Simian Immunodeficiency Virus DNA Research Articles

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates.

Antigen-specific CD8+ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

Therapeutic effect of (5R)-5-hydroxytriptolide (LLDT-8) in SIV infected rhesus monkeys

BackgroundsHuman immunodeficiency virus (HIV) infections induce robust, generalized inflammatory responses and lead to pathological systemic immune activation. This abnormal immune status persists despite successful antiretroviral therapy (ART). Immune modulating strategies in conjunction with ART were tried to reduce abnormal immune activation. Previously, we demonstrated that Tripterygium Wilfordii Hook F has been shown immunosuppressive activity in HIV patients. (5R)-5-hydroxytriptolide (LLDT-8), a new analog of triptolide, and the most active ingredient of Tripterygium Wilfordii Hook F, has been shown to have lower cytotoxicity. However, the role of LLDT-8 in HIV or simian immunodeficiency virus (SIV) needs to be explored. MethodsSix male adult Chinese rhesus monkeys were enrolled in our study. All of them were healthy and negative for SIV, and chronically SIVmac239 infected macaques were treated with LLDT-8 combined with ART (n = 4) or ART only (n = 2) after 14 weeks of infection. ART was determined at week 33, and LLDT-8 was continued until week 48. T cell immune activation and inflammation were compared during the period, and viral rebound time and reservoir were supervised after stopping ART. ResultsThe RNA level of the two groups continued to decline after initiating ART, RNA of 4 rhesus monkeys declined to the lower limit of detection at week 20. LLDT-8 administration combined with ART did not affect T cell activation and plasma levels of IL-6 and CRP. The viral load of all the macaques in both groups was rebounded 2 weeks after ART discontinuation. Furthermore, no significant decrease of SIV DNA was observed in the LLDT-8 treatment group. ConclusionsLLDT-8 administration during chronic SIV infection had no effect on T cell activation and plasma levels; Furthermore, LLDT-8 may not contribute to suppression of viral rebound and reservoir. These results suggest that LLDT-8 is unlikely to reduce immune activation and viral persistence without additional interventions.

DNA repair and gene editing: the director’s cut

Novelists and screenwriters have bombarded our imaginations with the idea of genetic engineering. From superhero origin stories to theme parks inhabited by dinosaurs, the prospect of re-writing the genetic code has inspired many and raised many ethical questions. The potential for these tools in medicine and biological sciences to prevent genetic diseases is readily being explored. Recent successes include destruction of simian immunodeficiency virus DNA from infected rhesus macaque monkeys (synonymous to the human immunodeficiency virus). The diverse power of these tools is also helping to control mosquito populations and supress the spread of malaria. New gene-editing tools have made genome editing faster, more accurate and cheaper than ever before, but how do they work? And how do we know whether the desired edits will be made?

Comparative Evaluation of Prophylactic SIV Vaccination Modalities Administered to the Oral Cavity.

Attempts to develop a protective human immunodeficiency virus (HIV) vaccine have had limited success, especially in terms of inducing protective antibodies capable of neutralizing different viral strains. As HIV transmission occurs mainly via mucosal surfaces, HIV replicates significantly in the gastrointestinal tract, and the oral route of vaccination is a very convenient one to implement worldwide, we explored three SIV vaccine modalities administered orally and composed of simian immunodeficiency virus (SIV) DNA priming with different boosting immunogens, with the goal of evaluating whether they could provide lasting humoral and cellular responses, including at mucosal surfaces that are sites of HIV entry. Twenty-four Cynomolgus macaques (CyM) were primed with replication-incompetent SIV DNA provirus and divided into three groups for the following booster vaccinations, all administered in the oral cavity: Group 1 with recombinant SIV gp140 and Escherichia coli heat-labile toxin adjuvant dmLT, Group 2 with recombinant SIV-Oral Poliovirus (SIV-OPV), and Group 3 with recombinant SIV-modified vaccinia ankara (SIV-MVA). Cell-mediated responses were measured using blood, lymph node, rectal and vaginal mononuclear cells. Significant levels of systemic and mucosal T-cell responses against Gag and Env were observed in all groups. Some SIV-specific plasma IgG, rectal and salivary IgA antibodies were generated, mainly in animals that received SIV DNA + SIV-MVA, but no vaginal IgA was detected. Susceptibility to infection after SIVmac251 challenge was similar in vaccinated and nonvaccinated animals, but acute infection viremia levels were lower in the group that received SIV DNA + SIV-MVA. Nonvaccinated CyM maintained central memory and total CD4+ T-cell levels in the normal range during the 5 months of postinfection follow-up as did the vaccinated animals, precluding evaluation of vaccine impact on disease progression. We conclude that the oral cavity vaccination tested in these regimens can stimulate cell-mediated immunity systemically and mucosally, but humoral response stimulation was limited with the doses and the vaccine platforms used.

Simian Immunodeficiency Virus-Infected Memory CD4+ T Cells Infiltrate to the Site of Infected Macrophages in the Neuroparenchyma of a Chronic Macaque Model of Neurological Complications of AIDS.

Simian immunodeficiency virus (SIV)-infected nonhuman primates can serve as a relevant model for AIDS neuropathogenesis. Current SIV-induced encephalitis (SIVE)/neurological complications of AIDS (neuroAIDS) models are generally associated with rapid progression to neuroAIDS, which does not reflect the tempo of neuroAIDS progression in humans. Recently, we isolated a neuropathogenic clone, SIVsm804E-CL757 (CL757), obtained from an SIV-infected rhesus macaque (RM). CL757 causes a more protracted progression to disease, inducing SIVE in 50% of inoculated animals, with high cerebral spinal fluid viral loads, multinucleated giant cells (MNGCs), and perivascular lymphocytic cuffing in the central nervous system (CNS). This latter finding is reminiscent of human immunodeficiency virus (HIV) encephalitis in humans but not generally observed in rapid progressor animals with neuroAIDS. Here, we studied which subsets of cells within the CNS were targeted by CL757 in animals with neurological symptoms of SIVE. Immunohistochemistry of brain sections demonstrated infiltration of CD4+ T cells (CD4) and macrophages (MΦs) to the site of MNGCs. Moreover, an increase in mononuclear cells isolated from the brain tissues of RMs with SIVE correlated with increased cerebrospinal fluid (CSF) viral load. Subset analysis showed a specific increase in brain CD4+ memory T cells (Br-mCD4), brain-MΦs (Br-MΦs), and brain B cells (Br-B cells). Both Br-mCD4s and Br-MΦs harbored replication-competent viral DNA, as demonstrated by virus isolation by coculture. However, only in animals exhibiting SIVE/neuroAIDS was virus isolated from Br-MΦs. These findings support the use of CL757 to study the pathogenesis of AIDS viruses in the central nervous system and indicate a previously unanticipated role of CD4s cells as a potential reservoir in the brain.IMPORTANCE While the use of combination antiretroviral therapy effectively suppresses systemic viral replication in the body, neurocognitive disorders as a result of HIV infection of the central nervous system (CNS) remain a clinical problem. Therefore, the use of nonhuman primate models is necessary to study mechanisms of neuropathogenesis. The neurotropic, molecular clone SIVsm804E-CL757 (CL757) results in neuroAIDS in 50% of infected rhesus macaques approximately 1 year postinfection. Using CL757-infected macaques, we investigate disease progression by examining subsets of cells within the CNS that were targeted by CL757 and could potentially serve as viral reservoirs. By isolating mononuclear cells from the brains of SIV-infected rhesus macaques with and without encephalitis, we show that immune cells invade the neuroparenchyma and increase in number in the CNS in animals with SIV-induced encephalitis (SIVE). Of these cells, both brain macrophages and brain memory CD4+ T cells harbor replication-competent SIV DNA; however, only brain CD4+ T cells harbored SIV DNA in animals without SIVE. These findings support use of CL757 as an important model to investigate disease progression in the CNS and as a model to study virus reservoirs in the CNS.

Experimental Treatment of SIV-Infected Macaques via Autograft of CCR5-Disrupted Hematopoietic Stem and Progenitor Cells

Hematopoietic stem cell (HSC)-based gene therapy targeting CCR5 represents a promising way to cure human immunodeficiency virus type 1 (HIV-1) infection. Yet the preclinical animal model with transplantation of autologous CCR5-ablated HSCs remains to be optimized. In this study, four Chinese rhesus macaques of simian immunodeficiency virus (SIV) chronic infection were given long-term antiretroviral therapy (ART), during which peripheral CD34+ hematopoietic stem and progenitor cells (HSPCs) were purified and infected with CCR5-specific CRISPR/Cas9 lentivirus (three monkeys) or GFP lentivirus (one monkey). After non-myeloablative conditioning, the CCR5-modified or GFP-labeled HSPCs were autotransplanted to four recipients, and ART was withdrawn following engraftment. All of the recipients survived the process of transplantation. The purified CD34+ HSPCs harbored an undetectable level of integrated SIV DNA. The efficiency of CCR5 disruption in HSPCs ranges from 6.5% to 15.6%. Animals experienced a comparable level of hematopoietic reconstuction and displayed a similar physiological homeostasis Despite the low-level editing of CCR5 in vivo (0.3%–1%), the CCR5-disrupted cells in peripheral CD4+ Effector Memory T cell (TEM) subsets were enriched 2- to 3-fold after cessation of ART. Moreover, two of the three treated monkeys displayed a delayed viral rebound and a moderately recovered immune function 6 months after ART withdrawal. This study highlights the importance of improving the CCR5-editing efficacy and augmenting the virus-specific immunity for effective treatment of HIV-1 infection.

Arsenic Trioxide Impacts Viral Latency and Delays Viral Rebound after Termination of ART in Chronically SIV-Infected Macaques.

The latent viral reservoir is the source of viral rebound after interruption of antiretroviral therapy (ART) and is the major obstacle in eradicating the latent human immunodeficiency virus‐1 (HIV‐1). In this study, arsenic class of mineral, arsenic trioxide, clinically approved for treating acute promyelocytic leukemia, is demonstrated to reactivate latent provirus in CD4+ T cells from HIV‐1 patients and Simian immunodeficiency virus (SIV)‐infected macaques, without significant systemic T cell activation and inflammatory responses. In a proof‐of‐concept study using chronically SIVmac239‐infected macaques, arsenic trioxide combined with ART delays viral rebound after ART termination, reduces the integrated SIV DNA copies in CD4+ T cells, and restores CD4+ T cells counts in vivo. Most importantly, half of arsenic trioxide‐treated macaques show no detectable viral rebound in the plasma for at least 80 days after ART discontinuation. Mechanistically, the study reveals that CD4 receptors and CCR5 co‐receptors of CD4+ T cells are significantly downregulated by arsenic trioxide treatment, which reduces susceptibility to infection after provirus reactivation. Furthermore, an increase in SIV‐specific immune responses after arsenic trioxide treatment may contribute to suppression of viral rebound. This work suggests that arsenic trioxide in combination with ART is a novel regimen in down‐sizing or even eradicating latent HIV‐1 reservoir.

Low level expression of the Mitochondrial Antiviral Signaling protein (MAVS) associated with long-term nonprogression in SIV-infected rhesus macaques

BackgroundAbnormally increased immune activation is one of the main pathological features of acquired immunodeficiency syndrome (AIDS). This study aimed to determine whether long-term nonprogression (LTNP) suppresses the upregulation of immune activation and to elucidate the mechanisms whereby the LTNP state is maintained.MethodsFor this study we selected 4 rhesus macaques(RMs) infected with simian immunodeficiency virus (SIV) that were long-term nonprogressors (LTNP); for comparison we chose 4 healthy RMs that were seronegative for SIV (hereafter referred to as the Control group), and 4 progressing infection (Progressive group) SIV RMs. We observed these animals for 6 months without intervention and explored the immunological and pathological differences among the 3 groups. A series of immune activation and inflammation markers—such as C- C chemokine receptor type 5 (CCR5), beta 2- microglobulin (β2-MG), Human Leukocyte Antigen - antigen D Related (HLA-DR), CD38, the levels of microbial translocation (LPS -binding protein), and MAVS—and histological features were monitored during this period.ResultsBoth SIV RNA and SIV DNA in the plasma and lymph nodes (LNs) of the LTNP group were at significantly lower levels than those of the Progressive group (P < 0.05). The CD4/CD8 ratio and CD4 cell count and proportion in the LTNP group were between those of the Progressive and Control groups (P < 0.05): that is, they were higher than in the Progressive group and lower than in the Control group. The LTNP macaques manifested slow progression and decreased immune activation and inflammation; they also had lower levels of CCR5, LPS-binding protein, and β2-MG than the Progressive RMs (P < 0.05). Activation of LTNP in both CD4+ and CD8+ T cells was significantly lower than in the Progressive group and closer to that in the Control group. The histological features of the LTNP macaques were also closer to those of the Control group, even though they had been infected with SIV 4 years earlier. These data point to low viral replication in the LTNP macaques but it is not static. The expression of MAVS in peripheral blood and LNs was lower in the LTNP group than that in the Progressive group (P < 0.01), and MAVS was positively correlated with SIV DNA in LNs (P < 0.05). This may reflect the low activation of T lymphocytes. It was speculated that MAVS may be the link between innate and acquired antiviral immunity in SIV infection.ConclusionsThe LTNP RMs in our study were in a relatively stable state of low activation and inflammation, some biological progression with no disease events. This may have been associated with their low levels of the mitochondrial antiviral signaling protein (MAVS).

Liver macrophage-associated inflammation correlates with SIV burden and is substantially reduced following cART.

Liver disease is a leading contributor to morbidity and mortality during HIV infection, despite the use of combination antiretroviral therapy (cART). The precise mechanisms of liver disease during HIV infection are poorly understood partially due to the difficulty in obtaining human liver samples as well as the presence of confounding factors (e.g. hepatitis co-infection, alcohol use). Utilizing the simian immunodeficiency virus (SIV) macaque model, a controlled study was conducted to evaluate the factors associated with liver inflammation and the impact of cART. We observed an increase in hepatic macrophages during untreated SIV infection that was associated with a number of inflammatory and fibrosis mediators (TNFα, CCL3, TGFβ). Moreover, an upregulation in the macrophage chemoattractant factor CCL2 was detected in the livers of SIV-infected macaques that coincided with an increase in the number of activated CD16+ monocyte/macrophages and T cells expressing the cognate receptor CCR2. Expression of Mac387 on monocyte/macrophages further indicated that these cells recently migrated to the liver. The hepatic macrophage and T cell levels strongly correlated with liver SIV DNA levels, and were not associated with the levels of 16S bacterial DNA. Utilizing in situ hybridization, SIV-infected cells were found primarily within portal triads, and were identified as T cells. Microarray analysis identified a strong antiviral transcriptomic signature in the liver during SIV infection. In contrast, macaques treated with cART exhibited lower levels of liver macrophages and had a substantial, but not complete, reduction in their inflammatory profile. In addition, residual SIV DNA and bacteria 16S DNA were detected in the livers during cART, implicating the liver as a site on-going immune activation during antiretroviral therapy. These findings provide mechanistic insights regarding how SIV infection promotes liver inflammation through macrophage recruitment, with implications for in HIV-infected individuals.

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

AbstractSequestering 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.

Early Sites of Virus Replication After Oral SIVmac251 Infection of Infant Macaques: Implications for Pathogenesis.

Despite optimization of preventative measures for vertical HIV-1 transmission, daily, roughly 400 infants become HIV infected, most of them through breastfeeding. Viral entry has been presumed to occur in the gastrointestinal tract; however, the exact entry site(s) have not been defined. Therefore, we quantified simian immunodeficiency virus (SIV) RNA and DNA in oral, intestinal, and systemic tissues of 15 infant macaques within 48-96 h after oral SIVmac251 exposure. SIV DNA was detected as early as 48 h, whereas SIV RNA was typically detected at later time points (72-96 h). Transmitted founder viruses were identical or very similar to a single genotype in the SIVmac251 challenge stock. SIV RNA and DNA were most frequently found in lymph nodes (LNs) draining the oral cavity and in the ileum. Using in situ hybridization, SIV-infected cells in LNs were exclusively represented by CD3+ T cells. SIV RNA and DNA were also detected in the lungs of 20% of the animals, and 60% of the animals had detectable SIV DNA in the cerebrum. The early detection of viral RNA or DNA in lung and brain tissues emphasizes the need for early treatment of pediatric HIV infection to prevent damage not only to the immune system but also to the respiratory tract and central nervous system.

Early treatment with reverse transcriptase inhibitors significantly suppresses peak plasma IFNα in vivo during acute simian immunodeficiency virus infection

Innate interferons (IFN) are comprised of multiple Type I and III subtypes. The in vivo kinetics of subtype responses during human immunodeficiency virus (HIV) infection is not well defined. Using the acute simian immunodeficiency virus (SIV) infection model, we show that plasma IFNα levels peak at day 10 post-infection (pi) after which they rapidly declined. The mRNA expression of Type I and III IFN subtypes were significantly elevated in the lymph nodes (LN) at day 10 pi. Though the expression levels of all subtypes declined by day 14–31 pi, numerous subtypes remained elevated suggesting that ongoing viral replication in LN continues to drive induction of these subtypes. Interestingly, treatment with reverse transcriptase (RT) inhibitors at day 7 pi significantly suppressed plasma IFNα responses by day 10 pi that significantly correlated with cell-associated SIV DNA loads suggesting that RT byproducts such as viral DNA likely plays a role in driving IFN responses during acute SIV infection. Quantification of Type I and III subtype transcripts in sorted subsets of LN CD4+ and CD8+ T cells, CD14+/CD14− monocytes/macrophages, and total CD11c/CD123+ dendritic cells (DC) at day 10 pi showed that DC expressed ∼3–4 log more subtype transcripts as compared to the other subsets. Taken together, our results provide new insights into the kinetics of innate interferon responses during early stages of infection, and provide evidence that DC’s are a major in vivo source of innate IFN during acute SIV infection.

Antiretroviral Therapy in Simian Immunodeficiency Virus-Infected Sooty Mangabeys: Implications for AIDS Pathogenesis.

Simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) do not develop AIDS despite high levels of viremia. Key factors involved in the benign course of SIV infection in SMs are the absence of chronic immune activation and low levels of infection of CD4(+) central memory (TCM) and stem cell memory (TSCM) T cells. To better understand the role of virus replication in determining the main features of SIV infection in SMs, we treated 12 SMs with a potent antiretroviral therapy (ART) regimen for 2 to 12 months. We observed that ART suppressed viremia to <60 copies/ml of plasma in 10 of 12 animals and induced a variable decrease in the level of cell-associated SIV DNA in peripheral blood (average changes of 0.9-, 1.1-, 1.5-, and 3.7-fold for CD4(+) transitional memory [TTM], TCM, effector memory [TEM], and TSCM cells, respectively). ART-treated SIV-infected SMs showed (i) increased percentages of circulating CD4(+) TCM cells, (ii) increased levels of CD4(+) T cells in the rectal mucosa, and (iii) significant declines in the frequencies of HLA-DR(+) CD8(+) T cells in the blood and rectal mucosa. In addition, we observed that ART interruption resulted in rapid viral rebound in all SIV-infected SMs, indicating that the virus reservoir persists for at least a year under ART despite lower infection levels of CD4(+) TCM and TSCM cells than those seen in pathogenic SIV infections of macaques. Overall, these data indicate that ART induces specific immunological changes in SIV-infected SMs, thus suggesting that virus replication affects immune function even in the context of this clinically benign infection. Studies of natural, nonpathogenic simian immunodeficiency virus (SIV) infection of African monkeys have provided important insights into the mechanisms responsible for the progression to AIDS during pathogenic human immunodeficiency virus (HIV) infection of humans and SIV infection of Asian macaques. In this study, for the first time, we treated SIV-infected sooty mangabeys, a natural host for the infection, with a potent antiretroviral therapy (ART) regimen for periods ranging from 2 to 12 months and monitored in detail how suppression of virus replication affected the main virological and immunological features of this nonpathogenic infection. The observed findings provide novel information on both the pathogenesis of residual immunological disease under ART during pathogenic infection and the mechanisms involved in virus persistence during primate lentiviral infections.

Initiation of Antiretroviral Therapy Restores CD4+ T Memory Stem Cell Homeostasis in Simian Immunodeficiency Virus-Infected Macaques.

Treatment of human immunodeficiency virus (HIV) infection with antiretroviral therapy (ART) has significantly improved prognosis. Unfortunately, interruption of ART almost invariably results in viral rebound, attributed to a pool of long-lived, latently infected cells. Based on their longevity and proliferative potential, CD4(+) T memory stem cells (TSCM) have been proposed as an important site of HIV persistence. In a previous study, we found that in simian immunodeficiency virus (SIV)-infected rhesus macaques (RM), CD4(+) TSCM are preserved in number but show (i) a decrease in the frequency of CCR5(+) cells, (ii) an expansion of the fraction of proliferating Ki-67(+) cells, and (iii) high levels of SIV DNA. To understand the impact of ART on both CD4(+) TSCM homeostasis and virus persistence, we conducted a longitudinal analysis of these cells in the blood and lymph nodes of 25 SIV-infected RM. We found that ART induced a significant restoration of CD4(+) CCR5(+) TSCM both in blood and in lymph nodes and a reduction in the fraction of proliferating CD4(+) Ki-67(+) TSCM in blood (but not lymph nodes). Importantly, we found that the level of SIV DNA in CD4(+) transitional memory (TTM) and effector memory (TEM) T cells declined ∼100-fold after ART in both blood and lymph nodes, while the level of SIV DNA in CD4(+) TSCM and central memory T cells (TCM-) did not significantly change. These data suggest that ART is effective at partially restoring CD4(+) TSCM homeostasis, and the observed stable level of virus in TSCM supports the hypothesis that these cells are a critical contributor to SIV persistence. Understanding the roles of various CD4(+) T cell memory subsets in immune homeostasis and HIV/SIV persistence during antiretroviral therapy (ART) is critical to effectively treat and cure HIV infection. T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into other memory T cell subsets, such as central and transitional memory T cells (TCM and TTM, respectively). CD4(+) TSCM are disrupted but not depleted during pathogenic SIV infection. We find that ART is partially effective at restoring CD4(+) TSCM homeostasis and that SIV DNA harbored within this subset contracts more slowly than virus harbored in shorter-lived subsets, such as TTM and effector memory (TEM). Because of their ability to persist long-term in an individual, understanding the dynamics of virally infected CD4(+) TSCM during suppressive ART is important for future therapeutic interventions aimed at modulating immune activation and purging the HIV reservoir.

Adipose Tissue Is a Neglected Viral Reservoir and an Inflammatory Site during Chronic HIV and SIV Infection.

Two of the crucial aspects of human immunodeficiency virus (HIV) infection are (i) viral persistence in reservoirs (precluding viral eradication) and (ii) chronic inflammation (directly associated with all-cause morbidities in antiretroviral therapy (ART)-controlled HIV-infected patients). The objective of the present study was to assess the potential involvement of adipose tissue in these two aspects. Adipose tissue is composed of adipocytes and the stromal vascular fraction (SVF); the latter comprises immune cells such as CD4+ T cells and macrophages (both of which are important target cells for HIV). The inflammatory potential of adipose tissue has been extensively described in the context of obesity. During HIV infection, the inflammatory profile of adipose tissue has been revealed by the occurrence of lipodystrophies (primarily related to ART). Data on the impact of HIV on the SVF (especially in individuals not receiving ART) are scarce. We first analyzed the impact of simian immunodeficiency virus (SIV) infection on abdominal subcutaneous and visceral adipose tissues in SIVmac251 infected macaques and found that both adipocytes and adipose tissue immune cells were affected. The adipocyte density was elevated, and adipose tissue immune cells presented enhanced immune activation and/or inflammatory profiles. We detected cell-associated SIV DNA and RNA in the SVF and in sorted CD4+ T cells and macrophages from adipose tissue. We demonstrated that SVF cells (including CD4+ T cells) are infected in ART-controlled HIV-infected patients. Importantly, the production of HIV RNA was detected by in situ hybridization, and after the in vitro reactivation of sorted CD4+ T cells from adipose tissue. We thus identified adipose tissue as a crucial cofactor in both viral persistence and chronic immune activation/inflammation during HIV infection. These observations open up new therapeutic strategies for limiting the size of the viral reservoir and decreasing low-grade chronic inflammation via the modulation of adipose tissue-related pathways.

Differential Impact of In Vivo CD8+ T Lymphocyte Depletion in Controller versus Progressor Simian Immunodeficiency Virus-Infected Macaques.

Numerous studies have demonstrated that CD8(+) T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8(+) T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIVmac239. At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8(+) T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8(+) T-bet(+) lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4(+) central memory T lymphocytes (TCM) and CD4(+) effector memory T lymphocytes (TEM) in progressor RMs but decreased in the CD4(+) TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4(+) T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8(+) T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8(+) T lymphocytes. In this study, we further dissect the impact of CD8(+) T lymphocytes on HIV/SIV replication during SIV infection. CD8(+) T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8(+) T lymphocyte depletion resulted in a more prominent increase in viral loads and CD4(+) T lymphocyte activation in controllers than in progressors. Interestingly, we found T-bet expression on CD8(+) T lymphocytes to be the best predictor of viral load increase following depletion. The levels of SIV DNA increase postdepletion in both CD4(+) TCM and TEM in progressor RMs but decrease in the CD4(+) TCM of controllers. The findings described in this study provide key insights into the differential functions of CD8(+) T lymphocytes in controller and progressor RMs.

Detecting the Simian Immunodeficiency Virus Reservoir as Viral DNA and RNA in Situ Simultaneously as a Model to Monitor Viral Latency

HIV and simian immunodeficiency virus (SIV) establish latent infection in long-lived cells that form a reservoir of virus that persists in infected individuals even after years of treatment with highly active antiretroviral therapy (HAART). We present here an innovative technique that can be used to detect latent and active infected cells in tissues. The HIV/SIV reservoir is not fully characterized and to cure HIV we need to determine where the virus is hiding either latently integrated into the host DNA or replicating at very low levels producing viral RNA. The SIV infection model currently used in the Tulane National Primate Research Center (TNPRC) is suitable to study the latency and emergence of the virus in the presence of treatment and/or after lifting the pressure imposed by HAART to remain latent. Figure 1 demonstrates simultaneous detection and colocalization of SIV RNA (red) and DNA (blue) in an infected multinuclear giant cell in the lung of an SIV-infected macaque. The image reveals that not all the nuclei have integrated the SIV. This method demonstrates that SIV-infected cells are relatively rare in vivo. The technique consists of double in situ hybridization: RNA hybridization and color development and after an RNase step a DNA hybridization developed with other color. The procedure combines previously described elements.1–3 This technique could be used to detect the virus integrated in cells that are not actively producing virus. This would be revealed by cells that are DNA positive, but RNA negative. Approaches identifying the expression of viral proteins or RNA will not work to identify latently infected cells. Only viral DNA detection can allow the identification of latently infected cells because they are defined by a lack of viral gene expression. This approach shows great promise to identify the location of cells, latently infected with SIV, and provide critical information to advance the efforts to develop a cure for HIV infection. FIG. 1. The image shows the identification of simian immunodeficiency virus (SIV) nucleic acids in the lung of an SIV-infected macaque. In situ hybridization of SIV RNA (red) and DNA (blue) identifies individual infected cells within tissue. All nuclei in the ...

Resistance to Infection, Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251Viremia, and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4β7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

Immunogenicity of a Vaccine Regimen Composed of Simian Immunodeficiency Virus DNA, rMVA, and Viral Particles Administered to Female Rhesus Macaques via Four Different Mucosal Routes

A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4(+) and CD8(+) T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8(+) granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.