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

Abstract

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.

Viral rebound kinetics following single and combination immunotherapy for HIV/SIV

“…We believe our approach to modeling reactivation from latency had several advantages over simpler models upon which it was based (e.g. [13,14,46,48,56]). Firstly, our model coherently captures the relationship between reservoir size and rebound, including interpatient variability in both quantities, when reactivation is rare and when it is common, without making an assumption a priori about which regime the kinetics are in.…”

Virus dynamics and phyloanatomy: Merging population dynamic and phylogenetic approaches

“…In the context of viruses, genetic tags can be engineered into noncoding regions of the viral genome . This approach has been recently adopted for SIV in order to study the dynamics of the latent reservoir . A 34‐nucleotide‐long cassette containing a 10‐random nucleotide barcode was inserted between the vpx and vpr genes of SIVmac239, yielding a theoretical upper bound of 4 10 unique barcodes.…”

“…Analyzing the structure of the rebounding viral population in terms of its barcode composition allowed Fennessey et al . to calculate the rate of reactivation from the latent reservoir.…”

“…In the context of viruses, genetic tags can be engineered into noncoding regions of the viral genome. 70,[73][74][75] This approach has been recently adopted for SIV in order to study the dynamics of the latent reservoir. 74 From the perspective of population dynamical modeling, bottlenecks are often equivalent to low migration rates between compartments in these systems.…”

“…These engineered viruses were used to challenge rhesus macaques that were subsequently put on antiretroviral treatment with multiple drugs. Treatment was then withdrawn after varying times, and rebound virus was analyzed.Analyzing the structure of the rebounding viral population in terms of its barcode composition allowed Fennessey et al74 to calculate the rate of reactivation from the latent reservoir. The reactivation rates they estimate range from 1 every 3 days to 1 every 1.5 days after 300 or more days of treatment.…”