Antiretroviral therapy (ART) has prolonged the life expectancy of persons living with HIV, the majority of whom are now older than 50 years. Aging people with HIV are at increased risk for cardiovascular events driven by HIV-related inflammation and hypercoagulation. Apixaban is a factor Xa inhibitor that reduces cardiovascular risks and treats stroke, deep vein thrombosis, and pulmonary embolism. We assessed apixaban's impact on key parameters of HIV/SIV pathogenesis in SIV-infected, aged rhesus macaques (RMs) receiving ART. Inflammation, coagulation, T cell subsets, B cells, and macrophages and their immune activation status were monitored throughout the study. We found no significant differences between the apixaban-treated and control groups forvirus replication or CD4+ T cell recovery in blood and tissues after ART. Apixaban did not significantly affect D-dimer, immune activation, or inflammation of SIV-infected, ART-treated RMs. Apixaban-treated RMs experienced multiple bleeding episodes, tissue hemorrhages, and myocardial infarctions, as demonstrated by pathological examination of necropsy-collected tissues. Given apixaban's lack of effect on immune activation, CD4+ T cell restoration, and inflammation, along with increased risk of hemorrhage, factor Xa inhibition may not be an efficient or safe option to target and prevent cardiovascular events in aging people with HIV.

HIV infection accelerates biological aging, but the contribution of the host's age to this process is unknown. We investigated the influence of SIV infection in macaques (SIVmac) on the risk of comorbidities and aging in young and old rhesus macaques (RMs) by assessing pathogenesis markers, DNA methylation-based epigenetic age (EA), and EA acceleration (EAA) in blood and tissues. Initially, upon SIV infection, the young RMs showed greater resilience to CD4+ T cell depletion, better control of T cell activation, hypercoagulation, and excessive inflammation, yet this resilience was progressively lost in the advanced stages of infection. During the late stages of infection, the young RMs, but not the aged ones, showed an increase in EA in PBMCs; also, EAA in the cerebellum and heart of young RMs was higher compared with old RMs. SIV infection was more pathogenic in aged animals in early stages, leading to a more rapid disease progression; however, accelerated aging mostly affected young animals, so that the levels of multiple key pathogenesis markers in the young RMs converged toward those specific to aged ones in the late stages of infection. We conclude that SIV infection-driven age acceleration is tissue specific, and that host age influences the susceptibility of different tissues to enhanced aging.

IntroductionLipid profiles change in human immunodeficiency virus (HIV) infection and correlate with inflammation. Lipidomic alterations are impacted by multiple non-HIV-related behavioral risk factors; thus, use of animal models in which these behavioral factors are controlled may inform on the specific lipid changes induced by simian immunodeficiency virus (SIV) infection and/or antiretroviral therapy (ART).MethodsUsing ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy, we assessed and compared (ANOVA) longitudinal lipid changes in naïve and ART-treated SIV-infected pigtailed macaques (PTMs). Key parameters of infection (IL-6, TNFa, D-dimer, CRP and CD4+ T cell counts) were correlated (Spearman) with lipid concentrations at critical time points of infection and treatment.ResultsSphingomyelins (SM) and lactosylceramides (LCER) increased during acute infection, returning to baseline during chronic infection; Hexosylceramides (HCER) increased throughout infection, being normalized with prolonged ART; Phosphatidylinositols (PI) and lysophosphatidylcholines (LPC) decreased with SIV infection and did not return to normal with ART; Phosphatidylethanolamines (PE), lysophosphatidylethanolamines (LPE) and phosphatidylcholines (PC) were unchanged by SIV infection, yet significantly decreased throughout ART. Specific lipid species (SLS) were also substantially modified by SIV and/or ART in most lipid classes. In conclusion, using a metabolically controlled model, we identified specific lipidomics signatures of SIV infection and/or ART, some of which were similar to people living with HIV (PWH). Many SLS were identical to those involved in development of organ dysfunctions encountered in virally suppressed individuals. Lipid changes also correlated with markers of disease progression, inflammation and coagulation.DiscussionOur data suggest that lipidomic profile alterations contribute to residual systemic inflammation and comorbidities seen in HIV/SIV infections and therefore may be used as biomarkers of SIV/HIV comorbidities. Further exploration into the benefits of interventions targeting dyslipidemia is needed for the prevention HIV-related comorbidities.

African green monkeys (AGMs) are natural hosts of SIV whose infection does not progress to AIDS. Since early events of infection may be critical to pathogenesis in nonnatural hosts, we investigated early SIV infection in 29 adult male AGMs intrarectally inoculated with SIVsab92018 (SIVsab) and serially sacrificed throughout acute into early chronic infection to understand patterns of viral establishment, dissemination, and their effect on disease progression. Using this model, we showed that foci of virus replication could be detected at the site of inoculation and in the draining lymphatics as early as 1-3 days postinfection (dpi). Furthermore, testing with ultrasensitive assays showed rapid onset ofviremia (2-4 dpi). After systemic spread, virus was detected in all tissues surveyed. Multiple transmitted/founderviruses were identified, confirming an optimal challenge dose, while demonstrating a moderate mucosal genetic bottleneck. Resident CD4+ T cells were the initial target cells; other immune cell populations were not significantly altered at the site of entry. Thus, intrarectal SIVsab infection is characterized by swift dissemination of the virus, a lack of major target cell recruitment, and no window of opportunity for interventions to prevent virus dissemination during the earliest stages of infection, similar to intrarectal transmission but different from vaginal transmission in macaques.

Understanding the underlying mechanisms of HIV pathogenesis is critical for designing successful HIV vaccines and cure strategies. However, achieving this goal is complicated by the virus’s direct interactions with immune cells, the induction of persistent reservoirs in the immune system cells, and multiple strategies developed by the virus for immune evasion. Meanwhile, HIV and SIV infections induce a pandysfunction of the immune cell populations, making it difficult to untangle the various concurrent mechanisms of HIV pathogenesis. Over the years, one of the most successful approaches for dissecting the immune correlates of protection in HIV/SIV infection has been the in vivo depletion of various immune cell populations and assessment of the impact of these depletions on the outcome of infection in non-human primate models. Here, we present a detailed analysis of the strategies and results of manipulating SIV pathogenesis through in vivo depletions of key immune cells populations. Although each of these methods has its limitations, they have all contributed to our understanding of key pathogenic pathways in HIV/SIV infection.

CD4 + T-cell depletion is a hallmark of HIV infection, leading to impairment of cellular immunity and opportunistic infections, but its contribution to SIV/HIV-associated gut dysfunction is unknown. Chronically SIV-infected African Green Monkeys (AGMs) partially recover mucosal CD4 + T-cells, maintain gut integrity and do not progress to AIDS. Here we assess the impact of prolonged, antibody-mediated CD4 + T-cell depletion on gut integrity and natural history of SIV infection in AGMs. All circulating CD4 + T-cells and >90% of mucosal CD4 + T-cells are depleted. Plasma viral loads and cell-associated viral RNA in tissues are lower in CD4 + -cell-depleted animals. CD4 + -cell-depleted AGMs maintain gut integrity, control immune activation and do not progress to AIDS. We thus conclude that CD4 + T-cell depletion is not a determinant of SIV-related gut dysfunction, when gastrointestinal tract epithelial damage and inflammation are absent, suggesting that disease progression and resistance to AIDS are independent of CD4 + T-cell restoration in SIVagm-infected AGMs.

The COVID-19 pandemic has highlighted the need for safe and effective vaccines to be rapidly developed and distributed worldwide, especially considering the emergence of new SARS-CoV-2 variants. Protein subunit vaccines have emerged as a promising approach due to their proven safety record and ability to elicit robust immune responses. In this study, we evaluated the immunogenicity and efficacy of an adjuvanted tetravalent S1 subunit protein COVID-19 vaccine candidate composed of the Wuhan, B.1.1.7 variant, B.1.351 variant, and P.1 variant spike proteins in a nonhuman primate model with controlled SIVsab infection. The vaccine candidate induced both humoral and cellular immune responses, with T- and B cell responses mainly peaking post-boost immunization. The vaccine also elicited neutralizing and cross-reactive antibodies, ACE2 blocking antibodies, and T-cell responses, including spike specific CD4+ T cells. Importantly, the vaccine candidate was able to generate Omicron variant spike binding and ACE2 blocking antibodies without specifically vaccinating with Omicron, suggesting potential broad protection against emerging variants. The tetravalent composition of the vaccine candidate has significant implications for COVID-19 vaccine development and implementation, providing broad antibody responses against numerous SARS-CoV-2 variants.

Whether CD8+ T lymphocytes control human immunodeficiency virus infection by cytopathic or non-cytopathic mechanisms is not fully understood. Multiple studies highlighted non-cytopathic effects, but one hypothesis is that cytopathic effects of CD8+ T cells occur before viral production. Here, to examine the role of CD8+ T cells prior to virus production, we treated SIVmac251-infected macaques with an integrase inhibitor combined with a CD8-depleting antibody, or with either reagent alone. We analyzed the ensuing viral dynamics using a mathematical model that included infected cells pre- and post- viral DNA integration to compare different immune effector mechanisms. Macaques receiving the integrase inhibitor alone experienced greater viral load decays, reaching lower nadirs on treatment, than those treated also with the CD8-depleting antibody. Models including CD8+ cell-mediated reduction of viral production (non-cytolytic) were found to best explain the viral profiles across all macaques, in addition an effect in killing infected cells pre-integration (cytolytic) was supported in some of the best models. Our results suggest that CD8+ T cells have both a cytolytic effect on infected cells before viral integration, and a direct, non-cytolytic effect by suppressing viral production.

HIV cure is sought after due to the prevalence of comorbidities that occur in persons with HIV. One of the most investigated HIV cure strategies is the “shock and kill” approach. ABSTRACT HIV persistence requires lifelong antiretroviral therapy (ART), calling for a cure. The histone deacetylase inhibitor, romidepsin, is used in the “shock and kill” approach with the goal of reactivating virus and subsequently clearing infected cells through cell-mediated immune responses. We tested serial and double infusions of romidepsin in a rhesus macaque (RM) model of SIV functional cure, which controls virus without ART. Off ART, romidepsin reactivated SIV in all RMs. Subsequent infusions resulted in diminished reactivation, and two RMs did not reactivate the virus after the second or third infusions. Therefore, those two RMs received CD8-depleting antibody to assess the replication competence of the residual reservoir. The remaining RMs received double infusions, i.e., two doses separated by 48-h. Double infusions were well tolerated, induced immune activation, and effectively reactivated SIV. Although reactivation was gradually diminished, cell-associated viral DNA was minimally changed, and viral outgrowth occurred in 4/5 RMs. In the RM which did not reactivate after CD8 depletion, viral outgrowth was not detected in peripheral blood mononuclear cells (PBMC)-derived CD4+ cells. The frequency of SIV-specific CD8+ T cells increased after romidepsin administration, and the increased SIV-specific immune responses were associated, although not statistically, with the diminished reactivation. Thus, our data showing sequential decreases in viral reactivation with repeated romidepsin administrations with all RMs and absence of viral reactivation after CD8+ T-cell depletion in one animal suggest that, in the context of healthy immune responses, romidepsin affected the inducible viral reservoir and gradually increased immune-mediated viral control. Given the disparities between the results of romidepsin administration to ART-suppressed SIVmac239-infected RMs and HIV-infected normal progressors compared to our immune-healthy model, our data suggest that improving immune function for greater SIV-specific responses should be the starting point of HIV cure strategies. IMPORTANCE HIV cure is sought after due to the prevalence of comorbidities that occur in persons with HIV. One of the most investigated HIV cure strategies is the “shock and kill” approach. Our study investigated the use of romidepsin, a histone deacetylase (HDAC) inhibitor, in our rhesus macaque model of functional cure, which allows for better resolution of viral reactivation due to the lack of antiretroviral therapy. We found that repeated rounds of romidepsin resulted in gradually diminished viral reactivation. One animal inevitably lacked replication-competent virus in the blood. With the accompanying enhancement of the SIV-specific immune response, our data suggest that there is a reduction of the viral reservoir in one animal by the cell-mediated immune response. With the differences observed between our model and persons living with HIV (PWH) treated with romidepsin, specifically in the context of a healthy immune system in our model, our data thereby indicate the importance of restoring the immune system for cure strategies.

Abstract The mechanisms of CD8+ T lymphocyte control of HIV infection are not fully understood. One hypothesis is that the main effect of CD8+ T-cells occurs before viral production. To settle this issue, we conducted an experiment where twenty SIVmac251-infected macaques received either CD8-depleting antibody (n=4); integrase inhibitor and CD8-depleting antibody (n=8); or only the inhibitor (n=8). We analyzed the ensuing viral dynamics with a mathematical model that included infected cells pre- and post- viral DNA integration to compare different possible immune effector mechanisms. Macaques receiving just integrase inhibitor experienced greater viral load decays than those treated also with CD8+ cell depleting antibody. A model including CD8+ cell effects in killing infected cells pre-integration (cytolytic) and reducing viral production (non-cytolytic) was found to best explain the viral profiles across all macaques. Our results suggest that CD8+ T-cells have both a cytolytic effect on infected cells before viral integration, which has not been seen before, and a non-cytolytic effect reducing viral production.

Unlike HIV infection, SIV infection is generally nonpathogenic in natural hosts, such as African green monkeys (AGMs), despite life-long high viral replication. Lack of disease progression was reportedly based on the ability of SIV-infected AGMs to prevent gut dysfunction, avoiding microbial translocation and the associated systemic immune activation and chronic inflammation. Yet, the maintenance of gut integrity has never been documented, and the mechanism(s) by which gut integrity is preserved are unknown. We sought to investigate the early events of SIV infection in AGMs, specifically examining the impact of SIVsab infection on the gut mucosa. Twenty-nine adult male AGMs were intrarectally infected with SIVsab92018 and serially sacrificed at well-defined stages of SIV infection, preramp-up (1-3 days post-infection (dpi)), ramp-up (4-6 dpi), peak viremia (9-12 dpi), and early chronic SIV infection (46-55 dpi), to assess the levels of immune activation, apoptosis, epithelial damage and microbial translocation in the GI tract and peripheral lymph nodes. Tissue viral loads, plasma cytokines and plasma markers of gut dysfunction were also measured throughout the course of early infection. While a strong, but transient, interferon-based inflammatory response was observed, the levels of plasma markers linked to enteropathy did not increase. Accordingly, no significant increases in apoptosis of either mucosal enterocytes or lymphocytes, and no damage to the mucosal epithelium were documented during early SIVsab infection of AGMs. These findings were supported by RNAseq of the gut tissue, which found no significant alterations in gene expression that would indicate microbial translocation. Thus, for the first time, we confirmed that gut epithelial integrity is preserved, with no evidence of microbial translocation, in AGMs throughout early SIVsab infection. This might protect AGMs from developing intestinal dysfunction and the subsequent chronic inflammation that drives both HIV disease progression and HIV-associated comorbidities.

HIV/SIV persistence in latent reservoirs requires lifelong antiretroviral treatment and calls for effective cure strategies. Romidepsin (RMD), a histone deacetylase inhibitor, was reported to reactivate HIV/SIV from reservoirs in virus-suppressed individuals. We characterized in detail the pharmacokinetics and safety profile of RMD in three SIV-naïve rhesus macaques which received two rounds of treatment. In plasma, RMD mean terminal half-life was 15.3 h. In comparison, RMD mean terminal half-life was much longer in tissues: 110 h in the lymph nodes (LNs) and 28 h in gastrointestinal tract. RMD administration was accompanied by transient liver and systemic toxicity. Isoflurane anesthesia induced near-immediate transient lymphopenia, which was further exacerbated and extended with the extensive immune modifications by RMD. The effect of RMD on circulating immune cells was complex: (i) slight increase in lymphocyte death rates; (ii) transient, robust increase in neutrophils; (iii) massive downregulation of lymphocyte surface markers; (iv) important migration of CD3+ T cells to the gut and LNs; and (v) hindrance to CD8+ T cell functionality, yet without reaching significance. Our results show that, in contrast to transient plasma concentrations, RMD has a long-term presence in tissues, with multiple immunomodulatory effects and minimal to moderate kidney, liver, and lymphocyte toxicities. As such, we concluded that RMD can be used for “shock and kill” approaches, preferentially in combination with other latency reversal agents or cytotoxic T lymphocyte boosting strategies with consideration taken for adverse effects.

Regulatory T cells (Tregs) may be key contributors to the HIV/SIV latent reservoir, since they harbor high levels of HIV/SIV; reverse CD4(+) T cell immune activation status, increasing the pool of resting CD4(+) T cells; and impair CD8(+) T cell function, favoring HIV persistence. We tested the hypothesis that Treg depletion is a valid intervention toward an HIV cure by depleted Tregs in 14 rhesus macaque (RM) controllers infected with SIVsab, the virus that naturally infects sabaeus monkeys, through different strategies: administration of an anti-CCR4 immunotoxin, two doses of an anti-CD25 immunotoxin (interleukin-2 with diphtheria toxin [IL-2-DT]), or two combinations of both. All of these treatments resulted in significant depletion of the circulating Tregs (>70%) and their partial depletion in the gut (25%) and lymph nodes (>50%). The fractions of CD4(+) T cells expressing Ki-67 increased up to 80% in experiments containing IL-2-DT and only 30% in anti-CCR4-treated RMs, paralleled by increases in the inflammatory cytokines. In the absence of ART, plasma virus rebounded to 103 vRNA copies/ml by day 10 after IL-2-DT administration. A large but transient boost of the SIV-specific CD8(+) T cell responses occurred in IL-2-DT-treated RMs. Such increases were minimal in the RMs receiving anti-CCR4-based regimens. Five RMs received IL-2-DT on ART, but treatment was discontinued because of high toxicity and lymphopenia. As such, while all treatments depleted a significant proportion of Tregs, the side effects in the presence of ART prevent their clinical use and call for different Treg depletion approaches. Thus, based on our data, Treg targeting as a strategy for HIV cure cannot be discarded. IMPORTANCE Regulatory T cells (Tregs) can decisively contribute to the establishment and persistence of the HIV reservoir, since they harbor high levels of HIV/SIV, increase the pool of resting CD4(+) T cells by reversing their immune activation status, and impair CD8(+) T cell function, favoring HIV persistence. We tested multiple Treg depletion strategies and showed that all of them are at least partially successful in depleting Tregs. As such, Treg depletion appears to be a valid intervention toward an HIV cure, reducing the size of the reservoir, reactivating the virus, and boosting cell-mediated immune responses. Yet, when Treg depletion was attempted in ART-suppressed animals, the treatment had to be discontinued due to high toxicity and lymphopenia. Therefore, while Treg targeting as a strategy for HIV cure cannot be discarded, the methodology for Treg depletion has to be revisited.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Consuming a high-fat diet (HFD) is a risk factor for obesity and diabetes; both of these diseases are also associated with systemic inflammation, similar to HIV infection. A HFD induces intestinal dysbiosis and impairs liver function and coagulation, with a potential negative impact on HIV/SIV pathogenesis. We administered a HFD rich in saturated fats and cholesterol to nonpathogenic (African green monkeys) and pathogenic (pigtailed macaques) SIV hosts. The HFD had a negative impact on SIV disease progression in both species. Thus, increased cell-associated SIV DNA and RNA occurred in the HFD-receiving nonhuman primates, indicating a potential reservoir expansion. The HFD induced prominent immune cell infiltration in the adipose tissue, an important SIV reservoir, and heightened systemic immune activation and inflammation, altering the intestinal immune environment and triggering gut damage and microbial translocation. Furthermore, HFD altered lipid metabolism and HDL oxidation and also induced liver steatosis and fibrosis. These metabolic disturbances triggered incipient atherosclerosis and heightened cardiovascular risk in the SIV-infected HFD-receiving nonhuman primates. Our study demonstrates that dietary intake has a discernable impact on the natural history of HIV/SIV infections and suggests that dietary changes can be used as adjuvant approaches for HIV-infected subjects, to reduce inflammation and the risk of non-AIDS comorbidities and possibly other infectious diseases.

ABSTRACT CD8+ cells play a key role in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, but their specific mechanism(s) of action in controlling the virus is unclear. Two-long-terminal-repeat (2-LTR) circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8+ cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RMs) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8+ cell depletion and received raltegravir (RAL) monotherapy or a combination of both. Blood, lymph nodes (LNs), and gut biopsy specimens were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from peripheral blood mononuclear cells (PBMCs) and LN lymphocytes were measured with quantitative reverse transcription-PCR (qRT-PCR). In the CD8 depletion group, an ∼1-log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8+ cell depletion group, an increase in pVLs following CD8 depletion similar to that in the CD8 depletion group was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best support an effect of CD8+ cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect but also allow for additional effects, which the data do not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8+ cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT. IMPORTANCE CD8+ T cells play an essential role in controlling HIV and SIV infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8+ cells, administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles, and a combination of the two treatments. We used a new mathematical model to help interpret the data, and the results suggest that CD8+ cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8+ cells in SIV infection. Confirmation of our results would be an important step in understanding immune control of HIV.

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation.

Current approaches do not eliminate all human immunodeficiency virus type 1 (HIV-1) maternal-to-infant transmissions (MTIT); new prevention paradigms might help avert new infections. We administered maraviroc (MVC) to rhesus macaques (RMs) to block CCR5-mediated entry, followed by repeated oral exposure of a CCR5-dependent clone of simian immunodeficiency virus (SIV) mac251 (SIVmac766). MVC significantly blocked the CCR5 coreceptor in peripheral blood mononuclear cells and tissue cells. All control animals and 60% of MVC-treated infant RMs became infected by the 6th challenge, with no significant difference between the number of exposures (P = 0.15). At the time of viral exposures, MVC plasma and tissue (including tonsil) concentrations were within the range seen in humans receiving MVC as a therapeutic. Both treated and control RMs were infected with only a single transmitted/founder variant, consistent with the dose of virus typical of HIV-1 infection. The uninfected RMs expressed the lowest levels of CCR5 on the CD4(+) T cells. Ramp-up viremia was significantly delayed (P = 0.05) in the MVC-treated RMs, yet peak and postpeak viral loads were similar in treated and control RMs. In conclusion, in spite of apparent effective CCR5 blockade in infant RMs, MVC had a marginal impact on acquisition and only a minimal impact on the postinfection delay of viremia following oral SIV infection. Newly developed, more effective CCR5 blockers may have a more dramatic impact on oral SIV transmission than MVC. IMPORTANCE We have previously suggested that the very low levels of simian immunodeficiency virus (SIV) maternal-to-infant transmissions (MTIT) in African nonhuman primates that are natural hosts of SIVs are due to a low availability of target cells (CCR5(+) CD4(+) T cells) in the oral mucosa of the infants, rather than maternal and milk factors. To confirm this new MTIT paradigm, we performed a proof-of-concept study in which we therapeutically blocked CCR5 with maraviroc (MVC) and orally exposed MVC-treated and naive infant rhesus macaques to SIV. MVC had only a marginal effect on oral SIV transmission. However, the observation that the infant RMs that remained uninfected at the completion of the study, after 6 repeated viral challenges, had the lowest CCR5 expression on the CD4(+) T cells prior to the MVC treatment appears to confirm our hypothesis, also suggesting that the partial effect of MVC is due to a limited efficacy of the drug. New, more effective CCR5 inhibitors may have a better effect in preventing SIV and HIV transmission.

CD8 (cid:2) cells play a key role in human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, but their specific mechanism(s) of action in controlling the virus is unclear. Two-long-terminal-repeat (2-LTR) circles are extrachromosomal products generated upon failed integration of HIV/SIV. To understand the specific effects of CD8 (cid:2) cells on infected cells, we analyzed the dynamics of 2-LTR circles in SIVmac251-infected rhesus macaques (RMs) treated with an integrase inhibitor (INT). Twenty RMs underwent CD8 (cid:2) cell depletion and received raltegravir (RAL) monotherapy or a combination of both. Blood, lymph nodes (LNs), and gut biopsy specimens were routinely sampled. Plasma viral loads (pVLs) and 2-LTR circles from peripheral blood mononuclear cells (PBMCs) and LN lymphocytes were measured with quantitative reverse transcription-PCR (qRT-PCR). In the CD8 depletion group, an (cid:3) 1-log increase in pVLs and a slow increase in PBMC 2-LTRs occurred following depletion. In the INT group, a strong decline in pVLs upon treatment initiation and no change in 2-LTR levels were observed. In the INT and CD8 (cid:2) cell depletion group, an increase in pVLs following CD8 depletion similar to that in the CD8 depletion group was observed, with a modest decline following INT initiation, and 2-LTR circles significantly increased in PBMCs and LNs. Analyzing the 2-LTR data across all treatment groups with a mathematical model indicates that the data best support an effect of CD8 (cid:2) cells in killing cells prior to viral integration. Sensitivity analyses of these results confirm that effect but also allow for additional effects, which the data do not discriminate well. Overall, we show that INT does not significantly increase the levels of 2-LTR circles. However, CD8 (cid:2) cell depletion increases the 2-LTR levels, which are enhanced in the presence of an INT. IMPORTANCE CD8 (cid:2) T cells play an essential role in controlling HIV and SIV infection, but the specific mechanisms involved remain poorly understood. Due to failed viral infection, HIV and SIV can form 2-LTR extrachromosomal circles that can be quantified. We present novel data on the dynamics of these 2-LTR forms in a SIV-infected macaque model under three different treatment conditions: depletion of CD8 (cid:2) cells, administration of the integrase inhibitor in a monotherapy, which favors the formation of 2-LTR circles, and a combination of the two treat-ments. We used a new mathematical model to help interpret the data, and the results suggest that CD8 (cid:2) cells exert a killing effect on infected cells prior to virus integration. These results provide new insights into the mechanisms of action of CD8 (cid:2) cells in SIV infection. Confirmation of our results would be an important step in understanding immune control of HIV. ultiple lines of demonstrate that are in immunodeficiency (HIV)/simian immunodeficiency virus infection: (i) a strong association exists between specific host major histocompatibility complex class I (MHC-I) alleles and HIV/SIV disease progression (1); (ii) a temporal association can be established between the postpeak decline in plasma viremia and the increase in virus-specific CD8 (cid:2) cytotoxic T lymphocyte (CTL) responses (2, 3); (iii) virus escape mutations consistently arise in the face of host cytotoxic responses during all stages of infection, starting from the time when cellular immune responses are generated (4–8); (iv) CD8 (cid:2) cell depletion in vivo results in a rapid and sustained increase in plasma viremia, which then returns to predepletion levels following the rebound of CD8 (cid:2) cells (9–21). Furthermore, CD8 (cid:2) T cells suppress HIV infection in vitro (22, 23). However, the specific mechanism(s) of action these cells take to control viral replication is poorly understood. To gain some insight, we assessed the role of CD8 (cid:2) cells in controlling two-long-terminal-repeat-positive (2-LTR (cid:2) ) cells, which we used as a surrogate for HIV-infected cells preintegration. A recent study showed that cells containing virus prior to integration can represent a sizeable fraction of the total population of infected cells (24). since cells are still de novo infected during therapy, thus allowing better detection of the effects of CD8 (cid:2) cells. We then compared our data to an extended model of viral dynamics to explore the effect of CD8 (cid:2) cells on 2-LTR circle dynamics. We report that the levels of 2-LTR circles modestly increase following CD8 (cid:2) cell depletion, are unchanged after RAL monotherapy, and dramatically increase following the combined CD8 (cid:2) cell depletion and RAL monotherapy. Using the model of viral dynamics that includes 2-LTR circles, we found that model predictions are most similar to the observed data when CD8 (cid:2) cell killing of infected cells occurs prior to viral integration. We present all results in blood as the number of 2-LTR circles/ml, which we calculated using the absolute count of PBMCs determined from the TruCounts. It is important to note that the number of 2-LTR circles/ml is a more faithful measure of the dynamics of 2-LTR circles than this number normalized by a million cells, because the latter also depends on the dynamics of cells without 2-LTR circles, which change with treatment. For tissue samples (such as LN biopsy specimens), we present the results as the number of 2-LTR circles/million CCR5 (cid:2) cells, because it is difficult to calculate absolute numbers of 2-LTR (cid:2) cells. Due to low cell counts in the jejunal biopsy specimens, we were unable to quantify the levels of 2-LTR circles in these samples. CA). However, we also tested the sensitivity , by repeating the described below with and/or 10 and p (cid:5) 10 4 virions/cell/day. The value of p had no effect on the fits or the conclusions presented On the other hand, (cid:4) influenced the quantitative values of the parameters but did not change the overall conclusions, although the predicted profiles of 2-LTR (cid:2) cells/ml were not as good. We then performed fits of the model to the changes in 2-LTR/ml to estimate (cid:5) and the effect of CD8 (cid:2) cell depletion. In these fits, we searched for the best set of parameters for all the data together, i.e., the 2-LTRs in the 20 RMs, normalized at the initial time and assuming five different effects of CD8 (cid:2) cell depletion (and their combinations two by two): (i) on the death of infected cells before integration, by reducing (cid:3) 1 in the model; (ii) on the death of productively infected cells by reducing (cid:3) 2 ; (iii) on the viral production, by increasing p ; (iv) on the infectivity of virus, by increasing (cid:4) ; and (iv) on the integration rate, by increasing k . The first three effects have been referred to in the literature of CD8 (cid:2) cell depletion studies as putative targets for the action of CD8 (cid:2) T cells (15, 16, 20, 42), and we include the last two for completeness. However, our interest is not in the specific values of the parameters but rather in the general behavior of the model and how it compares with our observed 2-LTR dynamics under different treatment conditions. In addition, in the current manuscript, we are studying only the 2-LTR dynamics and did not fit virus or T-cell dynamics. The model was simulated using R (https://cran.r-project.org/), and we used the DEoptim package of R for fitting. We compared models and selected the best one based on the corrected Akaike information criteria (AICc) (56). The best fits resulted systematically in (cid:5) (cid:5) 0/day or very near 0, indicating that R 2 (cid:5) 0 cells/ml. So, we also performed analyses imposing a lower bound of (cid:5) (cid:5) 0.1/day. Although these fits were not as good, the model may be more physiological.