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.

Transient viral replication during analytical treatment interruptions in SIV infected macaques can alter the rebound-competent viral reservoir.

Analytical treatment interruptions (ATIs) of antiretroviral therapy (ART) play a central role in evaluating the efficacy of HIV-1 treatment strategies targeting virus that persists despite ART. However, it remains unclear if ATIs alter the rebound-competent viral reservoir (RCVR), the virus population that persists during ART and from which viral recrudescence originates after ART discontinuation. To assess the impact of ATIs on the RCVR, we used a barcode sequence tagged SIV to track individual viral lineages through a series of ATIs in Rhesus macaques. We demonstrate that transient replication of individual rebounding lineages during an ATI can lead to their enrichment in the RCVR, increasing their probability of reactivating again after treatment discontinuation. These data establish that the RCVR can be altered by uncontrolled replication during ATI.

Genetically barcoded SIV reveals the emergence of escape mutations in multiple viral lineages during immune escape.

The rapidity of replication coupled with a high mutation rate enables HIV to evade selective pressures imposed by host immune responses. Investigating the ability of HIV to escape different selection forces has generally relied on population-level measures, such as the time to detectable escape mutations in plasma and the rate these mutations subsequently take over the virus population. Here we employed a barcoded synthetic swarm of simian immunodeficiency virus (SIV) in rhesus macaques to investigate the generation and selection of escape mutations within individual viral lineages at the Mamu-A*01-restricted Tat-SL8 epitope. We observed the persistence of more than 1,000 different barcode lineages following selection after acquiring escape mutations. Furthermore, the increased resolution into the virus population afforded by barcode analysis revealed changes in the population structure of the viral quasispecies as it adapted to immune pressure. The high frequency of emergence of escape mutations in parallel viral lineages at the Tat-SL8 epitope highlights the challenge posed by viral escape for the development of T cell-based vaccines. Importantly, the level of viral replication required for generating escape mutations in individual lineages can be directly estimated using the barcoded virus, thereby identifying the level of efficacy required for a successful vaccine to limit escape. Overall, assessing the survival of barcoded viral lineages during selection provides a direct and quantitative measure of the stringency of the underlying genetic bottleneck, making it possible to predict the ability of the virus to escape selective forces induced by host immune responses as well as during therapeutic interventions.

Rational design and in vivo selection of SHIVs encoding transmitted/founder subtype C HIV-1 envelopes.

Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 103 to 107 vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs.

In Vivo Validation of the Viral Barcoding of Simian Immunodeficiency Virus SIVmac239 and the Development of New Barcoded SIV and Subtype B and C Simian-Human Immunodeficiency Viruses.

Genetically barcoded viral populations are powerful tools for evaluating the overall viral population structure as well as assessing the dynamics and evolution of individual lineages in vivo over time. Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone simian immunodeficiency virus (SIV) SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences were within a 34-base barcode that could be tracked using next-generation deep sequencing. Here, we assessed the population size, distribution, and authenticity of individual viral clonotypes within this synthetic swarm using samples from 120 rhesus macaques infected intravenously. The number of replicating barcodes in plasma correlated with the infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of the inoculum size. Overall, 97% of detectable clonotypes in the viral stock were identified in the plasma of at least one infected animal. Additionally, we prepared a second-generation barcoded SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and an additional barcoded stock with suboptimal nucleotides corrected (SIVmac239Opt5M). We also generated four barcoded stocks from subtype B and C simian-human immunodeficiency virus (SHIV) clones. These new SHIV clones may be particularly valuable models to evaluate Env-targeting approaches to study viral transmission or viral reservoir clearance. Overall, this work further establishes the reliability of the barcoded virus approach and highlights the feasibility of adapting this technique to other viral clones.IMPORTANCE We recently developed and published a description of a barcoded simian immunodeficiency virus that has a short random sequence inserted directly into the viral genome. This allows for the tracking of individual viral lineages with high fidelity and ultradeep sensitivity. This virus was used to infect 120 rhesus macaques, and we report here the analysis of the barcodes of these animals during primary infection. We found that the vast majority of barcodes were functional in vivo We then expanded the barcoding approach in a second-generation SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and a barcoded stock of SIVmac239Opt5M whose sequence had 5 changes from the wild-type SIVmac239 sequence. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing a human immunodeficiency virus (HIV) type 1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.

Low-level alternative tRNA priming of reverse transcription of HIV-1 and SIV in vivo.

BACKGROUND: Reverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys3 is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys5, which has an acceptor stem sequence varying from tRNALys3 by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription. RESULTS: We undertook an unbiased approach, evaluating the primer binding site by deep-sequencing of HIV and SIV directly from the plasma of 15 humans and 11 macaques. We found that in humans there are low but measurable levels of viral RNA genomes harboring a PBS containing the noncanonical T at position 8 (PBS-Lys5) corresponding to the tRNAlys5 sequence and representing an average of 0.52% (range 0.07-1.6%) of the total viral population. This value is remarkably consistent with the proportion of PBS-Lys5 we identified in a cross-sectional assessment of the LANL HIV database (0.51%). In macaques chronically infected with SIVmac239, the PBS-Lys5 was also detected but at a frequency 1-log less than seen for HIV, with an average of 0.056% (range 0.01-0.09%). At this proportion, PBS-Lys5 was comparable to other transition mutations, making it impossible to determine whether the mutation observed is a result of use of tRNALys5 as an RT primer at very low levels or merely the product of in vitro cDNA synthesis/PCR error. We also identified two novel PBS sequences in HIV and SIV at low levels in vivo corresponding to tRNALys6 and tRNALys1,2, suggesting that these tRNAs may rarely also be used to prime RT. In vivo reversion of the PBS-Lys5 found in SIVmac239 was rapid and reached background levels by 30 days post-infection. CONCLUSIONS: We conclude that while alternative tRNAs can initiate reverse transcription of HIV and SIV in vivo, their overall contributions to the replicating viral population are small.

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.

Recombination Enhances HIV-1 Envelope Diversity by Facilitating the Survival of Latent Genomic Fragments in the Plasma Virus Population.

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation process including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.

Reduced evolutionary rates in HIV-1 reveal extensive latency periods among replicating lineages.

BACKGROUND: HIV-1 can persist for the duration of a patient's life due in part to its ability to hide from the immune system, and from antiretroviral drugs, in long-lived latent reservoirs. Latent forms of HIV-1 may also be disproportionally involved in transmission. Thus, it is important to detect and quantify latency in the HIV-1 life cycle. RESULTS: We developed a novel molecular clock-based phylogenetic tool to investigate the prevalence of HIV-1 lineages that have experienced latency. The method removes alternative sources that may affect evolutionary rates, such as hypermutation, recombination, and selection, to reveal the contribution of generation-time effects caused by latency. Our method was able to recover latent lineages with high specificity and sensitivity, and low false discovery rates, even on relatively short branches on simulated phylogenies. Applying the tool to HIV-1 sequences from 26 patients, we show that the majority of phylogenetic lineages have been affected by generation-time effects in every patient type, whether untreated, elite controller, or under effective or failing treatment. Furthermore, we discovered extensive effects of latency in sequence data (gag, pol, and env) from reservoirs as well as in the replicating plasma population. To better understand our phylogenetic findings, we developed a dynamic model of virus-host interactions to investigate the proportion of lineages in the actively replicating population that have ever been latent. Assuming neutral evolution, our dynamic modeling showed that under most parameter conditions, it is possible for a few activated latent viruses to propagate so that in time, most HIV-1 lineages will have been latent at some time in their past. CONCLUSIONS: These results suggest that cycling in and out of latency plays a major role in the evolution of HIV-1. Thus, no aspect of HIV-1 evolution can be fully understood without considering latency - including treatment, drug resistance, immune evasion, transmission, and pathogenesis.

No evidence for ongoing replication on ART in SIV-infected macaques.

The capacity of HIV-1 to replicate during optimal antiretroviral therapy (ART) is challenging to assess directly. To gain greater sensitivity to detect evolution on ART, we used a nonhuman primate (NHP) model providing precise control over the level of pre-ART evolution and more comprehensive analyses than are possible with clinical samples. We infected 21 rhesus macaques (RMs) with the barcoded virus SIVmac239M and initiated ART early to minimize baseline genetic diversity. RMs were treated for 285-1200 days. We used several tests of molecular evolution to compare 1352 near-full-length (nFL) SIV DNA single genome sequences from PBMCs, lymph nodes, and spleen obtained near the time of ART initiation and those present after long-term ART, none of which showed significant changes to the SIV DNA population during ART in any animal. To investigate the possibility of ongoing replication in unsampled putative tissue sanctuaries during ART, we discontinued treatment in four animals and confirmed that none of the 336 nFL SIV RNA sequences obtained from rebound plasma viremia showed evidence of evolution. The rigorous nature of our analyses reinforced the emerging consensus of a lack of appreciable ongoing replication on effective ART and validates the relevance of this NHP model for cure studies.

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Antibody-mediated depletion of viral reservoirs is limited in SIV-infected macaques treated early with antiretroviral therapy.

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4+ T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4+ T cell populations were then allowed 1 year on cART to recover. Despite profound CD4+ T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4-treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.

Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread.

A population at low census might go extinct or instead transition into exponential growth to become firmly established. Whether this pivotal event occurs for a within-host pathogen can be the difference between health and illness. Here, we define the principles governing whether HIV-1 spread among cells fails or becomes established by coupling stochastic modeling with laboratory experiments. Following ex vivo activation of latently infected CD4 T cells without de novo infection, stochastic cell division and death contributes to high variability in the magnitude of initial virus release. Transition to exponential HIV-1 spread often fails due to release of an insufficient amount of replication-competent virus. Establishment of exponential growth occurs when virus produced from multiple infected cells exceeds a critical population size. We quantitatively define the crucial transition to exponential viral spread. Thwarting this process would prevent HIV transmission or rebound from the latent reservoir.

Defining early SIV replication and dissemination dynamics following vaginal transmission.

Understanding HIV transmission is critical to guide the development of prophylactic interventions to prevent infection. We used a nonhuman primate (NHP) model with a synthetic swarm of sequence-tagged variants of SIVmac239 ("SIVmac239X") and scheduled necropsy during primary infection (days 3 to 14 after challenge) to study viral dynamics and host responses to the establishment and dissemination of infection following vaginal challenge. We demonstrate that local replication was initiated at multiple sites within the female genital tract (FGT), with each site having multiple viral variants. Local replication and spread in the FGT preceded lymphatic dissemination. Innate viral restriction factors were observed but appeared to follow viral replication and were ineffective at blocking initial viral establishment and dissemination. However, major delays were observed in time to dissemination in animals and among different viral variants within the same animal. It will be important to assess how phenotypic differences affect early viral dynamics.