Mother-to-child transmission of HIV-1 continues to cause new pediatric cases of infection through breastfeeding, a setting where it is not always possible to initiate early antiretroviral therapy (ART). Without novel interventions that do not rely on daily ART, HIV-1 infected children face lifelong medications to control infection. A detailed analysis of virus persistence following breastmilk transmission of HIV-1 and ART has not been performed. Here, we used infant rhesus macaques orally-infected with simian/human immunodeficiency virus (SHIV.C.CH505) to identify cellular and anatomical sites of virus persistence under ART. Viral DNA was detected at similar levels in blood and tissue CD4+ T cells after a year on ART, with virus in blood and lymphoid organs confirmed to be replication-competent. Viral RNA:DNA ratios were elevated in rectal CD4+ T cells compared to other sites (P≤0.0001), suggesting the gastrointestinal tract is an active site of virus transcription during ART-mediated suppression of viremia. SHIV.C.CH505 DNA was detected in multiple CD4+ T cell subsets, including cells with a naïve phenotype (CD45RA+CCR7+CD95-). While the frequency of naïve cells harboring intact provirus was lower than in memory cells, the high abundance of naïve cells in the infant CD4+ T cell pool made them a substantial source of persistent viral DNA (approximately 50% of total CD4+ T cell reservoir), with an estimated 1:2 ratio of intact provirus to total viral DNA. This viral reservoir profile broadens our understanding of virus persistence in a relevant infant macaque model and provides insight into targets for cure-directed approaches in the pediatric population. IMPORTANCE Uncovering the sanctuaries of the long-lived HIV-1 reservoir is crucial to develop cure strategies. Pediatric immunity is distinct from that of adults, which could alter where the reservoir is established in infancy. Thus, it is important to utilize pediatric models to inform cure-directed approaches for HIV-1-infected children. We used an infant rhesus macaque model of HIV-1 infection via breastfeeding to identify key sites of viral persistence under antiretroviral therapy (ART). The gastrointestinal tract was found to be a site for low-level viral transcription during ART. We also show naïve CD4+ T cells harbored intact provirus and were a major contributor to blood and lymphoid reservoir size. This is particularly striking as memory CD4+ T cells are generally regarded as the main source of latent HIV/SIV infection of adult humans and rhesus macaques. Our findings highlight unique features of reservoir composition in pediatric infection that should be considered for eradication efforts.

Understanding viral rebound in pediatric HIV-1 infection may inform the development of alternatives to lifelong antiretroviral therapy (ART) to achieve viral remission. We thus investigated viral rebound after analytical treatment interruption (ATI) in 10 infant macaques orally infected with SHIV.C.CH505 and treated with long-term ART. Rebound viremia was detected within 7-35 days of ATI in 9/10 animals, with post-treatment control of viremia seen in 5/5 Mamu-A*01 + macaques. Single-genome sequencing revealed initial rebound virus was similar to viral DNA present in CD4+ T cells from blood, rectum, and lymph nodes before ATI. We assessed the earliest sites of viral reactivation immediately following ATI using ImmunoPET imaging. The largest increase in signal that preceded detectable viral RNA in plasma was found in the gastrointestinal (GI) tract, a site with relatively high SHIV RNA/DNA ratios in CD4+ T cells prior to ATI. Thus, the GI tract may be an initial source of rebound virus but as ATI progresses, viral reactivation in other tissues likely contributes to the composition of plasma virus. Our study provides novel insight into the features of viral rebound in pediatric infection and highlights the application of a non-invasive technique to monitor areas of HIV-1 expression in children.