and miscarriage following infection. Our findings indicate that this ZIKV-VLP vaccine candidate protected pregnant macaques against fetal demise associated with highly pathogenic ZIKV challenge.

Zika virus (ZIKV) infection during pregnancy can cause a broad range of neurological birth defects, collectively named Congenital Zika Syndrome (CZS). We have previously shown that infection with the Puerto Rican isolate PRVABC59 (ZIKV-PR) results in abnormal oxygen transport in the placenta due to villous damage and uterine vasculitis in a nonhuman primate model. To investigate whether this type of damage occurs with endemically circulating strains in Thailand, we investigated a CZS case isolate, MU1-2017 (ZIKV-TH), in pregnant rhesus macaques. Pregnant animals (n = 3 per group) were infected subcutaneously with either ZIKV-PR or ZIKV-TH at ~50 days gestation (GD) and monitored for 40 days post-infection (GD90). Similar courses of viremia and immune activation were observed for both viruses when compared to uninfected controls. In addition, both viruses induced changes to the placental architecture, including spiral artery remodeling and the development of infarctions. Similar levels of viral RNA were detected at necropsy in maternal and fetal tissues. Overall, our results show that the ZIKV-TH strain MU1-2017 behaves similarly to the ZIKV-PR strain, and, importantly, provide evidence of in-utero infection with an additional contemporary strain of ZIKV.

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with Kaposi’s sarcoma and B cell malignancies. Like all herpesviruses, KSHV contains conserved envelope glycoproteins (gps) involved in virus binding, entry, assembly, and release from infected cells, which are also targets of the immune response. Due to the lack of a reproducible animal model of KSHV infection, the precise functions of the KSHV gps during infection in vivo are not completely known. Fortunately, a nonhuman primate (NHP) model of KSHV infection and disease has been established utilizing closely related rhesus macaque rhadinovirus (RRV) that naturally infects rhesus macaques (RM) and possesses analogous gps to KSHV. To address the roles conserved envelope gps gH and gL play during KSHV infection in vivo , we utilized the pathogenic RRV 17577 BAC to generate chimeric forms of RRV expressing KSHV gL or KSHV gH/gL, as well as an RRV mutant lacking gL expression. These viruses incorporate KSHV gH and gL into infectious virions, and although they display variable replication and differing plaque phenotypes in primary rhesus fibroblasts, they retain the ability to infect human B cells in vitro . Importantly, we also demonstrate that RRV gp chimeras can infect RM and induce the development of antibodies against KSHV. Overall, this work demonstrates that RRV gp chimeras can serve as important tools to assess the role of KSHV gH/gL in infection and disease while also providing an NHP model for testing the efficacy of KSHV gH and gL neutralizing antibodies and vaccine strategies to prevent and treat KSHV infection. IMPORTANCE Rhesus macaque rhadinovirus (RRV) is a rhesus macaque homolog of KSHV and serves as a model system for examining Kaposi’s sarcoma-associated herpesvirus (KSHV) infection and pathogenesis in vivo . KSHV and RRV both encode conserved herpesvirus envelope glycoproteins, including gH and gL, that are important for regulating entry into host cells. In this study, we utilized the RRV BAC system to generate chimeric forms of RRV expressing KSHV gH and gL, as well as a mutant form of RRV lacking gL expression. Although these mutant and chimeric viruses can replicate in vitro , they do display growth properties different from wild-type RRV. Importantly, we demonstrate that RRV gp chimeras are capable of infecting rhesus macaques in vivo , inducing B cell hyperplasia, and promoting the development of anti-viral antibody responses that can also recognize KSHV antigens. RRV gp chimeras provide a novel system that allows for the examination of the role of KSHV gH and gL during infection in vivo .

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.

O'nyong-nyong virus (ONNV) is a mosquito-borne alphavirus first isolated in Uganda in 1959. Since its discovery, ONNV has caused several outbreaks in Africa, manifesting clinically as fever, rash, and joint/muscle pain lasting months. Currently, we have a limited understanding of ONNV infection and disease in relevant animal models, which restricts the evaluation of vaccines and therapeutics. In 1967, Binn et al. reported that infection of rhesus macaques (RMs) with ONNV failed to induce viremia in two animals. This may be attributed to the potential attenuation of the virus through extensive passaging. To mitigate this issue, we constructed an infectious clone from the sequence of ONNV-UVRI0804 (ONNV 0804 ), a 2017 clinical isolate from a febrile patient in Uganda. This strain demonstrated high pathogenicity in immunocompetent mice, resulting in an earlier and more severe onset of disease and significantly higher viremia compared to a highly passaged control strain ONNV UgMP30 . In the current study, three male and three female rhesus macaques were subcutaneously inoculated with ONNV 0804 . All animals became viremic at 2 days post inoculation (dpi). Both classical and nonclassical monocytes were activated (CD169+), peaking at 3 dpi, which corresponded with the peak of viremia. Additionally, CD4+ and CD8+ effector memory T cells and memory B cells began proliferating in peripheral blood by day 7, peaking at day 10, which also corresponded to the timing of neutralizing antibody development, indicating a robust adaptive immune response to ONNV 0804 . Finally, key clinical disease manifestations were recapitulated, including lymphadenopathy and histological features of early-stage arthritis. Taken together, rhesus macaque infection with ONNV 0804 clinical isolate is a promising model for investigating immune responses to alphaviruses and evaluating vaccines to protect against future epidemics. Author summary:2. O'nyong-nyong virus (ONNV) is a mosquito-transmitted alphavirus that causes fever, rash, and prolonged joint and muscle pain, similar to chikungunya virus and other arthritogenic alphaviruses. Despite its capacity to cause outbreaks in Africa, ONNV remains understudied, and there are currently no approved vaccines or therapeutics to prevent or treat infection and disease. A major barrier to advancing ONNV research has been the lack of suitable animal models to study the virus and investigate host immune responses. We engineered an ONNV infectious clone of a recent clinical isolate sequenced from a patient in Uganda (ONNV 0804 ) that causes robust infection and disease in immunocompetent mice. In the current study, we provide data demonstrating that this contemporary ONNV strain infects rhesus macaques. Notably, rhesus macaques developed detectable viremia, rash, lymphadenopathy, joint and muscle inflammation, and strong innate and adaptive immune responses following subcutaneous ONNV 0804 infection. These findings suggest that ONNV 0804 infection in macaques is a promising model for studying ONNV pathogenesis and immunity. This model will be instrumental for evaluating future vaccine and therapeutic candidates aimed at preventing ONNV infection and related viral disease.

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.

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.

Immunocompromised individuals are at risk for developing lymphocryptovirus-associated lymphoproliferative diseases, such as Epstein Barr virus (EBV)-associated B cell lymphomas and post-transplant lymphoproliferative disorder (PTLD). We previously reported development of cynomolgus lymphocryptovirus (CyLCV)-associated PTLD in Mauritian cynomolgus macaques (MCMs) undergoing hematopoietic stem cell transplantation (HSCT), which mirrored EBV-PTLD in transplant patients. Here, we sought to develop a MCM model of lymphocryptovirus-associated lymphoproliferative disease in immunosuppressed MCMs without HSCT. Five simian immunodeficiency virus (SIV)-infected, CD8α+ cell-depleted MCMs received an infusion of autologous B-lymphoblastoid cells transformed with CyLCV, followed by varying degrees of immunosuppression. Four of five infused macaques developed masses coincident with increasing CyLCV plasma viremia, and necropsies confirmed the presence of multicentric lymphomas, which most commonly manifested in lymph nodes, gastrointestinal tract, adrenal glands, and pancreas. Affected tissues harbored neoplastic lymphocytes double-positive for CD20 and CyLCV EBNA2 antigen, large frequencies of proliferating B cells, and high levels of cell-associated CyLCV DNA. In addition, longitudinal 18F-fluorodeoxyglucose positron-emission tomography (18F-FDG PET) of one MCM successfully detected lymphoproliferative disease in the adrenal glands prior to clinical signs of disease. These data demonstrate successful induction of lymphocryptovirus-associated PTLD-like disease in 4 of 5 MCMs, and thus support the use of MCMs as a preclinical NHP model of EBV-associated lymphoproliferative disease that could be employed to test novel diagnostic and therapeutic modalities.

Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low.

Programmed cell death protein 1 (PD -1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV -infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD -1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD -1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2 -log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti- PD -1 CAR T cells depleted PD -1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Yellow fever virus (YFV) is endemic in >40 countries and causes viscerotropic disease with up to 20%-60% mortality. Successful live-attenuated yellow fever (YF) vaccines were developed in the mid-1930s, but their use is restricted or formally contraindicated in vulnerable populations including infants, the elderly, and people with compromised immune systems. In these studies, we describe the development of a next-generation hydrogen peroxide-inactivated YF vaccine and determine immune correlates of protection based on log neutralizing index (LNI) and neutralizing titer-50% (NT50) studies. In addition, we compare neutralizing antibody responses and protective efficacy of hydrogen peroxide-inactivated YF vaccine candidates to liveattenuated YFV-17D (YF-VAX) in a rhesus macaque model of viscerotropic YF. Our results indicate that an optimized, inactivated YF vaccine elicits protective antibody responses that prevent viral dissemination and lethal infection in rhesus macaques and may be a suitable alternative for vaccinating vulnerable populations who are not eligible to receive replicating live-attenuated YF vaccines.

IntroductionEpstein-Barr virus (EBV) is an oncogenic human herpesvirus associated with ~350,000 cases of lymphoid and epithelial malignancies every year, and is etiologically linked to infectious mononucleosis and multiple sclerosis. Despite four decades of research, no EBV vaccine candidate has yet reached licensure. Most previous vaccine attempts focused on a single viral entry glycoprotein, gp350, but recent data from clinical and pre-clinical studies, and the elucidation of viral entry mechanisms, support the inclusion of multiple entry glycoproteins in EBV vaccine design.MethodsHere we generated a modified vaccinia Ankara (MVA)-vectored EBV vaccine, MVA-EBV5-2, that targets five EBV entry glycoproteins, gp350, gB, and the gp42gHgL complex. We characterized the genetic and translational stability of the vaccine, followed by immunogenicity assessment in BALB/c mice and rhesus lymphocryptovirus-negative rhesus macaques as compared to a gp350-based MVA vaccine. Finally, we assessed the efficacy of MVA-EBV5-2-immune rhesus serum at preventing EBV infection in human CD34+ hematopoietic stem cell-reconstituted NSG mice, under two EBV challenge doses.ResultsThe MVA-EBV5-2 vaccine was genetically and translationally stable over 10 viral passages as shown by genetic and protein expression analysis, and when administered to female and male BALB/c mice, elicited serum EBV-specific IgG of both IgG1 and IgG2a subtypes with neutralizing activity in vitro. In Raji B cells, this neutralizing activity outperformed that of serum from mice immunized with a monovalent MVA-vectored gp350 vaccine. Similarly, MVA-EBV5-2 elicited EBV-specific IgG in rhesus macaques that were detected in both serum and saliva of immunized animals, with serum antibodies demonstrating neutralizing activity in vitro that outperformed serum from MVA-gp350-immunized macaques. Finally, pre-treatment with serum from MVA-EBV5-2-immunized macaques resulted in fewer EBV-infected mice in the two challenge experiments than pretreatment with serum from pre-immune macaques or macaques immunized with the monovalent gp350-based vaccine.DiscussionThese results support the inclusion of multiple entry glycoproteins in EBV vaccine design and position our vaccine as a strong candidate for clinical translation.

The rebound competent viral reservoir (RCVR)-virus that persists during antiretroviral treatment (ART) and can reignite systemic infection when treatment is stopped-is the primary barrier to eradicating HIV. We used time to initiation of ART during primary infection of rhesus macaques (RMs) after intravenous challenge with barcoded SIVmac239 as a means to elucidate the dynamics of RCVR establishment in groups of RMs by creating a multi-log range of pre-ART viral loads and then assessed viral time-to-rebound and reactivation rates resulting from the discontinuation of ART after one year. RMs started on ART on days 3, 4, 5, 6, 7, 9 or 12 post-infection showed a nearly 10-fold difference in pre-ART viral measurements for successive ART-initiation timepoints. Only 1 of 8 RMs initiating ART on days 3 and 4 rebounded after ART interruption despite measurable pre-ART plasma viremia. Rebounding plasma from the 1 rebounding RM contained only a single barcode lineage detected at day 50 post-ART. All RMs starting ART on days 5 and 6 rebounded between 14- and 50-days post-ART with 1-2 rebounding variants each. RMs starting ART on days 7, 9, and 12 had similar time-to-measurable plasma rebound kinetics despite multiple log differences in pre-ART plasma viral load (pVL), with all RMs rebounding between 7- and 16-days post-ART with 3-28 rebounding lineages. Calculated reactivation rates per pre-ART pVL were highest for RMs starting ART on days 5, 6, and 7 after which the rate of accumulation of the RCVR markedly decreased for RMs treated on days 9 and 12, consistent with multiphasic establishment and near saturation of the RCVR within 2 weeks post infection. Taken together, these data highlight the heterogeneity of the RCVR between RMs, the stochastic establishment of the very early RCVR, and the saturability of the RCVR prior to peak viral infection.

Expression of cell surface Fc gamma receptors on human cells