VRC01-class broadly neutralizing antibodies (bnAbs) are key targets for human immunodeficiency virus type 1 (HIV-1) vaccine design due to their specificity for the conserved CD4-binding site (CD4bs) on gp120. Germline-targeting immunogens, such as engineered outer domain germline targeting version 8 (eOD-GT8), have demonstrated potential in activating naïve B-cell precursors of VRC01-class bnAbs in people. eOD-GT8 is designed without a glycan on the perimeter of the CD4bs to enhance VRC01-class precursor activation. This glycan, located at position 276 on gp120, occurs in most HIV-1 strains and is accommodated by affinity-matured VRC01-class bnAbs. Consequently, booster immunogens will likely need to include this glycan to guide precursor B cells toward affinity maturation.

Here, we present initial insights from antibody responses in human-antibody transgenic mice induced by eOD-GT8 and its modified derivatives. Consistent with clinical trial findings, mice immunized twice with nanoparticle-displayed eOD-GT8 produced sera that modestly neutralized (~30%) a sentinel HIV-1 strain without the 276-glycan at the lowest serum dilution tested but failed to neutralize the same virus altered to contain the 276-glycan. In contrast, mice boosted with eOD-GT8 incorporating the 276-glycan produced antibodies that neutralized 276-glycan-bearing viruses (30-40% at 1:20 serum dilution). ELISA analyses showed that all mice develop antibodies to the nominal CD4bs as presented on eOD-GT8, with a more significant fraction of CD4bs-specific antibodies observed in those receiving modified eOD-GT8. Paired antibody sequence analyses from singly-sorted memory B cells indicate that eOD-GT8-immunized animals predominantly utilize VH3-23 (77%; n=34), associated with CDR H3-dominated CD4bs recognition. In contrast, mice boosted with modified eOD-GT8 exhibited a broader V-gene usage, including VH3-23 (52%), VH3-20 (20%) and VH1-24 (10-30%) (n=73). We are investigating the functional implications of these alternative V-gene-utilizing antibodies, particularly their potential ability to accommodate or circumvent the 276-glycan, to inform future immunogen design strategies to elicit VRC01-class bnAbs.

Introduction: Understanding the factors influencing HIV persistence is critical for developing curative strategies. We investigated sociodemographic, clinical, and immunogenetic correlates of HIV reservoir size and total proviral burden during antiretroviral therapy (ART).

Methods: We cross-sectionally studied 153 adults with HIV receiving ART. HIV reservoir size (genomically-intact proviruses/million CD4+ T cells) and total proviral burden (total HIV DNA copies/million CD4+ T-cells) were measured using the Intact Proviral DNA Assay, using custom primers/probes where required. HLA-I types were characterized by sequencing.

Results: Participants were 89% male with a median age of 54 (Q1-Q3:39-60) years. The median recent CD4+ T-cell count was 740 (Q1-Q3:565-935) cells/mm³, median nadir CD4+ T-cell count was 220 (Q1-Q3:110-413) cells/mm³, and median recent CD4/CD8 ratio was 0.89 (Q1-Q3:0.64-1.24). Participants had received triple-ART for a median 10 (Q1-Q3:5.5-19.4) years, and 66% were receiving integrase inhibitor-based regimens at sampling. Most (91%) had subtype-B HIV. The median reservoir size and total proviral burden were, respectively, 76 (Q1-Q3:26-176) and 922 (Q1-Q3:408-2102) HIV copies/million CD4+ T-cells. These measurements correlated strongly with one another (Spearman ρ=0.69, p<0.0001). The table lists factors significantly associated with intact reservoir size and/or total proviral burden in univariate analyses. Notably, the relationship between numerous variables and reservoir size/proviral burden differed based on HIV acquisition era, indicating that the latter exerted strong modifying effects.

Conclusions: Associations between low CD4-related measures and a larger reservoir emphasize the importance of early ART initiation. HLA associations, notably between the protective allele HLA-B*27:05 and a smaller reservoir, support host genetic effects on HIV persistence.

Background: In Sub-Saharan Africa, the most common form of injectable contraceptive is Depot MedroxyProgesterone Acetate (DMPA). There is controversy in the field as some studies have associated DMPA use with increased risk for HIV acquisition. Natural Killer (NK) cells are among the immune system's first responders and may have a protective role against HIV infection. NK cells represent 10% of peripheral blood mononuclear cells (PMBCs) and are classically divided into two subgroups based on their expression of CD56 and CD16. The CD56dimCD16bright NK cells are known for their cytolytic activity, while the CD56brightCD16dim NK cells play a crucial role in cytokine production. This study aims to assess the effects of DMPA on NK cell phenotypes.
Methods: Blood was collected from women from the Sex Worker Outreach Project (SWOP) in Nairobi, Kenya. The case group were participants using DMPA for at least two cycles. Controls included non-hormonal contraceptive users (non-HC), with samples collected during both the follicular and luteal phases. NK cell functions were evaluated by flow staining for CD107a (a marker of degranulation), NKG2D (an activation receptor), and IFN-γ following stimulation with either PMA/ionomycin or K562 cells.
Results: The cytokine-producing NK cells from DMPA users exhibited lower per cell IFN-gamma production following PMA/ionomycin stimulation than non-HC over the menstrual cycle (luteal p=0.011; follicular p=0.005). When stimulated with K562 cells, these NK cells from DMPA users showed increased degranulation compared to those from non-HC collected at the follicular phase of the menstrual cycle (p=0.007).
Conclusion: Our analysis shows that DMPA use correlates with NK cell functions. While the cytokine NK cells are traditionally regarded as only cytokine producers, our findings suggest this subset in women using DMPA has a heightened capacity for cytolytic function. Whether this phenotypic effect is protective, or a byproduct of increased HIV risk, remains to be determined.

Studying specific responses in blood and lymphoid organs is essential for vaccine development and diagnosis of infectious diseases. Following antigenic stimulation, the expression of molecules such as CD69, CD137/4-1BB and CD154/CD40L enables the detection of specific T lymphocytes (activation-induced markers/AIM assay) by flow cytometry. This assay has been applied in contexts like SARS-CoV-2 and HIV infection and vaccination.
HIV and its simian counterpart (SIV) induce immune dysfunction by triggering apoptosis in CD4 T lymphocytes, which are pivotal to adaptive immune responses. The infection also disrupts other components of the immune system, such as the production of interleukin-12 (IL-12), a cytokine that plays a role in preventing CD4 T cell apoptosis. Other cells including B and CD8 T are also altered, further emphasizing the impact of HIV/SIV on the development and maintenance of vaccine-induced responses.
Our objective was to optimize methods for monitoring vaccine responses in humans (PBMC) and in a macaque model infected with SIV (PBMC and peripheral lymph nodes). Using the AIM assay and an in vitro proliferation assay following antigen stimulation, we observed increased basal expression of activation markers, likely reflecting the chronic immune activation associated with SIV infection. Following stimulations, we also detected exacerbated cell apoptosis, suggesting activation-induced cell death (AICD). A combined AIM and Annexin V assay demonstrated that cells expressing activation markers are more prone to die, making their detection more complex. These findings underscore the complexities of studying antigen-specific responses in the context of chronic viral infections like HIV/SIV.
Therefore, optimizations to minimize T cell apoptosis are ongoing to improve the sensitivity of these analyses. The improved methods adapted to the macaque model will allow for more precise studies of vaccine immune responses, particularly in lymphoid organs that are difficult to access in human studies.

The use of dolutegravir (DTG) in modern antiretroviral regimens has significantly improved HIV-1 management. However, the virus has developed resistance mechanisms, observed especially among subtype D-infected individuals, exacerbating the global challenge of controlling HIV. Resistance to DTG has been linked to key integrase polymorphisms that reshape HIV-1 reservoirs, fostering the emergence of more dynamic and resilient viral populations. This study investigates how DTG-resistant mutations in subtype D integrase influence integration site preferences under therapeutic conditions. HIV-1 chimera viruses were constructed using the subtype B NL4-3 backbone incorporating subtype D integrase with key DTG-resistant integrase mutations, such as R263K or N155H. Control viruses without DTG-resistant integrase mutations were also generated for comparison. These viruses were used to infect cells cultured with or without DTG, and integration site libraries were prepared for Illumina sequencing and analyzed using the Barr Lab Integration Site Pipeline. Under both untreated and DTG-treated conditions, the HIV-1 chimeric viruses demonstrated preferential integration into specific genes, including MROH1, COLEC11 and SCN5A, which have been implicated in previous studies as potential contributors to HIV-1 reservoir formation. Notably, viruses carrying the N155H and R263K integrase mutations also showed significantly higher integration in long interspersed nucleotide elements (LINEs) and near short interspersed nucleotide elements (SINEs) compared to non-mutant controls. This preference was amplified under DTG treatment, potentially due to the presence of non-canonical B-form DNA structures, such as inverted repeats, at these integration sites. These findings reveal that DTG-resistant integrase mutations influence HIV-1 integration by targeting specific genes and regulated genomic features like SINEs and LINEs, particularly under therapeutic pressure. Further research into the biochemical mechanisms driving these patterns will inform the development of next-generation therapies to disrupt persistent HIV-1 reservoirs and help improve global HIV-1 treatment outcomes.

Introduction: People living with HIV (PLWH) receiving antiretroviral therapy (ART) can experience non-suppressible viremia (NSV), characterized by detectable plasma viral loads (pVL). Clinical guidelines state that NSV is caused by drug adherence/absorption issues or emergent HIV drug resistance, and recommend regimen modification. However, NSV can also be due to virus release from reservoir cells, which is not clinically actionable. We investigated the incidence and possible causes of NSV in a population-based cohort in British Columbia.

Methods: We retrospectively analyzed clinical histories and archived plasma from 2396 PLWH who donated their samples to research, and who were eligible to experience NSV on or after January 1, 2017. We defined an NSV episode as ≥2 consecutive pVL:50-1000 copies/mL ≥1 month apart that occurred after ≥4 months of ART-mediated suppression (pVL <50 copies/mL). NSV episodes ended upon pVL re-suppression (two consecutive pVL<50 copies/mL ≥4 months apart), or with virologic failure (pVL>1000 copies/mL), or the end of follow-up.

Results: Of 2396 participants, 245 (10.2%) experienced 289 NSV episodes, each lasting a median 232 (Q1-Q3:101-532) days. Overall, 75.8% of NSV episodes ended in re-suppression. We quantified antiretroviral levels during 125 NSV episodes from 184 plasma samples using a validated Mass Spectrometry assay: only 6.5% had low drug levels suggestive of incomplete adherence. HIV drug resistance genotyping during NSV for 88 participants revealed emergent drug resistance in only 8.0% (7/88) of cases. Of 43 NSV episodes with both drug levels and resistance data, 32 (74%) were not explainable by either low drug levels or emergent drug resistance.

Conclusions: NSV is relatively common, and typically lasts for >200 days. The NSV episodes described here are largely unexplained by emergent HIV drug resistance or adherence/absorption issues, suggesting viral reservoir release as the cause. Further research is needed to confirm these findings and inform updates to clinical guidelines.

Background and Objectives: Hepatitis C virus (HCV) infection is a serious comorbidity in people with HIV-1 (PWH). Antiretroviral therapy (ART) reduces HIV replication to undetectable levels but does not eliminate HIV reservoirs, which persist in memory CD4+ T-cells with diverse antigenic specificities. We investigated whether HCV-specific CD4+ T-cells are permissive to HIV and contribute to HIV persistence during ART, before/after spontaneous HCV clearance.

Methods: Memory CD4+ T-cells from chronically-infected HCV+ and HCV-negative controls (n=20/group) were infected with CCR5-tropic HIVNL4.3BaL in vitro. HIV integration and replication were measured by real-time PCR and HIV-p24 flow cytometry and ELISA, respectively. CFSE-based T-cell proliferation combined with intracellular HIV-p24 staining were used to examine the susceptibility of HCV-specific T-cells to HIVNL4.3BaL infection in HCV resolvers (n=8) and HCV chronic (n=2) participants. In one ART-treated PWH who naturally resolved HCV infection, Activation Induced Marker (AIM) assay was used to phenotype HCV-specific T-cells. Integrated HIV-DNA was measured in sorted HCV-specific T-cells. A monocyte-derived dendritic cell-based viral outgrowth assay (MDDC-VOA) was used to detect replication-competent HIV reservoirs.

Results: CD4+T-cells from chronically-infected HCV+ participants were more susceptible to HIVNL4.3BaL infection in vitro compared to HCV- controls, as demonstrated by intracellular (p=0.034) and soluble HIV-p24 quantification (p=0.030). HCV-specific T-cells proliferating in response to HCV-NS3 expressed CCR5 and supported productive HIV infection in vitro. In one ART-treated PWH acutely infected with HCV, the MDDC-VOA demonstrated that HCV-specific T-cells exhibit a follicular-helper phenotype and carry replication-competent HIV-DNA. Following spontaneous HCV clearance, the pool of HCV-specific T-cells decreased in frequency and shifted toward a Th17 phenotype, but remained highly enriched in integrated HIV-DNA.

Conclusion: Our results provide evidence that HCV-specific CD4+ T-cells are susceptible to HIV infection and may represent long-lived HIV reservoirs persisting during ART upon HCV spontaneous resolution. This raises awareness on the need to promptly treat HCV infection in PWH.

Introduction: Non-suppressible viremia (NSV) during ART can originate from clonally-expanded proviruses, but NSV's genetic composition, evolutionary dynamics and cellular sources remain incompletely understood. We phylogenetically characterized HIV diversity in four participants experiencing prolonged NSV.

Methods: Longitudinal single-genome partial pol sequencing was performed for 4-7 timepoints per participant, including pre-ART and during NSV. For two participants, near-full-length single-genome provirus and/or viral outgrowth sequencing from blood CD4+ T-cells was also performed.

Results: During NSV, which lasted a median 3.5 years, antiretrovirals were consistently detected in plasma and no emergent HIV drug resistance was observed. To date, 466 longitudinal plasma HIV sequences have been collected. Pre-ART HIV diversity was extensive in participants 1-3 who initiated ART in chronic infection, and minimal in participant 4 who started ART early. NSV sequences were all clonal. Participants 1 and 2 each experienced two NSV episodes, each featuring a different clone distinct from plasma sequences circulating immediately pre-ART. The phylogeny suggested the NSV sequences were archival. Viruses sampled longitudinally during participant 4's ~7-year NSV episode were clonal, and matched a plasma sequence present immediately pre-ART. Participant 3's ~5-year NSV episode featured at least three clones whose frequencies varied over time, where the dominant clone matched a plasma sequence isolated just after ART initiation. NSV sequences from participants 1, 2 and 4 had major splice donor site mutations that likely impair HIV replication. Despite >1000 near-full-length proviruses sequenced for participants 3 and 4, many of them clonal, we have not yet identified a blood provirus matching the NSV.

Conclusion: As reservoir-origin viremia is not clinically actionable, understanding NSV is paramount to avoid unnecessary ART regimen changes and anxiety. Results show that NSV commonly originates from clonally-expanded proviruses with 5' leader defects. In some cases, origin proviruses are either minority variants in blood or reside in other anatomical sites.