Introduction
The human herpes virus 8 (HHV-8) causes Kaposi Sarcoma (KS) and is linked with some type of B-cell proliferation disorders that develop upon aging and immunosuppression, including in people living with HIV (PLWH).
HHV-8 establishes latency in cellular and anatomical reservoirs in epithelial cells as well as B-cells. Factors controlling latency and replication remain seldomly described. We developed HHV-8 specific serological and virological assays to assess ex-vivo whether antibodies targeting HHV-8 could control replication.

Methods
Serum and cell lysate were obtained from cis and transgender men of the Engage cohort in Montreal. Participants with active KS (11 PWH on ART [HIV KS] and 11 HIV-negative classic KS [cKS]) were included from the McGill biobank. Circulating levels of IgG binding to BCBL1 HHV-8-infected cells were quantified by flow cytometry. Digital-droplet-PCR was used to assess HHV-8 DNA levels in blood extracts and biopsies.

Results
In absence of KS, HHV-8 seropositivity was higher in PWH than HIV-negative participants (67.5% vs. 41.8%, p<0.001). Moreover, in 106 HHV-8 seropositive participants, levels of anti-HHV-8 IgG were higher in PLWH than in 260 HIV-negative controls (median 1.5 vs 1.2AU, p=0.03). Levels of anti-HHV-8 IgG were negatively correlated with HHV-8 viremia in whole blood extracts (r=-0.48, p=0.004, n=46).
Patients with KS were all seropositive for HHV-8. HIV KS had higher levels of anti-HHV-8 IgG levels than cKS (3.7 vs. 1.8AU, p=0.02). Interestingly, HIV KS had lower circulating HHV-8 viremia than cKS (695 vs. 8405 copies/106 cells, p=0.04). However, HHV-8 DNA levels were similar in KS lesion biopsies for the 2 groups (HIV KS: 68.103 vs cKS: 323.103 copies/106 cells, p=0.89).

Conclusion
In both symptom-free carrier and KS patients, high levels of anti-HHV-8 IgG were associated with low circulating viremia. Whether higher anti-HHV-8 IgG levels contribute to prevent KS development should be further studied.

Background: The 2018 Botswana Tsepamo study reported an increased risk of NTDs in fetuses exposed to dolutegravir from conception. Folate deficiency in fetal development has been associated with NTDs. Annually, over 1 million people with HIV taking antiretroviral drugs become pregnant, making it critical to investigate potential interactions between INSTIs and folate transport pathways in the developing fetus. We investigated the in vitro effect of in utero exposure to INSTIs on the expression of placental proteins critical for fetal folate delivery.

Methodology: HTR8/SVneo and BeWo human placental cell lines representative of the first and third pregnancy trimester, respectively, were treated with clinically relevant doses of dolutegravir, bictegravir, cabotegravir or DMSO control for a period of 3, 6, 24, or 48 hours. mRNA and protein expression of folate receptor-α (FRα), and transporters, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), were assessed by qPCR and immunoblotting respectively. Functional assays to determine any changes in the activity of these folate receptor/transporters were performed.

Results: We observed a significant downregulation of the mRNA and protein expression of: i) FRα and PCFT in dolutegravir treated cells by 50%, ii) FRα, RFC, and PCFT in bictegravir treated cells by 20%, and iii) a very modest effect on FRα and RFC in cabotegravir treated cells. Functional assay data paralleled the expression findings. FRα and PCFT activity was lower in DTG treated cells, whereas activity was unaffected in bictegravir and cabotegravir treated cells.

Conclusion: Our findings suggest that the observed dysregulation of folate transporters in the placenta caused by INSTIs, particularly dolutegravir, could potentially result in an in utero folate deficiency that could place the fetus at increased risk for adverse birth outcomes. Future studies will further examine, in vivo, fetal folate levels and potential drug toxicity. (Supported by OHTN and CIHR).

Background: People with HIV (PWH) face accelerated comorbidities due to chronic inflammation. Cannabinoid-based medicines have potential anti-inflammatory benefits, suggesting their potential to reduce inflammation in PWH. Herein, we assessed changes in gene signature associated with immune cells and inflammatory pathways in PWH receiving oral cannabinoids under suppressive ART.
Methods: Ten participants were randomized to receive cannabidiol (CBD) only or CBD combined with Δ9-tetrahydrocannabinol (THC) capsules for 12 weeks, with dose titration. Single-cell RNA sequencing was performed on PBMCs from six individuals (3/arm) at baseline and week 12. Gene expression libraries were sequenced using Illumina NovaSeq, and differential gene expression (DEG) analysis was conducted with log2 fold-change > 0.25 and adjusted P<0.05. Gene Ontology Biological Process (GOBP) analysis assessed dysregulated pathways.
Results: CBD+THC treatment led to more DEGs than CBD-only, particularly in CD14+ classical monocytes and CD8 T-cells. Both treatments increased CD4+CD8+ double-positive T-cells and plasmacytoid dendritic cells (pDCs), with CBD-only reducing CD14+ and CD16+ monocytes. The rise in double-positive T-cells was linked to higher DNA repair and stress-response genes, while pDC increases were marked by reduced innate immune responses and migration. In the THC+CBD arm, CD14+ monocytes showed DEGs linked to circadian regulation (ARNTL, NR1D1), reduced pro-inflammatory cytokines (IL-6, IL-1β), and enhanced inhibition of TGF-β signaling pathway (SMAD6, SMAD7, SMURF1, BAMBI). GOBP analysis showed dysregulation in apoptosis, differentiation, and proliferation in CD14+ monocytes. CD8 T-cells showed higher expression of genes related to stress response and cell survival (CENPA, USP2, PPP1R15A, TNFAIP3, NR4A2) along with reduced immune activation (SH2D3C, ARHGEF3, TRAF3IP3) and inflammation (TNFAIP3, VDR, PLA2G4).
Conclusion: Oral cannabinoid treatment resulted in changes mostly in monocytes and CD8 T-cells profile and inflammation, while CBD+THC vs CBD alone had a more pronounced immune-regulatory effect. Findings suggest a potential role for oral cannabinoids as adjunct therapy for HIV-related inflammation and immune dysfunction.

Background:
HIV-1 persists during antiretroviral therapy (ART) and chronic immune activation/inflammation fuels comorbidities, such as cardiovascular disease (CVD). We previously demonstrated that myeloid cells expressing the enzyme ALDH (retinaldehyde dehydrogenase), which converts vitamin A into retinoic acid (RA), promote HIV-1 outgrowth in CD4+ T-cells via RA-dependent mechanisms. Thus, we hypothesized that ALDH activity is a novel functional marker of immunological dysfunction that predicts subclinical atherosclerosis in people with HIV (PWH).

Methods:
Studies were performed on PBMCs of ART-treated PWH (n=42) and uninfected controls (n=40) from the Canadian HIV/Aging Cohort Study (CHACS), with/without subclinical coronary artery atherosclerosis visualized as total plaque volume (TPV) and coronary artery calcium (CAC) score by computed tomography angiography. The ALDH activity was measured by flow cytometry on immune cells identified using lymphoid/myeloid lineage markers: CD4+CD3+ T-cells, dendritic cells (DC, CD3-HLA-DR+CD1c+), and monocytes (CD3-HLA-DR+CD1c-) subdivided into classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14-CD16+) monocytes.

Results:
The ALDH activity was predominantly detected in myeloid cells [i.e., dendritic cells (DCs), monocytes], mainly intermediate monocytes. The frequency of ALDH+ DCs and monocytes was significantly increased in PWH compared to controls, coinciding with an increased TPV (851 versus 143 mm3; p<0.0001). Counterintuitively, ALDH activity was slightly lower in TPV+ versus TPV- PWH. One explanation may be that a fraction of PWH (42%) received statins, which is known to decrease ALDH activity. Indeed, ALDH activity was significantly decreased in PWH receiving statins compared to statin-naive participants. Finally, the CAC score positively correlated with the frequency of ALDH+ monocytes in HIV+ART TPV+ participants (p=0.0390, r2=0.4534), suggesting a potential link between ALDH activity and plaque calcification.

Conclusion:
These studies point to ALDH activity in myeloid cells as a new functional marker of aberrant immune activation and potential predictor of CVD risk in PWH, and support the beneficial effects of statins in decreasing ALDH activity.

Background: Despite ART, people with HIV (PWH) are at higher risk of comorbidities including HIV-associated neurocognitive disorders (HAND). Worse HIV clinical outcomes are associated with (1) increased tryptophan (Trp) catabolism into Kynurenine (Kyn) via indolamine2,3-dioxygenase (IDO) expressed by myeloid cells, (2) imbalance in endocannabinoidome (eCBome) lipid mediators characterized by inverse relationship between N-acyl-ethanolamines (NAEs) vs 2-monoacylglycerols (MAGs), and (3) gut mucosal damage. Herein, we assessed the interplay between these perturbations in the context of HAND in PWH under ART.
Methods: Age- and sex-matched plasma samples from PWH on ART and clinically diagnosed with (n=40) and without (n=40) HAND, were obtained from the Brain Health Now cohort, in addition to 20 HIV- controls. Plasma eCBome mediators and tryptophan catabolites were measured by LC-MS/MS. Plasma inflammatory markers were quantified by Luminex or ELISA.
Results: Trp/Kyn ratios (IDO activity) were significantly increased in HAND+ compared to HIV- participants. Regardless of HAND status, significant increases in the Trp metabolites anthranilic acid, 5-hydroxy-indole-acetic acid were observed in PWH, while their levels of xanthurenic acid were decreased. HAND was associated with disrupted eCBome with increased levels of NAEs congeners PEA, SEA, OEA, LEA, and DPEA(n-6), but no change in MAGs. Significantly higher levels of MCP-1, TNF-α and sCD163 were found in HAND+ vs HIV-. Regardless of HAND, inflammatory markers IP-10, MIG and sTNFR-II, as well as I-FABP and Reg-3α (gut mucosal damage markers) and sCD14 (microbial translocation) were significantly elevated in PWH vs HIV-. No difference for IL-6 and IFN-γ were found among study groups.
Conclusion: HAND is associated with a peculiar immuno-metabolic signature characterized by increased IDO pathway activity, disrupted eCBome in favor of NAE congeners, and increased myeloid inflammatory markers MCP-1, TNF-α and sCD163.

Multiple genome-wide knockout/knockdown studies of viral infection have identified sets of host dependency factors (HDFs) that are essential for viral replication. Although these factors may be candidates for developing novel antivirals, defining candidates that do not lead to drug toxicity is challenging. One opportunity to identify targets is to leverage available human genome resources to determine which HDFs harbour homozygous loss of function polymorphisms in healthy people. To that end, we sought to combine data from 27 genome-wide host dependency factor screens that covered HIV, Hepatitis C, Hepatitis D, SARS-CoV-2, SARS-CoV, Ebola, Influenza A, Zika, Dengue and West Nile virus, with the genome aggregation database (gnomAD) including >125,000 human exome and >15,000 whole-genome sequences. We identified 2,907 unique HDFs combined across all viruses, including 353 which were essential for ≥2 viruses and 2 which were essential for 5 viruses. Of the combined list, 137 targets were deemed non-essential by the observed/expected constraint (O/E) score and the presence of homozygous loss-of-function variants found within the gnomAD control population. We identified AP1G2 (adaptor related protein complex 1 subunit gamma 2), a gene involved in clathrin-mediated transport within the trans-golgi network as a promising HDF target for HIV and SARS-CoV-2. AP1G2 has an O/E score of 0.96, placing it in the highly non-essential range (where 1.0 represents highly non-essential and 0 indicates essential). Currently CRISPR gene-editing in cell-lines is ongoing to confirm the HIV host dependency of AP1G2. We will assess HIV proliferation in AP1G2 -/- cell lines using X4 and R5-tropic HIV-1 strains and pinpoint the specific role of AP1G2’s in HIV replication. Future studies will be done to verify an impact on HIV and SARS-CoV-2 proliferation.