Early antiretroviral therapy in SIV-infected rhesus macaques reveals a multiphasic, saturable dynamic accumulation of the rebound competent viral reservoir.

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

Timing of initiation of anti-retroviral therapy predicts post-treatment control of SIV replication.

One approach to 'functional cure' of HIV infection is to induce durable control of HIV replication after the interruption of antiretroviral therapy (ART). However, the major factors that determine the viral 'setpoint' level after treatment interruption are not well understood. Here we combine data on ART interruption following SIV infection for 124 total animals from 10 independent studies across 3 institutional cohorts to understand the dynamics and predictors of post-treatment viral control. We find that the timing of treatment initiation is an important determinant of both the peak and early setpoint viral levels after treatment interruption. During the first 3 weeks of infection, every day of delay in treatment initiation is associated with a 0.22 log10 copies/ml decrease in post-rebound peak and setpoint viral levels. However, delay in initiation of ART beyond 3 weeks of infection is associated with higher post-rebound setpoint viral levels. For animals treated beyond 3 weeks post-infection, viral load at ART initiation was the primary predictor of post-rebound setpoint viral levels. Potential alternative predictors of post-rebound setpoint viral loads including cell-associated DNA or RNA, time from treatment interruption to rebound, and pre-interruption CD8+ T cell responses were also examined in the studies where these data were available. This analysis suggests that optimal timing of treatment initiation may be an important determinant of post-treatment control of HIV.

Preferential selection of viral escape mutants by CD8+ T cell 'sieving' of SIV reactivation from latency.

HIV rapidly rebounds after interruption of antiretroviral therapy (ART). HIV-specific CD8+ T cells may act to prevent early events in viral reactivation. However, the presence of viral immune escape mutations may limit the effect of CD8+ T cells on viral rebound. Here, we studied the impact of CD8 immune pressure on post-treatment rebound of barcoded SIVmac293M in 14 Mamu-A*01 positive rhesus macaques that initiated ART on day 14, and subsequently underwent two analytic treatment interruptions (ATIs). Rebound following the first ATI (seven months after ART initiation) was dominated by virus that retained the wild-type sequence at the Mamu-A*01 restricted Tat-SL8 epitope. By the end of the two-month treatment interruption, the replicating virus was predominantly escaped at the Tat-SL8 epitope. Animals reinitiated ART for 3 months prior to a second treatment interruption. Time-to-rebound and viral reactivation rate were significantly slower during the second treatment interruption compared to the first. Tat-SL8 escape mutants dominated early rebound during the second treatment interruption, despite the dominance of wild-type virus in the proviral reservoir. Furthermore, the escape mutations detected early in the second treatment interruption were well predicted by those replicating at the end of the first, indicating that escape mutant virus in the second interruption originated from the latent reservoir as opposed to evolving de novo post rebound. SL8-specific CD8+ T cell levels in blood prior to the second interruption were marginally, but significantly, higher (median 0.73% vs 0.60%, p = 0.016). CD8+ T cell depletion approximately 95 days after the second treatment interruption led to the reappearance of wild-type virus. This work suggests that CD8+ T cells can actively suppress the rebound of wild-type virus, leading to the dominance of escape mutant virus after treatment interruption.

Balancing Statistical Power and Risk in HIV Cure Clinical Trial Design.

BACKGROUND: Analytical treatment interruptions (ATI) are pauses of antiretroviral therapy (ART) in the context of human immunodeficiency virus (HIV) cure trials. They are the gold standard in determining if interventions being tested can achieve sustained virological control in the absence of ART. However, withholding ART comes with risks and discomforts to trial participant. We used mathematical models to explore how ATI study design can be improved to maximize statistical power, while minimizing risks to participants. METHODS: Using previously observed dynamics of time to viral rebound (TVR) post-ATI, we modelled estimates for optimal sample size, frequency, and ATI duration required to detect a significant difference in the TVR between control and intervention groups. Groups were compared using a log-rank test, and analytical and stochastic techniques. RESULTS: In placebo-controlled TVR studies, 120 participants are required in each arm to detect 30% difference in frequency of viral reactivation at 80% power. There was little statistical advantage to measuring viral load more frequently than weekly, or interrupting ART beyond 5 weeks in a TVR study. CONCLUSIONS: Current TVR HIV cure studies are underpowered to detect statistically significant changes in frequency of viral reactivation. Alternate study designs can improve the statistical power of ATI trials.

Impact of fluctuation in frequency of human immunodeficiency virus/simian immunodeficiency virus reactivation during antiretroviral therapy interruption.

Antiretroviral therapy (ART) provides effective control of human immunodeficiency virus (HIV) replication and maintains viral loads of HIV at undetectable levels. Interruption of ART causes rapid recrudescence of HIV plasma viremia due to reactivation of latently HIV-infected cells. Here, we characterize the timing of both the initial and subsequent successful viral reactivations following ART interruption in macaques infected with simian immunodeficiency virus (SIV). We compare these to previous results from HIV-infected patients. We find that on average the time until the first successful viral reactivation event is longer than the time between subsequent reactivations. Based on this result, we hypothesize that the reactivation frequency of both HIV and SIV may fluctuate over time, and that this may impact the treatment of HIV. We develop a stochastic model incorporating fluctuations in the frequency of viral reactivation following ART interruption that shows behaviours consistent with the observed data. Furthermore, we show that one of the impacts of a fluctuating reactivation frequency would be to significantly reduce the efficacy of 'anti-latency' interventions for HIV that aim to reduce the frequency of reactivation. It is therefore essential to consider the possibility of a fluctuating reactivation frequency when assessing the impact of such intervention strategies.

Estimating Initial Viral Levels during Simian Immunodeficiency Virus/Human Immunodeficiency Virus Reactivation from Latency.

Human immunodeficiency virus (HIV) viremia rebounds rapidly after treatment interruption, and a variety of strategies are being explored to reduce or control viral reactivation posttreatment. This viral rebound arises from reactivation of individual latently infected cells, which spread during ongoing rounds of productive infection. The level of virus produced by the initial individual reactivating cells is not known, although it may have major implications for the ability of different immune interventions to control viral rebound. Here we use data from both HIV and simian immunodeficiency virus (SIV) treatment interruption studies to estimate the initial viral load postinterruption and thereby the initial individual reactivation event. Using a barcoded virus (SIVmac239M) to track reactivation from individual latent cells, we use the observed viral growth rates and frequency of reactivation to model the dynamics of reactivation to estimate that a single reactivated latent cell can produce an average viral load equivalent to ∼0.1 to 0.5 viral RNA (vRNA) copies/ml. Modeling of treatment interruption in HIV suggests an initial viral load equivalent of ∼0.6 to 1 vRNA copies/ml. These low viral loads immediately following latent cell reactivation provide a window of opportunity for viral control by host immunity, before further replication allows viral spread. This work shows the initial levels of viral production that must be controlled in order to successfully suppress HIV reactivation following treatment interruption.IMPORTANCE Current treatment for HIV is able to suppress viral replication and prevent disease progression. However, treatment cannot eradicate infection, because the virus lies silent within latently infected cells. If treatment is stopped, the virus usually rebounds above the level of detection within a few weeks. There are a number of approaches being tested aimed at either eradicating latently infected cells or controlling the virus if it returns. Studying both the small pool of latently infected cells and the early events during viral reactivation is difficult, because these involve very small levels of virus that are difficult to measure directly. Here, we combine experimental data and mathematical modeling to understand the very early events during viral reactivation from latency in both HIV infection of humans and SIV infection of monkeys. We find that the initial levels of virus are low, which may help in designing therapies to control early viral reactivation.

Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy.

HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105 infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106 to <15 vRNA copies/mL by the time treatment was interrupted. Virus rapidly rebounded following treatment interruption and between 87 and 136 distinct clonotypes were detected in plasma at peak rebound viremia. This study confirmed that SIVmac239M viremia could be successfully curtailed with cART, and that upon cART discontinuation, rebounding viral variants could be identified and quantified. An additional 6 animals infected with SIVmac239M were treated with cART beginning on day 4 post-infection for 305, 374, or 482 days (study 2). Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33-0.70 events per day, likely representing reactivation from long-lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.

Modeling of Antilatency Treatment in HIV: What Is the Optimal Duration of Antiretroviral Therapy-Free HIV Remission?

A number of treatment strategies are currently being developed to promote antiretroviral therapy-free HIV cure or remission. While complete elimination of the HIV reservoir would prevent recurrence of infection, it is not clear how different remission lengths would affect viral rebound and transmission. In this work, we use a stochastic model to show that a treatment that achieves a 1-year average time to viral remission will still lead to nearly a quarter of subjects experiencing viral rebound within the first 3 months. Given quarterly viral testing intervals, this leads to an expected 39 (95% uncertainty interval [UI], 22 to 69) heterosexual transmissions and up to 262 (95% UI, 107 to 534) homosexual transmissions per 1,000 treated subjects over a 10-year period. Thus, a balance between high initial treatment levels, risk of recrudescence, and risk of transmission should be considered when assessing the "useful" or optimal length of antiretroviral therapy-free HIV remission to be targeted. We also investigate the trade-off between increasing the average duration of remission versus the risk of treatment failure (viral recrudescence) and the need for retreatment. To minimize drug exposure, we found that the optimal target of antilatency interventions is a 1,700-fold reduction in the size of the reservoir, which leads to an average time to recrudescence of 30 years. Interestingly, this is a significantly lower level of reduction than that required for complete elimination of the viral reservoir. Additionally, we show that when shorter periods are targeted, there is a real probability of viral transmission occurring between tests for viral rebound.IMPORTANCE Current treatment of HIV involves patients taking antiretroviral therapy to ensure that the level of virus remains very low or undetectable. Continuous therapy is required, as the virus persists in a latent state within cells, and when therapy is stopped, the virus rebounds, usually within 2 weeks. A major question is how to reduce the amount of persistent virus and therefore allow a delay or remission until the virus returns after ceasing therapy. In this work, we consider the probability that HIV will still rebound even after this reduction and ask what the likelihood of viral transmission would be in this case.

Correction: HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission.

[This corrects the article DOI: 10.1371/journal.ppat.1005000.][This corrects the article DOI: 10.1371/journal.ppat.1005740.][This corrects the article DOI: 10.1371/journal.ppat.1005679.].

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days--Implications for HIV Remission.

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to 'purge' the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different 'reactivation founder' viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.

Understanding the relationship between Plasmodium falciparum growth rate and multiplicity of infection.

Natural infections with Plasmodium falciparum are often composed of multiple concurrent genetically distinct parasite clones. Such multiclonal infections are more common in areas of high transmission, and the frequency of multiclonal infection also varies with age. A number of studies have suggested that multiclonal infection predicts the risk of subsequent clinical malaria. The multiplicity of infection is determined by the rate of new infections, the number of clones inoculated at each mosquito bite, and the duration of infections. Here, we used a mathematical modeling approach to understand how variation in the growth rate of blood-stage parasites affects the observed multiplicity of infection (MOI), as well as the relationship between the MOI and the risk of subsequent malaria. We then analyzed data from a study of multiclonal infection and malaria in an malaria-endemic area in Tanzania and show that the proportion of multiclonal infections varies with age and that the observed relationship between multiclonal infection and subsequent clinical events can be explained by a reduction in blood-stage parasite growth with age in this population.

Innate immunity induced by Plasmodium liver infection inhibits malaria reinfections.

Following transmission through a mosquito bite to the mammalian host, Plasmodium parasites first invade and replicate inside hepatocytes before infecting erythrocytes and causing malaria. The mechanisms limiting Plasmodium reinfections in humans living in regions of malaria endemicity have mainly been explored by studying the resistance induced by the blood stage of infection. However, epidemiologic studies have suggested that in high-transmission areas, preerythrocytic stages also activate host resistance to reinfection. This, along with the recent discovery that liver infections trigger a specific and effective type I interferon (IFN) response, prompted us to hypothesize that this pre-erythrocyte-stage-induced resistance is linked to liver innate immunity. Here, we combined experimental approaches and mathematical modeling to recapitulate field studies and understand the molecular basis behind such resistance. We present a newly established mouse reinfection model and demonstrate that rodent malaria liver-stage infection inhibits reinfection. This protection relies on the activation of innate immunity and involves the type I IFN response and the antimicrobial cytokine gamma IFN (IFN-γ). Importantly, mathematical simulations indicate that the predictions based on our experimental murine reinfection model fit available epidemiological data. Overall, our study revealed that liver-stage-induced innate immunity may contribute to the preerythrocytic resistance observed in humans in regions of malaria hyperendemicity.

Time-to-infection by Plasmodium falciparum is largely determined by random factors.

BACKGROUND: The identification of protective immune responses to P. falciparum infection is an important goal for the development of a vaccine for malaria. This requires the identification of susceptible and resistant individuals, so that their immune responses may be studied. Time-to-infection studies are one method for identifying putative susceptible individuals (infected early) versus resistant individuals (infected late). However, the timing of infection is dependent on random factors, such as whether the subject was bitten by an infected mosquito, as well as individual factors, such as their level of immunity. It is important to understand how much of the observed variation in infection is simply due to chance. METHODS: We analyse previously published data from a treatment-time-to-infection study of 201 individuals aged 0.5 to 78 years living in Western Kenya. We use a mathematical modelling approach to investigate the role of immunity versus random factors in determining time-to-infection in this cohort. We extend this analysis using a modelling approach to understand what factors might increase or decrease the utility of these studies for identifying susceptible and resistant individuals. RESULTS: We find that, under most circumstances, the observed distribution of time-to-infection is consistent with this simply being a random process. We find that age, method for detection of infection (PCR versus microscopy), and underlying force of infection are all factors in determining whether time-to-infection is a useful correlate of immunity. CONCLUSIONS: Many epidemiological studies of P. falciparum infection assume that the observed variation in infection outcomes, such as time-to-infection or presence or absence of infection, is determined by host resistance or susceptibility. However, under most circumstances, this distribution appears largely due to the random timing of infection, particularly in children. More direct measurements, such as parasite growth rate, may be more useful than time-to-infection in segregating patients based on their level of immunity.

Decreased growth rate of P. falciparum blood stage parasitemia with age in a holoendemic population.

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumulative exposure. The relative contribution of acquired immunity against various stages of the parasite life cycle is not well understood. In particular, it is not known whether changes in infection dynamics can be best explained by decreasing rates of infection, or by decreased growth rates of parasites in blood. Here, we analyze the dynamics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of different ages. We use both polymerase chain reaction (PCR) and microscopy detection of parasitemia in order to understand parasite growth rates and infection rates over time. The more sensitive PCR assay detects parasites earlier than microscopy, and demonstrates a higher overall prevalence of infection than microscopy alone. The delay between PCR and microscopy detection is significantly longer in adults compared with children, consistent with slower parasite growth with age. We estimated the parasite multiplication rate from delay to PCR and microscopy detections of parasitemia. We find that both the delay between PCR and microscopy infection as well as the differing reinfection dynamics in different age groups are best explained by a slowing of parasite growth with age.

Gammaherpesvirus latency induces antibody-associated thrombocytopenia in mice.

Human herpesviruses establish lifelong latency. Viral recrudescence can lead to the development of cancers, immunoproliferative disorders, transplantation complications, and thrombocytopenia. Although platelet-specific autoantibodies have been reported in patients infected with the Epstein-Barr virus (EBV), the mechanisms by which thrombocytopenia is induced remain unclear, as do the relative contributions of lytic viral replication and latent viral gene expression. The human gammaherpesviruses are tightly restricted in their ability to infect other mammals, so they are difficult to study in live animal models. Here we show that infection of mice with murine gammaherpesvirus-68 (γHV68), a rodent-specific pathogen closely related to EBV, induces the production of platelet-binding antibodies and causes thrombocytopenia. Infection of antibody-deficient mice does not lead to thrombocytopenia, indicating the platelet decrease is mediated by antibody. Additionally, infection with a latency-null recombinant γHV68 does not induce thrombocytopenia, suggesting factors associated with viral latency drive the infection-induced antibody-mediated thrombocytopenia. These studies describe an important animal model of gammaherpesvirus-induced autoimmune thrombocytopenia and demonstrate that this pathology is mediated by antibody and dependent on viral latency. This model will allow studies of the underlying mechanisms of disease progression and the testing of therapeutic strategies for the alleviation of virus-induced thrombocytopenia.

The dynamics of naturally acquired immunity to Plasmodium falciparum infection.

Severe malaria occurs predominantly in young children and immunity to clinical disease is associated with cumulative exposure in holoendemic settings. The relative contribution of immunity against various stages of the parasite life cycle that results in controlling infection and limiting disease is not well understood. Here we analyse the dynamics of Plasmodium falciparum malaria infection after treatment in a cohort of 197 healthy study participants of different ages in order to model naturally acquired immunity. We find that both delayed time-to-infection and reductions in asymptomatic parasitaemias in older age groups can be explained by immunity that reduces the growth of blood stage as opposed to liver stage parasites. We found that this mechanism would require at least two components - a rapidly acting strain-specific component, as well as a slowly acquired cross-reactive or general immunity to all strains. Analysis and modelling of malaria infection dynamics and naturally acquired immunity with age provides important insights into what mechanisms of immune control may be harnessed by malaria vaccine strategists.

CD8+ lymphocytes do not impact SIV reservoir establishment under ART.

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG.

Drug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIG's proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.

CD8+ T cells fail to limit SIV reactivation following ART withdrawal until after viral amplification.

To define the contribution of CD8+ T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8+ T cell depletion using a rhesusized anti-CD8ß monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8+ T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA+ cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8+ T cell-depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8+ T cell-depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8+ T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.