Simulating the entire natural course of HIV infection by extending the basic viral dynamics equations to include declining viral clearance.

The basic model of viral dynamics is a relatively simple set of equations describing the most essential features of the host-pathogen interactions. Coupled with data, it has been used extensively and successfully to reproduce and explain the features of the early acute phase of HIV infection and the effects of antiretroviral treatment, as well as to estimate the lifespan of infected cells, viral growth and clearance rates and predict early outcomes under different circumstances. However, it cannot reproduce the entire natural course of untreated HIV infection consistently with constant parameters. Here we show that it is possible to qualitatively reproduce the whole course of untreated HIV infection within the general framework of the basic model by assuming progressively declining viral clearance coupled with viral load. We discuss the interpretation of this model as proof-of-concept that may inspire further research into the role of viral clearance in HIV infection.

Correction to: Optima nutrition: an allocative efficiency tool to reduce childhood stunting by better targeting of nutrition-related interventions.

It has been highlighted that the original manuscript [1] contains a typesetting error in the name of Meera Shekar. This had been incorrectly captured as Meera Shekhar in the original article which has since been updated.

How should HIV resources be allocated? Lessons learnt from applying Optima HIV in 23 countries.

INTRODUCTION: With limited funds available, meeting global health targets requires countries to both mobilize and prioritize their health spending. Within this context, countries have recognized the importance of allocating funds for HIV as efficiently as possible to maximize impact. Over the past six years, the governments of 23 countries in Africa, Asia, Eastern Europe and Latin America have used the Optima HIV tool to estimate the optimal allocation of HIV resources. METHODS: Each study commenced with a request by the national government for technical assistance in conducting an HIV allocative efficiency study using Optima HIV. Each study team validated the required data, calibrated the Optima HIV epidemic model to produce HIV epidemic projections, agreed on cost functions for interventions, and used the model to calculate the optimal allocation of available funds to best address national strategic plan targets. From a review and analysis of these 23 country studies, we extract common themes around the optimal allocation of HIV funding in different epidemiological contexts. RESULTS AND DISCUSSION: The optimal distribution of HIV resources depends on the amount of funding available and the characteristics of each country's epidemic, response and targets. Universally, the modelling results indicated that scaling up treatment coverage is an efficient use of resources. There is scope for efficiency gains by targeting the HIV response towards the populations and geographical regions where HIV incidence is highest. Across a range of countries, the model results indicate that a more efficient allocation of HIV resources could reduce cumulative new HIV infections by an average of 18% over the years to 2020 and 25% over the years to 2030, along with an approximately 25% reduction in deaths for both timelines. However, in most countries this would still not be sufficient to meet the targets of the national strategic plan, with modelling results indicating that budget increases of up to 185% would be required. CONCLUSIONS: Greater epidemiological impact would be possible through better targeting of existing resources, but additional resources would still be required to meet targets. Allocative efficiency models have proven valuable in improving the HIV planning and budgeting process.

The global Optima HIV allocative efficiency model: targeting resources in efforts to end AIDS.

BACKGROUND: To move towards ending AIDS by 2030, HIV resources should be allocated cost-effectively. We used the Optima HIV model to estimate how global HIV resources could be retargeted for greatest epidemiological effect and how many additional new infections could be averted by 2030. METHODS: We collated standard data used in country modelling exercises (including demographic, epidemiological, behavioural, programmatic, and expenditure data) from Jan 1, 2000, to Dec 31, 2015 for 44 countries, capturing 80% of people living with HIV worldwide. These data were used to parameterise separate subnational and national models within the Optima HIV framework. To estimate optimal resource allocation at subnational, national, regional, and global levels, we used an adaptive stochastic descent optimisation algorithm in combination with the epidemic models and cost functions for each programme in each country. Optimal allocation analyses were done with international HIV funds remaining the same to each country and by redistributing these funds between countries. FINDINGS: Without additional funding, if countries were to optimally allocate their HIV resources from 2016 to 2030, we estimate that an additional 7·4 million (uncertainty range 3·9 million-14·0 million) new infections could be averted, representing a 26% (uncertainty range 13-50%) incidence reduction. Redistribution of international funds between countries could avert a further 1·9 million infections, which represents a 33% (uncertainty range 20-58%) incidence reduction overall. To reduce HIV incidence by 90% relative to 2010, we estimate that more than a three-fold increase of current annual funds will be necessary until 2030. The most common priorities for optimal resource reallocation are to scale up treatment and prevention programmes targeting key populations at greatest risk in each setting. Prioritisation of other HIV programmes depends on the epidemiology and cost-effectiveness of service delivery in each setting as well as resource availability. INTERPRETATION: Further reductions in global HIV incidence are possible through improved targeting of international and national HIV resources. FUNDING: World Bank and Australian NHMRC.

Optima Nutrition: an allocative efficiency tool to reduce childhood stunting by better targeting of nutrition-related interventions.

BACKGROUND: Child stunting due to chronic malnutrition is a major problem in low- and middle-income countries due, in part, to inadequate nutrition-related practices and insufficient access to services. Limited budgets for nutritional interventions mean that available resources must be targeted in the most cost-effective manner to have the greatest impact. Quantitative tools can help guide budget allocation decisions. METHODS: The Optima approach is an established framework to conduct resource allocation optimization analyses. We applied this approach to develop a new tool, 'Optima Nutrition', for conducting allocative efficiency analyses that address childhood stunting. At the core of the Optima approach is an epidemiological model for assessing the burden of disease; we use an adapted version of the Lives Saved Tool (LiST). Six nutritional interventions have been included in the first release of the tool: antenatal micronutrient supplementation, balanced energy-protein supplementation, exclusive breastfeeding promotion, promotion of improved infant and young child feeding (IYCF) practices, public provision of complementary foods, and vitamin A supplementation. To demonstrate the use of this tool, we applied it to evaluate the optimal allocation of resources in 7 districts in Bangladesh, using both publicly available data (such as through DHS) and data from a complementary costing study. RESULTS: Optima Nutrition can be used to estimate how to target resources to improve nutrition outcomes. Specifically, for the Bangladesh example, despite only limited nutrition-related funding available (an estimated $0.75 per person in need per year), even without any extra resources, better targeting of investments in nutrition programming could increase the cumulative number of children living without stunting by 1.3 million (an extra 5%) by 2030 compared to the current resource allocation. To minimize stunting, priority interventions should include promotion of improved IYCF practices as well as vitamin A supplementation. Once these programs are adequately funded, the public provision of complementary foods should be funded as the next priority. Programmatic efforts should give greatest emphasis to the regions of Dhaka and Chittagong, which have the greatest number of stunted children. CONCLUSIONS: A resource optimization tool can provide important guidance for targeting nutrition investments to achieve greater impact.

Relationship between Measures of HIV Reactivation and Decline of the Latent Reservoir under Latency-Reversing Agents.

Antiretroviral-free HIV remission requires substantial reduction of the number of latently infected cells and enhanced immune control of viremia. Latency-reversing agents (LRAs) aim to eliminate latently infected cells by increasing the rate of reactivation of HIV transcription, which exposes these cells to killing by the immune system. As LRAs are explored in clinical trials, it becomes increasingly important to assess the effect of an increased HIV reactivation rate on the decline of latently infected cells and to estimate LRA efficacy in increasing virus reactivation. However, whether the extent of HIV reactivation is a good predictor of the rate of decline of the number of latently infected cells is dependent on a number of factors. Our modeling shows that the mechanisms of maintenance and clearance of the reservoir, the life span of cells with reactivated HIV, and other factors may significantly impact the relationship between measures of HIV reactivation and the decline in the number of latently infected cells. The usual measures of HIV reactivation are the increase in cell-associated HIV RNA (CA RNA) and/or plasma HIV RNA soon after administration. We analyze two recent studies where CA RNA was used to estimate the impact of two novel LRAs, panobinostat and romidepsin. Both drugs increased the CA RNA level 3- to 4-fold in clinical trials. However, cells with panobinostat-reactivated HIV appeared long-lived (half-life > 1 month), suggesting that the HIV reactivation rate increased by approximately 8%. With romidepsin, the life span of cells that reactivated HIV was short (2 days), suggesting that the HIV reactivation rate may have doubled under treatment.IMPORTANCE Long-lived latently infected cells that persist on antiretroviral treatment (ART) are thought to be the source of viral rebound soon after ART interruption. The elimination of latently infected cells is an important step in achieving antiretroviral-free HIV remission. Latency-reversing agents (LRAs) aim to activate HIV expression in latently infected cells, which could lead to their death. Here, we discuss the possible impact of the LRAs on the reduction of the number of latently infected cells, depending on the mechanisms of their loss and self-renewal and on the life span of the cells that have HIV transcription activated by the LRAs.

Acute systemic DNA damage in youth does not impair immune defense with aging.

Aging-related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age-related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole-body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/6 mice to sublethal WBI (0.5-4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock-irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T- and B-cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.

Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

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.

Modeling the timing of antilatency drug administration during HIV treatment.

UNLABELLED: Latently infected cells are considered a major barrier to the cure of HIV infection, since they are long-lived under antiretroviral therapy (ART) and cause viral replication to restart soon after stopping ART. In the last decade, different types of antilatency drugs have been explored with the aim of reactivating and purging this latent reservoir and the hope of achieving a cure. Because of toxicity and safety considerations, antilatency drugs can only be given for a short time to patients on long-term ART, with little effect. We recently investigated the turnover of latently infected cells during active infection and have found that it was strongly correlated with viral load. This implies that although latently infected cells had long life spans in a setting of a low viral load (such as during ART), they turned over quickly under a high viral load. Possible reasons for this could be that an increased viral load causes increased activation or death of CD4(+) T cells, including those that are latently infected. Taking these results into account, we developed a mathematical model to study the most appropriate timing of antilatency drugs in relationship to the initiation of ART. We found that the best timing of a short-term antilatency drug would be the start of ART, when viral load, CD4(+) T cell activation, and latent cell turnover are all high. These results have important implications for the design of HIV cure-related clinical trials. IMPORTANCE: The antiretroviral therapy (ART) of HIV-infected patients currently needs to be lifelong, because the cells latently infected with HIV start new rounds of infection as soon as the treatment is stopped. In the last decade, a number of different types of antilatency drugs have been explored with the aim of "reactivating" and "purging" this latent reservoir and thus achieving a cure. These drugs have thus far been tested on patients only after long-term ART and have demonstrated little or no effect. We use mathematical modeling to show that the most efficacious timing of a short-term antilatency treatment may be the start of ART because of possible interactions of antilatency drugs with natural activation pathways.

Measuring turnover of SIV DNA in resting CD4+ T cells using pyrosequencing: implications for the timing of HIV eradication therapies.

Resting CD4+ T cells are a reservoir of latent HIV-1. Understanding the turnover of HIV DNA in these cells has implications for the development of eradication strategies. Most studies of viral latency focus on viral persistence under antiretroviral therapy (ART). We studied the turnover of SIV DNA resting CD4+ T cells during active infection in a cohort of 20 SIV-infected pigtail macaques. We compared SIV sequences at two Mane-A1*084:01-restricted CTL epitopes using serial plasma RNA and resting CD4+ T cell DNA samples by pyrosequencing, and used a mathematical modeling approach to estimate SIV DNA turnover. We found SIV DNA turnover in resting CD4+ T cells was slow in animals with low chronic viral loads, consistent with the long persistence of latency seen under ART. However, in animals with high levels of chronic viral replication, turnover was high. SIV DNA half-life within resting CD4 cells correleated with viral load (p = 0.0052) at the Gag KP9 CTL epitope. At a second CTL epitope in Tat (KVA10) there was a trend towards an association of SIV DNA half-life in resting CD4 cells and viral load (p = 0.0971). Further, we found that the turnover of resting CD4+ T cell SIV DNA was higher for escape during early infection than for escape later in infection (p = 0.0084). Our results suggest viral DNA within resting CD4 T cells is more labile and may be more susceptible to reactivation/eradication treatments when there are higher levels of virus replication and during early/acute infection.

Intracellular dynamics of HIV infection.

Early studies of HIV infection dynamics suggested that virus-producing HIV-infected cells had an average half-life of approximately 1 day. However, whether this average behavior is reflective of the dynamics of individual infected cells is unclear. Here, we use HIV-enhanced green fluorescent protein (EGFP) constructs and flow cytometry sorting to explore the dynamics of cell infection, viral protein production, and cell death in vitro. By following the numbers of productively infected cells expressing EGFP over time, we show that infected cell death slows down over time. Although infected cell death in vivo could be very different, our results suggest that the constant decay of cell numbers observed in vivo during antiretroviral treatment could reflect a balance of cell death and delayed viral protein production. We observe no correlation between viral protein production and death rate of productively infected cells, showing that viral protein production is not likely to be the sole determinant of the death of HIV-infected cells. Finally, we show that all observed features can be reproduced by a simple model in which infected cells have broad distributions of productive life spans, times to start viral protein production, and viral protein production rates. This broad spectrum of the level and timing of viral protein production provides new insights into the behavior and characteristics of HIV-infected cells.

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.

Standard trivalent influenza virus protein vaccination does not prime antibody-dependent cellular cytotoxicity in macaques.

Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011-2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.

Estimating the contribution of the gut to plasma viral load in early SIV infection.

BACKGROUND: There is significant debate about whether the gut plays a major role in viral replication and pathology in HIV infection. Here we aimed to estimate the contribution of the gut to the total virus observed in plasma, by comparing the frequency of different viral mutants in plasma and gut in SIV infection. RESULTS: We found that the maximum contribution of gut to plasma viral load estimated from rectal biopsy at day 28 post-infection had a median of 10%. The estimated values for individual animals ranged from nearly 100% to <3% in 4/14 animals. Importantly, these are maximum estimates, so that a value of 90%, for example, means that the real contribution may be anything between 0 and 90%, just not higher than 90%.We also studied the contribution of gut at the peak of plasma viral load (day 14). However, since there was very little escape in most animals at this time point, we could only estimate the maximal contribution of gut in 4 animals, in two of which it was <15%. CONCLUSIONS: The role of the gut in HIV is a controversial area, with many suggesting that it plays a dominant role in driving early infection. Our analysis suggests that, at least by day 28 post-infection, the gut is not contributing greatly to the plasma viral load.

Density-dependent blood stage Plasmodium falciparum suppresses malaria super-infection in a malaria holoendemic population.

Recent studies of Plasmodium berghei malaria in mice show that high blood-stage parasitemia levels inhibit the development of subsequent liver-stage infections. Whether a similar inhibitory effect on liver-stage Plasmodium falciparum by blood-stage infection occurs in humans is unknown. We have analyzed data from a treatment-time-to-infection cohort of children < 10 years of age residing in a malaria holoendemic area of Kenya where people experience a new blood-stage infection approximately every 2 weeks. We hypothesized that if high parasitemia blocked the liver stage, then high levels of parasitemia should be followed by a "skipped" peak of parasitemia. Statistical analysis of "natural infection" field data and stochastic simulation of infection dynamics show that the data are consistent with high P. falciparum parasitemia inhibiting liver-stage parasite development in humans.

The search for an HIV cure: tackling latent infection.

Strategies to eliminate infectious HIV that persists despite present treatments and with the potential to cure HIV infection are of great interest. One patient seems to have been cured of HIV infection after receiving a bone marrow transplant with cells resistant to the virus, although this strategy is not viable for large numbers of infected people. Several clinical trials are underway in which drugs are being used to activate cells that harbour latent HIV. In a recent study, investigators showed that activation of latent HIV infection in patients on antiretroviral therapy could be achieved with a single dose of vorinostat, a licensed anticancer drug that inhibits histone deacetylase. Although far from a cure, such studies provide some guidance towards the logical next steps for research. Clinical studies that use a longer duration of drug dosing, alternative agents, combination approaches, gene therapy, and immune-modulation approaches are all underway.

Trivalent live attenuated influenza-simian immunodeficiency virus vaccines: efficacy and evolution of cytotoxic T lymphocyte escape in macaques.

There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8(+) cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may assist in controlling viremia. We vaccinated 10 Mane-A1*08401(+) female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with SIV(mac251). Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced. There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed. All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a "fitness" cost to the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required to protect pigtail macaques from SIV.

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.

An "escape clock" for estimating the turnover of SIV DNA in resting CD4⁺ T cells.

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.