Antiretroviral therapy (ART) has helped HIV become a manageable chronic infection by repressing viremia to undetectable levels in People living with HIV (PLWH). However, this treatment is not curative, as treatment interruption causes viremia to rebound to pre-treatment levels. This rebound is driven by viral latency, a reversible, transcriptionally silent state of infection predominantly established in CD4⁺ T cells during acute infection. Since no viral proteins are produced, latently infected cells evade antiviral immune response and are not impacted by antiretroviral therapy. As a result, viral latency poses a significant barrier to achieving an HIV cure and requires a better understanding of latency maintenance to develop effective eradication measures.
The study of transcriptionally silent latently infected cells in vivo is highly challenging due to their rarity, with only about 1 in 105-107 CD4+ T cells being latently infected. Therefore, the development of techniques and tools that enable the identification, isolation and enrichment of these latent cells is crucial for advancing research in the field.
To address this, I generated a fluorescent protein-based HIV reporter system that allows for the characterization of the phenotypic and migratory characteristics of latent HIV-infected CD4+ T cells in vitro and in vivo. However, a key limitation of this model is the low frequency of latently infected cells generated, which prevents in-depth studies of these cells. To circumvent this limitation, I will be developing an in vitro culture system that supports the long-term maintenance and expansion of latent HIV-infected CD4+ T cells. This will enable us to do more in-depth studies to better understand the molecular and cellular mechanism that allows for the maintenance of latent infected CD4+ T cells in PLWH.

Background: HIV is associated with the secretion of plasma extracellular vesicles (EVs), including those containing viral protein Nef and cellular pro-inflammatory metalloproteinase ADAM17. Since EVs incorporate cell membrane proteins during their formation, characterizing EV surface markers may give insight into their cellular origins and inform our knowledge of latent HIV reservoirs.
Methods: Plasma of people with HIV (PWH) with undetectable viral loads on antiretroviral therapy (ART; n=20) and HIV-negative controls (n=20) was clarified using a 0.22 μm filter and EVs were isolated using the Total Exosome Kit (ThermoFisher). 37 surface markers on EV were profiled by flow cytometry using the MACSPlex EV kit (Miltenyi). Expression results were compared between groups (Mann-Whitney U test). Associations between selected EV markers and HIV reservoir size in PWH, as measured by the Intact Proviral DNA Assay in blood CD4+ T-cells, were explored using linear regression.
Results: EVs from PWH displayed significantly higher expression of CD29 (integrin beta 1) and CD62P (P-selectin) compared to controls (both p<0.0001). Smaller differences between groups were observed for CD3, CD41b, CD49e, CD69, CD81, and HLA-DR/DP/DQ (assessed in parallel by the MACSPlex assay). In linear regression models controlling for participant age, sex or time on ART, CD29 expression on EVs was significantly associated with intact HIV reservoir size (β=0.53-0.64; all p<0.02), while CD62P expression was not. In similar models, neither CD29 nor CD62P expression on EVs was associated with total proviral load.
Conclusions: CD29 expression is increased on plasma EVs from PWH and may be associated with intact HIV reservoir size. CD29 is a component of heterodimeric integrin receptors, and these data are consistent with the observation that VLA-4 (CD29/CD49d) is expressed on CD4 T cells harbouring translationally competent HIV proviruses. Ongoing studies are investigating the presence of Nef and other factors in CD29+ EVs.

Replication of human immunodeficiency virus (HIV) in the central nervous system (CNS) results in the release of proinflammatory factors and inflammasome induction. Activation of the inflammasome causes cleavage of gasdermin proteins, including gasdermin D (GSDMD) and gasdermin E (GSDME), which form transmembrane pores that facilitate cytokine release and pyroptotic cell death. Dimethyl fumarate (DMF) has potent anti-inflammatory effects and inhibits gasdermin oligomerization and pore formation. DMF may therefore be useful to combat the pathologic effects of HIV infection in the CNS. However, the role of gasdermin proteins on HIV infection in glial cells of the CNS and the effects of DMF on HIV replication in the CNS are currently unknown. We hypothesized that GSDMD and GSDME impair HIV replication, which will be enhanced by DMF treatment. We show that HIV replication and GSDMD expression are increased in the brain of individuals with neuroHIV, a subset of people with HIV that experience a greater burden of neuropsychiatric disorders. Infection of primary human microglia with HIV resulted in a profoundly altered cell proteome, including an increased abundance of inflammasome proteins, including caspase 1, GSDMD, and PYCARD. Despite apparent activation of the NLRP3 inflammasome, microglia do not undergo pyroptosis during HIV infection, which suggests an alternative role for gasdermins to control viral replication in microglia. Microglia treated with DMF had lower levels of HIV. However, in humanized mice infected with HIV, DMF treatment led to increased viral replication in the brain. Contrary to our hypothesis, DMF does not control HIV replication in vivo which may be due to the inhibitory effects of DMF on gasdermins. These findings enhance our understanding of how gasdermin proteins modulate HIV infection in the CNS and inform future therapeutic strategies.

Human immunodeficiency virus (HIV) infects cells of the central nervous system (CNS) establishing a viral reservoir that is difficult to treat and that may reseed peripheral immune cells when antiretroviral therapy is interrupted. Replication of HIV in the CNS occurs in microglia, where the virus can persist at low levels, and in astrocytes. Despite evidence for HIV infection in astrocytes in vivo, in vitro studies remain challenging due to the lack of necessary receptors for viral entry on astrocytes, except for CXCR4. It has been proposed that cell-to-cell contact between astrocytes and HIV-infected CD4+ T cells may facilitate transfer of the virus to astrocytes, but this remains to be determined using primary human cells and with non-permissive HIV isolates such as R5 strains. We hypothesized that infection of astrocytes depends on the tropism of the virus and the classical entry pathways could be circumvented through cell-to-cell contact with HIV-permissive cells, such as CD4+ T cells. Using immunofluorescence microscopy, we confirmed that HIV and SIV infect both microglia and astrocytes in vivo in humans and Rhesus macaques, respectively. In line with previous findings, we show robust infection of microglia with HIV BaL (an R5 isolate) that plateaus by day 8 post-infection. By contrast, astrocytes could not be infected with free HIV BaL. However, co-culturing astrocytes with HIV BaL-infected CD4+ T cells facilitated efficient viral transfer, with astrocyte infection levels correlating directly with the T cell-to-astrocyte ratio. Astrocytes could also be infected with HIV NL4-3 (an X4 isolate) and the viral particles produced from these cells could infect microglia. Therefore, the tropism of the virus can be overcome by cell-to-cell transfer from permissive cells that enter the CNS during infection. This aids our understanding of how HIV infects astrocytes and its contribution to the CNS viral reservoir, which will inform eradication strategies.

Background: Long-term non-progressors (LTNPs), representing approximately 1% of people with HIV (PWH), maintain undetectable viral loads without antiretroviral therapy (ART). Previous studies associate specific HLA alleles and immune receptor profiles with durable HIV control in LTNPs. Natural killer (NK) cells are early responders to HIV infection and regulate antiviral immunity through the balance of activating and inhibitory receptor signaling. Our objective is identifying NK cell populations associated with spontaneous HIV control.

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from LTNPs (viral load <50 copies/mL without ART; n = 12) through the Canadian Cohort of HIV Infected Slow Progressors, HIV viremic individuals (n = 16), ART-treated individuals (n = 22), and HIV-negative controls (n = 15). Cells were analyzed by 13-color flow cytometry. NK cells were defined as CD3⁻CD14⁻CD19⁻CD56dim/bright CD16⁻/dim/⁺ lymphocytes and assessed for expression of SLAM family activating receptors (SLAMF4, SLAMF6, SLAMF7) and inhibitory receptors (LAIR-1, TIGIT, PD-L1, CECAM).

Results: Median age was 50 (44-56), 29 (26-37), 50 (42-57), 51 (48-57) for LTNP, HIV-viremic individuals, ART-treated PWH and HIV-negative controls, respectively. Median (range) viral load for HIV viremic individuals was 59358 (831-10000000) copies/ml. Viremic PWH exhibited a significant reduction in a CD56dim NK cell subset lacking LAIR-1, TIGIT, and SLAMF4 compared with HIV-negative controls (p = 0.0133), ART-treated individuals (p = 0.0087), and LTNPs (p = 0.0285). This checkpoint-low NK cell subset was preserved in LTNPs and ART-treated individuals and did not differ from HIV-negative controls. LTNPs displayed increased inter-individual variability, with a subset showing elevated frequencies of this population.

Conclusions: Active HIV viremia is associated with loss of a checkpoint-low CD56dim NK cell subset, while preservation of this population characterizes LTNPs and ART-treated individuals. Maintenance of non-exhausted NK cell compartments may contribute to durable viral control and represents a potential target for HIV cure-directed immunotherapies.

HIV-1 remains a global health challenge, emphasizing the need to identify host genetic determinants that influence pathogenesis. We leveraged genotype and viral load data from 10,723 individuals of diverse genetic ancestries living with HIV-1 to perform a genome-wide association study (GWAS) of HIV-1 set-point viral load (spVL). We implicate the potential role of zinc finger proteins in pathogenesis through identification of a novel signal in the ZNF586 gene on chromosome 19, which is predicted to transcriptionally regulate RNA polymerase II. The top variant, (rs35962362-G) is intronic to the ZNF586 gene, and is associated with a 0.1 log increase in spVL per minor allele. Our subsequent transcriptome-wide association study (TWAS) along with expression quantitative trait loci (eQTL) data suggest that increased ZNF586 expression is associated with increased spVL in whole blood. Our findings implicate ZNF586 as a novel host factor for HIV-1 control and highlight the role of zinc finger proteins as potential contributors to viral persistence. This study represents the largest and most diverse GWAS of HIV spVL to date, integrating advanced statistical tools and data from multiple ancestries. This study also accounts for weaknesses in previous GWAS that have lacked power for discovery due to small sample size and lack of genetic diversity. By identifying genetic loci associated with spVL, the findings provide a foundation for understanding the genetic basis of HIV control and disease progression.

Human immunodeficiency virus type 1 (HIV-1) originated following cross-species transmission of simian immunodeficiency virus (SIV) infecting chimpanzees (SIVcpz). While SIV infection of its natural primate hosts is typically non-pathogenic, SIVcpz/HIV-1 is highly pathogenic. SIV/HIV pathogenesis is influenced by the viral accessory protein Nef, which manipulates several immune factors to facilitate disease progression. Specifically, Nef can modulate T cell activation by downregulating the T cell receptor subunit CD3ζ and the co-stimulatory receptor CD28. Several studies have demonstrated that while Nef downregulates CD28 from the T cell surface, this ability is not completely conserved throughout the SIV/HIV phylogeny. In comparison, Nef-mediated CD3 downregulation is highly conserved in SIV derived from lower-order primates yet completely absent from SIVcpz/HIV-1. Here, we used Nef proteins previously generated via an ancestral reconstruction pipeline, corresponding to common ancestors in the SIV/HIV phylogeny leading to HIV-1 group M, and evaluated their ability to downregulate CD3ζ and CD28 in both expression and infection systems. We observed that only the ancestral Nef of all primate lentiviruses efficiently downregulated both CD3ζ and CD28, while ancestors along the SIVcpz/HIV-1 lineage failed to downregulate CD3ζ and minimally recovered the ability to downregulate CD28 over time. Further, we showed that the loss of CD3ζ and CD28 downregulation was associated with increased expression of cell surface T cell activation markers and inflammatory cytokines, consistent with a pathogenic infection. Altogether, this study illustrates the evolutionary dynamics of Nef modulation of T cell activation and its impact on pathogenesis in the SIV/HIV lineage.

Introduction: Persistent detectable pVL during ART raises clinical concerns. We describe a case of prolonged (>6-year) detectable pVL attributable to a unique cause: the long-term presence − and subsequent RT-PCR-based amplification − of proviral DNA in plasma from a massively-expanded CD4+ T-cell clone harboring a defective provirus.

Methods: After ~10 years of pVL suppression, the participant experienced persistent detectable pVL (median 72; maximum 907 copies/mL) that was refractive to ART changes. Technical issues were ruled out. Antiretrovirals were consistently detected in plasma, and drug resistance genotypes performed three years apart returned genetically-identical, drug-susceptible protease/RT sequences, though integrase genotyping failed. We applied molecular and in vitro assays to plasma and cells collected before and during the detectable pVL, where plasma extracts were DNAse-treated to discriminate RNA from DNA.

Results: Following DNAse-treatment, we failed to amplify any HIV RNA despite testing >9mL plasma. In contrast, non-DNase-treated extracts readily amplified, though only from HIV's 5' half. Proviral quantification revealed an extraordinary >17,000 HIV copies/million CD4+ T-cells during the pVL-detectable period that was dominated by a single 3' defective population. This population was also abundant in plasma, yet represented <0.02% of blood proviruses pre-ART. Proviral sequencing from cells and plasma confirmed a massively-expanded clone with a deletion spanning HXB2 4891-9434, that exactly matched the clinical protease/RT genotypes (and explained why integrase genotyping had failed). The clone was integrated into exon 2 of NOTCH2NLC on chromosome 1. In vitro, the clone could produce trace amounts of p24, but no virions.

Conclusion: This case of persistently-detectable pVL featured no actual viremia, but rather prolonged proviral 'spillover' into plasma from a highly-expanded cell clone. Though the cause of clonal expansion remains unknown, the findings reassured participant and provider that ART was working as intended. Investigations of apparent nonsuppressible viremia should consider proviral DNAemia as a potential cause.

Introduction: While studying nonsuppressible viremia (NSV) during ART, we identified a participant with a blood reservoir exclusively comprising sequences from early infection, which is unusual because reservoirs are typically dominated by HIV sequences that circulated immediately pre-ART. We investigated using phylogenetic analyses.

Methods: The participant acquired HIV in 1988 and received nonsuppressive mono- and dual-therapy between 1993-1996. By the time triple-ART was initiated in 1997, all plasma viruses were AZT-resistant. ART was interrupted in 2000 and again in 2004; each time HIV rebounded rapidly. After ART re-initiation, the participant experienced two ~3.5-year-long NSV episodes. Using single-genome approaches, we characterized plasma HIV diversity at 10 timepoints spanning 1996-2025 (including all rebound and NSV episodes) and blood proviral diversity in 2025 (during the second NSV episode).

Results: The participant's phylogeny featured two distinct clades. The first was entirely AZT-resistant and contained the genetically-diverse plasma sequences circulating prior to triple-ART and the much-less-diverse sequences observed during the first rebound. All sequences from the second clade were entirely drug-susceptible, indicating they pre-dated monotherapy. This clade contained the genetically-restricted subset of sequences observed during the second rebound event, the distinct clonal sequences that emerged in the first and second NSV episodes, and all but one of the blood proviruses sampled in 2025 (the sole AZT-resistant provirus was defective due to a large internal deletion). The blood proviruses were highly clonal, with one (harboring a 5' leader defect) matching the second NSV viremia sequence exactly.

Conclusion: Clonally-expanded proviruses with 5' leader defects can cause NSV. The observation that the first rebound exclusively featured AZT-resistant viruses, yet archived, drug-susceptible sequences fueled the subsequent rebound and NSV events confirms that: 1) reservoir seeding begins in early infection; 2) reservoir composition can change dramatically during ART and 3) proviruses seeded early in infection can cause viral rebound and NSV.

Introduction: Female sex workers (FSWs) face a disproportionately high HIV risk, particularly in Sub-Saharan Africa, where HIV prevalence remains the highest. Alarmingly, some studies suggest that depot medroxyprogesterone acetate (DMPA), a widely used hormonal contraceptive in this region, may increase HIV acquisition risk. Our group has previously demonstrated that DMPA users have thinner cervical epithelial barriers and higher proportions of HIV target cells in the female genital tract. Although unprotected sex causes short-term vaginal inflammation, immune activation declines with prolonged engagement in sex work. HIV-exposed seronegative (HESN) FSWs in Nairobi, Kenya, exhibit a unique, less activated T-cell immune phenotype, termed immune quiescence. Interruptions in sex work have been associated with increased inflammation and elevated risk for seroconversion upon return. Here, we aim to use immunophenotyping of peripheral blood mononuclear cells (PBMCs) to characterize systemic T cell activation and functional responses in FSWs before, during, and after a one-month period of sexual abstinence.
Methods: This study will compare newly enrolled HIV-negative FSWs using DMPA or no hormonal contraception with HESN FSWs to assess how sex work interruption and DMPA use influence systemic immune responses. Multicolour flow cytometry panels will be optimized to characterize T-cell subsets and functional immune responses in PBMCs. We will identify Th17, Treg, and MAIT cells, as well as markers of immune activation and exhaustion. Functional responses will be assessed by recall-antigen stimulation with cytomegalovirus, Epstein–Barr virus, influenza, and human papillomavirus peptide pools. Memory and naïve T-cell populations, as well as cytokine production, will be assessed.
Significance: This approach will allow for the comprehensive examination of the effects of sex work interruption and DMPA use on systemic T-cell populations and HIV susceptibility in FSWs. These findings will advance the understanding of mechanism influencing HIV susceptibility and risk among key populations.

Effector memory (EM) and T follicular helper (TFH) CD4⁺ T cells represent key CD4⁺ T cell subsets, which are preferentially infected in visceral tissues despite early ART. Yet, their transcriptional profiles within the spleen remain incompletely characterized. Building on previous work from in SIV-infected rhesus macaques, we combined bulk and single-cell RNA sequencing to characterize splenic EM, and TFH in comparison to naïve CD4+ T cells. We compared their profile in healthy, SIV-infected, and SIV-infected under antiretroviral therapy (ART).
Bulk RNA sequencing was first used to define subset specific transcriptional signatures by comparing TFH and EM populations to naïve CD4⁺ T cells. These analyses identified core lineage-associated programs distinguishing EM and TFH CD4+ T cells. This approach also revealed that despite treatment, transcriptional profiles remain altered showing major reprograming related to immune activation, cytokine signaling and cellular metabolism. Single-cell transcriptomic profiling subsequently refined these profiles by revealing heterogeneity within the EM and TFH compartments, leading to the identification of at least three distinct subsets. In addition, RNA velocity analyses were performed to infer potential cellular trajectories and dynamic transcriptional changes, providing insights into putative state transitions within EM and TFH populations.
Together, this integrated bulk and single-cell transcriptomic framework provides a refined view of EM and TFH CD4⁺ T cell heterogeneity in the spleen across healthy, SIV infected, and ART treated SIV-infected RMs. Our findings establish a robust reference for CD4+ T cell subset-specific in the context of SIV infection.

Background

Persistent low-level HIV viremia (PLLV) during antiretroviral therapy (ART) can result from ART-related issues or drug resistance, which are clinically actionable, or viral release from clonally expanded proviruses, which is not clinically actionable. The latter is known as non-suppressible viremia (NSV). The impact of PLLV/NSV on systemic inflammation, a key driver of HIV comorbidities, remains unclear. We compared inflammatory markers during PLLV/NSV and periods of viral suppression (VS) to address this gap.

Methods

We studied 53 people living with HIV (PLWH) who experienced ≥1 episode of PLLV/NSV during ART, defined as ≥2 consecutive plasma viral loads (pVL) of 50–1,000 copies/mL at least one month apart following sustained suppression. Plasma levels of five pro-inflammatory cytokines (IFN-γ, TNF-α, IL-10, IL-6, and IL-8) were measured longitudinally: during pre-ART viremia (pVL >1,000 copies/mL); VS (pVL <50 copies/mL), and PLLV/NSV. HIV-negative controls (n=29; 76% male, median age 50 years) were included. Cytokine differences were evaluated using Friedman (paired) and Kruskal–Wallis (between-group) tests.

Results

The 53 PLWH were predominantly (77%) male, a median 56 years of age (Q1,Q3: 24,80), and 45% were receiving integrase-inhibitor-based ART at the time of PLLV/NSV. IFN-γ, TNF-α, and IL-10 levels were significantly higher during pre-ART viremia than during VS (all p<0.01), but did not differ between VS and PLLV/NSV. IL-6 and IL-8 levels did not vary across longitudinal time points. Compared with HIV-negative controls, all five cytokines were elevated in PLWH, even during VS on ART (all p<0.0001).

Conclusions

Consistent with previous reports, PLWH exhibited elevated pro-inflammatory cytokine levels compared to people without HIV, indicating that residual inflammation persists even during suppressive ART. Nevertheless, during PLLV/NSV, plasma cytokine levels did not differ significantly from those measured during VS, indicating that PLLV/NSV does not cause excess inflammation beyond that seen during periods of VS on ART.

*Equal contribution

Background

Understanding gut HIV reservoirs is critical to cure research, but sampling sufficient material for study is challenging in living donors. In a proof-of-concept study we applied the Intact Proviral DNA Assay (IPDA) and near full-length single-genome proviral sequencing to gut biopsy samples from 8 people living with HIV receiving suppressive ART.

Methods

For all participants, up to 60 biopsy samples were randomly collected during colonoscopy; for two participants up to 20 biopsies were also taken from the duodenum by upper endoscopy. Blood was collected by venipuncture. Tissue cell suspensions were generated by enzymatic digestion, and CD4+ T-cells were negatively selected from all samples using a kit designed for blood (STEMCELL technologies). Cellular purity was evaluated flow cytometrically post-hoc. Intact and defective proviruses were quantified by IPDA. For one participant, blood and gut proviruses were collected by near-full-length single-genome-sequencing.

Results

CD4+ T-cell purity was far higher in blood (~75%) than gut (~4%), where most recovered cells were epithelial. The IPDA failed in 3/8 participants due to HIV polymorphism. Though most proviruses detected by IPDA were defective, we observed intact proviruses in 4/5 colon samples (mean 36 copies/million cells; 23% of all proviruses) and 2/5 jejunum samples (mean 16 copies/million cells; 9% overall). Of note, intact proviruses comprised a slightly larger percentage of overall proviruses in colon than blood (mean 10% of all proviruses). All 18 proviruses isolated from gut by near-full-length sequencing were defective, mostly due to large deletions. No evidence of proviral genetic compartmentalization in blood and gut was observed.

Conclusions

Limited CD4+ T-cell recovery and HIV polymorphism-driven IPDA failures represent challenges when working with small tissue samples. Nevertheless, the observation of limited numbers yet relatively high percentages of intact proviruses in colon confirms the feasibility of these approaches and confirms gut as a major HIV reservoir.

Background
Tuberculosis (TB) screening is recommended for people living with HIV and other immunocompromising conditions, as well as during outbreak investigations. Diagnosis of latent TB infection (LTBI) remains challenging because affected individuals are asymptomatic. Interferon gamma release assays (IGRAs) are recommended for LTBI diagnosis, but they require prompt incubation of fresh blood with TB antigens, limiting their use in remote settings where timely sample transport is challenging. This proof-of-concept study evaluated the feasibility of stabilizing IGRA-stimulated blood on dried blood spot (DBS) cards for delayed shipment and analysis.

Methods
This study leveraged archived samples from a prior study in which QuantiFERON-TB IGRA tests were performed on blood collected from study participants with active TB (ATB, n=19), LTBI (n=21) or healthy controls (n=9). Frozen IGRA plasmas were thawed, mixed 1:1 with plasma-depleted whole blood from a healthy donor, and spotted onto Whatman 903 protein saver cards. Cards were dried for two hours and stored at 4°C with desiccant for up to two weeks. DBS punches were eluted overnight, and interferon gamma (IFNγ) levels were quantified using the QuantiFERON IFNγ ELISA. Plasma and DBS eluate TB-reactivity was scored then DBS results were compared to matched plasmas samples to assess assay performance.

Results
All 40 ATB/LTBI plasma samples were TB-reactive, and all 9 control samples were non-reactive. IFNγ was detectable in DBS eluates but at significantly lower concentrations than in plasma (p<0.0001). Using standard cutoffs, DBS sensitivity was 56.4% and specificity was 100%. A plasma-to-DBS IFNγ correction factor (mean ratio 6.32) was derived and applied, improving sensitivity to 97.5% while maintaining 100% specificity.

Conclusions and Impact
DBS-based stabilization of IGRA-stimulated blood is feasible and enables delayed sample transport without cold-chain requirements. This approach could substantially expand access to TB screening in remote and resource-limited settings.

Extracellular vesicles (EVs) are known for their ubiquitous presence in HIV-1 samples. Both EVs and HIV-1 particles are also known to exhibit similarities in size and surface protein compositions, making it challenging to discriminate them in lab assays. Distinguishing EVs from virus particles is important because cellular surface proteins present on both these particles are known to contribute to viral infection. However, an in-depth understanding of EV/virus specific contributions remains hindered due to limited tools available to distinguish them. Herein, we have expanded the applications of flow virometry (FV) to discern putative EVs and HIV-1 particles by developing dual staining approach for simultaneous targeting of surface antigens and internal virus capsid.

Infectious HIV-1 stocks were stained with PE-conjugated mAbs against a panel of shared surface proteins followed by intravirion/intravesicle staining for the HIV-1 capsid protein-Gagp24, and detection on the Beckman Coulter CytoFLEX (small particle cytometer). Staining of HIV-1 Gagp24, known to be minimally incorporated into EVs, was utilized as an EV/virus differentiating factor. Our data identified heterogeneity in antigen expression on putative EVs (Gagp24 negative) and viruses (Gagp24 positive), highlighting that a single surface antigen cannot serve as a reliable marker to differentiate EVs from viruses. Immunocapture assays targeting putative EVs and/or viruses were used to deplete different particles from virus stocks, and depletions were confirmed with this FV-based dual staining. Moreover, a loss of Gagp24 concentration and particle infectivity was observed largely in virus-depleted fractions, further supporting our discernment of putative EVs from viruses using this new dual staining approach.

This study provides an alternative approach to understand the breadth of particle heterogeneity in the EV and virus populations present within virus samples. These new approaches to discern EVs and viruses with FV assays can be used in future studies to characterize the contributions of EVs to HIV infection.

Background: The endocannabinoidome (eCBome) is a lipid-based signaling system which plays a key role in tissue homeostasis, metabolism and inflammation across various body systems, notably, in the liver. We recently reported an imbalance in plasma eCBome mediators in PWH characterized by an inverse relationship between anandamide and N-acyl-ethanolamines (NAEs) versus 2-AG and 2-monoacylglycerols (MAGs). In chronic liver diseases, the eCBome is reported to exert both pro- and anti-fibrogenic effects. However, the impact of HCV/HIV-coinfection and liver fibrosis on eCBome mediator levels remains understudied.
Methods: Plasma samples from ART-treated, HIV RNA suppressed HIV/HCV-coinfected participants including n=21 with, and n=51 without, liver fibrosis based on APRI score ≥1.5 and/or fibroscan median stiffness ≥7.2, were obtained longitudinally (pre- versus 6-12 months post-SVR) from the Canadian Coinfection Cohort. Levels of 23 eCBome mediators were measured by tandem mass spectrometry among two major families, the NAEs and MAGs, and the corresponding polyunsaturated fatty acid (PUFAs).
Results: In fibrotic versus non-fibrotic participants, we observed lower plasma levels of the NAE, LEA, both pre- and post-SVR, while the MAG, 2-SDG, and PUFA, arachidonic acid (AA), were lower only pre-SVR. SVR resulted in decreased levels of the NAE, DPA-EA(n-6), in both fibrotic and non-fibrotic groups, while levels of AEA, PEA, DHEA, the MAGs, 2-SDG, and the PUFA, DPA(n-6), were decreased in non-fibrotic participants. In fibrotic participants, pre-SVR levels of several NAEs, including AEA, LEA, and PEA, were negatively associated with median liver stiffness and BMI, while the PUFAs AA and DPA(n-6) were negatively associated with HCV viral load and BMI.
Conclusions: Among plasma eCBome mediators, HIV/HCV coinfection is associated with fibrosis-dependent (LEA, 2-SDG, and AA) and SVR-dependent (AEA, DHEA, PEA, DPA-EA(n-6), 2-SDG, and DPA(n-6)) alterations. These changes link metabolic regulation with liver disease severity, suggesting a potential interplay between eCBome and fibrosis pathogenesis in PWH with HCV coinfection.

The LTR region of HIV strains contains recognition sites for transcription factors such as Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB), Specificity Protein 1 (Sp1) and cAMP Response Element (CRE). Activating Transcription Factors (ATF) are members of the basic leucine zipper (bZIP) protein family. The Protein Kinase R (PKR), which activates the integrated stress response (ISR) by phosphorylating eIF2α (1), promotes translation of ATF4, capable to bind the CRE site, thereby linking viral sensing to host stress pathways (2). ATF5, which is homolog to ATF4, regulates mitochondrial stress and apoptosis, that remains poorly understood during lentiviral infections.
Thus, our objective is to characterize the role of ATF5 during HIV/SIV infection,.
Primary human monocytes were infected with HIVBaL to assess the expression of ATF family members by qPCR. We also analyzed the 5’LTR sequences of several HIV and SIV strains to identify conserved ATF motifs and potential stress-responsive elements. To assess transcriptional activity of ATF4 and ATF5, plasmids were developed to study LTR transcriptional activity including different fluorescent proteins.
We found that an early and transient expression of the ATF5 gene expression in monocytes infected with HIVBaL. Our results show the presence of an Amino Acid Response Element (AARE) site capable to bind ATF4 and ATF5, specifically in HIV-1 subtype B. These results provide a basis to explore the potential role of ATF5 , particularly in the context of HIV subtype B.
1) Pyburn J, J Virus Erad, 2025; 2) Corne A, Biology, 2024.

Human proteins incorporated into the HIV envelope can profoundly influence viral homing, attachment, and infectivity. Moreover, our lab has shown that this repertoire of surface proteins on viral particles is incredibly diverse, and that unique host protein profiles on viral particles could impact viral transmission and homing in vivo. However, it is extremely challenging to use conventional lab techniques to isolate phenotypically distinct virus subpopulations that remain functional for downstream experimental analyses. Therefore, the lack of high-throughput methodology capable of sorting viable HIV particles remains a critical barrier to understanding the impact of HIV subsets on transmission and disease.

Herein, we demonstrate high yield sorting of virus particles built on the principles of Flow Virometry (FV) our lab has developed. To evaluate the reliability and reproducibility of this novel strategy, we employed a biological reference particle to standardize measurements acquired by a range of commercially available flow cytometry analyzers from multiple manufacturers and compared data to a recently developed small particle sorter (Beckman Coulter, CytoFLEX-SRT). We optimized pre- and post- sort parameters to successfully purify HIV pseudoparticles and intact infectious virions. Importantly, we demonstrated that sorted particles remained viable for downstream analyses of viral proteins, viral genomes, and infectivity assays.

To our knowledge, this is the first study to achieve high-yield purification of functional HIV particles via standardized cytometry-based sorting methods. These novel virus sorting techniques will provide enhanced tools to study virus heterogeneity and the unique phenotypes of specific virus subpopulations. Future work will apply this tool to study virus subpopulations circulating in infected patients, discerning their contributions to disease progression and viral spread.

The critical role of the envelope (Env) protein of Human Immunodeficiency Virus-1 (HIV-1) in viral attachment and entry makes it a key target for vaccination. The low level of Env expression on the viral surface, as well as on infected cells, makes recognition by the host immune system difficult. Further, the heavily glycosylated nature of Env impairs induction of broadly neutralizing antibody (BnAb) responses by “shielding” interactions with cognate B cells. However, recent work has identified mutations in the Env protein which block N-linked surface glycosylation near conserved epitopes such as the CD4 binding site (CD4bs), promoting enhanced B cell interactions. Additionally, the Arts lab has discovered a unique N425K mutation which when present dramatically increases affinity for CD4. This study will use an Env-recombinant VSV vector based on the approved Zaire Ebolavirus vaccine. We have previously found that this vector provides modest immune protection in vaccinated mice and macaques. However, studies thus far have been limited to Env from only a single transmitted/founder (T/F) strain: A74, and there warrants inclusion of other subtype T/F viruses as well. Through optimization with the aforementioned mutations and T/F Env sequences, we hypothesize our VSV-HIV vector may preferentially select and expand B cells with anti-CD4bs activity. From this, we predict they will effectively neutralize most strains of wildtype HIV-1 and protect against novel infection. To address this, a two-fold method will be used: (1) a state-of-the-art human tonsil organoid model designed to test prime and boost immune responses to vaccines, and (2) human/murine CD4 transgenic mice. This project will identify which combination of mutations and which T/F Env is the “best” to elicit effective anti-HIV immunity and will justify future studies in primates to further develop the VSV-HIV vector and its potential use in a vaccine.

The Human Immunodeficiency Virus (HIV) infects CD4⁺ T lymphocytes primarily through the chemokine receptor namely CCR5 or CXCR4, whereas the Simian Immunodeficiency Virus (SIV) does not use CXCR4 for cell entry. The G protein–coupled receptor GPR15 has been shown to facilitate in SIV infection in certain cell lines; however, its role as a coreceptor in vivo remains to unclear. In this study, we sought to characterize the coreceptor expression on CD4⁺ T cells and to evaluate the contribution of these coreceptors to SIV infection.
Blood and lymphoid samples were collected from macaques infected with the SIVmac251 strain either treated or not with antiretroviral therapy. Phenotypic characterization of CD4⁺ T-cell subsets expressing these coreceptors was performed using multiparametric flow cytometry. In addition, CD4⁺ T cells were sorted based on coreceptor expression, and viral DNA was quantified by quantitative real-time PCR (qRT-PCR).
We found that distinct CD4⁺ T cell subsets express CCR5 and GPR15. Effector memory CD4⁺ T cells preferentially expressed CCR5, whereas central memory CD4⁺ T cells exhibited higher levels of GPR15 expression. During the acute phase of SIV infection, a transient depletion of CCR5- and GPR15-expressing CD4⁺ T cells was observed in macaques. Cell sorting experiments revealed the presence of HIV DNA within CCR5- and GPR15-expressing CD4⁺ T-cells indicating their susceptibility to infection.
Altogether, our findings highlight the contribution of GPR15 as a functional coreceptor for SIV infection in macaques. These results improve our understanding of alternative coreceptor usage that may play a role in viral dissemination in macaques.

Background: Sex chromosomes function as genetic mediators of immune activity, and sex hormones influence transcription among multiple immune cell types. Sex-specific effects on mucosal immune responses may have important implications for HIV transmission.

Methods: Rectal mucosal tissue explants from healthy individuals assigned male at birth (AMAB; n = 24) and assigned female at birth (AFAB; n=25) were challenged ex vivo with HIV-1 BaL and p24 production was quantified longitudinally post-infection (Days 3-14). Additionally, cytokine concentrations were quantified for a subset of participants (AFAB, n = 22 and AMAB, n = 17; IL-6, CCL2, G-CSF, IFN-a2, IL-2, IFN-g, IL-7, IL-1RA, IL-8, TNF-a, IP-10, MIP-1a, and IL-10). Values were normalized to biopsy weight and median Area Under Curve (AUC) was analyzed for comparison between AFAB and AMAB individuals.

Results: HIV-1 viral replication was higher in rectal tissues from AFAB compared to AMAB individuals (median AUC 1083 vs 553; p = 0.05). While pro-inflammatory cytokine concentrations were consistently higher in AFAB compared to AMAB, there were no statistically significant differences in median AUC cytokine concentrations based on sex. Multiple cytokines positively correlated with HIV-1 replication in rectal tissues, including IL-6 (r = 0.57, q = 0.0001), IL-8 (r = 0.54, q = 0.001), TNF-a (r = 0.56, q = 0.0002), IFN-g (r = 0.54, q = 0.0004), IL-1RA (r = 0.47, q = 0.002), IFN-a2 (r = 0.45, q = 0.004), MIP-1a (r = 0.45, q = 0.004), IL-2 (r = 0.44, q = 0.005), and IP-10 (r = 0.43, q = 0.007).

Discussion: This preliminary analysis demonstrates enhanced susceptibility to rectal HIV-1 transmission among AFAB compared to AMAB individuals. Heightened pro-inflammatory cytokine response may be an important contributing factor, yet further analyses of the transcriptome and microbiome are needed to better understand the immune environment and the role of tissue hormonal milieu.

Introduction: The most commonly used hormonal contraceptive by women in Africa is Depot medroxyprogesterone acetate (DMPA). Studies have shown its use may be associated with increased HIV acquisition. In 2024, of the ~1.3 million new HIV infections, 45% were in young women and girls, with 63% being from Sub-Saharan Africa. Therefore, we sought to get a better understanding of the impact of DMPA on the immune response among women using the product. An essential part of immune regulation is T cell exhaustion, however, this has not been assessed in women taking DMPA.
Method: A sub-study of the Longitudinal Assessment of Mucosal Immune Quiescence (LAM-IQ) study was conducted. Peripheral blood mononuclear cells (PBMC) were collected and cryopreserved from participants of the Sex Worker Outreach Program (SWOP) in Nairobi, Kenya who were either using DMPA or no hormonal contraception (HC). Those not using HC had samples taken during the follicular and luteal phases of their menstrual cycle. Flow cytometry was performed in Winnipeg, Canada to assess T cell phenotype; and exhaustion, activation and senescence were assessed in those T cell subsets.
Results: DMPA users had a higher blood proportion of LAG3 on multiple T cell subsets (CD4, CD8, MAIT, TH17, Tc17), and CD69 on Tc17 and T regulatory cells compared to women not taking HC. As well, the proportion of blood CCR5+iNKT cells, CCR5+MAIT cells and α4β7+CCR5+ CD4+ T cells was lower in women using DMPA compared to women not using HC.
Conclusions: This study shows that DMPA use is associated with a unique T cell phenotype with more exhausted T cells which may imply a reduced capacity to prevent the establishment of HIV infection. This could impact susceptibility to HIV for female sex workers. More studies are needed to understand the immunological impact of DMPA and their consequences on HIV risk.

Background: We previously identified Aryl Hydrocarbon Receptor (AhR) as a barrier to HIV-1 replication/outgrowth in CD4+ T-cells and revealed that the AhR pharmacological blockade modulated genes/pathways related to HIV-1 interactors and tissue migration/residency. Among differentially expressed genes, we demonstrated that Hypermethylated in Cancer 1 (HIC1), a repressor of Tat-mediated HIV-1 transcription and master regulator of tissue migration/residency, is a direct AhR target. Here, we investigated the capacity of AhR to act on HIV reservoirs and promote viral latency and tissue residency via HIC1-dependent mechanisms.
Methods: Memory CD4+ T-cells enriched from people with HIV-1 (PWH) receiving antiretroviral therapy (ART) and HIV-negative controls using magnetic beads were stimulated with CD3/CD28 antibodies and exposed to AhR agonist, FICZ and antagonist, CH223191. The expression of AhR and HIC1 was measured by real-time RT-PCR. HIC1 silencing was performed using the Dharmacon siRNA technology. HIV replication was monitored by HIV-p24 ELISA, flow cytometry intracellular HIV-p24 staining and HIV-DNA PCR upon in vitro infection with single-round VSV-G-pseudotyped HIV (HIVVSV-G) and viral outgrowth assay. Phenotypic analysis was performed by flow cytometry.
Results: AhR and HIC1 mRNA were overexpressed in CD3/CD28-activated CD4+ T cells of ART-treated PWH versus HIV-negative individuals, with FICZ and CH223191 upregulating and downregulating HIC1 mRNA expression, respectively. The CH223191-mediated AhR blockade upon HIVVSV-G integration increased intracellular HIV-p24 expression. HIC1 siRNA silencing increased HIVVSV-G replication in vitro and boosted viral outgrowth in CD4+ T-cells of ART-treated PWH, in the FICZ presence. The CH223191-mediated HIC1 downregulation led to a significant increase in markers associated with cell migration (CCR3, CCR6, CXCR5), while FICZ-mediated HIC1 upregulation increased gut-homing (ITGB7) and tissue residency (CXCR6) markers. Finally, CH223191 facilitated HIV outgrowth in CD4 + T-cells of ART-treated PWH.
Conclusions: Our findings point to AhR as transcriptional regulator of HIV latency and gut homing/residency of CD4+ T-cells in part via HIC1-dependent mechanisms.

Introduction: Depot medroxyprogesterone acetate (DMPA), an injectable progestin contraceptive, binds non-specifically to the glucocorticoid receptor (GR) with higher affinity than natural progesterone. Cortisol has anti-inflammatory properties and binds to GR. Our team has shown that DMPA use leads to higher cortisol levels compared to women not using hormonal contraception. This chronic exposure to cortisol could contribute to immune suppression, which has been linked to increased HIV susceptibility. This study examines how DMPA affects the T cell response to cortisol.
Methods: Peripheral blood mononuclear cells (PBMCs) were collected from female sex workers in Nairobi, Kenya. PBMCs from DMPA users (n=21) and FSWs not using hormonal contraceptives sampled during both the follicular (n=20) and luteal (n=20) phases, were stimulated with phytohemagglutinin-L (PHA-L) and treated with a serial dilution of hydrocortisone (10⁻⁵ to 10⁻¹⁰ M) for 48 hours to detect CD4 and CD8 cytokine production (IFN-γ, TNF-α, IL-2) by flow cytometry. Cortisol sensitivity was defined as the percent reduction in cytokine production after stimulation with varying hydrocortisone concentrations, relative to PHA alone.
Results: Following PHA-L stimulation, DMPA users showed lower expression of IFN-γ in both CD4+ (p=0.01) and CD8+ cells (p=0.013) compared with the luteal group. This trend was detected in activated T cell subsets: CD4+CD38+ (p=0.03), CD4+HLA-DR+ (p=0.02), CD8+CD38+ (p=0.01), and CD8+HLA-DR+ (p=0.02) cells. Cortisol sensitivity curves showed a significant interaction between group and hydrocortisone dose, with TNF-α inhibition in activated T cells varying by group across concentrations. Qualitatively, DMPA users showed greater cortisol responsiveness, with steeper inhibition slopes, while luteal-phase controls showed lower responsiveness and shallower slopes, particularly at hydrocortisone concentrations between 10⁻⁷ and 10⁻⁵ M.
Conclusion: This study indicated that DMPA may increase T cell responsiveness to glucocorticoids, potentially rendering cells more susceptible to HIV infection.

Background: The human immunodeficiency virus (HIV) reservoir persists even after antiretroviral therapy (ART) is initiated, which can contribute to chronic inflammation observed in people living with HIV (PLHIV). Monitoring the size of the HIV reservoir is of importance as reservoir eradication strategies become used as therapies in clinical settings. Typically, reservoir quantification studies focus on males. Over half of people living with HIV globally are females and there are known sex differences in HIV infection and disease progression, but there are still many knowledge gaps when it comes to the HIV reservoir in females. This study focuses on quantifying the HIV reservoir in female sex workers from Nairobi, Kenya prior to and post-ART initiation.

Methods: Using a nested quantitative polymerase chain reaction (qPCR) assay, the frequency of peripheral blood mononuclear cells (PBMC) containing total and integrated HIV DNA was determined. Standard curves of 3 to 300,000 copies of either HIV DNA or CD3 DNA were created using ACH-2 cells, which contain a single copy of integrated HIV DNA. Genomic DNA was isolated from PBMC at a concentration of 10⁶ cells/ml. In a pre-amplification polymerase chain reaction (PCR) assay, CD3 DNA and HIV DNA were co-amplified. The product was then added to two qPCR assays, one to quantify CD3 DNA and one to quantify HIV DNA. The standard curves were run alongside all samples.

Results: This assay has been optimized, with all standard curves having reaction efficiencies between 80 to 107%. All HIV positive samples tested for optimization fall onto the standard curves, indicating that the assay is suitable for quantifying the HIV reservoir in PBMC.

Conclusions: This assay can quantify the HIV reservoir in PBMC and will be applied to samples for this study. Testing and analysis of samples are ongoing. Preliminary results will be presented.

Despite antiretroviral therapy, HIV-1 persists in the brain and leading to ongoing neuroinflammation and neurological disease in persons with HIV (PWH). While viral proteins trigger neuronal injury, the specific molecular pathways governing inflammatory cell death in the central nervous system remain incompletely understood. This study investigates the role of Gasdermin B (GSDMB), a mediator of pyroptosis, in the pathogenesis of HIV-associated neurological disease.
Our findings reveal that GSDMB transcripts and proteins, specifically the cleaved GSDMB fragment, are highly upregulated in the brains of PWH with neurological disease. Immunofluorescence analysis localized this increased expression primarily to astrocytes, and to a lesser extent, microglia, within regions of active neuroinflammation. In vitro experiments using HIV-infected human astrocytes demonstrated proteolytic cleavage of GSDMB, resulting in the release of lactate dehydrogenase (LDH), a hallmark of plasma membrane rupture (PMR). Further mechanistic assays identified that the N-GSDMB domain containing exon-6 is the primary driver of PMR and concomitant mitochondrial damage, evidenced by the loss of membrane potential.
Interestingly, HIV-infected microglia exhibited minimal GSDMB expression and did not undergo PMR or LDH release, despite active viral replication.
HIV-1 infection induced chemokine release from both astrocytes and microglia compared to mock-infected cells, while Type III interferons and pro-inflammatory cytokines were released by infected astrocytes and microglia, respectively, with concomitant neurotoxicity caused by microglia-derived conditioned media. Proteomic analyses of HIV-infected astrocytes revealed downregulation of cell adhesion and extracellular matrix proteins. These findings were further validated in SIV-infected nonhuman primates, where GSDMB and cleaved GSDMB immunoreactivity were prominent in animals with encephalitis.
In conclusion, GSDMB serves as a cell-specific mediator of pyroptosis in HIV-infected astrocytes. The differential expression of GSDMB across neural cell types modulates their response to infection, identifying GSDMB-mediated astrocyte death as a critical contributor to the neuroinflammatory environment underlying HIV-associated neurological disease.

Background
Women represent a majority of global HIV-1 infections, contracted primarily through heterosexual transmission across the female reproductive tract (FRT). Although semen contains both R5 and X4 HIV-1 strains, initial infection in the FRT typically occurs through the R5 strain. We previously showed that genital epithelial cells (GECs) mount a strong antiviral response to X4 HIV-1, leading to its sequestration within the cells, whereas R5 HIV-1 crosses the epithelial barrier without triggering significant antiviral immunity. Since sex hormones regulate immune activity in the FRT and estradiol is known to enhance antiviral protection, this study examined how estradiol and progesterone influence GEC interactions with HIV-1.
Methods
Endometrial tissues were collected from women undergoing hysterectomy after informed consent. GECs were cultured either without hormones or with estradiol (10⁻⁹ M) or progesterone (10⁻⁷ M). HIV-1 translocation across GEC was assessed using TZMbl reporter cells or p24 ELISA in basolateral supernatants. Intracellular viral trafficking was visualized by immunofluorescence microscopy.
Results
In hormone-free conditions, GECs activated the TLR2 interferon signaling pathway in response to X4 but not R5 HIV-1. Estradiol exposure, however, induced comparable antiviral immunity to both strains. Estradiol-mediated TLR2 activation increased expression of antiviral factors BST-2 and ISG-15, promoting sequestration of both X4 and R5 HIV-1 in endosomal compartments and limiting viral passage. In contrast, progesterone suppressed interferon signaling in response to both strains, resulting in increased viral translocation. The use of hormone antagonists eliminated these effects, confirming that estradiol and progesterone directly modulate HIV-1 trafficking through GECs.
Conclusion
Without hormonal influence, only X4 HIV-1 elicits a strong antiviral response in GECs. Estradiol activated anti-viral immunity to both X4 and R5 strains, trapping them intracellularly. Progesterone diminishes antiviral immunity and enhances viral trafficking across the epithelium. These findings highlight the critical role of sex hormones in HIV-1 transmission and may inform prevention strategies.

Background
The vaginal microbiome (VMB) is a critical determinant of mucosal health and susceptibility to sexually transmitted infections (STIs). A eubiotic VMB, dominated by Lactobacillus crispatus (LC), is linked to reduced inflammation, enhanced barrier function, and protection against viral pathogens. In contrast, dysbiotic VMB enriched in anaerobes such as Gardnerella vaginalis (GV) is associated with biofilm formation, pro-inflammatory signaling, and increased STI risk. Extracellular DNA (eDNA), a major biofilm component, could influence epithelial immune responses, but its immunomodulatory role has not been examined. We report that eDNA from eubiotic and dysbiotic bacteria exerts opposing effects on vaginal epithelial immunity.
Methods
Vaginal epithelial cells (VK2) were treated with eDNA extracted from LC and GV bacterial cultures and RNA was extracted to analyze gene expression in VK2 cells. We also examined whether soluble factors and cytokines released by VK2 cells in response to bacterial eDNA could affect HIV-1 replication in target immune cells. To assess this, we used the indicator 1G5 Jurkat T cell line, which carries an integrated HIV-1 LTR-luciferase reporter as proxy for viral transcriptional activation.
Results
We found that L. crispatus eDNA suppressed pro-inflammatory cytokines, enhanced anti-inflammatory and antiviral responses in VK2 cells, while G. vaginalis eDNA triggered robust pro-inflammatory signaling. Both DNase digestion of eDNA and TLR-9 inhibition in VK2 cells attenuated these responses, confirming DNA-dependent signaling and establishing TLR-9 as a key sensor of bacterial eDNA. Furthermore, epithelial supernatants from L. crispatus eDNA-treated cells reduced HIV-1 long terminal repeat (LTR) activation in Jurkat T-cell reporter assays, suggesting that eubiotic eDNA can modulate epithelial signaling to limit HIV-1 replication.
Conclusion
These results show a previously unrecognized role for bacterial eDNA, as a differential regulator of inflammation and antiviral immunity in the vaginal tract, highlighting eubiotic eDNA as a novel therapeutic avenue for counteracting dysbiosis and enhancing mucosal protection.

Background: In a Canadian multi-centre prospective, observational cohort of people with HIV (PWH) receiving >2 COVID-19 vaccinations, we examined impact of COVID-19 vaccination on immunogenicity and durability. In this sub-study, we examined impact of repeated COVID-19 vaccination on HIV reservoir dynamics and inflammatory mediators relevant to immune activation, B cell function and intestinal integrity.

Methods: Data was collected between August 2021-April 2022. Participants had to have specimens available >2 time points to facilitate within-person change comparisons. Time points included V1-Screening/Baseline (before dose #1); V4 - 6 months after 2nd dose, and VB-1: 4 weeks after 3rd dose. Participants also had to remain naïve to natural COVID-19 infection at each time point. Total HIV DNA and cell-associated HIV RNA(LTR-gag) were quantified using real time PCR/qPCR. Plasma levels of A Proliferation-Inducing Ligand(APRIL), Soluble Tumor Necrosis Factor Receptor II(STNF sTNF-RII), and Regenerating islet-derived protein-α(REG3A) were assessed via ELISA to evaluate immune activation and microbial translocation.

Results: Fourteen participants (71% male) were included. Mean age was 53.4±15.1 years; half were >55 years of age. Average CD4 count was 608.4±251.3 cells/μL, and 35.7% had CD4 counts <500 cells/μL. The most common comorbidities were obesity(36%), hypertension(29%), and dyslipidemia(21%). All had suppressed viral load on ART. HIV DNA levels remained stable over time, and no consistent trend in HIV RNA transcriptional activity was observed post-vaccination. Soluble mediators did not significantly change between baseline and follow-up visits. Subgroup analysis suggested that individuals with CD4<500 cells/mm³ had mild increases in APRIL and REG3α, while those with fewer comorbidities showed more dynamic patterns of immune and inflammatory mediators.

Conclusion: Repeated COVID-19 vaccination did not significantly alter HIV reservoir size, transcriptional activity of HIV, or soluble marker levels in PLWH at the time points examined. Our results provide additional safety reassurance with regards to repeated vaccination in this population.

Introduction: Cervical cancer, which is caused by high-risk human papillomaviruses (hr-HPV), remains one of the leading causes of cancer-related mortality in women worldwide, with its impact being most severe in low-resource settings. While vaccination has significantly reduced the burden of cancers linked to HPV, uptake is still lower than needed to end cervical cancer. Therefore, understanding the immune mechanisms underlying the natural clearance of hr-HPV is critical for developing therapeutic interventions. Our study aims to investigate the role of innate immune cells, specifically natural killer (NK) cells, monocytes, and dendritic cells (DCs), in the natural clearance of hr-HPV among Female sex workers (FSWs) in Nairobi, Kenya.

Methods: We hypothesize that hr-HPV clearance is associated with increased NK activation and enhanced NK function, as well as increased activation of monocytes and DCs. Using a retrospective case-control study design, our study will analyze cryopreserved peripheral blood mononuclear cells (PBMCs) from the SWOP HPV cohort, comprising both HIV-positive and HIV-negative FSWs. Flow cytometry will assess activation, inhibition, and exhaustion markers in these innate immune cells at baseline (hr-HPV detection) and at clearance or 12-month follow-up (in the absence of clearance). A functional assay using target cell lines (K562 and 721.221) will be used to measure NK degranulation (CD107a) and cytokine production (IFN-γ, TNF-α).

Current progress: Optimization of flow cytometry panels is underway, including antibody titration and voltage optimization for the detection of markers of interest. Target cell lines are being cultured for the NK functional assay. Once optimized, participant samples will be analyzed to compare immune profiles between those who cleared hr-HPV and those with persistent infection.

Conclusion: Identifying immune correlates of protection could ultimately reduce disparities in cervical cancer outcomes globally by improving our understanding of the immune mechanisms driving natural clearance in high-risk groups.

Background: Cervical cancer rates are 2–6 times higher among people living with HIV (PLWH) than in HIV uninfected individuals, due to reduced control of high-risk Human Papilloma Virus (HPV). While most HPV infections clear spontaneously, the immune mechanisms driving this control remain unclear. Studying HPV-specific T-cell responses in Kenyan female sex workers (FSWs) may inform therapeutic HPV vaccine development and strengthen cervical cancer prevention strategies, particularly for PLWH.

Methods: A longitudinal cohort of 702 FSWs (18% PLWH) from the Sex Worker Outreach Program (SWOP) HPV cohort in Nairobi, Kenya, was followed bi-monthly for 12 months. HPV-specific cellular immunity will be assessed by flow cytometry, measuring T-cell activation (CD69) and function (IFN-γ) after stimulation of PBMCs with overlapping 15-mer peptide pools (HPV16/18 E6 and E7) at varying concentrations. To establish this assay using control samples, the in-house HPV16 E6 peptide pool was compared with a commercial pool (Miltenyi, 130-095-997). HPV-specific humoral immunity was evaluated using a multiplex bead array against L1 antigens of HPV types 6/11/16 and 18 among FSWs from the SWOP HPV cohort and healthy controls from Winnipeg, Canada.

Results: Optimization of the flow cytometry panel enabled detection of HPV-specific CD4⁺ and CD8⁺ T-cell activation and function, with antigen-induced CD69 and IFN-γ responses observed across peptide concentrations. The in-house HPV16 E6 peptides pool showed comparable stimulation to a commercial HPV16 E6 peptide pool. Multiplex bead array analysis detected HPV6/11/16/18 L1-specific IgG responses and distinguished antibody levels among SWOP HPV participants and healthy controls.

Conclusion: In this optimization study, the pooled HPV peptides elicited measurable HPV 16/18E6/E7-specific CD4 and CD8 T-cell activation and functional responses. The multiplex bead array detected HPV 6/11/16 and 18 L1-specific antibodies among participants. These assays provide a platform for comparing humoral and cellular correlates of HPV control versus persistence among FSW living with HIV.

Background: Despite effective viral suppression with antiretroviral therapy (ART), persistent central nervous system (CNS) inflammation remains a major concern in people living with HIV (PLWH). Induced inflammatory signalling at the blood–brain barrier (BBB) is known to compromise barrier integrity, promote immune cell infiltration, and contribute to cognitive dysfunction. Tenofovir alafenamide (TAF) or tenofovir disoproxil fumarate (TDF), with emtricitabine (FTC), are common NRTI backbones in first-line HIV regimens. Emerging evidence suggests that NRTIs exert anti-inflammatory effects independent of antiviral activity, supporting investigation into whether NRTIs attenuate inflammatory responses at the BBB.
Methods: Immortalized human brain endothelial cells (hCMEC/D3), an in vitro BBB model, were pre-treated for 6 hours with NRTI backbone combinations (TAF or TDF±FTC) at therapeutic and supratherapeutic concentrations (TAF: 600–2400; TDF: 200–1800 ng/mL), followed by 18 hours of co-treatment with the ER stress inducer Tunicamycin (Tuni, 1.5 µg/mL). Gene expression of inflammatory cytokines (IL6, IL23A), chemokines (CXCL2, CXCL8) and ER stress markers (HSPA5, DDIT3, ATF4, PPP1R15A) was quantified using qPCR.
Results: Tunicamycin significantly upregulated inflammatory and ER stress markers compared to vehicle controls, an effect attenuated by both NRTI backbones. In Tuni-treated cells, TAF+FTC treatment demonstrated dose-dependent reduction of key pro-inflammatory cytokine/chemokine gene expression, including IL6 (18-38%), CXCL2 (2-14%), CXCL8 (31-43%), and IL23A (18-41%) as well as ER-stress markers, including HSPA5 (14-28%), DDIT3 (18-24%). Similarly, TDF (200 ng/mL) +FTC suppressed gene expression of both inflammation (IL6, 20%; CXCL2, 21%; CXCL8, 25%; IL23A, 18%) and ER-stress (HSPA5, 21%; DDIT3, 16%) markers.
Conclusion: Our findings suggest that TAF or TDF+FTC-based backbones partially mitigate inflammatory and ER stress signalling in vitro. While these data suggest a potential role for NRTI regimens in modulating CNS inflammation, further work in relevant in vivo models is needed and will be the focus of future studies.

Objectives:
Human immunodeficiency virus (HIV) polymerase chain reaction (PCR) testing in British Columbia (BC) was previously centralized at the public health laboratory, BC Centre for Disease Control (BCCDC). In cases of unknown birth-parent HIV status at delivery, infants are often started on prophylactic antiretrovirals (ARVs) awaiting test results. We aimed to implement rapid HIV PCR testing at BC Women’s Hospital/Children’s Hospital (BCWH/BCCH) to reduce test turn-around time (TAT) and unnecessary infant ARV exposure.
Methods:
GeneXpert Qualitative HIV-1 PCR testing was implemented at BCWH/BCCH in June 2025. Parallel HIV PCR testing at BCCDC was done on samples. Time to result from sample collection and sample receipt by lab were recorded. Use of ARVs for prevention of perinatal HIV transmission was monitored. Post-implementation data was compared to historical provincial data from 2018-2023.
Results:
Thirty rapid HIV PCR tests were run on 23 unique patients from June 1 to October 3, 2025. Mean TAT from time of collection was 5.0 hours and from receipt by laboratory was 3.5 hours. Four (13.3%) tests had TAT of >6 hours from receipt by lab due to being run the following morning. The mean TAT from laboratory receipt for 19 parallel samples tested at BCCDC was 57.1 hours (range 24.5 to 118.7 hours (p <0.0001). Use of infant ARVs decreased from an average of 1.2 cases/month to 0.5 cases per month (Mann–Whitney U test: p = 0.231). Four infants during this period were started on ARVs for unknown HIV status while awaiting test results and received a mean of 1 day therapy, compared to a mean of 7.49 days therapy from 2018-2023 data.
Conclusions:
Implementation of rapid HIV PCR testing at BCWH/BCCH significantly decreased test TAT and reduced infant exposure to prophylactic ARVs in the setting of unknown birth parent HIV status at delivery.

Background: HIV drug resistance genotyping cannot be performed when viral loads are undetectable. Proviral DNA genotyping offers an alternative, but may not fully capture historic drug resistance and cannot assess whether resistance mutations are present on intact proviruses capable of reseeding viremia. We analyzed on-ART proviral landscapes in seven individuals with complex HIV drug resistance histories, and asked: 1) to what extent is historic drug resistance preserved on intact versus defective proviruses? and 2) to what extent does bulk proviral genotyping reflect within-host drug resistance history?

Methods: One-time proviral genotyping of protease/reverse transcriptase and integrase by bulk PCR/Sanger sequencing was performed, along with in-depth proviral landscape characterization by near-full-length-single-genome sequencing (Illumina). Proviral genotypes were compared to those historically obtained through clinical drug resistance testing (Stanford HIVdb interpretation).

Results: Seven participants, who had received ART for a median 21 (IQR:10-24) years, had historic resistance to a median 2 (IQR:1-2) drug classes. After collapsing the 2,649 single-genome proviruses collected to distinct sequences containing pol, 56 intact and 1,442 defective proviruses were analyzed. Proviral drug resistance mutation distribution was highly heterogeneous. In 5/6 participants for whom we isolated intact proviruses, drug resistance mutations were unequally distributed on intact and defective proviruses, though differentially (i.e., sometimes intact genomes preserved more, sometimes vice-versa). Bulk proviral genotyping largely captured historic drug resistance in only 3/7 participants. Hypermutated proviruses harboring APOBEC-driven mutations at key resistance sites were observed, but at prevalences too low to be captured by bulk genotyping. Despite complex drug resistance histories, 4/7 participants retained substantial numbers of fully drug-susceptible proviruses.

Conclusion: A single bulk proviral genotype performed during long-term ART incompletely captures historic drug resistance in most individuals. The observation that intact proviruses don't reliably preserve all historic drug resistance mutations helps explain rare cases where resistant HIV doesn't re-emerge when regimens are recycled.

Background: Timely HIV viral load (VL) testing is central to effective HIV care, supporting viral suppression and reduced transmission. Decentralized HIV VL testing expands access for key populations by bringing services closer to where care is accessed. During the COVID-19 pandemic, over 200 community-based sites across Canada implemented the GeneXpert molecular platform, presenting an opportunity to leverage existing infrastructure for HIV VL monitoring. However, plasma separation remains a major barrier in these settings due to limited access to centrifugation equipment. This study evaluated low-barrier plasma separation approaches for community-based settings to reduce barriers and support equitable access to HIV VL testing.
Methods: Two low-barrier plasma separation methods were evaluated: passive separation and microcentrifugation using a compact, low-cost centrifuge, with standard centrifugation as the reference method. Separation efficiency was assessed by measuring residual blood components in healthy whole blood using hematology analysis and qPCR. Microcentrifugation was evaluated at time 0, while passive separation was assessed at multiple time points over 24 hours. Removal of white blood cells was a key outcome, given their potential to contain proviral HIV DNA and affect VL quantification. HIV-spiked whole blood was used to assess the impact of each method on HIV VL using the Xpert® HIV-1 VL XC assay.
Results: Microcentrifugation and 24-hour passive separation produced plasma comparable to standard centrifugation with equivalent HIV-1 VL XC assay performance, while earlier passive separation time points led to incomplete removal of white blood cells.
Conclusion: Low-barrier plasma separation provides a means for HIV VL testing in community-based settings. Passive separation reduces equipment needs but showed greater residual components at earlier time points. Additional investigations using HIV-positive clinical specimens are required to determine the potential impact on VL. Presently, compact microcentrifuges with brief spin times offer a practical alternative that balances assay performance with accessibility in resource-limited settings.

Chronic HIV infection and its simian counterpart (SIV) reshape immune homeostasis, compromising the development and durability of vaccine immune responses. One hallmark of this dysfunction is the extensive apoptosis of CD4 T lymphocytes, which are essential in coordinating adaptive immunity. Indeed, CD4 T cells are essential for B cell differentiation and maturation and for CD8 T cells in which a defect in CD4 T cells leads to their exhaustion. During HIV/SIV infection, CD4 T cells are described as being more prone to activation-induced cell death (AICD).

Our objective was to optimize the monitoring of vaccine T cell responses in people living with HIV (PLWH) and in rhesus macaques infected with SIV. The methodology was based on the activation-induced marker (AIM) assay using flow cytometry. Thus, the upregulation of CD69, CD137/4-1BB and CD154/CD40L enabled the detection of antigen-specific T cells. Given the potential impact of AICD on specific immune response during HIV/SIV infections, a cocktail of inhibitors was used to prevent antigen-induced cell death during stimulation. Antigen-specific responses were consistently quantified relative to unstimulated controls.

We demonstrated that the cocktail of inhibitors prevents specifically the death of activated CD4 T cells (AICD) without affecting the death of exhausted T cells (PD1 high) in PLWH. Although individual responses varied, the cocktail enhanced the detection of antigen-specific T cells in the effector memory (EM) T cell subsets involved in vaccine immune responses. Thus, we demonstrated that two types of T cells are more prone to die either spontaneously or after AICD. These subsets are responding in a different way in which blocking AICD enhances AIM detection in EM.

Further optimizations are ongoing to improve the detection of antigen-specific responses in the non-human primate model, which will contribute to a better understanding of vaccine immune response orchestration in the lymphoid organs.

Elevated cervicovaginal cytokines/chemokines and epithelial disruption are associated with HIV risk. The Toronto Sex, Couples, and Science (SECS) Study demonstrated that penile-vaginal sex induces cervicovaginal inflammation and epithelial disruption, but there was considerable interindividual heterogeneity in the degree of post-sex inflammation among participants, and the concentrations of vaginal proinflammatory cytokines present before sex were highly variable. In this study, we quantified HIV associated immune factors in cervicovaginal secretions of HIV-uninfected women before and 1hr after penile-vaginal sex. We demonstrate that penile-vaginal sex induced the greatest degree of immune change among women with the lowest levels of HIV-associated markers at baseline, although this effect was only observed for proinflammatory cytokines (IL-1α, IL-6, IL-8, IL-17) and epithelial markers (sEcad, MMP-9), but not chemokines (MIP-1β, MIP-3α, MIG). Cervicovaginal IgA and IgG increased after sex, and baseline IgA was associated with inflammation and epithelial disruption. Using a multi-cytokine score to define vaginal inflammation, we also show that women with baseline inflammation usually exhibited persistent inflammation after sex, and unsupervised hierarchical clustering demonstrated that post-sex inflammation was more common in a participant group with higher baseline concentrations of inflammatory cytokines. We conclude that inflammation in the female genital tract after penile-vaginal sex, during the window when most HIV transmission occurs, is dependent on the baseline vaginal immune milieu, and that several phenotypes of sex-induced inflammation may exist, with potentially important implications for our understanding of HIV susceptibility.

The major barrier to curing HIV-1 infection is the persistence of latent reservoirs formed following integration of viral DNA into the host genome. These transcriptionally silent proviruses evade immune detection yet retain the capacity to reactivate, leading to viral rebound and immune decline. Although integration is essential for viral persistence, the influence of local genomic context on HIV-1 integration site (IS) selection and latency establishment remains incompletely understood.
Recent work from the Barr Lab indicates that HIV-1 exploits host proteins and secondary DNA structures to preferentially target integration sites that favor latency. G-quadruplexes (G4s), four-stranded DNA structures formed in guanine-rich genomic regions, have emerged as key determinants of this process. G4s are prevalent throughout the genome and are bound by G4-binding proteins (G4BPs), which modulate transcription through steric hindrance of RNA polymerase II and recruitment of epigenetic regulators. We previously demonstrated that chemical stabilization of G4s during HIV-1 infection increases the proportion of latently infected cells.
Using mass spectrometry, we identified three host G4BPs—Nucleolin, Nucleophosmin-1, and hnRNPA1—that associate with integrase from HIV-1 subtypes A, B, and D during infection of U87.CD4+.CXCR4+ cells. Knockdown of these proteins significantly reduced integration near G4-enriched regions, indicating a role for G4BPs in directing integration site selection. To further characterize this effect, we generated ATAC-seq libraries from U87.CD4+.CXCR4+ cells and cross-referenced chromatin accessibility with subtype A, B and D integration sites. Preliminary data shows reduced G4BP expression was associated with a shift in integration toward more transcriptionally dormant genomic regions for subtypes A and D.
Together, these findings identify G4 DNA and G4BPs as novel host determinants of HIV-1 integration targeting and suggest a mechanistic link between integration site selection and the establishment of viral latency.

HIV-1 infection continues to be considered as a global health problem, immunologically characterized in patients by the progressive loss of virus-specific memory CD4 T-cells (MEM) despite suppressive antiretroviral therapy (ART). Indeed, previous experiments have shown that MEM from patients under ART display higher sensitivity both to Fas- and reactive oxygen species (ROS)-mediated apoptosis, when compared to naturally protected elite controllers (EC) and HIV-uninfected control donors (HIV-). Although these immunological defects are well documented, it is not the case for the underlying molecular events responsible for them, and it is unclear if we can restore better resistance to death in MEM during chronic HIV-1 infection.
In this context, the recent works in my laboratory confirm that MEM from patients under ART display lower NRF2 activity than those of EC and HIV- donors, resulting in low BCL2 expression and defective mitochondrial integrity along with impaired anti-ROS program. Interestingly, our data also vouch for our ability to restore proper BCL2 expression and mitochondrial status, leading to higher MEM resistance to Fas-, H2O2- and NO-mediated apoptosis in the range of what is found with EC and HIV- donors. This can be achieved by using specific NRF2 drug inducers such as Ki696, 4-octyl itaconate or dihydro-CDDO-trifluoroethyl amide. In summary, by using several experimental approaches such as quantitative PCR, Imaging and multi-parameter flow cytometry along with confocal microscopy, we show that artificially targeting NRF2 activity in Mem from patients under ART restore proper cell resistance to death, revealing a new enticing therapeutic venue for better virus-specific CD4 immunity.

Introduction:
Women living with HIV have shorter life and health spans compared to men with and women without HIV. Previous studies show that women living with HIV have lower mitochondrial DNA (mtDNA) content than women without HIV. MtDNA content can be altered by ART, particularly earlier NRTIs such as AZT and d-drugs. However, less is known about the effects on mtDNA content from current NRTIs or INSTIs. Here, we investigate a contemporary cohort of women living with and without HIV.
Methods:
Participants in this cross-sectional study are from the BCC3 cohort, a holistic community-based study of healthy aging in women living with and without HIV in British Columbia. Blood mtDNA content was measured using monochrome multiplex qPCR and reported as mtDNA/nDNA ratio. Comparisons between groups were done using Chi-squared or Mann-Whitney tests.
Results:
For this preliminary analysis, we measured mtDNA content in 102 women living with (90% undetectable) and 163 without HIV. Both groups were similar with respect to age, ethnicity, body mass index, smoking, and opioid use (See Table1). MtDNA content did not differ between women with 156 [131-180] vs. without HIV 148 [124-171], p=0.11.
Conclusion:
These preliminary results suggest that mtDNA content for women living with HIV is not significantly different to women without HIV. An important consideration is that our participants are socio-demographically similar and in the 2020’s ART era where medications may be more mitochondria-friendly. Following assay completion for all participants, multivariable analysis will investigate factors associated with mtDNA, adjusting for potential confounders.

T follicular helper cells, which are essential for B cells, represent HIV-1 reservoirs in human visceral lymphoid organs. However, the consequence of viral persistence on B cell differentiation is largely so far unknown as sampling spleen and mesenteric lymph nodes in humans is impractical.
Therefore, we addressed this question in rhesus macaques (RMs) infected with simian immunodeficiency virus (SIV).
We demonstrated by flow cytometry an altered B cell differentiation in visceral lymphoid organs of ART-treated SIV-infected RMs with an expansion of activated memory CD21-CD27+ B cells expressing CD95 and PD-1. Transcriptomic analysis showed that splenic CD21-CD27+CD95+ cells exhibit a B-1-like cell signature characterized by an IGHM profile and the expression of transcriptional factor genes such as TOX, TOX2, SOX5 and TBX21. The coculture of sorted CD21-CD27+CD95+ B cells with CD4+ T cells demonstrates their lower capacity to support T cell proliferation as compared to naïve B cells.
Altogether, our results highlight the immune consequences of viral persistence in visceral lymphoid organs of SIV-infected RMs.