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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.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 10 5 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

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.

RNA 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. Here 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. We 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 stimulation. Combining 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.

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/SIV-Gag vaccine group showed a significant delay in time to measurable viral rebound compared to controls, and manifested lower plasma viral loads (PVL) 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 to 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 post-rebound, highlighting the need for adjunctive therapies to improve the durability of virologic control elicited by CD8+ T cell-targeting vaccines.

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.sup.+] 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.sup.+] memory T cells in blood and tissues, including lymph node [CD4.sup.+] 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.sup.+] memory T cells. These data indicate anti-PD-1 CAR T cells depleted [PD-1.sup.+] T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

[This corrects the article DOI: 10.1371/journal.ppat.1009565.].

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.

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 ), 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 . In the current study, three male and three female rhesus macaques were subcutaneously inoculated with ONNV . 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 . Finally, key clinical disease manifestations were recapitulated, including lymphadenopathy and histological features of early-stage arthritis. Taken together, rhesus macaque infection with ONNV clinical isolate is a promising model for investigating immune responses to alphaviruses and evaluating vaccines to protect against future epidemics. 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 ) 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 infection. These findings suggest that ONNV 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.

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.