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1. Barcoded HIV-1 reveals viral persistence driven by clonal proliferation and distinct epigenetic patterns

   https://doi.org/10.1038/s41467-025-56771-4  

   Zhang, Tian-hao; Shi, Yuan; Komarova, Natalia L; Wordaz, Dominik; Kostelny, Matthew; Gonzales, Alexander; Abbaali, Izra; Chen, Hongying; Bresson-Tan, Gabrielle; Dimapasoc, Melanie; et al (December 2025, Nature Communications)

   Abstract The HIV reservoir consists of infected cells in which the HIV-1 genome persists as provirus despite effective antiretroviral therapy (ART). Studies exploring HIV cure therapies often measure intact proviral DNA levels, time to rebound after ART interruption, or ex vivo stimulation assays of latently infected cells. This study utilizes barcoded HIV to analyze the reservoir in humanized mice. Using bulk PCR and deep sequencing methodologies, we retrieve 890 viral RNA barcodes and 504 proviral barcodes linked to 15,305 integration sites at the single RNA or DNA molecule in vivo. We track viral genetic diversity throughout early infection, ART, and rebound. The proviral reservoir retains genetic diversity despite cellular clonal proliferation and viral seeding by rebounding virus. Non-proliferated cell clones are likely the result of elimination of proviruses associated with transcriptional activation and viremia. Elimination of proviruses associated with viremia is less prominent among proliferated cell clones. Proliferated, but not massively expanded, cell clones contribute to proviral expansion and viremia, suggesting they fuel viral persistence. This approach enables comprehensive assessment of viral levels, lineages, integration sites, clonal proliferation and proviral epigenetic patterns in vivo. These findings highlight complex reservoir dynamics and the role of proliferated cell clones in viral persistence. 

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2. Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

   https://doi.org/10.1073/pnas.2006816117  

   Simonetti, Francesco R.; White, Jennifer A.; Tumiotto, Camille; Ritter, Kristen D.; Cai, Mian; Gandhi, Rajesh T.; Deeks, Steven G.; Howell, Bonnie J.; Montaner, Luis J.; Blankson, Joel N.; et al (August 2020, Proceedings of the National Academy of Sciences)

   A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1 + adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10 6 CD4 + T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10 6 CD4 + T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART. 

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3. Synergistic Chromatin-Modifying Treatments Reactivate Latent HIV and Decrease Migration of Multiple Host-Cell Types

   https://doi.org/10.3390/v13061097  

   Blanco, Alexandra; Mahajan, Tarun; Coronado, Robert A.; Ma, Kelly; Demma, Dominic R.; Dar, Roy D. (June 2021, Viruses)

   Upon infection of its host cell, human immunodeficiency virus (HIV) establishes a quiescent and non-productive state capable of spontaneous reactivation. Diverse cell types harboring the provirus form a latent reservoir, constituting a major obstacle to curing HIV. Here, we investigate the effects of latency reversal agents (LRAs) in an HIV-infected THP-1 monocyte cell line in vitro. We demonstrate that leading drug treatments synergize activation of the HIV long terminal repeat (LTR) promoter. We establish a latency model in THP-1 monocytes using a replication incompetent HIV reporter vector with functional Tat, and show that chromatin modifiers synergize with a potent transcriptional activator to enhance HIV reactivation, similar to T-cells. Furthermore, leading reactivation cocktails are shown to differentially affect latency reactivation and surface expression of chemokine receptor type 4 (CXCR4), leading to altered host cell migration. This study investigates the effect of chromatin-modifying LRA treatments on HIV latent reactivation and cell migration in monocytes. As previously reported in T-cells, epigenetic mechanisms in monocytes contribute to controlling the relationship between latent reactivation and cell migration. Ultimately, advanced “Shock and Kill” therapy needs to successfully target and account for all host cell types represented in a complex and composite latency milieu. 

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4. Predictive Value of CD8+ T cell and CD4/CD8 Ratio at Two Years of Successful ART

   Serrano-Villar S.; Wu K.; Hunt P.W.; Lok J.J.; Ron R.; Sainz T.; Moreno S.; Deeks S.G.; Bosch R.J. (April 2022, EBioMedicine)

   Background While increased CD8 counts and low CD4/CD8 ratio during treated HIV correlate with immunosenescence, their additional predictive values to identify individuals with HIV at higher risk of clinical events remain controversial. Methods We selected treatment-naive individuals initiating ART from ACTG studies 384, 388, A5095, A5142, A5202, and A5257 who had achieved viral suppression at year 2. We examined the effect of CD8+ T cell counts and CD4/CD8 at year 2 on the probability of AIDS and serious non-AIDS events in years 3
   7. We used inverse probability weighting methods to address informative censoring, combined with multivariable logistic regression models. Findings We analyzed 5133 participants with a median age of 38 years; 959 (19%) were female, pre-ART median CD4 counts were 249 (Q1-Q3 91
   372) cell/µL. Compared to participants with CD8 counts between 500/µL and 1499/µL, those with >1500/µL had a higher risk of clinical events during years 3
   7 (aOR 1.75; 95%CI 1.33
   2.32). CD4/CD8 ratio was not predictive of greater risk of events through year 7. Additional analyses revealed consistent CD8 count effect sizes for the risk of AIDS events and noninfectious non-AIDS events, but opposite effects for the risk of severe infections, which were more frequent among individuals with CD8 counts <500/µL (aOR 1.70; 95%CI 1.09
   2.65). Interpretation The results of this analysis with pooled data from clinical trials support the value of the CD8 count as a predictor of clinical progression. People with very high CD8 counts during suppressive ART might benefit from closer monitoring and may be a target population for novel interventions. 

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5. Prevalence and factors associated with self-reported HIV testing among adolescent girls and young women in Rwanda: evidence from 2019/20 Rwanda Demographic and Health Survey

   https://doi.org/10.1186/s12889-022-13679-8  

   Musekiwa, Alfred; Silinda, Patricia; Bamogo, Assanatou; Twabi, Halima S; Mohammed, Mohanad; Batidzirai, Jesca Mercy; Matsena_Zingoni, Zvifadzo; Singini, Geoffrey Chiyuzga; Moyo, Maureen; Mchunu, Nobuhle Nokubonga; et al (December 2022, BMC Public Health)

   Abstract BackgroundHIV/AIDS remains a major public health problem globally. The majority of people living with HIV are from Sub-Saharan Africa, particularly adolescent girls and young women (AGYW) aged 15-24 years. HIV testing is crucial as it is the gateway to HIV prevention, treatment, and care; therefore this study determined the prevalence and factors associated with self-reported HIV testing among AGYW in Rwanda. MethodsWe conducted secondary data analysis on the AGYW using data extracted from the nationally representative population-based 2019/2020 cross-sectional Rwanda Demographic and Health Survey (DHS). We described the characteristics of study participants and determined the prevalence of HIV testing and associated factors using the multivariable logistic regression model. We adjusted all our analyses for unequal sampling probabilities using survey weights. ResultsThere were a total of 5,732 AGYW, with the majority (57%) aged 15-19 years, 83% were not living with a man, 80% were from rural areas, 29% were from the East region, and 20% had a history of pregnancy. Self-reported HIV testing prevalence was 55.4% (95%CI: 53.7 to 57.0%). The odds of ever having an HIV test were significantly higher for those aged 20-24 years (aOR 2.87, 95%CI: 2.44 to 3.37); with higher education (aOR 2.41, 95%CI:1.48 to 3.93); who were rich (aOR 2.06, 95%CI:1.57 to 2.70); with access to at least one media (aOR 1.64, 95%CI: 1.14 to 2.37); who had ever been pregnant (aOR 16.12, 95%CI: 9.60 to 27.07); who ever had sex (aOR 2.40, 95%CI: 1.96 to 2.95); and those who had comprehensive HIV knowledge (aOR 1.34, 95%CI: 1.17 to 1.54). ConclusionsWe report an unmet need for HIV testing among AGYW in Rwanda. We recommend a combination of strategies to optimize access to HIV testing services, especially among the 15-19 years adolescent girls, including facility-based testing, school and community outreach, awareness campaigns on HIV testing, and home-based testing through HIV self-testing. 

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