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.

The development of cervical cancer and its precursors are linked to persistent infection with oncogenic types of human papillomavirus (HPV). Host immune responses seem to be determinants of risk for this disease. However, little is known about the immunologic determinants of HPV persistence. Here, we examined the association between lymphoproliferative responses to antigens/mitogens and persistent HPV infection in women older than 45 years. Women included in this study were participants in a 10,000-woman population-based cohort study of cervical neoplasia in Costa Rica. Women older than 45 years and HPV DNA positive at a screening visit were selected as cases (n = 283). We selected a comparably sized control group of HPV DNA-negative women, matched to cases on age and time since enrollment (n = 261). At an additional clinical visit, women were cytologically and virologically rescreened, and cervical and blood specimens were collected. Proliferative responses to phytohemagglutinin (PHA), influenza virus (Flu), and HPV16 virus-like particle (VLP) were lower among women with persistent HPV infection [median counts per minute (cpm): 72,849 for PHA, 1,241 for Flu, and 727 for VLP] than for the control group (median cpm: 107,049 for PHA, 2,111 for Flu, and 2,068 for VLP). The decreases were most profound in women with long-term persistence and were only observed for the oldest age group (z65 years). Our results indicate that an impairment in host immunologic responses is associated to persistent HPV infection. The fact that effects were evident for all studied stimuli is suggestive of a generalized effect.

Plasma viremia decreases coincident with the appearance of virus-specific CD8+ T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8+ T cell responses and transient increase in plasma viremia after depletion of CD8+ T cells in SIV-infected monkeys strongly suggest a role for CD8+ T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8+ T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8+ T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8+ T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for &gt;6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8+ T cells demonstrated an escape mutation in this epitope &lt;3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8+ T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.