Background: Since the May 2022 Mpox outbreak, 1,837 cases and 51 hospitalizations have been reported in Canada. The outbreak disproportionately affected people living with HIV (PWH). There are significant gaps in understanding how HIV affects immune responses to Mpox infection in PWH. Understanding the effect of HIV on immune response following Mpox infection may inform development of vaccines and treatments.

Methods: Peripheral Blood Mononuclear Cells (PBMCs) from 13 individuals enrolled in the Monkeypox Prospective Observational Cohort Study were used. Participants included HIV+ and HIV- individuals at least 3 months post Mpox-infection as well as Mpox-naïve HIV+ control individuals. Differences in proportions of peripheral CD4+ and CD8+ T cells, Natural Killer (NK) cells, Monocytes, Dendritic Cells (DC), and Innate Lymphoid cells (ILCs) were enumerated using spectral flow cytometry (SONY Spectral ID7000).

Results: All participants were male with median age of 40. PLWH participants (n=9) had an average CD4+ T cell count of 799 cells/mm3, and all were on ART. No significant changes were observed between HIV+ vs HIV- groups post Mpox infection in the proportions of B cells, NK cells, monocytes, and DC. We found a significant decrease in CD4+ T cell counts in PWH post-Mpox infection compared with both HIV- individuals post-Mpox infection (p=0.036) and Mpox-naïve HIV+ control patients (p=0.01). We also observed a significant decrease in the proportions of ILC2 in PWH post-Mpox infection compared to HIV- patients post-Mpox infection (p=0.012).

Conclusions: The lack of differences in immune proportions post-Mpox infection in HIV+ vs HIV- individuals may relate to the small sample size or normalization of immune cell numbers by time of sample collections. Furthermore, as the HIV+ participants were not immunocompromised, this may have precluded us from observing significant differences. Work is ongoing to investigate changes in different T cell subsets, cytokine profiles and HIV reservoir size.

There are around 39 million people worldwide living with HIV, with a significant portion of this population residing in Sub-Saharan Africa (UNAIDS, 2024). HIV set-point viral load (spVL) is a critical indicator of disease progression and transmission risk, making it a key target for genetic studies. This project aims to identify genetic variants influencing HIV spVL through a multi-ancestry genome-wide association study (GWAS) and fine-mapping, leveraging data from approximately 13,000 individuals across African, European, African-American, and Hispanic ancestries. Genotype data from 23 cohorts were obtained through the International Collaboration for the Genomics of HIV (ICGH). Following the processing of each cohort for quality control, principal component analysis, and genotype imputation, a linear mixed-model regression was performed and results were aggregated via meta-analysis to determine the impact of each single nucleotide polymorphism (SNP) on spVL. While confirming the findings of previous GWAS, novel findings of the meta-analysis include association of the SNP rs35962362 in the ZNF586 gene as an HIV susceptibility marker. Fine-mapping each individual risk locus identified putatively causal SNPs, and provided insight into the heterogeneity of both novel and known HIV risk loci. 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 HIV spVL, the findings provide a foundation for understanding the genetic basis of HIV control and disease progression.

Despite the success of antiretroviral therapy, the life-long use of antiretroviral treatment requires a high financial investment, results in an increase of antiretroviral resistance and may have side effects. Thus, novel treatment strategies are emerging based on therapeutic vaccination of people living with HIV (PLHIV), where viral replication will be controlled at undetectable levels by the host's immune system after antiretroviral treatment discontinuation. Thus, the present study longitudinally evaluated the HIV-1 proviral epitopes inferred from near full-length genome (NFLG) sequences with high affinity to the most frequent HLA-A, -B and -C alleles of PLHIV from two Brazilian cities, Rio de Janeiro (RJ) and Rio Grande (RS). Overall, 86 PLHIV (RJ = 46; RS = 40) were recruited and had their peripheral whole blood collected. HIV-1 proviral NFLG were PCR-amplified and ultradeep-sequenced in an Illumina MiSeq platform. Results were analyzed in Geneious package and T-cell epitopes were predicted using the MHC-I Binding Prediction Tool. All samples included in the first timepoint were sequenced and 96.5% (83/86) had the NFLG determined. Six epitopes with high affinity to the most frequent HLA alleles were selected among viral sequences from RJ (RTLNAWVKV-Gag, HQKEPPFLW-Pol, KHQKEPPFL-Pol, TQDFWEVQL-Pol, VLDVGDAYF-Pol and VNTPPLVKL-Pol) and three from RS (KHQKEPPFL-Pol, TQDFWEVQL-Pol and VLDVGDAYF-Pol). Twenty RJ individuals had a second timepoint collected after two years of follow-up and the six selected HIV proviral epitopes remained in the peripheral blood compartment of 19 (95%) samples. Altogether, a set of HIV epitopes were selected in two conserved regions of HIV proteins. These epitopes showed high affinity to the most frequent HLA class I alleles, were highly conserved among circulating viruses and highly stable across time in the samples analyzed. Therefore, these are promising immunogen candidates for the development of therapeutic vaccines to counteract HIV-1 infection and disease progression in those regions of Brazil.

Introduction
Viral latency remains a significant barrier to achieving an HIV cure, but mechanisms underlying latency and reactivation are not fully understood. In this study, we investigated cellular gene expression profiles associated with spontaneous and prostratin-induced HIV reactivation using a latent T cell line model.
Methods
A CEM-derived CD4+ T cell clone (C-Lat) harbouring a transcriptionally silent full-length HIV provirus encoding a Nef-GFP reporter gene was used as a latency model. Cells were stimulated with prostratin (a protein kinase C agonist) or left unstimulated. HIV-expressing (GFP+) cells, reflecting spontaneous or prostratin-induced viral reactivation, and matched non-expressing (GFPneg) cells were isolated using flow cytometry. Bulk RNA sequencing was performed to quantify gene expression in each cell population. Differentially expressed genes were identified based on fold change. Gene Set Enrichment Analysis (GSEA) was used to investigate underlying biological pathways.
Results
In total, 31 genes exhibited at least five-fold induction in both spontaneous and prostratin-induced HIV-expressing (GFP+) cell populations (compared to their respective GFPneg controls); while 124 genes displayed at least a three-fold increase over controls. GSEA revealed the enrichment of inflammatory and immune activation gene sets in both spontaneous and prostratin-induced reactivated cells. Most notable of these was the MSigDB Hallmark TNF-α signalling via NF-κB pathway, which has been associated previously with the induction of HIV. Within this pathway, seven genes (EFNA1, BCL3, EGR2, CD69, MAP3K8, BCL6, and PLEK) demonstrated at least a three-fold increase in expression in reactivated cells compared to GFPneg controls.
Conclusions
This study provides new insight into cellular gene expression and signalling events that may contribute to HIV reactivation. Significant enrichment of inflammatory pathways, most notably for TNF-α, was observed in CEM T cells undergoing both spontaneous and prostratin-induced HIV reactivation. Our results highlight potential gene targets that can inform therapeutic strategies to eliminate latent HIV reservoirs.

Background
SAMHD1 limits HIV-1 replication by restricting dNTP pools. Agonists of the aryl hydrocarbon receptor (AhR), specifically 6-formylindolo [3,2-b] carbazole (FICZ), has been shown to decrease SAMHD1 phosphorylation through the transcriptional repression of cyclin-dependent kinases 1/2 (CDK1/2), enhancing its antiviral activity Conversely, our research indicates that retinoic acid (RA) enhances HIV-1 replication in monocyte-derived macrophages (MDM) by promoting SAMHD1 phosphorylation. Given the significance of macrophages in HIV-1 infection in areas rich in both AhR ligands and RA, such as the intestine and placenta, we investigated the interplay between these pathways in regulating SAMHD1 activity and HIV-1 replication in macrophages.
Methods
MDM were treated with all-trans RA (ATRA) and/or FICZ and then exposed to single-round VSV-G-pseudotyped HIV-1. HIV replication was assessed using HIV-1 p24 ELISA and nested real-time PCR for reverse transcripts and integrated HIV-DNA. Western immunoblotting for SAMHD1/pSAMHD1 and RT-PCR for validating AhR-related genes were conducted.
Results
FICZ inhibited HIV-1 replication in MDM at multiple levels, including reverse transcription and integration, but this effect was negated in the presence of RA, which significantly boosted HIV-1 replication. FICZ reduced SAMHD1 phosphorylation, but RA countered this effect by increasing CDK1 expression. Additionally, analysis of AhR-associated genes in RA-treated MDMs showed downregulation of the AhR nuclear translocator (ARNT) and upregulation of the AhR repressor (AHRR), indicating a shift towards non-genomic AhR functions.
Conclusions
Our findings highlight opposing roles of the AhR and RA pathways in regulating SAMHD1's antiviral activity, pointing to a diminished AhR-mediated antiviral response in MDMs when RA is present. This underscores the potential for natural SAMHD1 modulators as innovative therapeutic targets for HIV-1.

Introduction: HIV viral stocks contain an abundance of extracellular vesicles (EVs) that can share size, density, and surface protein composition to viral particles. Differentiating viruses from EVs remains a key challenge given the overlapping structural properties of these nanoparticles. Herein, we are developing flow virometry approaches to detect viral structural proteins present abundantly inside virus particles to overcome this problem. Drawing from the well-established technique of intracellular staining in flow cytometry, here, we have developed intravirion staining techniques targeting the capsid protein of HIV-1, Gag p24, known to be minimally incorporated in EVs.

Methods: HIV-1 pseudoviruses were permeabilized, fixed and stained with 2 different anti-p24 antibodies, namely, clone KC57 conjugated to R-PE or FITC, and clone 28B7 conjugated to APC. Unpermeabilized viruses were used as controls. We also tested the impact of Amicon-based filtration to remove PFA from staining reactions prior to permeabilization and intravirion staining. We also performed a simultaneous surface stain and intravirion stain of pseudovirus particles expressing different human proteins. Optimized intravirion staining protocols were then performed on HIV-1 propagated in PBMCs to demonstrate utility in biologically relevant virus model systems.

Results and Conclusions: Our results showed optimal intravirion staining of Gag p24 with PFA fixation and saponin permeabilization in staining reactions with and without filtration to remove PFA. The KC57-FITC anti-p24 antibody yielded the highest levels of intravirion staining that was demonstrated to be specific, with no staining observed on unpermeabilized viruses. Our results demonstrate for the first time that simultaneous detection of surface and intravirion antigens can be achieved with flow virometry methods. Intravirion staining using flow virometry can have a wide range of applications including accurate discernment of viruses from EVs, identification of virion-incorporated cytoplasmic proteins and/or nucleic acids or to characterize the incorporation of host restriction factors and/or antiviral molecules in virus particles.

Reverse cDNA transcripts of HIV-1 are detectable by the intracellular dsDNA sensor, cyclic GMP-AMP synthase (cGAS), upon HIV-1 capsid core disassembly in macrophages. The induction of the cGAS-stimulator of interferon genes (STING) pathway leads to the activation of the transcription factor, interferon regulatory factor 3 (IRF3), and the production of interferons to counteract viral infections. Liquid-liquid phase separation (LLPS) is emerging as a key mechanism to organize cellular processes such as the innate immune response mediated by cGAS. cGAS and dsDNAs assemble into membrane-less organelles through the process of LLPS, and cGAS-mediated immune responses are enhanced by Ras GTPase-activating protein-binding protein 1 (G3BP1). As shown by our laboratory, proteins that make up the HIV-1 capsid core also co-condense and phase separate (Cell Reports 2023). In this work, we propose that HIV-1 interferes with the LLPS of cGAS to repress innate immunity and antiviral activity.

Using purified HIV-1 or HIV-1 with fluorescently-labelled proteins (IN-eGFP), microscopy and proximity ligation assay techniques, we show that intact HIV-1 capsid cores and cGAS are in proximity early post-infection in macrophages. Yet, the immune response remains low despite hyper-stimulation of cGAS. Viral capsid cores, identified by IN-eGFP-labelled cores or with an anti-NC antibody, are found within clouds of cGAS condensates. HIV-1 infection also significantly reduced cellular stress responses in macrophages by reducing the abundance of G3BP1 condensates. This work reveals that HIV-1 infectivity is assured via interactions of critical immune sensors within biomolecular condensates and by suppressing stress responses post-infection. A future experiment will determine if HIV-1 alters the phase separation of cGAS, notably through capsid’s interaction with G3BP1, to repress the cGAS-mediated antiviral response.

This project highlights the role of LLPS in innate responses to virus infection, presenting it as a potential tool to enhance host antiviral responses against HIV-1.

Background: Cannabinoid-based medicines (CBM) have garnered attention due to their anti-inflammatory potential in people with HIV (PWH), whose comorbidities are driven by chronic inflammation. The expanded endocannabinoid system [or endocannabinoidome (eCBome)] and gut microbiota serve as regulators of many homeostatic processes and inflammation, but their cross-talk in PWH has not been examined. In a prospective, randomized pilot clinical trial involving PWH on antiretroviral therapy (ART) who were randomly assigned to cannabidiol (CBD) ± Δ9-tetrahydrocannabinol (THC) capsules for 12 weeks, titrating doses as tolerated, we examined the impact of CBM on plasma eCBome mediators and gut microbiota.
Methods: Ten individuals were randomized, 5 to the CBD+THC arm and 5 to the CBD-only arm, and eight individuals completed the study. Plasma for THC and CBD metabolites was collected at each visit and measured in batch by LC-MS/MS. eCBome mediators were measured at each visit, whereby fecal microbiota was assessed by 16S rDNA sequencing at treatment initiation and at the end of the treatment period.
Results: Plasma concentrations of THC and CBD metabolites varied throughout the course of the study. Capsule administration resulted in a significant decrease in the mono-acyl-glycerols (MAG) 2-eicosapentaenoylglycerol (2-EPG) and 2-oleoylglycerol (2-OG). No changes were observed in other eCBome mediators measured. PWH in the distinct treatment arms had different fecal bacterial taxa at baseline. These differences persisted through the course of the study and were not altered by capsule administration. However, Coprobacillus and Lachnospiraceae UCG001 abundance was lower in the THC/CBD arm, while Collinsella abundance was higher compared to the CBD arm.
Conclusion: The 2-MAGs 2-EPG and 2-OG were reduced following capsule administration. No changes in fecal bacterial taxa were observed following 12 weeks of treatment. Findings suggest that CBM may have promise to reduce some eCBome mediators in PWH, although larger studies are needed to confirm observations.

Activating Transcription Factors (ATFs), which belong to the basic leucine zipper group, play a crucial role in the regulation of gene expression by binding to DNA at cyclic AMP response element (CRE) motifs (1). Disruption of the symbiosis between the endoplasmic reticulum and mitochondria may contribute to cell death induced by human immunodeficiency virus (HIV) infection (2). Among the ATFs, ATF5 regulates mitochondrial stress, cell survival and cell death, but its functions remain poorly understood in the context of host-pathogen interactions.
Therefore, our main objective was to clarify the role of ATF5 during HIV/SIV infection.
We used primary human monocyte and lymphocytes infected with HIVBaL. We sorted cell populations of rhesus macaques either non infected (n=2) or infected with SIVmac251 (n=4). We performed qPCR and bulk RNAseq for analyzing the expression of the members of the ATF family. We also generated a plasmid encoding for atf5.
Our results show an early and transient expression of ATF5 gene expression in HIV-infected monocytes. Furthermore, we found higher expression of ATF5 in hepatic CD4 T cells. Further analyses are underway to elucidate the signaling cascades induced by ATF5 to highlight the critical nodes responsible for cell death, as well as the effect of ATF5 during viral infection.
1) Corne A Biology 2024
2) Petit J Biol Chem. 2002

Human immunodeficiency virus type 1 (HIV-1) uses the Vpu accessory protein to evade immune surveillance. Vpu modulates BST-2, CD4, and HLA-C to enhance viral egress, evade antibody-mediated cellular cytotoxicity, and prevent cytotoxic T cell responses, respectively. These functions are dependent on Vpu amino acid motifs that interact with host cell proteins to subvert trafficking pathways. However, it is difficult to decipher specific Vpu motifs as a 83 base pair overlap between the vpu and env ORFs exists in the bicistronic mRNA encoding these two proteins, affecting 27 amino acids of Vpu. Accordingly, this has precluded the identification of sequence determinants that define Vpu function, specifically at its C-terminal end, in a fully infectious context. To address this, we created a vector encoding the full infectious molecular clone of the transmitted/founder HIV-1 strain CH58, with the vpu and env ORFs separated entirely. Furthermore, we flanked vpu with restriction digest target sequences, allowing for cloning of whole Vpu proteins from additional strains into the infectious backbone. To validate this approach, we inserted the NL4-3 Vpu protein into this backbone and tested constructs for their ability to downregulate BST-2, CD4, and HLA-C in primary CD4+ T cells. Both CH58 Vpu and NL4-3 Vpu downregulated BST-2 and CD4; however, NL4-3 Vpu was unable to downregulate HLA-C. We also demonstrated that the N-terminus of Vpu is required for HLA-C downregulation by Vpu, as replacing the first 27 amino acids of CH58 Vpu with NL4-3 Vpu in this backbone blocked HLA-C downregulation. Taken together, our molecular clone will be used to further elucidate how Vpu downregulates host protein targets in a fully infectious context. Given the importance of Vpu during HIV-1 infection, a full understanding of its functions in a relevant context may inform potential therapeutic strategies to help PLWH.

The surface protein profile of HIV-1 is incredibly diverse, as these enveloped virions can incorporate a wide range of human proteins through budding from an infected cell. Our research demonstrates many of these newly acquired proteins can influence viral homing, attachment, and inflammatory responses. Despite its low transmission efficiency, HIV’s ability to modulate its surface protein composition could offer significant adaptive advantages, aiding its persistence and spread. Given that just one virus, among a myriad of diverse particles, is required to establish an initial infection, the contributions of individual virion phenotypes become crucial in understanding infection and designing more effective vaccines. Our lab has been pioneering new methods in Flow Virometry (FV) to perform high-throughput quantification of surface proteins on individual virions. In this study we developed techniques for multi-colour immunophenotyping of HIV-1, to detect the co-incorporation of multiple antigens on single virus particles. First, we employed HIV pseudoviruses as controlled virus model systems to simultaneously stain multiple proteins on individual particles. We then applied these techniques to HIV isolates propagated in CD4+ T cell lines and PBMCs, observing natural patterns of co-incorporation. In parallel, ongoing studies are optimizing virus sorting protocols to purify and isolate homogenous virus subpopulations, representing a new approach for studying viral heterogeneity. The isolation of uniform virus subpopulations will enable controlled biological studies to better understand how distinct virion phenotypes influence HIV biology and disease. The development of multi-colour immunophenotyping and virus sorting protocols will yield new knowledge of HIV biology that could inform novel therapeutic and vaccine approaches.

The Human Immunodeficiency Virus Type 1 (HIV-1) Nef accessory protein is expressed early during HIV-1 infection and contributes significantly to evasion of host immune surveillance. Nef modulates cellular trafficking pathways to downregulate the expression of various immune effectors on the surface of host cells, including the HIV-1 entry receptor, Cluster of Differentiation 4 (CD4). Another protein downregulated in this manner is Serine Incorporator 5 (SERINC5), a multi-pass transmembrane protein that incorporates into progeny virions during egress and inhibits fusion with target cells, thereby reducing infectivity. Nef restores infectivity by engaging cellular adaptor proteins (APs) to internalize SERINC5 via clathrin-mediated endocytosis. An endocytic dileucine sorting motif ([D/E]xxxL[L/I]₁₆₅) in the C-terminal region of Nef is critical for establishing the association with APs necessary for antagonism of SERINC5 and CD4, suggesting equivalent pathways are involved in the downregulation of both proteins.

We previously identified a Nef DN₁₆₄ND polymorphism located within the dileucine motif that ablates SERINC5 downregulation while preserving CD4 downregulation. We hypothesize that the DN₁₆₄ND polymorphism alters the interaction between Nef and APs involved in cellular trafficking, due to its location within the dileucine motif, which ultimately uncouples the two pathways. We used bimolecular fluorescence complementation (BiFC) with confocal microscopy to discern the interaction between Nef and SERINC5 within the endocytic pathway of transfected cells. We aim to further characterize the subcellular compartments and APs involved in the functional uncoupling to elucidate the pathway through which SERINC5 is trafficked during Nef-mediated downregulation. The results of this investigation may have important implications for the development of novel therapeutics that target Nef. If administered during the early stages of infection, such drugs could reduce HIV-1 proliferation and improve health outcomes in people living with HIV-1.

HIV specifically infects T lymphocytes expressing CD4 molecules and a chemokine receptor namely CCR5 or CXCR4. Unlike HIV, CXCR4 is not used by the simian immunodeficiency virus (SIV) to infect T cells while SIV infects cell lines expressing the chemokine receptor GPR15. However, its role in vivo remains elusive.

Our hypothesis is that GPR15 may contribute to SIV infection. In this study, we determined i) the dynamics of CD4 T cells expressing CCR5 and GPR15 and ii) whether these cells are infected establishing viral reservoirs after antiretroviral therapy (ART).

Rhesus macaques (RMs) were infected with SIVmac251 (20 AID50). ART was administrated at day 4 post-infection (tenofovir/emtricitabine/raltegravir or dolutegravir/ ritonavir). After necropsy, lymphoid tissues, including the spleen, mesenteric and axillary/inguinal lymph nodes, as well peripheral blood were recovered. The phenotype of CD4 T cells expressing CCR5 and GPR15 were analyzed by flow cytometry. CCR5 and GPR15 T cell subsets were sorted by flow cytometry and viral RNA and DNA were quantified by qRT-PCR.

Our results demonstrate that CD4 T cells expressing CCR5 and GPR15 are distinct CD4 T cell subsets in RMs. Whereas CCR5 is expressed by effector memory and terminal differentiated CD4 T cells, GPR15 is mainly expressed by central memory T cells. We observed a transient depletion of CD4 T cells expressing CCR5 and GPR15 during the acute phase of infection both in the blood and peripheral lymph nodes. This depletion is prevented by ART. In chronically SIV-infected RMs, a decrease in the percentage of CD4 T cells expressing these coreceptors was also noticed in mesenteric lymph nodes. Sorting of CD4 T cell subsets expressing GPR15 and CCR5 demonstrated the presence of cell-associated SIV DNA.

Altogether, our results describe the impact of GPR15 as a coreceptor for SIV participating in the establishment of viral reservoirs in RMs.

Human immunodeficiency virus 1 (HIV-1) remains a global health challenge, affecting ~39 million people. New HIV-1 infections are typically established by a single transmitted/founder (T/F) virus, with the heavily glycosylated HIV-1 envelope protein gp120 playing a key role in transmission. We hypothesized that specific N-glycosylation patterns are associated with the successful transmission of T/F viruses, while chronic viruses develop more diverse glycosylation profiles to evade antibody neutralization.
To evaluate gp120 glycosylation patterns between T/F and chronic virions, solubilized viral lysates were visualized on SDS-PAGE gels, gp120 bands were excised, digested with trypsin and chymotrypsin, and resulting glycopeptides were subjected to electron-transfer/higher-energy collision dissociation mass spectrometry analysis. Glycopeptide analysis software analysed site specific gp120 N-glycosylation across 24-27 N-glycosylation sites using a library of 55 glycoforms representing common glycans added to gp120 in the endoplasmic reticulum (high mannose), trans-Golgi and Golgi (complex). Preliminary findings indicate the proportion of complex glycans on the T/F isolate, B4, is higher than the chronic isolate, Q0. Despite having fewer N-glycosylation sites, B4 has more than double the amount of glycosylation than Q0. Equivalent N-glycosylation sites between B4 and Q0 were aligned based on sequence and structure position. At most equivalent sites, B4 had a higher count of glycosylation and high mannose glycoforms.
Transmission fitness of the B4 and Q0 was assessed via a multi-virus competition on human cervical tissue under treatment conditions that saturate C-type lectin receptors and mannose glycans on gp120. Results indicated that B4 had significantly higher replication fitness on cervical tissue. However, in Th1 and Th17 cell lines, Q0 outcompeted B4, suggesting cervical tissue may benefit T/F viruses in establishing infection.
Insights into T/F and chronic gp120 glycosylation profiles may identify features essential for HIV-1 transmission, potentially serving as robust vaccine targets and enhancing the understanding of gp120 glycosylation’s role in transmission.

Despite effective antiretroviral therapy (ART), HIV-1 persistence is the major obstacle to a functional cure. Understanding the tissue microenvironment associated with persistence during ART is key. We have developed immunoPET/CT-guided-omics allowing the identification and subsequent interrogation of foci of viral gene expression using the SIV/rhesus macaque model. Here we compare the spatial transcriptomics (10x Visium) of the local gut tissue neighborhood of the rebound eclipse-phase foci (4-6 days post-ATI) from animals initiating ART 4 days (short lifespan reservoir ) or 10 weeks (persistent reservoir) after high-dose challenge. Adjacent tissue sections containing foci of rebounding virus were evaluated with SIV proviral PCR, immunofluorescence, and spatial transcriptomics. SIV presence in both short lifespan and persistent conditions was associated with higher transcriptional levels, up-regulation of genes related to SIV infection, and activation of innate immune responses. Notably, we also detected significant differences between the short lifespan and persistent reservoirs. The biomarkers and pathways for the persistent reservoir revealed the activation of the integrated stress response (ISR) with its characteristic translational reprograming and cellular adaptation. Pathway analyses reveal many parallels between the persistent reservoir foci of virus rebounding 6 days after ATI and the tumor microenvironment (TME) which is the contextual basis of modern cancer research. SIV in persistent reservoirs was distinctly associated with IgA plasma cells, monocytes, and cycling gamma-delta T-cells, while in short lifespan reservoir was associated with IgG plasma cells, Th17 cells, and DCs. Additional signatures of epithelial cells combined with pathway analysis suggest that persistent reservoirs are associated with gut tertiary lymphoid aggregates and characterized by a regional ISR adaptation to restore cellular homeostasis. We propose that the integrated stress response facilitates the development of a viral microenvironment very analogous to the well-defined TME suggesting both utilize the altered microenvironment as a physiological sanctuary to persist while evading immune responses.

Introduction: Efficient diagnostic workflows for HIV are critical to ensure timely treatment while avoiding unnecessary confirmatory testing. HIV-2, though rare, presents diagnostic challenges due to overlapping reactivity patterns with HIV-1. This study evaluates the rate of HIV-2 indeterminate samples detected between 2016 and 2023 in Alberta using the ARCHITECT HIV Ag/Ab Combo assay combined with the Bio-Rad GeeniusTM HIV-1/2 Supplemental Assay testing algorithm.

Methods: A total of 8,360 samples were tested on ARCHITECT Assay, with COI values stratified into defined ranges. Reactive sample results were confirmed with the GeeniusTM assay as HIV-1 positive, HIV-2 indeterminate, HIV-2 positive, or HIV-positive untypable. Statistical significance of assay performance across COI ranges was assessed using Chi-square tests.

Results: Of 3,400 ARCHITECT reactive samples, 2,208 were confirmed as HIV-1 positive, 41 as HIV-2 indeterminate, 1 as HIV-2 positive, and 2 as HIV-positive untypable on the GeeniusTM assay. Of the 3,400 reactive samples, 936 (27.5%) had COI values between 1-5 and were more likely to be false positives: 896/936 (95.7%) were negative for HIV, 38 (4.1%) were HIV-2 indeterminate (IND), and 7 (0.7%) were HIV-1 positive by GeeniusTM. For COI values ≥500, nearly all samples (99.8%) were HIV-1 positive. Of the 24 unique patients testing HIV-2 IND, 18 (75%) had adequate follow-up testing for HIV-2, of which, all (100%) were confirmed to be negative for HIV-2 despite being IND on the GeeniusTM.

Conclusion: There is high agreement between the ARCHITECT and the GeeniusTM for HIV-1, but highlights limited use in sending HIV-2 indeterminate samples for confirmatory testing, as none were confirmed to be HIV-2 positive in this analysis. The ARCHITECT assay demonstrates strong concordance between high COI values (≥500) and HIV-1 confirmation. Additionally, the false positive rate for samples with COI values of <5 highlights the need for continual confirmation testing in that range.

BACKGROUND: Vaccination has been crucial in the prevention and control of SARS-CoV-2, but immunocompromised patients, such as people living with HIV or end-stage renal disease (ESRD), remain at risk. Studies have shown ESRD patients have reduced antibody production and memory B-cell differentiation in response to COVID-19 vaccination. However, little is known about how early innate immune responses contribute to these differences. Further, sex-stratification is limited in COVID-19 vaccination studies despite knowledge of sexual dimorphism in immune responses.

METHODS: Blood samples were collected from individuals before and 1-4 days post-dose 1 of the BNT162b2 vaccine. RNA sequencing was performed in ESRD (n=10 female, n=7 male) and healthy controls (HC) (n=19 female, n=9 male) and 19 plasma cytokines were quantified in ESRD (n=8 female, n=7 male) and HC (n=12 female, n=8 male). Detailed enrollment and follow-up questionnaires were collected and individuals with prior COVID-19 were excluded from the analysis.

RESULTS: Females were more reactogenic to COVID-19 vaccination irrespective of population, displaying increases in IL-10 (ESRD log2 fold-change (FC)=3.18, Wilcoxon p<0.01; HC FC=3.95, p<0.001), IL-2 (ESRD FC=4.72, p<0.0001; HC FC=3.42, p<0.0001), and IL-13 (ESRD FC=2.35, p<0.01; HC FC=1.85, p<0.0001). HC females showed increased macrophage derived cytokines in response to vaccination such as IL-1β (FC=3.97, p<0.0001), IL-6 (FC=2.65, p<0.0001), and TNF-α (FC=1.57, p<0.001). No significant differences in cytokine concentrations were observed when comparing females in both groups post-dose 1.

CONCLUSIONS: Overall, females displayed greater reactogenicity to vaccination. Qualitative differences like pre-existing inflammation in people living with ESRD influenced early innate immune responses. Females with ESRD exhibited non-significant increases in macrophage derived cytokines, known markers of inflammasome activation, in response to vaccination. These findings highlight the importance of sex-stratified immunological research to uncover unique vaccine responses in both healthy and immunocompromised populations. Such insights could inform population- and sex-specific vaccination strategies.

Introduction: Over half of people living with HIV globally are women, mostly of reproductive age. In Canada, 25% of people with HIV are women. In 2023, people living with HIV gave birth, to 239 children, and 96% had received antiretroviral therapy during pregnancy. While there are several classes of antiretrovirals, integrase strand transfer inhibitors (InSTIs) are commonly used given their high tolerability and efficacy, including during pregnancy, despite limited or mixed safety data such as mitochondrial dysregulation in vitro and in animal models.

Methods: Human embryonic stem cell lines CA1S and H9 were exposed to InSTIs (raltegravir, bictegravir, dolutegravir, cabotegravir) at 0.01, 0.1, 0.5, and 1x Cmax (peak plasma concentration) for 3.5 days, then assessed by flow cytometry for cellular health, including pluripotency, apoptosis, and mitochondrial reactive oxygen species (mtROS). Multidimensional reduction with supervised X-shift clustering was used to analyze the co-expression of cellular health markers. Differences between drug exposures and 0.1% DMSO control were determined by Wilcoxon-rank sum test.

Results: Bictegravir exposure was associated with spontaneously differentiating apoptotic cells with low mtROS (p=0.005). Both dolutegravir and cabotegravir exposure also increased the number of spontaneously differentiating cells with low mtROS levels (p=0.013 and p=0.008, respectively), and decreased the number of early apoptotic pluripotent cells (p=0.005 and p=0.020, respectively). Raltegravir exposure was not significantly different from the DMSO control.

Conclusions: Bictegravir, dolutegravir, and cabotegravir induced a loss of pluripotency, with a shift toward spontaneously differentiated cells, alongside a reduction in mtROS levels. These findings suggest potentially detrimental effects on early development with InSTI exposure during embryogenesis.

Adolescent girls and young women (AGYW) in generalized HIV epidemics experience increased risk of HIV acquisition, but it is unknown the extent to which biological mechanisms contribute. Studying the genital immune cell milieu among AGYW is critical to better understanding whether age is associated HIV transmission-related factors, controlling for other predictors of HIV risk such as other STIs, sexual behaviour, contraceptives, and other possible confounders.
We examined the immune cell frequency and abundance at baseline in the genital tract of AGYW aged 15-24 in fishing villages near Entebbe, Uganda. A total of 277 participants were enrolled in this prospective cohort, with girls aged 15-17 (n = 114), 18-20 (n = 63), and 21-24 years (n = 100). A high prevalence rates of STIs was observed within this cohort, including human papillomavirus (HPV) (40%), Chlamydia trachomatis (CT) (14%), Neisseria gonorrhoeae (NG) (2%), Trichomonas vaginalis (TV) (9%), and herpes simplex virus (HSV) seroprevalence (45.6%).
We observed a significant increase in the abundance of CD4+ T-cells among younger participants (aged 15-19) [β = 0.25 ,95% CI (0.05 - 0.44), p=0.014] suggesting an age-related associations with genital immune cell abundance. Age remained a predictive factor for immune cell abundance in multivariable linear regression models adjusting for the presence of STIs [β = 0.24 95% CI (0.02 - 0.47) p=0.03], HPV, and HSV-2 serology, highlighting the potential for immunological shifts to influence susceptibility among younger AGYW.
Our ongoing analyses will further explore the role of soluble inflammatory markers, microbiome, and their relationship with genital immune cell populations. These findings will inform strategies to optimize prevention and care interventions tailored to the unique immunological needs of AGYW in high-burden settings.

BACKGROUND/AIMS
The persistence of a latent HIV reservoir within the population of CD4 T cells fails combined with antiretroviral therapy (cART). The elimination of this latent viral reservoir is a crucial aspect of current strategies for an HIV-1 cure. Our previous non-infectious HIV/Virus-like particle (HLP) specifically reactivated the latent viral pool in HIV-specific CD4+ T cells of individuals treated for 5-20 years on stable cART during acute and chronic HIV infection. For improved safety, HLP production was derived from near full-length HIV-1 genome (HP) mutated to prevent genomic RNA packaging. However, without gRNA, the HLP showed a lower latency reversal level than an HP in the CD4+ T cells. In this study, we hypothesize that by co-opting the existence of HIV genome with novel RNA motifs, we can preferentially package the RNA adjuvant into HLP which can enhance HIV latency reversal effectiveness.
METHODS
Encapsidated HLP was generated by co-transfecting Multi RNA adjuvant and HLP plasmids into HEK-293T cells. Both RNA copies of the novel adjuvant and HLP were quantified by qRT-PCR. Next, we used THP-1 dual cell lines and human PBMCs to test the immunostimulatory responses. Nested-PCR and RT/nested-PCR were used to amplify proviral and latent reactivated viral genomes. RNA viral copies released into supernatant were quantified by qRT-PCR, viral diversity by next-generation sequencing using Oxford Nanopore.
RESULTS
A 4:1 plasmid ratio expressing RNA adjuvant versus the HLP is optimal to generate a newly encapsidated HLP that almost completely excludes remnant genomic HIV RNA. Our encapsidated HLP can activate NF-kB significantly more than HLP alone. The encapsidated HLP had a higher level of viral RNA copies released after latency reversal than HLP alone.
CONCLUSION
The encapsidated HLP could induce immunostimulatory effects and enhance the HIV latency reversal within CD4+ T cells in individuals on long-term cART.

HIV-1 acquires its lipid envelope during egress from infected cells. In doing so, virions incorporate cellular proteins from the plasma membrane into nascent viral envelopes. Our lab has been extensively characterizing these virion-incorporated host proteins as they can remain functional and alter virus biology. For example, our recent works demonstrated the exploitation of PSGL-1 as a viral attachment factor, and the utilization of virion-incorporated CD14 to shuttle bacterial LPS. Beyond functionality, incorporated proteins also offer researchers a tool to discern virus origins. We hypothesize that HIV-1 from different tissues may incorporate proteins that reflect the unique producer cell types, thereby allowing the inference of the cellular source of HIV-1 reservoirs.

Herein, we propagated HIV-1BaL in donor-matched peripheral-blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) isolated from three different donors. Virus-containing supernatants were then analyzed by mass spectrometry. We discovered that, while MDM- and PBMC-derived HIV-1 share a large fraction of identified proteins, there were statistically significant differences in the enrichment of those proteins between the two groups. This differential enrichment drove PBMC and MDM clusters by principal components analysis, and hierarchical clustering. Volcano plots identified differentially expressed proteins, and a curated list was validated through flow virometry, immunoprecipitation and western blots.

A key challenge in HIV cure strategies is identifying the key viral reservoirs to target and eliminate infectious virus from systemic shedding. The goal of this work is to determine how characterizing the human proteins on viral particles might improve our understanding of HIV-1 reservoirs. Our results highlight the ability to discern PBMC- and MDM-derived HIV-1 based solely on these virion-incorporated human proteins, reinforcing our hypothesis that virions emerge from infected cells with a unique protein fingerprint that can inform on cellular origin. Ongoing studies are extending these findings to additional cell types and tissue sources, including clinical samples.

Background: Persistent central nervous system (CNS) inflammation in people living with HIV (PLWH) remains a critical concern despite effective viral suppression achieved through antiretroviral therapy (ART). CNS inflammation at the blood-brain barrier (BBB) can impair barrier integrity, facilitating immune cell infiltration and contributing to cognitive dysfunction. Clinically, dolutegravir (DTG), although effective, has been associated with neuropsychiatric adverse effects. Our group demonstrated that DTG disrupts the BBB by inducing pro-inflammatory cytokines and altering tight junction protein expression, leading to increased permeability. However, the modulatory effects of nucleoside reverse transcriptase inhibitors (NRTIs), such as tenofovir alafenamide (TAF) and emtricitabine (FTC), administered with DTG on CNS inflammation remain unexplored. NRTIs, primarily known for their antiviral activity, have been reported to suppress inflammasome activation and reduce inflammatory signaling pathways, including cytokines and chemokines. This study investigates whether NRTI backbones, in combination with DTG, reduce inflammatory responses at the BBB.

Methods: Primary cultures of mouse brain microvascular endothelial cells (BMECs), an in vitro BBB model, were cultured and treated with DTG (5000 ng/mL) alone or in combination with NRTI backbone (TAF 300 ng/mL + FTC 4000 ng/mL) for 24 hours. Control groups included DMSO (vehicle) treatment. Inflammatory cytokines and chemokines mRNA expression levels (e.g., Il6, Ccl2, Cxcl1, Cxcl2, Il23a, Il12b) were quantified using qPCR analysis

Results: NRTI backbone treatment in combination with DTG significantly reduced the expression of key pro-inflammatory markers, including Il6 (∼20% reduction), Cxcl2 (∼30% reduction), and Il23a (∼40% reduction), compared to DTG-treated cells alone. This reduction indicates a potential modulatory role of NRTIs in mitigating DTG-induced inflammatory responses at the BBB.

Conclusion: Our findings demonstrate that NRTI backbone, in combination with DTG, exhibit anti-inflammatory properties that could mitigate dolutegravir-induced BBB inflammation. This highlights the potential of NRTI-associated regimens to address ART-associated CNS inflammation and improve neurological outcomes in PLWH.

HIV set-point viral load (spVL) is a strong predictor of disease progression and transmission risk, with approximately 25% of variance attributable to host genetic factors. A genome-wide association study identified a novel region on chromosome 1 significantly associated with decreased HIV spVL and in vitro knockout of CHD1L increased HIV replication in myeloid but not Jurkat T cells. However, the relationship between spVL-associated variants, CHD1L expression, and spVL remains unclear. This study explored the relationship between spVL-associated variants and CHD1L expression in monocytes and CD4+ T cells to assess cell-type-specific responses. Analysis of CHD1L expression in CD4+ T cells from people living with HIV did not differ by genotype. However, in monocytes, individuals heterozygous for protective spVL alleles exhibited significantly lower CHD1L expression compared to homozygous reference individuals as determined by qPCR, suggesting lower CHD1L expression corresponds to lower HIV spVL. Next, we utilized transcriptomic profiles of healthy African American individuals from the Immune Variation project using DESeq2 to test whether HIV spVL-associated variants influenced genes downstream of CHD1L. While no genes were differentially expressed based on genotype in CD4+ T cells, 27 genes were downregulated amongst individuals with protective spVL alleles in monocytes. Pathway analysis revealed individuals with protective spVL-associated alleles exhibited lower expression of genes involved in cytokine and interferon signaling. While a strong interferon response restricts HIV replication in vitro, increased expression of interferon stimulating genes has been associated with increased HIV set-point viral load previously. While further work is needed to determine whether transcriptome changes are directly mediated by CHD1L, these results suggest that HIV spVL-associated variants modulate overall immune activation which may affect HIV pathogenesis differently during acute or chronic infection. Overall, these findings highlight new insights into genetically regulated control of HIV and HIV pathogenesis.

The persistence of latent HIV-1 reservoirs following integration into host cells is the main barrier to curing HIV-1. Despite antiretroviral therapy, latently infected proviruses can reactivate, leading to CD4+ T cell decline and AIDS. The site of integration and the DNA structures that surround it are critical to establishing latent infection. G-Quadruplex DNA (G4s) have been identified as DNA structures that influence HIV-1 integration and latency. G4s are poly-guanine tracts that form stable 4-stranded helices and it has been established that stabilizing G4s promotes integration near G4s and increases the proportion of latently infected cells. Moreover, liquid chromatography mass spectrometry identified three host G4-binding proteins (G4BPs) associated with the HIV-1 pre-integration complex (PIC): Nucleolin, Nucleophosmin-1, and hnRNPA1. Based on these findings, we hypothesized that G4s, in conjunction with PIC-associated G4BPs, guide the HIV-1 PIC to specific genomic regions, facilitating integration site selection and favoring proviral latency.
To test this hypothesis, cells were treated with small interfering RNAs to reduce the expression of the identified G4BPs. Then, cells were infected with HIV-1 with a subtype B backbone possessing either subtype B or D integrase. Genomic DNA was harvested and analyzed using a custom bioinformatics tool, the Barr Lab Integration Site Identification Pipeline. In cells with reduced G4BP expression, integration near genes and transcription start sites—both regions enriched in G4s—was significantly diminished for both HIV-1 subtypes. A clinically relevant integration site hotspot was also identified in control samples but was lost when G4BP expression was reduced. Additionally, changes in G4BP expression influenced the orientation of integrated proviruses relative to gene targets, which is known to affect proviral transcriptional activity. These findings identify G4s and G4BPs as new players in HIV-1 integration site targeting and suggest that G4BPs may serve as promising therapeutic targets for disrupting latent HIV-1 reservoirs.

Antiviral resistant HIV-1 remains a significant challenge in the pursuit of a cure. Raltegravir, an integrase inhibitor, blocks HIV-1 integration into the host genome, but resistant strains retain the ability to integrate. Whether raltegravir-resistant strains alter their integration site preferences, which could influence their pathogenesis, remains under explored. This study investigates how HIV-1 subtype D integrases adapt their integration site profiles under sub-inhibitory raltegravir concentrations in vitro. Resistant HIV strains were selected by progressively increasing raltegravir concentrations over time. Genomic DNA was extracted from HIV-infected cells at various time points, and integration site profiles were analyzed using the Barr Lab Integration Site Identification Pipeline (BLISIP). Early infection stages showed a predominant preference for integration within genes. However, as raltegravir pressure increased, integration frequency near genes decreased, while integration within long interspersed nuclear elements (LINEs) became significantly enriched during later infection stages. LINEs are known to influence chromatin structure and transcriptional activity, and their enrichment at integration sites may impact proviral and/or nearby host gene expression. These findings suggest that raltegravir-resistant HIV adapts its integration site preferences over time, with decreased targeting of genes and increased targeting of LINEs, potentially contributing to altered viral pathogenesis. This shift highlights a possible mechanism by which resistant HIV-1 evades therapeutic pressure, offering new insights into the behaviour of drug-resistant HIV-1 integration. Understanding these changes in integration dynamics may inform the development of strategies to address raltegravir resistance and improve therapeutic outcomes.

The phylogenetic closeness of non-human primates to humans makes them attractive models for assessing the pathogenicity related to infectious diseases, such as HIV infection. Rhesus macaques (RMs) offer the opportunity to decipher (i) cellular and anatomical reservoirs and (ii) the immune consequences associated with viral persistence in tissues despite early antiretroviral therapy (ART) (1-3). Among the infected CD4 T cell subsets, we and others have reported that T follicular helper (Tfh) cells represent one of the main viral reservoirs in lymphoid tissues of SIV-infected RMs.
Therefore, we decided to explore in greater depth gene profile of Tfh cells compared to those expressed in effector memory (TEM) and naïve CD4 T cells; in particular, given the nature of Tfh cells, which are mostly localized within B cell follicles in lymphoid tissues.
By combining cell sorting with flow cytometry and bulk RNA-sequencing, we analyzed the profiles of these T cell populations isolated from the spleen and mesenteric lymph nodes of RMs, either infected with the SIVmac251 strain (20 AID50) or treated with an early ART (day 4 post-infection)(1-3).
One of our main results indicates that Tfh cells who display a distinct transcriptomic profile compared to TEM and naive cells, demonstrate an altered profile in SIV-infected RMs that persists despite early ART administration. Such profile is associated with a partial restoration of Tfh cells in lymphoid tissues. Single cell transcriptomic analysis reveals the presence of heterogeneous Tfh cell subsets.
Given the importance of Tfh cells for B cell and vaccine immune responses, understanding more in deep their gene profile in lymphoid tissues is of main importance. Strategies aiming to restore and rejuvenate Tfh cells may contribute to improve immunity and vaccine immune responses in people living with HIV.

1 Rabezanahary Mucosal Immunol. 2020.
2 Rabezanahary J Virol. 2020
3 Clain JCI Insight. 2023