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.

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.

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.

Derivation and Characterization of Pathogenic Transmitted/Founder Molecular Clones from Simian Immunodeficiency Virus SIVsmE660 and SIVmac251 following Mucosal Infection.

UNLABELLED: Currently available simian immunodeficiency virus (SIV) infectious molecular clones (IMCs) and isolates used in nonhuman primate (NHP) models of AIDS were originally derived from infected macaques during chronic infection or end stage disease and may not authentically recapitulate features of transmitted/founder (T/F) genomes that are of particular interest in transmission, pathogenesis, prevention, and treatment studies. We therefore generated and characterized T/F IMCs from genetically and biologically heterogeneous challenge stocks of SIVmac251 and SIVsmE660. Single-genome amplification (SGA) was used to identify full-length T/F genomes present in plasma during acute infection resulting from atraumatic rectal inoculation of Indian rhesus macaques with low doses of SIVmac251 or SIVsmE660. All 8 T/F clones yielded viruses that were infectious and replication competent in vitro, with replication kinetics similar to those of the widely used chronic-infection-derived IMCs SIVmac239 and SIVsmE543. Phenotypically, the new T/F virus strains exhibited a range of neutralization sensitivity profiles. Four T/F virus strains were inoculated into rhesus macaques, and each exhibited typical SIV replication kinetics. The SIVsm T/F viruses were sensitive to TRIM5α restriction. All T/F viruses were pathogenic in rhesus macaques, resulting in progressive CD4(+) T cell loss in gastrointestinal tissues, peripheral blood, and lymphatic tissues. The animals developed pathological immune activation; lymphoid tissue damage, including fibrosis; and clinically significant immunodeficiency leading to AIDS-defining clinical endpoints. These T/F clones represent a new molecular platform for the analysis of virus transmission and immunopathogenesis and for the generation of novel "bar-coded" challenge viruses and next-generation simian-human immunodeficiency viruses that may advance the HIV/AIDS vaccine agenda. IMPORTANCE: Nonhuman primate research has relied on only a few infectious molecular clones for a myriad of diverse research projects, including pathogenesis, preclinical vaccine evaluations, transmission, and host-versus-pathogen interactions. With new data suggesting a selected phenotype of the virus that causes infection (i.e., the transmitted/founder virus), we sought to generate and characterize infectious molecular clones from two widely used simian immunodeficiency virus lineages (SIVmac251 and SIVsmE660). Although the exact requirements necessary to be a T/F virus are not yet fully understood, we generated cloned viruses with all the necessary characteristic of a successful T/F virus. The cloned viruses revealed typical acute and set point viral-load dynamics with pathological immune activation, lymphoid tissue damage progressing to significant immunodeficiency, and AIDS-defining clinical endpoints in some animals. These T/F clones represent a new molecular platform for studies requiring authentic T/F viruses.

Generation and characterization of a SIVmac239 clone corrected at four suboptimal nucleotides.

BACKGROUND: SIVmac239 is a commonly used virus in non-human primate models of HIV transmission and pathogenesis. Previous studies identified four suboptimal nucleotides in the SIVmac239 genome, which putatively inhibit its replicative capacity. Since all four suboptimal changes revert to the optimal nucleotide consensus sequence during viral replication in vitro and in vivo, we sought to eliminate the variability of generating these mutations de novo and increase the overall consistency of viral replication by introducing the optimal nucleotides directly to the infectious molecular clone. RESULTS: Using site directed mutagenesis of the full-length/nef-open SIVmac239 clone, we reverted all four nucleotides to the consensus/optimal base to generate SIVmac239Opt and subsequently tested its infectivity and replicative capacity in vitro and in vivo. In primary and cell line cultures, we observed that the optimized virus displayed consistent modest but not statistically significant increases in replicative kinetics compared to wild type. In vivo, SIVmac239Opt replicated to high peak titers with an average of 1.2 × 10(8) viral RNA copies/ml at day 12 following intrarectal challenge, reaching set-point viremia of 1.2 × 10(6) viral RNA copies/ml by day 28. Although the peak and set point viremia means were not statistically different from the original "wild type" SIVmac239, viral load variation at set point was greater for SIVmac239WT compared to SIVmac239Opt (p = 0.0015) demonstrating a greater consistency of the optimized virus. Synonymous mutations were added to the integrase gene of SIVmac239Opt to generate a molecular tag consisting of ten genetically distinguishable viral variants referred to as SIVmac239OptX (Del Prete et al., J Virol. doi: 10.1128/JVI.01026-14 , 2014). Replication dynamics in vitro of these optimized clones were not statistically different from the parental clones. Interestingly, the consistently observed rapid reversion of the primer binding site suboptimal nucleotide is not due to viral RT error but is changed post-integration of a mismatched base via host proofreading mechanisms. CONCLUSIONS: Overall, our results demonstrate that SIVmac239Opt is a functional alternative to parental SIVmac239 with marginally faster replication dynamics and with increased replication uniformity providing a more consistent and reproducible infection model in nonhuman primates.

Molecularly tagged simian immunodeficiency virus SIVmac239 synthetic swarm for tracking independent infection events.

Following mucosal human immunodeficiency virus type 1 transmission, systemic infection is established by one or only a few viral variants. Modeling single-variant, mucosal transmission in nonhuman primates using limiting-dose inoculations with a diverse simian immunodeficiency virus isolate stock may increase variability between animals since individual variants within the stock may have substantial functional differences. To decrease variability between animals while retaining the ability to enumerate transmitted/founder variants by sequence analysis, we modified the SIVmac239 clone to generate 10 unique clones that differ by two or three synonymous mutations (molecular tags). Transfection- and infection-derived virus stocks containing all 10 variants showed limited phenotypic differences in 9 of the 10 clones. Twenty-nine rhesus macaques were challenged intrarectally or intravenously with either a single dose or repeated, limiting doses of either stock. The proportion of each variant within each inoculum and in plasma from infected animals was determined by using a novel real-time single-genome amplification assay. Each animal was infected with one to five variants, the number correlating with the dose. Longitudinal sequence analysis revealed that the molecular tags are highly stable with no reversion to the parental sequence detected in >2 years of follow-up. Overall, the viral stocks are functional and mucosally transmissible and the number of variants is conveniently discernible by sequence analysis of a small amplicon. This approach should be useful for tracking individual infection events in preclinical vaccine evaluations, long-term viral reservoir establishment/clearance research, and transmission/early-event studies. Importance: Human immunodeficiency virus type 1 transmission is established by one or only a few viral variants. Modeling of limited variant transmission in nonhuman primates with a diverse simian immunodeficiency virus isolate stock may increase the variability between animals because of functional differences in the individual variants within the stock. To decrease such variability while retaining the ability to distinguish and enumerate transmitted/founder variants by sequence analysis, we generated a viral stock with 10 sequence-identifiable but otherwise genetically identical variants. This virus was characterized in vitro and in vivo and shown to allow discrimination of distinct transmission events. This approach provides a novel nonhuman primate challenge system for the study of viral transmission, evaluation of vaccines and other prevention approaches, and characterization of viral reservoirs and strategies to target them.