The HIV latent reservoir in resting memory CD4 ⁺ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo , the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4 ⁺ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR ⁺ CD4 ⁺ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.

Significant advances in the field of HIV-1 therapeutics to achieve antiretroviral treatment (ART)-free remission and cure for persons living with HIV-1 are being made with the advent of broadly neutralizing antibodies and very early ART in perinatal infection. The need for HIV-1 remission and cure arises due to the inability of ART to eradicate the major reservoir for HIV-1 in resting memory CD4+ T cells (the latent reservoir), and the strict adherence to lifelong treatment. To measure the efficacy of these cure interventions on reservoir size and to dissect reservoir dynamics, assays that are sensitive and specific to intact proviruses are critical. In this review, we provided a broad overview of some of the key interventions underway to purge the reservoir in adults living with HIV-1 and ones under study in pediatric populations to reduce and control the latent reservoir, primarily focusing on very early treatment in combination with broadly neutralizing antibodies. We also summarized assays currently in use to measure HIV-1 reservoirs and their feasibility and considerations for studies in children.

HIV-1 establishes latency in resting memory CD4+ T cells, creating a long-lived reservoir that poses a barrier to HIV-1 cure. This reservoir is thought to form by initial infection of antigen-activated CD4+ T-cells that then transition into quiescent memory CD4+ T cells. However, naïve CD4+ T cells have been shown to harbor integrated HIV-1 in adults with non-perinatally acquired HIV-1, and more recently in young children. Given that perinatal HIV-1 transmission is established in an immune environment dominated by naïve CD4+ T cells, we hypothesized that naïve CD4+ T cells may be a substantial reservoir in young adults with longstanding perinatal infection. To investigate this, we studied a cohort of 10 adolescents and young adults (AYA) living with well-controlled, perinatally-acquired HIV-1. We quantified and characterized intact and defective HIV-1 proviruses and their location in sorted naïve and memory CD4+ T cells using an intact proviral DNA assay, near full-length single genome sequencing, and integration site profiling. We found that a median of 93.5% of HIV-1 proviruses resided in memory CD4+ T cells in AYA with perinatal HIV-1, with a few participants also having intact proviruses in naïve CD4+ T cells, but at substantially lower frequencies. HIV-1 integration site analysis, using the unbiased ligation-mediated PCR method, showed that 78.0% of proviruses were located within active transcription units in introns and 46.9% of integration sites were clonal, with detection in multiple cells. Overall, similar to adults with non-perinatally acquired HIV-1, integration sites were enriched in regions with epigenetic marks for active enhancers and transcription. Therapies to attain HIV-1 ART-free remission and cure for this population will need to consider reservoirs in non-canonical T cells subsets, as well as substantial HIV-1 infected cells arising from cellular proliferation.

Background: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels (occult infection), with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections. Methods: The analytical sensitivity and specificity of an HIV-1 DNA droplet digital PCR (ddPCR) assay was determined, across different HIV-1 subtypes, using serial dilutions of a plasmid containing a 160-base pair sequence of the HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. Results: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. Conclusions: This assay has applications for detecting occult HIV-1-infection that may occur in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in both infants and adults, and in HIV-1 cure interventions, particularly with perinatal infections.

HIV-1 establishes latency in resting memory CD4+ T cells, creating a long-lived reservoir that poses a barrier to HIV-1 cure. This reservoir is thought to form by initial infection of antigen-activated CD4+ T-cells that then transition into quiescent memory CD4+ T cells. However, naïve CD4+ T cells have been shown to harbor integrated HIV-1 in adults with non-perinatally acquired HIV-1, and more recently in young children. Given that perinatal HIV-1 transmission is established in an immune environment dominated by naïve CD4+ T cells, we hypothesized that naïve CD4+ T cells may be a substantial reservoir in young adults with longstanding perinatal infection. To investigate this, we studied a cohort of 10 adolescents and young adults (AYA) living with well-controlled, perinatally-acquired HIV-1. We quantified and characterized intact and defective HIV-1 proviruses and their location in sorted naïve and memory CD4+ T cells using an intact proviral DNA assay, near full-length single genome sequencing, and integration site profiling. We found that a median of 93.5% of HIV-1 proviruses resided in memory CD4+ T cells in AYA with perinatal HIV-1, with a few participants also having intact proviruses in naïve CD4+ T cells, but at substantially lower frequencies. HIV-1 integration site analysis, using the unbiased ligation-mediated PCR method, showed that 78.0% of proviruses were located within active transcription units in introns and 46.9% of integration sites were clonal, with detection in multiple cells. Overall, similar to adults with non-perinatally acquired HIV-1, integration sites were enriched in regions with epigenetic marks for active enhancers and transcription. Therapies to attain HIV-1 ART-free remission and cure for this population will need to consider reservoirs in non-canonical T cells subsets, as well as substantial HIV-1 infected cells arising from cellular proliferation.

HIV-1 establishes latency in resting memory CD4+ T cells, creating a long-lived reservoir that poses a barrier to HIV-1 cure. This reservoir is thought to form by initial infection of antigen-activated CD4+ T-cells that then transition into quiescent memory CD4+ T cells. However, naïve CD4+ T cells have been shown to harbor integrated HIV-1 in adults with non-perinatally acquired HIV-1, and more recently in young children. Given that perinatal HIV-1 transmission is established in an immune environment dominated by naïve CD4+ T cells, we hypothesized that naïve CD4+ T cells may be a substantial reservoir in young adults with longstanding perinatal infection. To investigate this, we studied a cohort of 10 adolescents and young adults (AYA) living with well-controlled, perinatally-acquired HIV-1. We quantified and characterized intact and defective HIV-1 proviruses and their location in sorted naïve and memory CD4+ T cells using an intact proviral DNA assay, near full-length single genome sequencing, and integration site profiling. We found that a median of 93.5% of HIV-1 proviruses resided in memory CD4+ T cells in AYA with perinatal HIV-1, with a few participants also having intact proviruses in naïve CD4+ T cells, but at substantially lower frequencies. HIV-1 integration site analysis, using the unbiased ligation-mediated PCR method, showed that 78.0% of proviruses were located within active transcription units in introns and 46.9% of integration sites were clonal, with detection in multiple cells. Overall, similar to adults with non-perinatally acquired HIV-1, integration sites were enriched in regions with epigenetic marks for active enhancers and transcription. Therapies to attain HIV-1 ART-free remission and cure for this population will need to consider reservoirs in non-canonical T cells subsets, as well as substantial HIV-1 infected cells arising from cellular proliferation.