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. Surprisingly, AP1G2 knockout resulted in up to a 43-fold increase in HIV infection. Validation experiments are underway to investigate the underlying mechanism, including potential effects on CD4 or MHC-I trafficking. These findings may redefine AP1G2’s role in HIV replication and inform host-targeted therapeutic strategies.

Background: Current HIV prevention approaches - condoms, pre-exposure prophylaxis (PrEP), and antiviral microbicides - primarily target viral exposure or replication and do not address host determinants of susceptibility, such as inflammation, which increases the density and activation of CD4⁺ HIV target cells at the female genital tract (FGT). This study evaluated whether acetylsalicylic acid (ASA or aspirin) could induce a state of lower level of T cell immune activation (called immune quiescence) at the FGT of female sex workers (FSWs) from Nairobi, Kenya as a novel HIV prevention approach.
Methods: The Inducing Immune Quiescence (IIQ) study was a CIHR-funded randomized, open-label study assessing the ASA dose that optimally reduces CD4⁺ HIV target cells. FSWs were randomized to receive 81mg ASA, 325mg ASA, or no drug, daily for five months. Peripheral blood and endocervical cytobrush samples were collected at baseline and monthly visits. Flow cytometry was done to immunophenotype CD4+ T cells at baseline, month 2, and month 5.
Results: 312 FSWs were enrolled (104 per arm); 199 (62.8%) completed follow-up. No serious adverse events occurred. Common adverse events were heartburn (46%), headache (14.9%), dizziness/nausea/vomiting (7.3%), and loss of appetite (1.3%). The incidence of sexually transmitted infections was similar between study arms during follow-up. At month 5, there were no significant differences across arms in endocervical CD4⁺ cells expressing CD69 or CCR5, nor in α4β7⁺, Th17, or Th17⁺CCR5⁺ subsets. In blood, both ASA doses were associated with post-treatment increases in CD4⁺CCR5⁺ (p=0.002), Th17 (p=0.001), and Th17⁺CCR5⁺ cells (p=0.007).
Conclusion: Oral ASA did not significantly alter genital HIV target cell proportions. The post-treatment systemic increase in target cells suggests that ASA may suppress immune activation during active use with quicker systemic than mucosal rebound. Further analyses will clarify ASA’s potential role as an adjunct HIV prevention strategy during bacterial vaginosis or STIs.

Many HIV controllers, who naturally suppress viral replication without therapy, display HLA-B57-restricted CD8 T cells against Gag KF11 (KAFSPEVIPMF). We identified two T cell receptors (TCRs), Gp3x1 and Gp3x2, that recognize KF11 presented on both HLA-B57 and HLA-E. These TCRs differ by 7 amino acids in their CDR3ß, but Gp3x2 demonstrates higher sensitivity and cross-reactivity. We performed functional assays and structural modelling to investigate the basis for this difference.
TCR genes (and mutants) were synthesized, cloned and expressed in Jurkat-CD8 cells to assess their function using an NFAT luciferase reporter assay. Structures of TCR-peptide/HLA (pHLA) complexes were predicted and analyzed using TCRdock, an AlphaFold-based computational tool refined using validated TCR-pHLA data. Structure analyses, including contact mapping, distance measurements, and hydrogen-bonding, identified contact points that may contribute to TCR sensitivity.
TCR reporter assays confirmed KF11 recognition on HLA-B57 and HLA-E, with Gp3x2 exhibiting higher sensitivity for both targets. Gp3x1 sensitivity was enhanced by Gly substitutions replacing CDR3ß residue 6 (Pro) or 7 (Asp), suggesting these amino acids inhibited TCR function. Structure models predicted that both TCRs engage KF11 residues 7-10 (VIPM). Consistent with its enhanced sensitivity, six contacts were predicted between Gp3x2 and KF11, compared with 3 between Gp3x1 and KF11. This difference was largely due to Gp3x2 CDR3ß residue 6 (Asn), which formed two hydrogen bonds with KF11 residue 8 (Ile).
By combining functional assessments with structural modelling, we can identify features that modulate TCR sensitivity and HLA recognition. For Gp3x1 and Gp3x2 TCRs, we identified CDR3β positions 6 and 7 as determinants of pHLA binding, with close proximity and hydrogen-bonding driving affinity. Ongoing studies may identify new features that modulate TCR cross-recognition of molecules. Determining the molecular basis of protective immune responses, particularly in the context of non-classical HLA-E, could inform vaccine and therapeutics development.

Invariant Natural Killer T (iNKT) cells are dynamic lymphocytes critical in anti-HIV immunity. Immune dysfunction persists in people living with HIV (PLWH) despite ART initiation and viral suppression but is different in immunological responders (IR) versus non-responders (INR), defined as CD4/CD8 of ≥ 1 or <1, respectively. Development of immune checkpoint inhibitors (ICIs) targeting immune checkpoints (ICs) PD-1 and LAG-3 warrants research elucidating IC-related dysfunction of iNKT cells in PLWH, and in vitro assessment of LAG-3 and PD-1 ICIs on iNKT cell function. The overall hypothesis is that ICs LAG-3 and PD-1 regulate iNKT cell activation in healthy conditions and are associated with iNKT cell dysfunction in chronic HIV infection, and by blocking LAG-3 and/or PD-1 with ICIs in vitro, iNKT cell function will be enhanced. Studies utilizing peripheral blood mononuclear cells (PBMCs) from healthy donors led to the development a novel in vitro CD1d-tetramer iNKT stimulation which was as effective as the ⍺-GalCer gold-standard iNKT stimulant, as well as determined iNKT cell PD-1 and LAG-3 expression peaked after 2-4 days of activation and was inversely related to cell function. Utilizing PBMCs from PLWH (n=38), there was a unique ex vivo CD4+ iNKT cell phenotype in the INR subgroup and a dysfunctional state of CD4- CD8- double-negative (DN) iNKT cells upon stimulation in the INR subgroup. There was significantly enhanced proliferation and activation of iNKT cells in response to the in vitro dual LAG-3+PD-1 ICIs. Overall, this study provides evidence of targeting immune checkpoints LAG-3 and PD-1 in enhancing in vitro iNKT cell function in the context of HIV-1 infection. Future directions include understanding the effects of harnessing iNKT cell function in PLWH as a feasible immunotherapeutic option and its potential as a focus of future clinical investigation.

Immunization strategies capable of eliciting VRC01-class broadly neutralizing antibodies (bnAbs), which target the conserved CD4-binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env), are a key goal in HIV-1 vaccine development. VRC01-class germline precursors are sterically hindered from binding most HIV-1 Envs by a conserved glycan at Asn276 near the CD4bs periphery. To circumvent this barrier, the VRC01 germline-targeting immunogen eOD-GT8 is engineered without the Asn276 glycan. Since affinity-matured VRC01-class bnAbs accommodate this glycan, its reintroduction in booster immunogens is considered necessary to support proper maturation pathways. However, the effect of sequential immunization with glycan-modified variants on downstream antibody responses remains unclear.

Here, we present initial findings from antibody responses elicited by eOD-GT8 and glycan-modified variants in human-antibody transgenic TRIANNI mice. Mice immunized twice with the eOD-GT8 60mer produced sera that neutralized an HIV-1 strain lacking the Asn276 glycan (~30% at 1:20 dilution) but failed to neutralize the same virus modified to include the Asn276 glycan. In contrast, mice boosted with an Asn276 glycan-bearing eOD-GT8 variant produced sera that modestly neutralized HIV-1 strains regardless of glycan status (30–40% at a 1:20 dilution), suggesting improved neutralization breadth. These findings suggest that exposure to a glycan-modified eOD-GT8 immunogen may shift serum antibody responses toward more native-like HIV-1 Env recognition.

To further investigate these responses, antibodies were cloned from singly sorted eODGT8pos/eODGT8KOneg memory B cells. ELISA analyses so far show that approximately one-third of the cloned antibodies bind eOD-GT8–lumazine synthase. VH gene usage does not distinguish binders from non-binders, as both predominantly use VH3-23. However, binding antibodies exhibited restricted VH3/VK3 pairing and 15 to 16-residue HCDR3s, whereas non-binders were more diverse in both pairing and HCDR3 length. Ongoing characterization aims to define features of productive antigen engagement to inform alternate immunogen design strategies that may support antibody precursor maturation toward desirable bnAbs.

T-cell function remains impaired in people with HIV (PWH), even following extended antiretroviral therapy (ART), partially due to metabolic dysfunction and inability to induce autophagy. We have found elevated levels of the autophagy inhibitor acyl coA binding protein (ACBP) in the plasma of ART-treated PWH as compared to controls. Herein, we assessed the influence of extracellular ACBP on T-cell function in PWH on ART.

Methods
Plasma ACBP were quantified by ELISA in 50 PWH on effective ART (mean duration of 14.7 years) and 30 controls with similar age. In vitro, recombinant ACBP (recACBP) was added at increasing concentrations up to 10 µg/mL on PBMCs from PWH on ART and controls. Lipid uptake and T-cell responses were assessed by flow cytometry. A synthetic acyl-coA was added to cell culture at up to 10 µg/mL.

Results
Plasma ACBP levels were higher in PWH on ART compared to controls (median 127.5 vs 78.1 ng/mL, p=0.03), independently of age and sex.
To mimic the effect of extracellular ACBP, we added recACBP to PBMCs from ART-treated PWH or controls before stimulating them. RecACBP prevented CD4 and CD8 T-cell cytokine production and secretion as well as cell proliferation in response to phorbol-myristate acetate, anti-CD3 antibodies or HIV peptides.
RecACBP addition decreased expression of autophagy markers Beclin1, p62 and ULK1 in anti-CD3 stimulated CD4 and CD8 T-cells. Treatment with autophagy inducers spermidine or dimethyl-oxoglutarate did not rescue T-cell responses.
RecACBP treatment increased the lipid uptake as observed with BODIPY lipid probes. Addition of acyl-CoA lipids only did not influence T-cell responses. However, recACBP and acyl-CoA addition totally blocked T-cell responses and induced cell death.

Conclusion
Extracellular ACBP increase T-cell lipid uptake, leading to metabolic irresponsiveness and T-cell death, warranting the development of ACBP inhibitors to enhance anti-HIV T-cell responses in PWH, towards an HIV cure.

Natural Killer (NK) cells and interferon-alpha (IFN-α) are important innate immune defences against HIV-1 infection. NK-cell-based immunotherapies represent a potential frontier for HIV-1 eradication. However, a hallmark of HIV-1 pathogenesis is profound immune impairment, which can impact NK cell function. Humans produce 12 IFN-α subtypes that display distinct potencies against different viral infections. We have found that the IFN-α14 subtype is more effective than the clinically approved IFN-α2 subtype at suppressing HIV-1 infection. Additionally, preliminary data suggest that the potency of the IFN-α14 subtype may involve enhancement of NK cell function. In this investigation, we augmented NK cell development in the C57BL/6 Rag-/-yc-/-CD47-/- triple knockout - bone marrow, liver, and thymus (TKO-BLT) humanized mouse model via adeno-associated virus (AAV)-mediated interleukin-15 (IL-15) delivery. Using this TKO-BLT15 mouse model, we evaluated the efficacy of the IFN-α14 subtype to enhance NK cell function relative to clinically approved IFN-α2. Daily administration of IFN-α14 for 10 days post-infection significantly suppressed HIV-1 viremia and invigorated NK cell effector functions, as evidenced by marked upregulation of the proliferation marker Ki-67 and the degranulation marker CD107a. Furthermore, IFN-α14 treatment significantly increased expression of the cytotoxic ligand TRAIL and normalized levels of the exhaustion marker TIGIT to uninfected levels. Ex vivo assays against isogeneic HIV-1-infected CD4+ T cells confirmed that NK cells from IFN-α14-treated animals exhibited increased direct cytotoxicity and modest enhancement of antibody-dependent cellular cytotoxicity (ADCC) compared to NK cells from IFN-α2-treated mice. Overall, the data suggest that IFN-α14 has beneficial effects on NK-cells during acute HIV-1 infection, warranting further investigation into its use as an immunotherapeutic for the elimination of HIV-1 reservoirs.

Background: In people with HIV (PWH), liver dysfunction and chronic inflammation may persist despite HCV sustained virologic response (SVR) due to liver tissue damage. Chronic inflammation and gut mucosal damage in PWH, alongside enhanced tryptophan (Trp) catabolism to kynurenine (Kyn) via indoleamine 2,3-dioxygenase (IDO) induced by IFN-γ and TNF-α signaling, are associated with worse clinical outcomes. We evaluated the impact of liver fibrosis and SVR on systemic inflammation, Kyn pathway, and gut mucosal damage in HIV/HCV coinfection.
Methods: Plasma samples from ART-treated HIV/HCV-coinfected participants (pre- versus 6-12 months post-SVR) with (n=21) or without liver fibrosis (n=51) were obtained from the Canadian Coinfection Cohort, along with n=20 HIV-uninfected controls from the Canadian HIV Aging Cohort. Inflammatory cytokines and gut mucosal damage markers were quantified by Luminex or ELISA. Trp catabolites were measured by LC-MS/MS.
Results: Compared to uninfected participants, HCV-coinfected PWH exhibited higher plasma levels of IP-10, MIG, TNF-α, and sTNFRII, while elevated IFN-γ and IL-6 were observed exclusively in fibrotic individuals. Coinfection was associated with upregulation of the Kyn pathway, including increased levels of Kyn, 3-hydroxykynurenine (3-H-Kyn), quinolinic acid (QA), picolinic acid (PA), increased IDO and Kynurenine-3-monooxygenase activities and reduced kynurenine aminotransferase activity. Fibrotic participants displayed higher IL-6, TNF-α, sTNFRII, and downstream Kyn metabolites 3-HAA, QA, PA. Following SVR, inflammatory markers decreased in both groups but normalized only in non-fibrotic participants. SVR normalized terminal Kyn metabolites (3-HAA, QA, PA) but not upstream dysregulation. Markers of gut mucosal damage (REG-3α, sCD14) remained elevated irrespective of SVR or fibrosis status.
Conclusion: HCV/HIV coinfection is associated with increased proinflammatory IFN-γ and TNF-α signaling, normalized after SVR only in non-fibrotic individuals. The Kyn pathway was also upregulated in HIV/HCV coinfection, while SVR resulted in only partial normalization of terminal metabolites. Altogether, liver fibrosis is associated with persistent immuno-metabolic dysregulation in PWH, even after SVR.