Barriers to breastfeeding and targeted breastfeeding social transfer programs in São Paulo, Brazil: a qualitative study.

HIV eradication is challenging because of the persistence of a viral reservoir that evades both immune clearance and antiretroviral therapy (ART). The best characterized persistent reservoir in people with HIV (PWH) is found in long-lived HIV-infected memory CD4 T cells, a highly heterogeneous cell population circulating systemically (e.g., in peripheral blood mononuclear cells, PBMC) or residing in secondary lymphoid tissues. Estimates suggest that ART-treated PWH with well-suppressed viral replication have less than one in a million circulating CD4 T cells harboring a replication-competent provirus. Latently infected cells are heterogeneous cell populations that harbor integrated HIV proviruses, which do not produce detectable viral proteins. The lack of viral production is due to post-integration latency resulting from epigenetic silencing of initially productive proviruses as well as from immediate early non-productive infections arising directly after integration. These non-productively infected cells (NP cells) resulting from immediate early latency fail to produce viral proteins despite successful proviral integration. Most of the HIV latency transcriptomes have been generated from in vitro cell culture models in which primary human CD4 T cells are infected with single LTR-dependent reporter viruses and FACS-sorted for productive infection, then cultured for two to eight weeks until epigenetic silencing progressively converts productively infected cells into non-productive ones. While these benchmark studies have greatly advanced our understanding of HIV latency, they do not provide information on the properties of primary human CD4 T cells harboring non-productive proviruses established immediately after integration. While reporter viruses are widely used to study the latent reservoir in cell culture model systems, next-generation sequencing (NGS) approaches have provided important insights into the composition of the long-lived persistent HIV reservoirs in PWH, years or even decades after active replication was stopped. However, these approaches do not discriminate between proviruses that were initially productive but subsequently silenced through epigenetic means and proviruses that were never active, which define immediate early latent cells. To overcome these limitations, dual-reporter HIV constructs have been developed to allow direct and simultaneous in vitro detection of cells harboring a non-productive or a productive integrated provirus, based on LTR-dependent and LTR-independent markers. HIV-GKO was developed as a single-round virus lacking and was successfully used to characterize the latent reservoir in both peripheral blood and tonsils. HIV pMorpheus-V5 was developed to express all viral genes, including (except for ) and two separate reporter cassettes under the control of either the LTR viral promoter (HSA-mCherry-IRES-Nef cassette) or the cellular PGK promoter (PGK-V5-NGFR cassette). By including , the system allows the study of HIV latency under more physiological conditions, as this accessory protein interacts with multiple host factors. Previous studies demonstrated that pMorpheus-V5 efficiently infects primary CD4 T cells, with both productively and non-productively infected cells harboring integrated HIV-DNA. Furthermore, a percentage of non-productive pMorpheus-V5 proviruses remain inducible upon PMA/ionomycin stimulation. For these reasons, the pMorpheus-V5 construct has been widely used to dissect mechanisms of immediate early latency establishment and/or reversal. To date, however, only a few studies have used these dual reporter viruses to directly analyze the transcriptome of primary human CD4 T cells harboring non-productive proviruses established immediately after integration.