Recent innovations in vaccine design have reignited optimism about the feasibility of developing a safe and effective HIV vaccine. For instance, germline-targeting (GT) immunogens have been designed to selectively expand naïve B cell precursors with broadly neutraliing antibodies (bnAbs)-associated germline-encoded features, thereby priming them for structure-guided maturation. The frequencies and affinities of these precursors in the populations targeted for vaccination are critical to the success of GT vaccine strategies, but limited information is currently available across different geographies and demographics. We set up a study to characterise CD4 binding site-targeting VRC01-class bnAb precursor frequencies in 60 healthy Kenyan adults with varied levels of prior exposure to malaria. Naïve VRC01-class bnAb precursors were detected using the germline-targeting eOD-GT8 immunogen. The overall frequency of these precursors was similar to that reported in North American populations, with IGHV1-2 allelic composition rather than prior long-term exposure to malaria impacting the precursor frequency estimates. Our findings also revealed four new allelic variants of the IGHV1-2 gene that are not currently included in the immunogenetic reference database. Notably, a novel IGHV1-2 allele (IGHV1-2*06_S5416) demonstrated the ability to engage eOD-GT8, unlike the reference *06 allele. Overall, our results support the development of GT vaccines to induce VRC01-class bnAbs in this population and reiterate the need for participant genotyping in GT vaccine trials, particularly in African populations where genetic diversity is high yet understudied.

This target product profile (TPP) highlights the minimal and optimal characteristics for ex-vivo and in-vivo cell and gene therapy-based products aimed at achieving an HIV cure (ie, durable antiretroviral-free viral control). The need for an effective, safe, scalable, affordable, accessible, and acceptable cure for HIV infection remains a major global priority. The possibilities for cell and gene therapy-based products for an HIV cure are rapidly expanding. In a multi-stakeholder consensus process of clinical experts and civil society, including representatives from low-income and middle-income countries, participants generally agreed on the optimal targets, whereas consensus on the minimal targets was not reached on every parameter. There was less agreement on the minimal targets for ex-vivo than in-vivo therapies given the complexity of ex-vivo interventions. The TPP is planned to be updated at regular intervals. Building a TPP, such as this one, is an important process for stakeholder engagement and aligning ambitions for the development of products that are acceptable to both clinicians and civil society.

Curing HIV has become a scientific priority with the development of HIV cure-related research collaborations and increasing clinical efforts. However, for potential study communities the meaning of HIV cure-related research needs to be fully understood. We conducted qualitative research in rural KwaZulu-Natal, South Africa investigating the knowledge and understanding of HIV cure. We used deliberative approaches to facilitate in-depth discussions. Five deliberative group discussions were conducted in IsiZulu (the local language) with five participants in each group. Data were audio-recorded and translated verbatim and transcribed into English in anonymised format. Data were later analysed thematically with three main themes identified: knowledge of HIV cure, HIV cure terminology and HIV cure trials. Our findings showed that participants had a limited understanding of HIV cure-related research, a lack of trust regarding HIV cure science and participating in future cure trials. There were no local language terms used to describe HIV cure terminology, although several suggestions were shared in the discussions. Understanding the level of knowledge of rural populations regarding HIV cure-related research is essential for tailoring research and intervention strategies that meet their specific needs and circumstances. This can increase participation in the research and inform future HIV cure strategies.

Background: Genetic variation within HIV-1 subtype C (HIV-1C) long terminal repeat (LTR) transmitted/founder viruses influences transcription activation and clinical disease outcomes. The role of specific mutations such as thymine-to-adenine (T5A) mutation at position five of the Specificity protein 1 (Sp1) III motif (Sp1IIIT5A) remains underexplored. This study investigates the impact of Sp1IIIT5A on HIV-1C LTR transcription activity and Sp1 binding affinity. Methods: The Sp1IIIT5A mutant and consensus HIV-1C LTR sequences were cloned into the pGL3 Luciferase Basic reporter vector, sequenced, and transfected into SVG and Jurkat cell lines, independently. Transcription activity and Sp1 expression were assessed via luciferase assays and Western blot. Structural models of Sp1IIIT5A, consensus LTRs and Sp1 were generated, and docking scores calculated using HDOCK, HADDOCK, and pyDockDNA. Molecular dynamics simulations analyzed stability and interactions of Sp1IIIT5A LTR-Sp1 complexes. Results and Discussion: The Sp1III5A mutant significantly increased basal (SVG: p<0.0001; Jurkat: p=0.0052) and Tat-mediated (SVG and Jurkat: p<0.0001) HIV-1C LTR transcription activity in both cell lines, with stronger effects in SVG cells. Sp1 expression levels remained similar across cell lines (p=0.0814). Sp1III5A exhibited higher binding affinity (-332.7, -174.6, and -279.2 kcal/mol) than the canonical sequence (-311.4, -157.0, and -247.3 kcal/mol). Conclusion: The Sp1IIIT5A mutation significantly enhances HIV-1C LTR transcription activity and Sp1 binding affinity, indicating its potential tole in modulating HIV-1C transcription and pathogenesis. Further investigation is needed to elucidate its impact on HIV-1C latency.

Accurate timing estimates of when participants acquire HIV in HIV prevention trials are necessary for determining antibody levels at acquisition. The Antibody-Mediated Prevention (AMP) Studies showed that a passively administered broadly neutralizing antibody can prevent the acquisition of HIV from a neutralization-sensitive virus. We developed a pipeline for estimating the date of detectable HIV acquisition (DDA) in AMP Study participants using diagnostic and viral sequence data. Using a Bayesian strategy that combines three streams of data (REN [rev/vpu/env/Δnef] sequence, GP [gag/Δpol] sequence, and diagnostic) where their 95% credible intervals overlap based on pre-specified criteria and decision rules. We evaluated the performance of our AMP pipeline using PacBio viral sequence data from 41 participants across two prospective acute HIV acquisition cohort studies, FRESH and RV217, with twice-weekly sampling. These cohort studies enrolled young women in South Africa and men and women in Kenya and Thailand, respectively, with a high likelihood of HIV acquisition. In evaluating performance, "true DDA" was the center of bounds between last-negative and first-positive RNA diagnostic tests (median time 4 days, range 2-7 days); bias was the mean difference between estimated and true DDA. Using diagnostic data alone yielded timing estimates with a bias of 2.4 days and root mean square error (RMSE) of 7.9 days. These results were improved using sequence + diagnostic data (bias 1.5 days, RMSE 6.9 days), as well as by restricting sequence-based estimation to samples from ≤5 weeks post-DDA (bias 0.2 days, RMSE 7.8 days).IMPORTANCEIn HIV prevention trials, accurate timing estimates of when individual participants acquire HIV can be used to estimate antibody levels at the time of acquisition, which is useful for projecting antibody levels needed for prevention. The results we report here suggest that if sequence-based estimation of acquisition timing is used in future clinical trials of combination broadly neutralizing antibody (bnAb) regimens or multispecific bnAbs for HIV prevention, a sampling frequency of at least monthly is needed. Moreover, in the samples analyzed here, we observed less bias in sequence-based timing estimation for samples taken <5 weeks post-DDA. This observation is consistent with the timing of immune-driven selective pressures that may negatively impact the power to detect acquisition sieve effects.

Antiretroviral therapy (ART) can effectively control Human Immunodeficiency virus (HIV) replication, however lifelong treatment is required due to persistence of viral reservoirs which drive recrudescence once ART is stopped. This necessitates curative interventions which can achieve either eradication or durable remission off ART. Advances in technology have fostered development of multi-omic techniques encompassing Polymerase Chain Reaction (PCR) and sequencing, spatial transcriptomic and proteomic analysis, immunofluorescent imaging, and Artificial intelligence (AI)-driven multi-modal data analysis, to understand the HIV reservoir biology and mechanisms of persistence. These have informed the investigation of therapeutic interventions including broadly neutralizing antibodies, latency reversal, immune cell argumentation, antiviral compounds, and gene therapy. Between April 7–10 2025, experts in the field convened at the Keystone Symposia conference - “HIV Cure: Antiretroviral Therapy (ART)-Free Control of HIV Infection in Durban, South Africa, to discuss novel strategies for eradication and/or durable ART-free control of HIV.

After sporadic reports of post-treatment control of HIV in children who initiated combination anti-retroviral therapy (cART) early, we prospectively studied 284 very-early-cART-treated children from KwaZulu-Natal, South Africa, after vertical HIV transmission to assess control of viremia. Eighty-four percent of the children achieved aviremia on cART, but aviremia persisting to 36 or more months was observed in only 32%. We observed that male infants have lower baseline plasma viral loads (P = 0.01). Unexpectedly, a subset (n = 5) of males maintained aviremia despite unscheduled complete discontinuation of cART lasting 3-10 months (n = 4) or intermittent cART adherence during 17-month loss to follow-up (n = 1). We further observed, in vertically transmitted viruses, a negative correlation between type I interferon (IFN-I) resistance and viral replication capacity (VRC) (P < 0.0001) that was markedly stronger for males than for females (r = -0.51 versus r = -0.07 for IFN-alpha). Although viruses transmitted to male fetuses were more IFN-I sensitive and of higher VRC than those transmitted to females in the full cohort (P < 0.0001 and P = 0.0003, respectively), the viruses transmitted to the five males maintaining cART-free aviremia had significantly lower replication capacity (P < 0.0001). These data suggest that viremic control can occur in some infants with in utero-acquired HIV infection after early cART initiation and may be associated with innate immune sex differences.

Background:On January 24, 2025, the United States government issued an executive order to freeze all foreign aid programs, including The President's Emergency Plan for AIDS Relief (PEPFAR), for 90 days. A limited waiver option became available, but its implementation remains incomplete. We estimated the impact of these policy changes on HIV deaths and new infections in seven sub-Saharan African (SSA) countries-Ethiopia, Kenya, Malawi, South Africa, Tanzania, Zambia, and Zimbabwe -, which together account for about half of all people living with HIV in SSA. Methods:We used STDSIM, an established individual-based simulation model, and previously published quantifications for the seven countries. We predicted changes in HIV deaths and new infections over the period 2025-2030 for four scenarios: (1) Executive order-proportional, where treatment disruption was proportional to the country-specific PEPFAR's share of total HIV funding; (2) Executive order-realistic, assuming near-total system collapse due to program dependencies; and (3-4) Waiver scenarios where treatment was resumed after 4 or after 8 weeks. Resumptions of programs accounted for delays due to organizational and logistical challenges. Findings:A 90-day funding freeze would result in 60 thousand [95% UI: 49-71 thousand] excess HIV deaths for the Executive order-proportional scenario. This number would increase to 74 thousand excess HIV deaths [95% UI: 63-89 thousand] for the Executive order-realistic scenario. Under a 4-week and 8-week waiver scenario, projected excess HIV deaths ranged between 21 thousand [95% UI: 15-28 thousand] and 28 thousand [95% UI: 22-36 thousand] respectively. Excess new infections ranged between 35 and 103 thousand for the different scenarios. Interpretation:The sudden cessation of PEPFAR funding likely results in tens of thousands of HIV deaths and new infections. These losses of life and health should compel the United States government to rapidly and fully re-instate one of the most successful health programs in history. Funding:None.

BackgroundHIV remains a major challenge in KwaZulu-Natal, South Africa, particularly for young women who face disproportionate risks and barriers to prevention and treatment. Most HIV cure trials, however, occur in high-income countries.ObjectiveTo examine the perspectives of young women diagnosed with acute HIV in a longitudinal study, focusing on their perceptions on ATI-inclusive HIV cure trials and the barriers and facilitators to participation.Materials and MethodsBetween October 2022 and February 2024, we conducted closed-ended surveys and in-depth interviews with 20 women aged 19-33 living with HIV, who were willing but ineligible or unable to participate in an HIV cure trial.ResultsMany participants reported mental health challenges, including major depression (40%), moderate to severe anxiety (35%), and low self-esteem (35%). While women diagnosed during acute HIV supported pausing antiretroviral treatment (ART) during analytical treatment interruption (ATI) to advance HIV cure research, concerns about health risks and HIV-related stigma were significant barriers to enrollment. Trust in the research team and close monitoring were seen as positive factors, while fears around sharing of HIV/ATI status and transmission to sex partners complicated decision-making. Participants expressed a need for psychological counseling and access to community resources to manage ATI-related stressors.ConclusionsUnderstanding women's perspectives on HIV cure research, especially ATI trials, is vital. Building trust and addressing psychosocial challenges through a healing-centered approach can facilitate trial participation. Socio-behavioral research before and during HIV cure trials will be essential to inform participant-centered protocol design.

Uncertainty persists regarding the contribution of tissue macrophages to HIV reservoirs, largely due to insufficient characterization of these reservoirs within their native tissue microenvironments. This study aimed to characterize and quantify macrophage reservoirs in human lymph node (LN) tissues in terms of their phenotype, location, and their potential for sustained productive infection during suppressive antiretroviral therapy. We examined the topology, nature, and size of macrophage reservoirs in lymph nodes (LNs) from 45 PLWH subtype C on suppressive ART and 14 matched controls using in situ imaging and multiplexed immunofluorescence microscopy. Germinal center CD68+ macrophages harbored HIV gag-pol DNA, HIV gag-pol RNA and Gagp24 protein. Digital droplet PCR confirmed the presence of proviral reservoirs in myeloid cells within LNs. High-resolution imaging techniques revealed that infected macrophages within GCs displayed distinct morphological characteristics, featuring larger and irregular shapes. In contrast, phagocytic macrophages exhibited intracellular staining for CD4+ T cells, had regular shapes, and were predominantly found outside the GCs. Our findings provide detailed quantitative, spatial, and phenotypic characterization of macrophage reservoirs in LNs, offering a clear estimation of the extent to which macrophages contribute to persistent HIV reservoirs in these tissues. These findings establish a basis for developing targeted strategies aimed at the elimination of these reservoirs in LN tissues. ### Competing Interest Statement The authors have declared no competing interest.

Previously, a slot blot or an indirect enzyme-linked immunosorbent assay (ELISA) using a synthetic or purified MTP antigen, conceptually demonstrated IgG antibody induction in pulmonary TB patients, albeit with small sample sizes and differing sensitivity. Therefore, we evaluated an IgG MTP ELISA in larger populations from The Gambia (n = 549), Uganda (n = 161), and South Africa (n = 193), comprising human immunodeficiency virus (HIV) positive and negative, with microbiologically confirmed active TB. The association between the IgG level and demographic characteristics was determined by multivariate logistic regression. The sensitivity (44.8-61.2 %) and specificity (33.4-78.5 %) varied in the three cohorts. Anti-MTP antibody titres differed between the TB positive and negative groups within the South African and The Gambian cohorts (p < 0.001), but not in Uganda (p = 0.35). Antibodies were detected in HIV positive and negative patients and were reduced at 6-month follow-up after treatment (p > 0.067). The study verified previous findings that anti-MTP antibodies, and therefore MTP antigen, are produced during active TB. However, the accuracy of the MTP-IgG ELISA was low, and is therefore not suitable as a target product profile in the high burden TB areas investigated. Further studies are needed to clarify the variable reactivities in different geographical areas.

Sub-Saharan Africa accounts for almost 70 % of people living with HIV (PLWH) worldwide, with the greatest numbers centred in South Africa where 98 % of infections are caused by subtype C (HIV-1C). However, HIV-1 subtype B (HIV-1B), prevalent in Europe and North America, has been the focus of most cure research and testing despite making up only 12 % of HIV-1 infections globally. Development of latency models for non-subtype B viruses is a necessary step to address this disproportionate focus. Furthermore, the impact of genetic variation between viral subtypes, specifically within the long terminal repeat (LTR) element of the viral transcriptional promoter on latency reversal, remains unclear. To address this scientific gap, we constructed a minimal genome retroviral vector expressing HIV-1C consensus transactivator of transcription protein (Tat) and green fluorescent protein (GFP) under the control of either HIV-1C consensus LTR (C731CC) or the transmitted/founder (T/F) LTRs derived from PLWH (CT/F731CC), produced corresponding LTR pseudotyped viruses using a vesicular stomatitis virus (VSV-G) pseudotyped Envelope vector and the pCMV Delta R8.91 packaging vector containing HIV-1 accessory and rev genes. Viruses produced in this way were used to infect Jurkat E6 and primary CD4+ T cells in vitro. By enriching for latently infected cells, and treating them with different latency reversing agents, we developed an HIV-1C latency model that demonstrated that the HIV-1C consensus LTR has lower reactivation potential compared to its HIV-1B counterpart. Furthermore, HIV-1C T/F LTR pseudotyped proviral genetic variants exhibited a heterogenous reactivation response which was modulated by host cell (genetic) variation. Our data suggests that genetic variation both within and between HIV-1 subtypes influences latency reversal. Future studies should investigate the specific role of variation in host cellular environment on reactivation differences.

From the perspective of developing relevant interventions for treating HIV and controlling its spread, it is particularly important to comprehensively understand the underlying diversity of the virus, especially in countries where the virus has been present and evolving since the cross-species transmission event that triggered the global pandemic. Here, we generate and phylogenetically analyse sequences derived from the gag-protease (2010 bp; n = 115), partial integrase (345 bp; n = 36), and nef (719 bp; n = 321) genes of HIV-1 group M (HIV-1M) isolates sampled between 2000 and 2022 from two cosmopolitan cities and 40 remote villages of Cameroon. While 52.4% of all sequenced viruses belonged to circulating recombinant form (CRF) 02_AG (CRF02_AG), the remainder were highly diverse, collectively representing seven subtypes and sub-subtypes, eight CRFs, and 36 highly divergent lineages that fall outside the established HIV-1M classification. Additionally, in 77 samples for which at least two genes were typed, 31% of the studied viruses apparently had fragments from viruses belonging to different clades. Furthermore, we found that the distribution of HIV-1M populations is similar between different regions of Cameroon. In contrast, HIV-1M demographics in Cameroon differ significantly from those in its neighbouring countries in the Congo Basin (CB). In phylogenetic trees, viral sequences cluster according to the countries where they were sampled, suggesting that while there are minimal geographical or social barriers to viral dissemination throughout Cameroon, there is strongly impeded dispersal of HIV-1M lineages between Cameroon and other locations of the CB. This suggests that the apparent stability of highly diverse Cameroonian HIV-1M populations may be attributable to the extensive mixing of human populations within the country and the concomitant trans-national movements of major lineages with very similar degrees of fitness; coupled with the relatively infrequent inter-national transmission of these lineages from neighbouring countries in the CB.

Introduction HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C. Methods Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability. Results The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2–6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region. Conclusions We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related lactobacilli, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the vaginal microbiota and enhances bacterial fitness by biochemically sequestering OA in a derivative form only ohyA-harboring organisms can exploit. OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro BV model, suggesting a metabolite-based treatment approach.

Analytical treatment interruption (ATI) is widely acknowledged as an essential component of studies to advance our understanding of HIV cure, but discussion has largely been focused on adults. To address this gap, we reviewed evidence related to the safety and utility of ATI in paediatric populations. Three randomised ATI trials using CD4 T-cell and clinical criteria to guide restart of antiretroviral therapy (ART) have been conducted. These trials found low risks associated with ATI in children, including reassuring findings pertaining to neurocognitive outcomes. Similar to adults treated during acute infection, infants treated early in life have shifts in virological and immunological parameters that increase their likelihood of achieving ART-free viral control. Early ART limits the size and diversity of the viral reservoir and shapes effective innate and HIV-specific humoral and cellular responses. Several cases of durable ART-free viral control in early treated children have been reported. We recommend that, where appropriate for the study question and where adequate monitoring is available, ATI should be integrated into ART-free viral control research in children living with HIV. Paediatric participants have the greatest likelihood of benefiting and potentially the most years to prospectively realise those benefits. Excluding children from ATI trials limits the evidence base and delays access to interventions.