Distinct SIV-specific CD8+ T cells in the lymph node exhibit simultaneous effector and stem-like profiles and are associated with limited SIV persistence.

Human immunodeficiency virus (HIV) cure efforts are increasingly focused on harnessing CD8+ T cell functions, which requires a deeper understanding of CD8+ T cells promoting HIV control. Here we identifiy an antigen-responsive TOXhiTCF1+CD39+CD8+ T cell population with high expression of inhibitory receptors and low expression of canonical cytolytic molecules. Transcriptional analysis of simian immunodeficiency virus (SIV)-specific CD8+ T cells and proteomic analysis of purified CD8+ T cell subsets identified TOXhiTCF1+CD39+CD8+ T cells as intermediate effectors that retained stem-like features with a lineage relationship with terminal effector T cells. TOXhiTCF1+CD39+CD8+ T cells were found at higher frequency than TCF1-CD39+CD8+ T cells in follicular microenvironments and were preferentially located in proximity of SIV-RNA+ cells. Their frequency was associated with reduced plasma viremia and lower SIV reservoir size. Highly similar TOXhiTCF1+CD39+CD8+ T cells were detected in lymph nodes from antiretroviral therapy-naive and antiretroviral therapy-suppressed people living with HIV, suggesting this population of CD8+ T cells contributes to limiting SIV and HIV persistence.

CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

CTLA-4+PD-1- Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques.

Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.

Chronic immune activation and gut barrier dysfunction is associated with neuroinflammation in ART-suppressed SIV+ rhesus macaques.

HIV-associated neurocognitive disorders (HAND) affect ~40% of virally suppressed people with HIV (PWH), however, the precise viral dependent and independent changes to the brain are unclear. Here we characterized the CNS reservoir and immune environment of SIV-infected (SIV+) rhesus macaques during acute (n = 4), chronic (n = 12) or ART-suppressed SIV infection (n = 11). Multiplex immunofluorescence for markers of SIV infection (vRNA/vDNA) and immune activation was performed on frontal cortex and matched colon tissue. SIV+ animals contained detectable viral DNA+ cells that were not reduced in the frontal cortex or the gut by ART, supporting the presence of a stable viral reservoir in these compartments. SIV+ animals had impaired blood brain barrier (BBB) integrity and heightened levels of astrocytes or myeloid cells expressing antiviral, anti-inflammatory or oxidative stress markers which were not abrogated by ART. Neuroinflammation and BBB dysfunction correlated with measures of viremia and immune activation in the gut. Furthermore, SIV-uninfected animals with experimentally induced gut damage and colitis showed a similar immune activation profile in the frontal cortex to those of SIV-infected animals, supporting the role of chronic gut damage as an independent source of neuroinflammation. Together, these findings implicate gut-associated immune activation/damage as a significant contributor to neuroinflammation in ART-suppressed HIV/SIV infection which may drive HAND pathogenesis.

Prolonged Mpox Disease in People With Advanced HIV: Characterization of Mpox Skin Lesions.

We report 3 complicated and prolonged cases of mpox in people with advanced human immunodeficiency virus (HIV) not on antiretroviral therapy (ART) at mpox diagnosis. Multiple medical countermeasures were used, including prolonged tecovirimat treatment and immune optimization with ART initiation. Immunofluorescence of skin biopsies demonstrated a dense immune infiltrate of predominantly myeloid and CD8+ T cells, with a strong type I interferon local response. RNAscope detected abundant replication of monkeypox virus (MPXV) in epithelial cells and dendritic cells. These data suggest that prolonged mpox in people with advanced HIV may be due to ongoing MPXV replication, warranting aggressive medical countermeasures and immune optimization.

Regional Analysis of Intact and Defective HIV Proviruses in the Brain of Viremic and Virally Suppressed People with HIV.

Here, we provide the first regional analysis of intact and defective HIV reservoirs within the brain. Brain tissue from both viremic and virally suppressed people with HIV (PWH) harbored HIV pol DNA in all regions tested, with lower levels present in basal ganglia and cerebellum relative to frontal white matter. Intact proviruses were primarily found in the frontal white matter but also detected in other brain regions of PWH, demonstrating frontal white matter as a major brain reservoir of intact, potentially replication competent HIV DNA that persists despite antiretroviral therapy. ANN NEUROL 2023;94:798-802.

Tissue myeloid cells in SIV-infected primates acquire viral DNA through phagocytosis of infected T cells.

The viral accessory protein Vpx, expressed by certain simian and human immunodeficiency viruses (SIVs and HIVs), is thought to improve viral infectivity of myeloid cells. We infected 35 Asian macaques and African green monkeys with viruses that do or do not express Vpx and examined viral targeting of cells in vivo. While lack of Vpx expression affected viral dynamics in vivo, with decreased viral loads and infection of CD4⁺ T cells, Vpx expression had no detectable effect on infectivity of myeloid cells. Moreover, viral DNA was observed only within myeloid cells in tissues not massively depleted of CD4⁺ T cells. Myeloid cells containing viral DNA also showed evidence of T cell phagocytosis in vivo, suggesting that their viral DNA may be attributed to phagocytosis of SIV-infected T cells. These data suggest that myeloid cells are not a major source of SIV in vivo, irrespective of Vpx expression.

Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection.

In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein 1 (XBP-1s). XBP-1s is also present in large amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and, thus, contribute to the pathogenesis of KSHV MCD. KSHV thymidine kinase (TK), the ORF21 gene product, can enhance the production of dTTP and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced upregulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominantly found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and, thus, contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and valganciclovir in this disease.IMPORTANCE Spliced X-box binding protein 1 (XBP-1s), part of the unfolded protein response and expressed in developing germinal center B cells, can induce Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of dTTP for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused, in part, by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD.

Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV.

Viruses have colonized the germ line of our ancestors on several occasions during evolution, leading to the integration in the human genome of viral sequences from over 30 retroviral groups and a few nonretroviruses. Among the recently emerged viruses infecting humans, several target the testis (e.g., human immunodeficiency virus [HIV], Zika virus, and Ebola virus). Here, we aimed to investigate whether human testicular germ cells (TGCs) can support integration by HIV, a contemporary retrovirus that started to spread in the human population during the last century. We report that albeit alternative receptors enabled HIV-1 binding to TGCs, HIV virions failed to infect TGCs in vitro Nevertheless, exposure of TGCs to infected lymphocytes, naturally present in the testis from HIV+ men, led to HIV-1 entry, integration, and early protein expression. Similarly, cell-associated infection or bypassing viral entry led to HIV-1 integration in a spermatogonial cell line. Using DNAscope, HIV-1 and simian immunodeficiency virus (SIV) DNA were detected within a few TGCs in the testis from one infected patient, one rhesus macaque, and one African green monkey in vivo Molecular landscape analysis revealed that early TGCs were enriched in HIV early cofactors up to integration and had overall low antiviral defenses compared with testicular macrophages and Sertoli cells. In conclusion, our study reveals that TGCs can support the entry and integration of HIV upon cell-associated infection. This could represent a way for this contemporary virus to integrate into our germ line and become endogenous in the future, as happened during human evolution for a number of viruses.IMPORTANCE Viruses have colonized the host germ line on many occasions during evolution to eventually become endogenous. Here, we aimed at investigating whether human testicular germ cells (TGCs) can support such viral invasion by studying HIV interactions with TGCs in vitro Our results indicate that isolated primary TGCs express alternative HIV-1 receptors, allowing virion binding but not entry. However, HIV-1 entered and integrated into TGCs upon cell-associated infection and produced low levels of viral proteins. In vivo, HIV-1 and SIV DNA was detected in a few TGCs. Molecular landscape analysis showed that TGCs have overall weak antiviral defenses. Altogether, our results indicate that human TGCs can support HIV-1 early replication, including integration, suggesting potential for endogenization in future generations.

Fingolimod retains cytolytic T cells and limits T follicular helper cell infection in lymphoid sites of SIV persistence.

Lymph nodes (LN) and their resident T follicular helper CD4+ T cells (Tfh) are a critical site for HIV replication and persistence. Therefore, optimizing antiviral activity in lymphoid tissues will be needed to reduce or eliminate the HIV reservoir. In this study, we retained effector immune cells in LN of cART-suppressed, SIV-infected rhesus macaques by treatment with the lysophospholipid sphingosine-1 phosphate receptor modulator FTY720 (fingolimod). FTY720 was remarkably effective in reducing circulating CD4+ and CD8+ T cells, including those with cytolytic potential, and in increasing the number of these T cells retained in LN, as determined directly in situ by histocytometry and immunohistochemistry. The FTY720-induced inhibition of T cell egress from LN resulted in a measurable decrease of SIV-DNA content in blood as well as in LN Tfh cells in most treated animals. In conclusion, FTY720 administration has the potential to limit viral persistence, including in the critical Tfh cellular reservoir. These findings provide rationale for strategies designed to retain antiviral T cells in lymphoid tissues to target HIV remission.

Kynurenine 3-Monooxygenase Inhibition during Acute Simian Immunodeficiency Virus Infection Lowers PD-1 Expression and Improves Post-Combination Antiretroviral Therapy CD4+ T Cell Counts and Body Weight.

The kynurenine pathway (KP) is a key regulator of many important physiological processes and plays a harmful role in cancer, many neurologic conditions, and chronic viral infections. In HIV infection, KP activity is consistently associated with reduced CD4 T cell counts and elevated levels of T cell activation and viral load; it also independently predicts mortality and morbidity from non-AIDS events. Kynurenine 3-monooxygenase (KMO) is a therapeutically important target in the KP. Using the nonhuman primate model of SIV infection in rhesus macaques, we investigated whether KMO inhibition could slow the course of disease progression. We used a KMO inhibitor, CHDI-340246, to perturb the KP during early acute infection and followed the animals for 1 y to assess clinical outcomes and immune phenotype and function during pre-combination antiretroviral therapy acute infection and combination antiretroviral therapy-treated chronic infection. Inhibition of KMO in acute SIV infection disrupted the KP and prevented SIV-induced increases in downstream metabolites, improving clinical outcome as measured by both increased CD4+ T cell counts and body weight. KMO inhibition increased naive T cell frequency and lowered PD-1 expression in naive and memory T cell subsets. Importantly, early PD-1 expression during acute SIV infection predicted clinical outcomes of body weight and CD4+ T cell counts. Our data indicate that KMO inhibition in early acute SIV infection provides clinical benefit and suggest a rationale for testing KMO inhibition as an adjunctive treatment in SIV/HIV infection to slow the progression of the disease and improve immune reconstitution.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Ex Vivo Manipulation.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

Correction: Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

[This corrects the article DOI: 10.1371/journal.ppat.1006902.].

Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT02452268.

Differential impact of transplantation on peripheral and tissue-associated viral reservoirs: Implications for HIV gene therapy.

Autologous transplantation and engraftment of HIV-resistant cells in sufficient numbers should recapitulate the functional cure of the Berlin Patient, with applicability to a greater number of infected individuals and with a superior safety profile. A robust preclinical model of suppressed HIV infection is critical in order to test such gene therapy-based cure strategies, both alone and in combination with other cure strategies. Here, we present a nonhuman primate (NHP) model of latent infection using simian/human immunodeficiency virus (SHIV) and combination antiretroviral therapy (cART) in pigtail macaques. We demonstrate that transplantation of CCR5 gene-edited hematopoietic stem/progenitor cells (HSPCs) persist in infected and suppressed animals, and that protected cells expand through virus-dependent positive selection. CCR5 gene-edited cells are readily detectable in tissues, namely those closely associated with viral reservoirs such as lymph nodes and gastrointestinal tract. Following autologous transplantation, tissue-associated SHIV DNA and RNA levels in suppressed animals are significantly reduced (p ≤ 0.05), relative to suppressed, untransplanted control animals. In contrast, the size of the peripheral reservoir, measured by QVOA, is variably impacted by transplantation. Our studies demonstrate that CCR5 gene editing is equally feasible in infected and uninfected animals, that edited cells persist, traffic to, and engraft in tissue reservoirs, and that this approach significantly reduces secondary lymphoid tissue viral reservoir size. Our robust NHP model of HIV gene therapy and viral persistence can be immediately applied to the investigation of combinatorial approaches that incorporate anti-HIV gene therapy, immune modulators, therapeutic vaccination, and latency reversing agents.

Gammaherpesvirus infection and malignant disease in rhesus macaques experimentally infected with SIV or SHIV.

Human gammaherpesviruses are associated with malignancies in HIV infected individuals; in macaques used in non-human primate models of HIV infection, gammaherpesvirus infections also occur. Limited data on prevalence and tumorigenicity of macaque gammaherpesviruses, mostly cross-sectional analyses of small series, are available. We comprehensively examine all three-rhesus macaque gammaherpesviruses -Rhesus rhadinovirus (RRV), Rhesus Lymphocryptovirus (RLCV) and Retroperitoneal Fibromatosis Herpesvirus (RFHV) in macaques experimentally infected with Simian Immunodeficiency Virus or Simian Human Immunodeficiency Virus (SIV/SHIV) in studies spanning 15 years at the AIDS and Cancer Virus Program of the Frederick National Laboratory for Cancer Research. We evaluated 18 animals with malignancies (16 lymphomas, one fibrosarcoma and one carcinoma) and 32 controls. We developed real time quantitative PCR assays for each gammaherpesvirus DNA viral load (VL) in malignant and non-tumor tissues; we also characterized the tumors using immunohistochemistry and in situ hybridization. Furthermore, we retrospectively quantified gammaherpesvirus DNA VL and SIV/SHIV RNA VL in longitudinally-collected PBMCs and plasma, respectively. One or more gammaherpesviruses were detected in 17 tumors; generally, one was predominant, and the relevant DNA VL in the tumor was very high compared to surrounding tissues. RLCV was predominant in tumors resembling diffuse large B cell lymphomas; in a Burkitt-like lymphoma, RRV was predominant; and in the fibrosarcoma, RFHV was predominant. Median RRV and RLCV PBMC DNA VL were significantly higher in cases than controls; SIV/SHIV VL and RLCV VL were independently associated with cancer. Local regressions showed that longitudinal VL patterns in cases and controls, from SIV infection to necropsy, differed for each gammaherpesvirus: while RFHV VL increased only slightly in all animals, RLCV and RRV VL increased significantly and continued to increase steeply in cases; in controls, VL flattened. In conclusion, the data suggest that gammaherpesviruses may play a significant role in tumorogenesis in macaques infected with immunodeficiency viruses.

Simian Immunodeficiency Virus Persistence in Cellular and Anatomic Reservoirs in Antiretroviral Therapy-Suppressed Infant Rhesus Macaques.

Worldwide, nearly two million children are infected with human immunodeficiency virus (HIV), with breastfeeding accounting for the majority of contemporary HIV transmissions. Antiretroviral therapy (ART) has reduced HIV-related morbidity and mortality but is not curative. The main barrier to a cure is persistence of latent HIV in long-lived reservoirs. However, our understanding of the cellular and anatomic sources of the HIV reservoir during infancy and childhood is limited. Here, we developed a pediatric model of ART suppression in orally simian immunodeficiency virus (SIV)-infected rhesus macaque (RM) infants, with measurement of virus persistence in blood and tissues after 6 to 9 months of ART. Cross-sectional analyses were conducted to compare SIV RNA and DNA levels in adult and infant RMs naive to treatment and on ART. We demonstrate efficient viral suppression following ART initiation in SIV-infected RM infants with sustained undetectable plasma viral loads in the setting of heterogeneous penetration of ART into lymphoid and gastrointestinal tissues and low drug levels in the brain. We further show reduction in SIV RNA and DNA on ART in lymphoid tissues of both infant and adult RMs but stable (albeit low) levels of SIV RNA and DNA in the brains of viremic and ART-suppressed infants. Finally, we report a large contribution of naive CD4+ T cells to the total CD4 reservoir of SIV in blood and lymph nodes of ART-suppressed RM infants that differs from what we show in adults. These results reveal important aspects of HIV/SIV persistence in infants and provide insight into strategic targets for cure interventions in a pediatric population.IMPORTANCE While antiretroviral therapy (ART) can reduce HIV replication, the virus cannot be eradicated from an infected individual, and our incomplete understanding of HIV persistence in reservoirs greatly complicates the generation of a cure for HIV infection. Given the immaturity of the infant immune system, it is critically important to study HIV reservoirs specifically in this population. Here, we established a pediatric animal model to simulate breastfeeding transmission and study SIV reservoirs in rhesus macaque (RM) infants. Our study demonstrates that ART can be safely administered to infant RMs for prolonged periods and that it efficiently controls viral replication in this model. SIV persistence was shown in blood and tissues, with similar anatomic distributions of SIV reservoirs in infant and adult RMs. However, in the peripheral blood and lymph nodes, a greater contribution of the naive CD4+ T cells to the SIV reservoir was observed in infants than in adults.

Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs.

The sexual transmission of viruses is responsible for the spread of multiple infectious diseases. Although the human immunodeficiency virus (HIV)/AIDS pandemic remains fueled by sexual contacts with infected semen, the origin of virus in semen is still unknown. In a substantial number of HIV-infected men, viral strains present in semen differ from the ones in blood, suggesting that HIV is locally produced within the genital tract. Such local production may be responsible for the persistence of HIV in semen despite effective antiretroviral therapy. In this study, we used single-genome amplification, amplicon sequencing (env gene), and phylogenetic analyses to compare the genetic structures of simian immunodeficiency virus (SIV) populations across all the male genital organs and blood in intravenously inoculated cynomolgus macaques in the chronic stage of infection. Examination of the virus populations present in the male genital tissues of the macaques revealed compartmentalized SIV populations in testis, epididymis, vas deferens, seminal vesicles, and urethra. We found genetic similarities between the viral strains present in semen and those in epididymis, vas deferens, and seminal vesicles. The contribution of male genital organs to virus shedding in semen varied among individuals and could not be predicted based on their infection or proinflammatory cytokine mRNA levels. These data indicate that rather than a single source, multiple genital organs are involved in the release of free virus and infected cells into semen. These findings have important implications for our understanding of systemic virus shedding and persistence in semen and for the design of eradication strategies to access viral reservoirs.IMPORTANCE Semen is instrumental for the dissemination of viruses through sexual contacts. Worryingly, a number of systemic viruses, such as HIV, can persist in this body fluid in the absence of viremia. The local source(s) of virus in semen, however, remains unknown. To elucidate the anatomic origin(s) of the virus released in semen, we compared viral populations present in semen with those in the male genital organs and blood of the Asian macaque model, using single-genome amplification, amplicon sequencing (env gene), and phylogenetic analysis. Our results show that multiple genital tissues harbor compartmentalized strains, some of them (i.e., from epididymis, vas deferens, and seminal vesicles) displaying genetic similarities with the viral populations present in semen. This study is the first to uncover local genital sources of viral populations in semen, providing a new basis for innovative targeted strategies to prevent and eradicate HIV in the male genital tract.

Central Nervous System Inflammation and Infection during Early, Nonaccelerated Simian-Human Immunodeficiency Virus Infection in Rhesus Macaques.

Studies utilizing highly pathogenic simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) have largely focused on the immunopathology of the central nervous system (CNS) during end-stage neurological AIDS and SIV encephalitis. However, this may not model pathophysiology in earlier stages of infection. In this nonaccelerated SHIV model, plasma SHIV RNA levels and peripheral blood and colonic CD4+ T cell counts mirrored early human immunodeficiency virus (HIV) infection in humans. At 12 weeks postinfection, cerebrospinal fluid (CSF) detection of SHIV RNA and elevations in IP-10 and MCP-1 reflected a discrete neurovirologic process. Immunohistochemical staining revealed a diffuse, low-level CD3+ CD4- cellular infiltrate in the brain parenchyma without a concomitant increase in CD68/CD163+ monocytes, macrophages, and activated microglial cells. Rare SHIV-infected cells in the brain parenchyma and meninges were identified by RNAScope in situ hybridization. In the meninges, there was also a trend toward increased CD4+ infiltration in SHIV-infected animals but no differences in CD68/CD163+ cells between SHIV-infected and uninfected control animals. These data suggest that in a model that closely recapitulates human disease, CNS inflammation and SHIV in CSF are predominantly mediated by T cell-mediated processes during early infection in both brain parenchyma and meninges. Because SHIV expresses an HIV rather than SIV envelope, this model could inform studies to understand potential HIV cure strategies targeting the HIV envelope.IMPORTANCE Animal models of the neurologic effects of HIV are needed because brain pathology is difficult to assess in humans. Many current models focus on the effects of late-stage disease utilizing SIV. In the era of antiretroviral therapy, manifestations of late-stage HIV are less common. Furthermore, new interventions, such as monoclonal antibodies and therapeutic vaccinations, target HIV envelope. We therefore describe a new model of central nervous system involvement in rhesus macaques infected with SHIV expressing HIV envelope in earlier, less aggressive stages of disease. Here, we demonstrate that SHIV mimics the early clinical course in humans and that early neurologic inflammation is characterized by predominantly T cell-mediated inflammation accompanied by SHIV infection in the brain and meninges. This model can be utilized to assess the effect of novel therapies targeted to HIV envelope on reducing brain inflammation before end-stage disease.