Background: Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) suffer from a high burden of infectious and non-infectious pulmonary diseases, suggesting that their lung immunity is not fully restored. Cytotoxic CD8 T-cells are essential in controlling chronic viral infections. However, excessive CD8 T-cell activation during HIV infection can contribute to lung mucosal tissue damage. Furthermore, tobacco smoking is part of the lifestyle of many PLWH that changes the lung environment, promoting pulmonary inflammation. Herein, we first validated CD8 tissue-resident memory (Trm) and non-Trm markers from animal models in the human lung and used them to characterize the effects of HIV and smoking on pulmonary cytotoxic CD8 T-cells.

Methods: Bronchoalveolar lavage (BAL) fluid and matched blood were obtained from asymptomatic ART-treated PLWH (smokers: n=4; non-smokers: n=7) and seronegative controls (smokers: n=4; non-smokers: n=8). Lymphocytes were isolated and CD8 subsets were characterized by multiparametric flow cytometry.

Results: Human lung Trm consisted of primarily CD69+ cell subsets expressing CD103 and/or CD49a. CD8Trm were largely CXCR6+CXCR3+, while CD8 non-Trm were largely CXCR6-CXCR3+. Levels of CX3CR1 and KLRG1 were highest in CD8 Non-Trm. Most airway CD8 T-cells produced GzmA/B but little Perforin. Both smoking and HIV infection were independently associated with a significant increase in total CD8 T-cell frequencies in BAL. HIV and smoking were associated with increased expression of Perforin and GzmB respectively. GzmA+ and GzmB+ CD8 T-cells showed higher expression of CD103 and CXCR6 in smokers and higher frequencies of CX3CR1+KLRG1+ and Ki67+ cells in PLWH.

Conclusion: Smoking and HIV could promote cytotoxic CD8 T-cell retention in small airways through different mechanisms. While smoking likely increases recruitment and retention of CD8Trm via CXCR6 and CD103 respectively, HIV is associated with CD8 non-Trm recruitment via CX3CR1 from the periphery, which could contribute to increased tissue damage in PLWH despite ART.

Background: Of the People Living with HIV (PLWH) enrolled in the Canadian HIV and Aging Cohort Study (CHACS), 94% were co-infected with cytomegalovirus (CMV), which drives the expansion of NKG2C+CD57+ adaptive-like Natural Killer (adapNK) cells. Frequencies of adapNK cells did not differ in CMV+PLWH and CMV+HIV- persons enrolled in the CHACS (groups 1/2) in contrast to reports by others where the frequency of adapNK cells was higher in CMV+PLWH than in CMV+HIV- persons. We questioned whether the discrepancy between our results and those of others was due to differences in age or time on-ART

Methods: We evaluated the frequency of CD3-CD14-CD19-CD56dimNKG2C+CD57+ adapNK cells in 3 groups of CMV+PLWH and 2 groups of CMV+HIV- persons. Group 3/4 were Primary infection (PI) cohort participants.

Groups 1
CMV+PLWH
Older 2
CMV+HIV-
Older 3
CMV+PLWH
Younger 4
CMV+PLWH
Older 5
CMV+HIV-
Younger
Age 55.7(51.1,59.3) 57.7(50.8,62.4) 33.2(30.8,38.2)
54.1(52.9,60.1) 31.5(25.2,36.6)
ART duration Long
None Short Short None
Years on-ART
16.0(8.6,19.1) 1.4(1.0, 2.2) 1.94(1.8,2.0)
% adapNK cells 15.9(5.8,39.4) 13.7(3.7,33.9) 39.8(18.8,58.2) 27.5(18.8,52.85) 15.6(2.5,33.3)


Results: AdapNK cell frequency was similar in groups 1 and 2, group. Group 3 had a higher frequency of adapNK cells than group 5 (p=0.003, Mann-Whitney). Groups 1 and 4 were of similar age and significantly older than group 3. Groups 3 and 4 were on-ART for a similar duration that was of shorter than group 1. The frequency of adapNK cells was similar in groups 3 and 4 and higher than in group 1. Time on-ART was negatively correlated with adapNK cell frequency

Conclusion: The absence of significant differences in the frequency of adapNK cells in older CMV+PLWH and CMV+HIV individuals (groups 1 and 2) and in groups 3 and 4 of different ages but similar time on-ART was due a time on-ART dependent decline in the frequency of these cells in group 1

Background and Objective: Development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV vaccine and targeting the important regions of HIV-1 envelope (Env) are very promising at optimizing HIV-specific immune responses. The aim of this study is to develop an Ebola virus glycoprotein (EboGP) mucin-like domain (MLD) replacement system as a new DC-targeting vaccine approach against HIV-1.
Methods: We have developed an EboGP-based chimeric protein technology by replacing the MLD of EboGP with HIV C2-V3-C3 (134 aa) (EboGP∆M-V3) or C2-V3-C3-V4-C4-V5-C5 (243 aa) (EboGP∆M-V3-5) polypeptides. Virus-like particles (VLPs) were produced in 293T cells transfected with EboGP∆M-V3, or EboGP∆M-V3-5 expressing plasmids and packaging plasmid (Δ8.2). Furthermore, we have produced a recombinant vesicular stomatitis virus (rVSV) co-expressing HIVenv/EboGPΔM-V3 chimeric protein. Above VLPs or rVSV were used to immunize BALB/c mice and at 35 days post immunization, the sera, vaginal fluid and spleens from immunized mice were collected and measured for anti-HIVgp120 humeral or cellular responses.
Results: The results showed that both EbGPΔM-V3 and EbGPΔM-V3-5 incorporated in the HIV VLPs maintained the high efficiency of EboGP-mediated viral entry into human macrophages and dendritic cells (DCs). Animal studies revealed that immunization with VLPs containing the above chimeric proteins, especially EbGPΔM-V3, induced significantly stronger anti-HIV antibodies than HIV gp120 alone in mouse serum and vaginal fluid. Moreover, the splenocytes isolated from mice immunized with EbGPΔM-V3 VLPs produced significantly high levels of IFN- γ, IL-2, IL-4, IL-5, and MIP-1a. Additionally, we have demonstrated that rVSV-HIVenv/EboGPΔM-V3 elicited robust anti-HIV antibody responses that strongly cross-reacted with HIV gp120 recombinant proteins derived from clades B (IIIB), C (C.1086D7), and AE (AE.A244D11).
Conclusion: Our study demonstrated the feasibility of this novel DC-targeting vaccine approach in delivering various large heterologous polypeptides of HIV-1 and/or other emergent infections to DCs for eliciting efficient immune responses against disease.

Background
Invariant Natural Killer T (iNKT) cells are innate lymphocytes critical in combatting viral infection. Our lab showed in HIV infection, expression of lymphocyte-activation-gene-3 (LAG-3), an inhibitory checkpoint marker, is increased on iNKT cells and correlates with decreased function. Another checkpoint, PD-1, also correlates with decreased function. LAG-3 and PD-1 expression and their relationship to iNKT cellular function is not well characterized, and we hypothesize that blocking PD-1 alone, or in conjunction with LAG-3 via immunotherapeutic blockades, will enhance iNKT function.

Methods
Utilizing peripheral blood mononuclear cells from HIV-uninfected donors (n=4), iNKT expression of LAG-3 and PD-1 and cytokine production was assessed via a multi-day (2, 4, 7, 10 day) in vitro stimulation assay. Efficacy of anti-LAG-3 and anti-PD-1 antibody blockades were assessed via a 10-day proliferation assay (n=9).

Results
LAG-3 and PD-1 proportion and median fluorescence intensity (MFI) peaked at Day 4 and 7, respectively (LAG-3: 88.5%, 6163.8 MFI; PD-1: 80.5%, 7731.8 MFI), with a steep decrease by Day 10, when iNKT proliferation peaked, representing a relationship between LAG-3 and PD-1 expression and iNKT cell activation. Single PD-1 and dual PD-1+LAG-3 blockades showed enhanced proliferation with means of 6 and 6.29 log2 fold-change compared to the stimulation without blockade control (3.07 log2 fold-change) (p=0.0005), with follow-up analysis showing the dual PD-1+LAG3 blockade system significantly enhanced iNKT cell proliferative capacity compared to the single PD-1 blockade (p=0.013).

Significance
This study is the first to report iNKT cell LAG-3 and PD-1 expression kinetics and provide proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets enhancing iNKT proliferative ability. This blockade system will be applied in vitro to samples from people living with HIV to assess if HIV-mediated dysregulation of iNKT function can be reversed, to ameliorate immune responses to various opportunistic infections and boost viral control in a functional HIV cure approach.

One of the major complications of human immunodeficiency virus type-1 (HIV-1) infection in the post-antiretroviral therapy era is the development of neurological dysfunctions known as HIV-1-associated neurological disorders (HAND), which could affect up to 50% of people living with HIV.
Microglia are long-lived cells, which may allow them to act as a viral reservoir of active and latent infections. Given that dysregulated microglial functions have emerged as possible causes for various CNS disorders, we postulated that HIV-induced microglial inflammation is an important mediator of HAND.
We used human primary fetal microglia (HPFM) to study the consequences of chronic HIV-1 infection on neuroinflammation. We observed a sustained viral production under antiretroviral treatment in HPFM, correlated with inflammatory responses characterized by an increased CXCL10 secretion, and a potent LPS-induced IL-1β secretion combined with a reduced IL-10 response. In addition, RNA expression for some inflammation related genes was deregulated in infected cells. The TREM-2-CD33 axis was also deregulated, with a significantly reduced TREM-2 expression.
We further analyzed the mechanisms triggering IL-1β secretion. We measured a very modest basal secretion but a robust LPS-induced secretion only in HIV-infected cells, suggesting that HIV possess weak priming but potent signal 2 inducing abilities. HIV-induced IL-1β secretion appears to be only partially dependent on NLRP3 and caspase 1. Preliminary data suggest that secretion mechanisms involve glycogen synthase kinase-3β mediated autophagy inhibition. Our results also suggest that the inflammatory response is independent on Nef and gp120 but at least partially dependent on Vpr.
Finally, our results evoke a microglia-astrocyte interplay whereby astrocyte derived soluble factors increase both HIV-1 viral production and IL-1β secretion, and conversely infected microglia conditioned medium induce a proinflammatory phenotype in astrocytes.
In conclusion our data strongly suggest that HIV productive infection in microglia is responsible to some extent of the neuroinflammation leading to HAND.

Background: Dolutegravir (DTG) is a well-tolerated drug used in first-line combination antiretroviral therapy. However, DTG has been associated with oxidative stress in clinical and in vitro studies, a pathway that may contribute to the small increased risk of neural tube defects (NTD) reported. We investigated the effect of DTG on oxidative stress pathways using a murine pregnancy model.

Methods: C57BL/6 mice were mated and randomly assigned to daily treatment with either control (water, N=10), 1xDTG (2.5mg/kg DTG+33.3/50mg/kg emtricitabine (E)/tenofovir disoproxil fumarate (T), N=10), yielding therapeutic levels of DTG, or 5xDTG (12.5mg/kg+33.3/50mg/kg E/T, N=10), yielding supratherapeutic levels of DTG. Placentas were collected at gestational day 9.5. Gene expression of oxidative stress markers was assessed by RT-PCR with HPRT as the housekeeping gene using 2 placentas/litter. Linear regression analyses were used to examine relationships between log-transformed factors and treatment. Percentage differences (95% confidence intervals) were calculated by exponentiating the coefficients.

Results: Compared to control, in both therapeutic and supratherapeutic DTG-treatment groups we observed a significant decrease in expression of catalase (1xDTG vs. control: -69% (-53%, -79%), p<0.001; 5xDTG vs. control: -58% (-37%, -72%), p<0.001) and NADPH-oxidase 1 (1xDTG vs. control: -78% (-61%, -88%), p<0.001; 5xDTG vs. control: -83% (-69%, -90%), p<0.001). Expression levels of superoxide dismutase 1 (SOD1) were similar between groups, and SOD2 was marginally lower in the 1xDTG group vs. control (p=0.046). Expression of the angiogenic factor VEGF was also downregulated in both DTG groups (-65% (-45%, -77%), p<0.001; -53% (-27%, 70%), p=0.001).

Conclusion: DTG was associated with significant downregulation of the placental antioxidant gene catalase which may suggest increased oxidative stress, a cellular imbalance common to many pathways leading to congenital defects. We also observed downregulation of the ROS producing NADPH-oxidase, as well as VEGF. These data may suggest that DTG influences angiogenic pathways in the placenta.

The hallmark of HIV-1 infection is rapid and irreversible damage to the immune system. Despite efficient suppression of viral replication by antiretroviral therapy (ART), immune dysfunction persists in people living with HIV (PLWH), leading to comorbidities and vulnerability to co-infections. A strong predictor of viral rebound and immune deficiency is the enrichment of extracellular vesicles (EVs) with microRNA 155 (miR-155). In addition, miR-155 enriched EVs have been shown to enhance HIV-1 infection. MiR 155 is a multifunctional microRNA that regulates genes involved in many immune functions such as immune cell development, activation, and survival. We therefore hypothesized that miR-155 enriched EVs increase HIV 1 infection by promoting a pro-inflammatory state in infected cells.
We used two viral preparations (NL4.3BE with or without miR 155 enriched EVs) to infect human peripheral blood mononuclear cells (PBMCs). RT-qPCR was used to measure the expression of lamin B1, a structural component of the nucleus, and SOCS1 (suppressor of cytokine signaling 1), a regulator of cytokine-mediated cell activation, both targets of miR-155 in infected PBMCs. We also measured IL-15 expression in PBMCs by RT-qPCR and quantified IL-8 in the cell-free supernatant by ELISA. Finally, we infected PMBCs with or without IL-15 to determine if this cytokine had an effect on HIV-1 infection.
Lamin B1 and SOCS1 were downregulated in the presence of miR-155 enriched EVs. Infected PBMCs that received miR-155 enriched EVs overexpressed IL-8 and IL-15. We measured a higher viral load when we added IL-15 to PBMCs during the infection.
Vesicular miR-155 promotes cytokine production in immune cells. The addition of IL-15 to infected cells increased viral replication. These results suggest that the vesicular miR-155 increase HIV-1 infection and viral integration by increasing the production of inflammatory cytokines. This study highlights a potential mechanism for the role of vesicular miR 155 in viral rebound.

The heavily glycosylated HIV-1 envelope glycoprotein (Env) is the sole viral antigen present at the surface of virions and infected cells, thus representing the main target for antibody responses. New small molecules entry inhibitors are being used for the treatment of HIV-1 infected-individuals. The FDA-approved HIV-1 attachment inhibitor, temsavir (BMS-626529), is one of them and binds to the gp120 preventing the interaction between Env and the host cell receptor CD4. This molecule also stabilizes Env in a prefusion “closed” conformation. This conformation, known as State 1, is preferentially targeted by broadly neutralizing antibodies (bNAbs). We recently reported that temsavir (TMR) impacts the overall glycosylation of Env but also decreases its cleavage, resulting in a change in Env antigenicity. Here we extended these results to a panel of primary Envs and molecular infectious clones (IMCs). We observed that TMR affects Env antigenicity of most of them but to different extents. Interestingly, this phenotype seems to be linked to the capacity of TMR to impair Env cleavage. Finally, we observed that the effect of TMR on Env processing and bNAb recognition correlate well with the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Overall, our results suggest that the impact of TMR on Env processing and antigenicity should be considered for the development of new antibody-based approaches for temsavir-treated individuals.

Introduction :
Studies of the immune system show differences in the composition and proportion of lymphocytes in the peripheral blood and lymphoid organs. During an HIV infection, immunological perturbations occur, including transcriptomic changes in immune cells. Understanding these changes in different organs is necessary to develop personalized treatment. We chose to study SIVmac251 infection in Macaca mulata, a relevant model for AIDS studies.

Objectives:
We want to characterize specific or common transcriptomic signatures of lymphoid populations in different organs and create an ATLAS with our dataset and associated bioinformatics analyses.

Methods :
We have uninfected, infected macaques (SIVmac251) and infected individuals who received early antiretroviral therapy (ART). Our laboratory has a cell library and lymph library of these different individuals. Samples include lymphoid tissues such as the spleen, the mesenteric and axillary/inguinal lymph nodes, and the blood. Non-lymphoid tissues such as the liver, lung, or brain are also available. With these samples, the cell sorting by flow cytometry allows us to target specific cell types and subtypes of these cells. By Bulk RNAseq analysis, we obtained the specific transcriptome of these cells. To interpret these results, we have developed a pipeline allowing quality control of reads, alignment with the reference genome, and quantification. For differential expression analysis, we used the DESeq2 package. Furthermore, these approaches are complemented by unsupervised single-cell analysis techniques.

Results:
We have already established transcriptomes for the spleen and mesenteric lymph nodes (for myeloid cells, B and T cells). Among the latter, we have already studied some subpopulations like CD4, CD8, and Tfh for T cells.

Conclusion:
This ATLAS will allow us to create a map of transcriptomic expressions for immune cells in different lymphoid organs under multiple conditions.

While it has long been known that enveloped viruses incorporate cellular proteins as they bud through the plasma membrane of infected cells, the impact that incorporated proteins have on viral pathogenesis is often undervalued. For example, some cellular proteins such as tetherin or SERINC5 inhibit HIV infection, whereas others like ICAM-1 or LFA-1 can confer advantages to virions and enhance viral infectivity. While a selection of cellular proteins have been characterized for the roles they play in HIV pathogenesis, many remain unknown or understudied. Mass spectrometry is one technique that has been used previously to identify proteins associated with HIV. Although this technique is powerful and has identified many targets with high sensitivity, it does not determine whether the proteins are inside the virion or on the virion surface. This is an important caveat since several proteins on the HIV surface are known to affect viral adhesion and homing, thus determining protein localization within HIV can provide context as to the role the protein might play during in vivo infection. As a new method for specifically phenotyping the surface of HIV virions, we adapted a commercially available flow cytometry-based cell surface screening kit for use in staining HIV particles. Using this method, we screened for over 300 antigens on the surface of 4 different HIV isolates propagated in primary cells. The screen unveiled over 30 novel proteins in the envelope which were widely reproducible across all viral strains tested and, to our knowledge, have not been previously described in the literature. Bioinformatics analyses show that these novel candidates play roles in adhesion, cell activation and proliferation. Importantly, the screen detected many proteins that are already known to be incorporated in HIV while also yielding many undetectable proteins, which collectively provided a strong validation for protein specificity with this technique.

Background: Tissue-resident macrophages (TRM) exist as either embryonically-derived long-lived TRMs with the capacity for self-renewal and/or short-lived TRMs derived from infiltrating monocytes. Difficulties in accessing deep tissue limit functional studies on mechanisms that govern HIV-1 infection/persistence in distinct TRM subsets of people living with HIV (PLWH) receiving antiretroviral therapy (ART). Term placenta is a transitory organ rich in both embryonically-derived (fetal) and monocyte-derived (maternal) TRMs called Hofbauer cells (HbC) and decidual macrophages (dMF), respectively. Thus, term placentae may represent a model to parse out mechanisms of HIV-1 persistence within TRMs.

Methods: Term placentae from HIV-1-negative women were collected within 2 hours of delivery. Placenta was dissected to separate the maternal decidual tissues from the fetal villous tissues. The two compartments were mechanically and enzymatically digested to isolate leukocyte populations. A cocktail of fluorochrome-conjugated antibodies was designed for flow cytometry (BD Symphony A5) identification of myeloid and lymphocyte subsets. In parallel, a fraction of cells was exposed to transmitted founder THRO HIV-1 strain (HIVTHRO) and cultured in the presence of M-CSF for 23 days. HIV-1 integration and replication was measured by PCR and ELISA, respectively.

Results: Our preliminary results indicate that maternal decidual and fetal villous tissues exhibited similar frequencies of helper (CD4+CD3+) and cytotoxic (CD8+CD3+) T cells, NK cells (CD56+), B cells (CD19+) and myeloid cells (CD14+CD3-). Moreover, specific markers were used to distinguish HbC (FOLR2+HLA-DR-) from dMF (FOLR2-HLA-DR+). Finally, we observed more robust viral replication in maternal (peak of 33, 672 pg/ml HIV-p24 at day 13 post-infection) compared to the fetal leukocytes (peak of 20,150 pg/ml HIV-1 p24 at day 17 post-infection), thus raising questions on molecular mechanisms underlying these differences.

Conclusion: The placenta represents an accessible experimental model to study mechanisms of HIV persistence in embryonically and versus monocyte-derived TRMs and explore consequences on placental homeostasis.

Background: People living with HIV (PLWH) experience accelerated cellular and immunological aging relative to their HIV-negative peers. This may be influenced by co-infection with other chronic/latent viruses (including CMV, EBV, HHV-8, HSV-1, HSV-2, HCV, and HBV) that are known to be associated with markers of aging or age-associated diseases. Our aim was to determine associations between sex, HIV status, number of chronic/latent viral infections, and markers of immune aging, in a cohort of PLWH versus controls.

Methods: CARMA cohort participants, balanced for sex (n=26 female, n=25 male) and HIV status (n=25 HIV+, n=26 HIV-), with a broad range of viral coinfections were selected for this analysis, with age being balanced throughout [20-76y]. Infection status for CMV, EBV, HHV-8, HSV-1, and HSV-2 was determined serologically; HIV, HCV, and HBV were self-reported. Stored live PBMCs were used to assess the CD4:CD8 and proliferation-competent:senescent CD8 T-cell (CD8+,CD28+:CD28-) ratios via flow cytometry. Participants were dichotomized by sex, HIV status, and above and below median number of non-HIV chronic/latent viral infections. Associations between number of viruses, HIV status, and age were assessed using Mann-Whitney and Spearman’s correlation.

Results: Female participants exhibited higher CD4:CD8 (1.8[0.9-2.7] vs 1.1[0.3-1.7], p=0.019 and CD8+,CD28+:CD28- ratios (1.4[0.9-2.7] vs 0.9[0.3-1.5], p=0.012) than males. These ratios were also lower in PLWH vs. controls CD4:CD8 (0.7[0.3-1.2] vs 2.0[1.7-3.3], p<0.001 and CD8+,CD28+:CD28- (0.9[0.3-1.4] vs 1.6[0.7-2.7], p=0.0198). Individuals with above-median chronic/latent viral infections exhibited lower CD4:CD8 (1.0[0.5-1.8] vs 1.7[1.0-3.1], p=0.021), but no difference in CD8+,CD28+:CD28- ratio. No correlation was observed with age.

Discussion: These preliminary data suggest that male sex and HIV status may be associated with unfavourable immune cell ratios. Increasing our sample size to >300 CARMA participants with well-balanced age, sex, and HIV status is planned and will allow multivariable analysis and identification of independent associations; thus increasing our understanding of immune aging.

BACKGROUND
Rates of sexually transmitted and blood borne infections (STBBIs) are rising in Canada. Both structural and individual barriers exist in obtaining testing for STBBIs. Dried blood spots (DBS) are a proven method of blood collection and are used in lower resourced settings to diagnose STBBIs but are not validated within Alberta-based diagnostic laboratories. Blood collection for DBS is also simpler, requiring only a few drops from a lanced finger rather than venipuncture, and scalable. We hypothesized that DBS samples would render similar results to traditionally collected blood samples for STBBIs.

METHODS
Up to 50 participants with each of known HIV, Hepatitis B (HBV) and C (HCV) or, past syphilis infections will be recruited from local subspecialty clinics to provide phlebotomy-collected whole blood samples. From each tube of whole blood, 3-4 DBS cards were prepared, dried, punched, and eluted according to predefined standards. The eluate and plasma samples were then tested for HIV, HCV and syphilis antibodies and HBV surface antigen, using the Abbott Alinity chemiluminescent immunoassay serology platform. HIV+ eluates were confirmed by the Geenius immunoblot.

RESULTS
Preliminary results of 8 participants include 5 having known HIV and 3 having recent syphilis. Among known HIV+ participants, the results of all 5 DBS samples were concordant with matched plasma samples on both the primary and confirmatory serologic assays. Among the 3 syphilis samples, the results were again concordant between DBS and matched traditional serology testing. Recruitment of additional patients with HIV, HBV, HCV, and syphilis is ongoing.

CONCLUSION
These limited data are encouraging and suggest DBS will have a role in combating rising STBBI rates via new testing options in Alberta, however, additional study is required to confirm test performance and population benefits.

Background: Despite the success of antiretroviral therapy (ART) in reducing viral replication, people with HIV (PWH) continue to suffer from chronic inflammation leading to accelerated comorbidities, such as atherosclerosis and coronary artery disease (CAD). Inflammatory monocytes play an important role in the development of atheromatous plaques, but their role in CAD during chronic HIV inflammation remains understudied.

Methods: Blood specimens were obtained from ART-treated PWH with (HIV+CAD+; n=38) and without subclinical CAD (HIV+CAD-; n=40) and uninfected controls with (HIV-CAD+; n=19) and without subclinical CAD (HIV-CAD-; n=24) from the Canadian HIV and Aging Cohort. CAD was determined by the presence of coronary artery plaques measured by cardiac computed tomography angiography in all participants. Monocyte subsets were identified as classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14-CD16+) and their effector functions were assessed ex vivo by expression of CD80, CD86, CD62L, CD163, CCR2 and CX3CR1 using flow cytometry. Regulatory T-cells (Tregs) were characterized as CD4+CD25highCD127lowFoxP3+ cells.

Results: HIV+CAD+ participants showed significantly higher frequencies of classical monocytes and lower non-classical monocyte frequencies compared to HIV+CAD- individuals. In all monocyte subsets, expression of plaque homing marker CX3CR1 was reduced in HIV+CAD+ individuals, while their proportion of CX3CR1-CCR2+ subset was higher versus HIV-CAD- controls. No changes in other monocyte markers were observed between study groups. Moreover, classical monocyte frequencies correlated positively with Treg frequencies in CAD+ groups, whereas non-classical monocyte frequencies negatively correlated with Tregs only in HIV+CAD+ individuals. In HIV+CAD+ participants, frequencies of classical monocytes correlated negatively with age, LDL and LDL/HDL ratio, while their non-classical monocyte frequencies correlated positively with these parameters.

Conclusions: PWH with subclinical CAD display a unique monocyte signature, characterized by an increase in classical and decrease in non-classical monocyte frequencies, and an altered monocyte homing capacity which were linked to their lipid metabolism and Treg frequencies.

Activating transcription factors, ATFs, are a group of basic-region leucine zipper (bZIP) factors, which bind to cyclic AMP response element (CRE) in various promoters and play major role in the UPR (Unfolded Protein Response) process in cells. During HIV and SIV infections, cellular stress have been described associated with mitochondrial and lysosome damages that can lead to cell death (1, 2). Furthermore, with viral HIV Tat, ATF4 binds to a ATF/CREB site in the long terminal repeat (LTR) and thereby increase replication. Whereas a similarity of functions between ATF4 and its paralogue ATF5 has been described in response to celluar stress, it is unknown so far whether ATF5 may also favor HIV/SIV replication. Thus, the main objective of this study is to determine the role of ATF5 in the context of HIV/SIV infection.
We analyzed by q-PCR ATF4 and ATF5 gene expressions and the downstream effector genes both in purified human CD4 T cells and monocytes infected in vitro with HIVBaL. Viral replication and production were assessed by measuring p24 production by ELISA and viral RNA by q-PCR. Our results indicated distinct dynamics of ATF4 and ATF5 gene expression after HIV infection. Furthermore, the dynamics was different comparing CD4 T cells and monocytes. We then analyzed gene expression in lymphoid and non lymphoid organs of SIV-infected rhesus macaques. Sorted CD4 T cell susbsets and monocytes were also analyzed. Interestingly, we found a difference in the expression of ATF4 and ATF5 in SIV-infected RMs in comparison to uninfected RMs. Additionnal experiments are actually performed to decipher the role of ATF4 and ATF5 in the context of viral replication.
This project may be ultimately allow to the use of ATF inhibitors as therapeutic targets to control viral replication.

1) Laforge M, Limou S, Harper F, Casartelli N, Rodrigues V, Silvestre R, Haloui H, Zagury JF, Senik A, Estaquier J. DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV. PLoS Pathog. 2013. 9:e1003328.

2) Petit F, Arnoult D, Lelièvre JD, Moutouh-de Parseval L, Hance AJ, Schneider P, Corbeil J, Ameisen JC, Estaquier J. Productive HIV-1 infection of primary CD4+ T cells induces mitochondrial membrane permeabilization leading to a caspase-independent cell death. J Biol Chem. 2002. 277:1477-87.

It is well known that HIV-1 latency is a major hurdle in antiretroviral treatment (ART). Several latency reversing agent (LRA) strategies combined with ART failed in eradicating latently infected reservoirs. The main barrier is the heterogeneity of the latent HIV-1 reservoirs as well as the molecular complexity of latency mechanisms.

Although it is widely known that host transcriptional silencing machinery plays the principal role in HIV-1 latency, it now appears that the rapid evolution of HIV-1 viruses favors the establishment of proviral latency as immune escape mechanism. HIV-1 genetic variants result from error-prone Reverse Transcriptase (RT), APOBEC-3, as well as recombination.

Here we show that G-to-A mutations in the Long Terminal Repeat (LTR) region of the HIV-1 genome introduced by viral RT errors and host APOBEC3 enzymatic activity generate HIV-1 sequences that can display a reversible, latency-like phenotype.

These mutated latency prone viruses (LPVs) display reduced transcription upon infection monitored by reporter eGFP expression, however bounce back up to 30-fold in Jurkat cells and 3-fold in primary CD4+ T cells following LRA induction. In addition, in a longitudinal virus adaptation assay, 5 replicative LPVs displayed the potential to evolve and regain viral fitness following two rounds of LRA activation cycles.

This project also demonstrates that some 5’-LTR mutations in the viral promoter of LPVs mapped to transcription factor biding sites SP-1, NF-kB, NFAT and TAR. These appear to be modulating the viral promoter’s ability to respond to transactivation and therefore promote and maintain proviral latency when mutated.

A significant roadblock to the production of an HIV-1 vaccine has been the inability to elicit cross-clade neutralizing antibody responses that can protect against the exceptionally high level of HIV-1 genetic diversity circulating in human populations. However, in targeting transmitted/founder (T/F) viruses, which are HIV-1 variants with high transmission fitness, we aim to exploit their unique phenotypic properties for anti-HIV-1 prophylactic vaccine development. By eliciting antibodies capable of targeting and neutralizing T/F viruses before infection occurs, we hypothesize that we will generate enhanced vaccine protection compared to a vaccine designed to target the entire quasi-species. To that end, we used a reverse vaccinology approach, which involved the screening of thousands of HIV-1 subtype B sequences within the Los Alamos database, to identify viruses which exhibit characteristics for enhanced transmission fitness. We aim to use these viral sequences in combination with our established virus-like-particle (VLP) vaccine platform to deliver these T/F envelope (Env) glycoproteins as vaccine immunogens. Our VLPs are genome-less assemblies of HIV structural proteins which are morphologically indistinguishable from HIV-1. We have already published data showing our VLP immunogens possess in-vitro immune priming and recall capabilities. Furthermore, to complement this approach we have structurally engineered mutations in the glycan shield of our Env immunogens to elicit a targeted humoral immune response against sites known to be highly vulnerable to neutralizing antibody mediated attack i.e., the CD4 binding site (CD4bs). These resulting viruses have been shown to exhibit increased vulnerability to neutralization by a variety of known broadly neutralizing antibodies (BnAbs) and changes in viral kinetics. We aim to screen the antigenicity of our immunogens using a murine B cell line before proceeding to animal studies. By generating cross-clade neutralizing antibodies through vaccination, this research will have overcome some of the most significant roadblocks to HIV vaccine development.

The HIV-1 envelope glycoprotein (Env) trimer mediates viral entry. Env samples different conformations, in its unliganded form it samples a “closed” conformation which resistant to non-neutralizing antibodies (nnAbs). Small-molecule CD4 mimetics (CD4mc) such as BNM-III-170 and MCG-IV-210 sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) mediated by nnAbs present in plasma from people living with HIV (PLWH). Structural studies revealed that the new family of MCG-IV-210 CD4mc derivatives bind within the Phe43 cavity in close proximity to the highly-conserved Asp368 residue. We speculated that further optimized MCG-IV-210 analogs capable of forming H-bonds with Asp368 could gain breadth and ADCC potency.
We optimized MCG-IV-210 piperidine nitrogen substituent to develop new CD4mcs. High resolution structures of complexes formed by new analogs and a gp120CRF01_AE coree were solved by X-ray crystallography to provide insight into interactions within the CD4-binding cavity and Asp CD4-binding cavity in close proximity to the highly-conserved Asp368. Their capacity to neutralize viral particles and sensitize infected cells to ADCC was measured. The best performing analogs in neutralization and ADCC assays were subjected to structural analyses that confirmed their binding within the Phe43 cavity in a manner similar to MCG-IV-210. In addition, several modifications of the piperidine core improved the position of the new CD4mcs in the Phe43 pocket and surrounding vestibule. Interestingly, two analogs (DL-I-101 and Dl-I-102) formed an H-bond with the α-carboxylic acid group of Asp368 and presented improved neutralization and ADCC activities. Overall, the new structural and biological attributes of these molecules make them good candidates for HIV-1 eradication strategies.

Transient viremia reported following COVID-19 mRNA vaccination in ART-suppressed PWH suggests a stimulatory effect on the HIV reservoir. Another recent study also reported that Nef-specific CD8+ T cells increased and acquired granzyme-B effector function after COVID-19 mRNA vaccination, where this correlated with markers of immune-mediated suppression of HIV-transcribing cells. However, that study did not investigate HIV viremia, nor detect significant reservoir size changes (n=13). We investigated HIV viremia and reservoir size following COVID-19 mRNA vaccination in 62 ART-treated PWH.

Participants (89% male) were sampled pre-vaccination, and one month following the first two vaccine doses. Plasma HIV loads (pVL) were measured using the Cobas 6800 (LLOQ:20 copies/mL). Intact and total HIV copies/million CD4+ T cells were measured using the Intact Proviral DNA Assay. Anti-SARS-CoV-2 S serum antibody concentrations were measured using the Roche Elecsys Anti-S assay.

Pre-vaccination, 82% of participants had pVL<20 copies/mL (max:110 copies/mL). No significant differences in pVL were observed post-vaccination (all p>0.4): one month post-first and second doses, 79% and 85% of participants had pVL<20 copies/mL (max:183 and 79 copies/mL), respectively. Pre-vaccination, the median intact reservoir size was 80 (IQR:28-197; n=46) HIV copies/million CD4+ T cells. Intact reservoir size did not change significantly post-vaccination (all p>0.2): one month post-first and second doses, medians were 85 (IQR:29-184; n=46) and 65 (IQR:22-168; n=29) copies/million CD4+ T cells, respectively. No significant changes in total, nor 5'/3' defective proviral burdens were observed post-vaccination (all p>0.1), nor in any outcome upon stratification by sex, vaccine regimen, or ART type (multiple comparisons addressed using q-values). No correlations were observed between SARS-CoV-2 anti-S antibody concentration post-vaccination, and change in reservoir size, nor observation of detectable viremia (all p>0.2).

While COVID-19 mRNA vaccination may modestly stimulate the HIV reservoir in some individuals, we observed no measurable changes in reservoir size nor lasting plasma viremia following immunization.

Biological functions fluctuate in a circadian manner to align with day/night environmental changes. In HIV-uninfected individuals, daily variations were reported for cortisol, melatonin and CD4+ T-cell counts in blood. HIV-1 infection is characterized by alterations in CD4+ T-cell homeostasis, chronic immune activation and gut barrier damage. Daily variations in immunological/virological parameters in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART) remain poorly investigated.
Eleven ART-treated PLWH (median age: 57 years; CD4 counts: 606 cells/µl; time since infection: 242 months; aviremia under ART: 216 months) were admitted for 40 hours. Blood was collected, before food intake, every 4 hours, for 24 hours (8:00, 12:00, 16:00, 20:00, 24:00, 4:00, 8:00). Plasma levels of cortisol/melatonin and markers of intestinal permeability (FABP2, LBP) were measured by ELISA. Flow cytometry allowed the quantification and characterization of leukocyte subsets. CD4+ T-cells were isolated and matched RNA/DNA served to quantify cell associated integrated HIV DNA and LTR-Gag HIV-RNA levels by nested PCR/RT-PCR. Peak/nadir terms were used to define maximal/minimal levels.
Plasma cortisol and melatonin levels peaked at 8:00 and 4:00, respectively. Peak plasma FABP2 levels coincided with nadir LBP levels at 4:00. Memory/naive/regulatory CD4+ T-cells counts peaked between 20:00-4:00, with nadir counts at 12:00. The expression of the HIV-1 co-receptors CCR5/CXCR4 and gut-homing molecules CCR6/integrinβ7 on memory T-cells expression levels peaked between 20:00-4:00. Integrated HIV DNA levels in CD4+ T-cells revealed minor daily fluctuations. The HIV-RNA/DNA ratio (surrogate marker of HIV-1 transcription) peaked at 4am.
Daily variations in melatonin/cortisol levels, T-cell counts, mucosal permeability markers and key molecules involved in HIV replication/pathogenesis were observed in ART-treated PLWH. The peak of HIV transcription in CD4+ T-cells, at 4:00, coincided with peak FABP2 and melatonin and nadir LBP levels. These findings should inform therapeutic interventions on HIV-1 cure/remission on the importance of selecting the optimal time of treatment/monitoring.

HIV-1 Envelope (Env) conformation determines the susceptibility of infected CD4+ T cells to Antibody Dependent Cellular Cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more “open” conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu and Env. Limited data exists however of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu and Env are all required to efficiently downregulate CD4 on infected CD4+T cells, we show here that any combination of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared to CD4+T cells. However, treatment of infected macrophages with small CD4-mimetics expose vulnerable CD4-induced Env epitopes and sensitize them to ADCC.

Introduction: Immunometabolism, the crosstalk between immunology and metabolism, has provided new promising therapeutic venues for treating HIV-1. Our laboratory has shown that antiviral T-cell functions in PLWH (people living with HIV) were mediated through autophagy-dependent energy production and could be a target to boost protective immunity in these individuals. Furthermore, Autophagy provides different sources of nutrients (lipids and amino acids for CD8 and CD4 T-cells, respectively) to ensure optimal energy production. However, it remains critical to have a complete picture of how antiviral T-cells use nutrients and metabolic enzymes. Here, we aimed to assess the role of mitochondrial HSP60 in memory T-cell energy production and function.
Goals and Methods: We assessed the expression levels and mitochondrial localization of HSP60 within T-cells following their cellular activation by multiparametric and imaging Flow Cytometry. Blockade of HSP60 within memory T-cells by gene silencing was followed by evaluating expression levels of metabolic enzymes and mitochondrial energy production using western blotting and seahorse metabolic analyzer, respectively. Finally, evaluating the effector function of T-cells after HSP60 blockade by multiparametric Flow Cytometry.
Results: The results show an increase in mitochondrial HSP60 expression in an HSF-1-dependent manner within memory T cells following cell activation. We found that mitochondrial HSP60 was critical to ensure optimal energy production by stabilizing the expression of several enzymes and nutrient transporters, which were involved in lipid and glutamine catabolism. Finally, our data vouched for our ability to rescue the energy-dependent antiviral immunity of T-cells when HSP60 expression is impaired with alpha-ketoglutarate supplementation.

Conclusion: Overall, our study demonstrates a new metabolic mechanism involving the vital role of HSP60 in providing the energy in Memory T-cells against Viruses which could be a novel therapeutic tool for PLWH. In addition, we show that alpha-ketoglutarate supplementation may be beneficial for boosting T-cell immunity against viruses including HIV-1.

SARS-CoV-2 has infected more than 658 million people worldwide causing more than 6.7 million confirmed deaths since the beginning of the COVID-19 pandemic. Immunocompromised individuals in particular are at an increased risk of infection, severe disease, and death. Vaccines have been instrumental in stemming the worst of the COVID-19 pandemic; however, the dynamics of the vaccine induced immune responses in immunocompromised individuals remains a key area of research. Studies have shown end stage renal disease (ESRD) patients have significantly reduced humoral responses and memory B cell formation in responses to COVID-19 vaccination; however, early innate immune responses have yet to be elucidated. To investigate the early innate immune response to COVID-19 vaccination, blood was collected before (BD1) and 1-4 days post dose 1 (PD1) of BNT162b2 vaccination (n=17 ESRD; 28 healthy controls). RNA-Sequencing was performed and differential gene expression and pathway analyses were used to compare groups, controlling for age, sex, and batch. Preliminary analyses of vaccine responses identified 125 significantly differentially expressed genes (DEG) (1.17E-08 < padj < 0.04) in ESRD patients and 107 DEGs (3.13E-03 < padj < 0.05) in healthy controls. 71 DEGs were significant in both populations, 54 unique to ESRD patients and 36 unique to healthy controls. Detailed enrolment and follow-up questionnaires (demographics, medical history, COVID-19 history, medications, etc.) were collected from participants and cellular and antibody responses are being measured at 2 weeks, 6 months, and one-year post vaccination. Long-term immunological impacts will be tested for association with early innate immune responses to COVID-19 vaccination. We show ESRD patients elicit a unique immune response to COVID-19 vaccination compared to healthy controls. These data can help inform ongoing vaccination strategies for all immunocompromised populations, including people newly diagnosed and living with HIV.

Background: Hepatitis C virus (HCV) co-infection is a challenging comorbidity in people living with HIV, as reflected by larger HIV reservoir size in HCV/HIV co-infected compared to HIV mono-infected individuals. While Direct Acting Antivirals (DAA) cure HCV, HIV reservoirs persist under antiretroviral therapy (ART). Here, we investigated the impact of DAA-mediated HCV cure on HIV-DNA reservoir size and residual HIV transcription in HCV/HIV co-infected subjects.
Method: To measure changes in HIV reservoir markers before and after DAA treatment, total CD4 T cells were enriched from HCV/HIV co-infected individuals (n=20) before DAA (baseline), at the end of treatment (EOT), and three months post-DAA (follow-up). Total DNA and RNA were simultaneously extracted from purified CD4 T cells. Integrated HIV-DNA was measured using quantitative real-time alu-gag nested PCR. Nested RT-PCR was used to measure un-spliced LTR-gag and pol HIV transcripts as well as multiply spliced tat/rev transcripts. The HIV-RNA/DNA ratio was used as a surrogate marker for HIV transcription.
Result(s): We observed a significant reduction in integrated HIV-DNA levels following DAA treatment (approximately 0.7-fold decrease, p= 0,0026). Interestingly, at the follow-up time point post-DAA, patients who had HCV prior to HIV infection showed a more robust significant decrease in integrated HIV-DNA than those who were first infected with HIV (p= 0.0172), raising the possibility that HIV infects HCV-specific T-cells. Although some donors showed a transient elevation of un-spliced HIV RNA at EOT, at follow-up there were no significant differences from baseline. Multiply spliced HIV RNA and HIV-RNA/DNA ratios were not significantly affected by DAA-mediated HCV cure.
Conclusion(s): Together, these results highlight the beneficial impact of DAA on reducing HIV reservoir markers in HCV/HIV co-infected subjects. Future studies should determine whether HCV-specific CD4 T-cells are permissive to HIV infection and contribute to HIV-DNA reservoir persistence during ART.
Keywords: DAA, HCV/HIV co-infection, ART, HIV reservoir

Background: Modified autologous lymphocytes as treatment against many diseases is the focus of increasing interest. In the context of HIV, this approach is costly, time-consuming, and, to date, has not been particularly effective. The creation of an “off-the-shelf’ therapy against HIV may have the best chance of facilitating a scalable cure. Delta One T (DOT) cells, an expanded population of gamma delta 1 (Vd1) T cells, have previously been used in cancer studies and do not appear to induce graft-versus-host-disease (GvHD). Furthermore, they have been shown to kill/suppress target cells both in vitro and in vivo through a wide variety of cytotoxic receptors such as NKG2D and NKG2C which should be able to recognize ligands on HIV-infected cells.

Objective: Determine if DOT cells preferentially enact effector functions against HIV-infected CD4+ T cells.

Methods: First, alpha/beta T cells will be depleted from peripheral blood mononuclear cells (PBMC) using magnetic beads. The remaining cells will be stimulated with a cytokine cocktail according to the DOT cell protocol as published (Almeida et al., 2016). Following expansion, DOT cells will be co-cultured with HIV-infected J1.1 cells or primary isolated CD4+ T cells. Cell death of HIV-infected versus their uninfected counterparts will be assessed by annexin V via flow cytometry. To assess viral replication, a p24 ELISA will be used. To observe DOT cell migration, a transwell assay will be used. In brief, wells will be seeded with HIV-infected or uninfected macrophages, and a suspension of DOT cells will be placed above in a 0.8um filter. Migrated DOT cells will be counted via flow cytometry.

Results: DOT cells have been generated and this protocol is being optimized. HIV-infected cell killing experiments are ongoing.

Conclusions: This project proposes a novel immunotherapeutic “off-the-shelf” cure strategy using activated Vd1 T cells to potentially target and eradicate HIV-infected cells.

HIV-1 persistence is thought to be the consequence of viral latency in T CD4+ cell populations. It is postulated that viral reactivation combined with antiretroviral treatment would allow clearance of latently infected cells. This "shock and kill" strategy relies on the use of latency reversing agents (LRAs). These non-discriminant agents were reported to reverse latency of T cells in vivo. However, knowledge regarding their effect on other latently infected populations such as macrophages is scarce. Therefore, we aimed to monitor the impact of 3 different classes of LRA agents on macrophage’s physiology, susceptibility to HIV-1 infection and viral production.

Primary human monocyte-derived macrophages (MDMs) were exposed for 6 to 24h with optimal doses of LRAs either used alone or in dual combinations. Studied LRAs were bryostatin-1, JQ-1 and romidepsin. Selected cytokines secretion and expression were monitored by ELISA and RT-qPCR, respectively. Cell viability, phagocytosis, endocytosis, efferocytosis and susceptibility to HIV-1 infection were quantified by flow cytometry. Viral production was assessed by ELISA or Western blot of the viral capsid.

While LRAs did not alter cell viability, bryostatin-1 tend to increase inflammatory cytokines expression and secretion but slightly decreases phagocytosis and endocytosis while romidepsin decreases efferocytosis. Bryostatin-1 or romidepsin stimulation prior to HIV-1 inoculation decreased infection rate. This could be linked to the downregulation of CD4 and SAMHD-1 inactivated form induced by bryostatin-1 and romidepsin, respectively. Furthermore, treatment with bryostatin-1 after HIV-1 infection induced a strong decrease in viral and cell-associated CAp24 and p17.

None of the LRA tested was able to increase HIV-1 production in vitro. Our data indicate that bryostatin-1 stimulation of infected macrophages dramatically decreases HIV-1 production while inducing a pro-inflammatory state. Thus, our data suggest that LRAs treatments have distinct outcomes in macrophages and T cells, which need to be better deciphered to achieve an HIV-1 cure.

Background:
HIV+ individuals have a relative high risk of folate deficiency, with the incidence being even higher amongst those with neuropsychiatric symptoms. Folate transport into central nervous system is mainly mediated by reduced folate carrier (SLC19A1/RFC), proton-coupled folate transporter (SLC46A1/PCFT) and folate receptor alpha (Folr1/FRα). Previous findings from our group suggested a potential interaction between DTG and folate transporters at the placenta. We hypothesized that such interactions could also occur at brain barriers including choroid plexus (CP) and blood-brain barrier (BBB).

Methods:
Human cerebral microvascular endothelial cells (hCMEC/D3) and primary cultures of mouse microvascular endothelial cells (in vitro BBB models) were treated with clinical relevant concentrations of DTG (2000, 5000ng/ml) or BTG (3000, 6000ng/ml) for 24h. Isolated mouse brain capillaries were treated ex vivo with DTG (5000ng/ml) or BTG (6000ng/ml) for 5h. C57BL/6 mice were administered orally either DTG (5mg/kg/day); BTG (5mg/kg/day); DTG + backbone (tenofovir alafenamide (50mg/kg/day) + emtricitabine (33.3mg/kg/day) or BTG + backbone; for 14 days. Slc19a1, Slc46a1, Folr1 gene expression was quantified by qPCR in cell cultures, CP and brain capillaries.

Results:
DTG exposure significantly downregulated SLC19A1/Slc19a1, SLC46A1/Slc46a1 gene expression in hCMEC/D3 and mouse BBB cells. DTG exposure robustly downregulated Slc19a1, Slc46a1 and Folr1 while BTG significantly downregulated Slc19a1 in isolated mouse brain capillaries. Chronic treatment of DTG in mice significantly downregulated Folr1, while BTG downregulated Slc19a1, Slc46a1 and Folr1 expression in brain capillaries. Administration of DTG and BTG + backbone in mice significantly downregulated Slc19a1 and Slc46a1 expression at the CP, BTG + backbone also downregulated Folr1 in brain capillaries.

Conclusions:
Our findings suggest a potential interaction between DTG or BTG-based antiretroviral therapy (cART) with cerebral folate transport pathways, which could potentially increase the risk of cerebral folate deficiency among HIV+ individuals. These interactions may also contribute to cART-associated neuropsychiatric adverse effects observed in the clinic.

Background:
Autophagy, a cytosolic-structure degradation pathway producing energy, allows efficient anti-HIV T-cell responses through the production of IL21 in CD4 T-cells. Intracellular acyl-CoA-binding protein (ACBP) favors autophagy whereas secreted extracellular ACBP inhibits autophagy. Herein, we assessed intra- and extracellular ACBP levels in people living with HIV (PLWH) under antiretroviral therapy (ART).
Methods:
Plasma ACBP and cytokine levels were assessed by ELISA in 60 long-term (median 14.7 years) ART-treated PLWH and 30 uninfected controls. Intracellular ACBP levels were assessed by flow cytometry in PBMC. Metabolomic analyses were performed on serum samples by LC-MS.
Results:
ACBP levels were higher in ART-treated PLWH compared to controls (medians 127.5 vs 78.1 ng/mL, p=0.03), independently of age, sex and weight. Intracellular ACBP was detected in all leukocytes in both groups. Plasma ACBP levels correlated negatively with its intracellular levels in T-cells (r=-0.9, p=0.02) and monocytes (r=-0.9, p=0.08) suggesting that low plasma levels of ACBP are associated with cellular retention of the protein.
In ART-treated PLWH, plasma ACBP levels were neither associated with CD4 nor CD8 T-cell counts, but correlated with levels of growth factors (EGF, G-CSF, GRO), pro-inflammatory cytokines (IFNα2, IFNγ, IL1β) and homeostatic factors (IL7 and IL15) (r>0.3, p<0.05 for all comparisons). Plasma ACBP levels were inversely associated with plasma IL21 levels (r=-0.54, p<0.01). PLWH with plasma ACBP levels above the median had two-fold higher levels of glutamic acid (p=0.02), a higher glutamic acid/glutamine ratio (p=0.03) and tended to have higher levels of α-ketoglutarate (1.5-fold difference, p=0.09) in their serum.
Conclusion:
Higher plasma levels of ACBP in ART-treated PLWH were associated with inflammation, oxidative stress, and markers of T cell dysfunction. Our findings indicate that circulating ACBP might weaken anti-HIV T cell functions in an IL21 dependent manner. The ACBP pathway might constitute a target for improving anti-HIV T-cell responses.

Introduction: Viruses require host cell components to establish and maintain infection. Multiple genome-wide knockout/knockdown studies of HIV and other viruses have identified sets of host dependency factors (HDFs) that are essential for viral replication. Although these factors may be candidates for development of novel antivirals, defining targets that do not lead to drug toxicity is challenging. One opportunity to identify good targets is to define which HDFs harbour homozygous loss of function (LoFs) polymorphisms in healthy people.

Methods: We performed a literature review to identify genome-wide studies of viral HDFs for multiple viruses. We identified 27 studies covering HIV, Hepatitis C, Hepatitis D, SARS-CoV-2, SARS-CoV, Ebola, Influenza A, Zika, Dengue and West Nile virus. These HDFs were intersected with the genome aggregation database (gnomAD), a resource containing >125,000 human exome and >15,000 whole-genome sequences, to identify HDFs that harbour homozygous LoFs in healthy individuals.

Results: We identified 2898 unique HDFs across all viruses. 326 of these were implicated in more than 1 virus and 2 HDFs were implicated in 5 viruses. Using gnomAD data, we found that HDFs implicated in more than one virus tend to be highly intolerant to a LoF mutation suggesting they are highly conserved within the host. Six candidate HDFs that intersect with HIV were narrowed down for CRISPR gene editing, as potential broad-acting drug targets (Table 1).

Conclusion: In silico and in vitro screening of HDFs harbouring homozygous LoFs in healthy people may aid in the development of novel broad acting antivirals.

Objective: Limited data exist regarding the immune benefits of fourth COVID-19 vaccine doses in people living with HIV (PLWH) receiving ART, particularly now that most have experienced a SARS-CoV-2 infection. We quantified wild-type (WT)-, Omicron-BA.5- and Omicron-BQ.1-specific neutralization up to one month post-fourth COVID-19 vaccine dose in 63 (19 SARS-CoV-2-naive and 44 SARS-CoV-2-experienced) PLWH.
Design: Longitudinal observational cohort.
Methods: Quantification of wild-type (WT)-, Omicron-BA.5- and Omicron-BQ.1-specific neutralization using live virus assays.
Results: Participants received monovalent (44%) and bivalent (56%) mRNA fourth doses. In COVID-19-naive PLWH, fourth doses enhanced WT- and Omicron-BA.5-specific neutralization modestly above three-dose levels (p=0.1). In COVID-19-experienced PLWH, fourth doses enhanced WT-specific neutralization modestly (p=0.1) and BA.5-specific neutralization significantly (p=0.002). Consistent with humoral benefits of 'hybrid' immunity, COVID-19-experienced PLWH exhibited the highest neutralization post-fourth dose, where those with Omicron-era infections exhibited higher WT-specific (p=0.04) but not BA.5- or BQ.1-specific neutralization than those with pre-Omicron-era infections. BA.5-specific neutralization was significantly lower than WT in everyone regardless of COVID-19 experience, with BQ.1-specific neutralization lower still (all p<0.0001). In multivariable analyses, fourth dose valency did not affect neutralization magnitude. Rather, an mRNA-1273 fourth dose (versus a BNT162b2 one) was the strongest correlate of WT-specific neutralization, while prior COVID-19, regardless of era, was the strongest correlate of BA.5 and BQ.1-specific neutralization post-fourth dose.
Conclusions: Fourth COVID-19 vaccine doses, irrespective of valency, benefit PLWH regardless of prior SARS-CoV-2 infection. Results support existing recommendations that all adults receive a fourth COVID-19 vaccine dose within 6 months of their third (or their most recent SARS-CoV-2 infection).

TILRR, a co-receptor of IL-1R1, plays an important role in enhancing IL-1/IL-1R1/TLR-NFκB inflammation responses through its association with IL-1R1. TILRR is an important modulator of many genes involved in inflammatory responses and promotes inflammatory cytokine secretion by epithelial cells. It promotes immune cell migration through the induction of soluble inflammatory mediators. TILRR protein is not only expressed in PBMCs and tissues but also circulates in blood. The plasma TILRR protein levels among individuals vary greatly (<2.38ng/ml to >5μg/ml) and are positively correlated with several proinflammatory cytokines. Thus, TILRR not only modulates inflammatory responses in cells and tissues but may also modulate systemic inflammation.
Inflammation is a double-edged sword and underlies a wide variety of physiological and pathophysiological processes. Chronic and persistent inflammation is pathologic and associated with a number of human infectious and chronic diseases, such as HIV, SARS-CoV-2, H1N1, obesity, diabetes, atherosclerosis, asthma and neurodegenerative diseases. The great variations in plasma TILRR protein levels among individuals and its positive correlations with several pro-inflammatory cytokines suggest that high plasma TILRR protein level could be a high risk factor for infectious diseases.
We analyzed TILRR protein and proinflammatory cytokines of 941 archived HIV negative plasma samples from 390 women who were HIV negative when they were enrolled in the Pumwani cohort. We find that women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels <100 ng/ml (p<0.0001; RR=3.72 and OR=15.29). Our pilot study also showed that median plasma TILRR protein levels in the COVID-19 ICU patients are 38-fold higher than its level in the COVID-19 patients with mild symptoms (P<0.00001).
Further studies on the influence of high plasma TILRR protein level as a high risk factor for infectious and chronic diseases would help to develop better diagnostics and better treatment.

Introduction
While mRNA SARS-CoV-2 vaccination elicits strong humoral responses in healthy individuals, humoral responses in People living with HIV (PLWH) remains to be clarified. Here, we conducted a longitudinal study of vaccine immunogenicity elicited after three doses of mRNA SARS-CoV-2 vaccine in PLWH stratified by their CD4 count.

Methods
We measured the capacity of antibodies to recognize the full SARS-CoV-2 Spikes from different variants of concern (VOC) using a cell-based assay and flow cytometry. We also measured the Fc-mediated effector functions (ADCC) by the capacity of PBMCs to kill CEM.NKr expressing stable SARS-CoV-2 Spikes in presence of plasma. We measured the relative capacity of antibodies to mediate neutralization of authentic SARS-CoV-2 viruses after the third dose.

Results
After 3 doses of an mRNA vaccine, PLWH having CD4≤250 cells/mm3, had around 2-fold lower levels of antibodies able to recognize the Delta, BA.1 and BA.2 Spikes compared to PLWH having more than 250 CD4 cells/mm3. Albeit the differences were not significant, Fc-effector were lower in individuals having CD4≤250 compared to those with CD4≥250. Strikingly, no major differences were observed in the capacity of plasma from these individuals to neutralize authentic SARS-CoV-2 after the third dose. All groups showed increased recognition of SARS-CoV-2 Spikes after vaccination with antibody levels decreasing over-time.

Conclusions
Our results emphasize the heterogeneity of vaccine immunogenicity in PLWH based on CD4 count. This study will help determine the need for additional boosts according to the populations to be targeted.

In a previous study (Mohammadzadeh, et al. mBio. 2021), we reported the persistence of HIV-encoded DNA, RNA and proteins in the brain despite concurrent effective antiretroviral therapy (ART) with undetectable plasma and cerebrospinal fluid viral RNA levels, which was often in association with neurocognitive impairments. Although the determinants of HIV persistence have garnered attention, the expression and regulation of antiretroviral host restriction factors (RFs) in the brain during HIV or SIV infections remain unknown. We investigated the transcriptomic profile of antiretroviral RF genes by RNA deep sequencing with confirmation by qRT-PCR in cerebral cortex from uninfected persons (HIV[-]), HIV-infected without pre-mortem brain disease (HIV[+]), HIV-infected with neurocognitive disorders (HIV[+]/HAND), and neurocognitive disorders with encephalitis (HIV[+]/HIVE). We observed significant increases in RF expression in brains of HIV[+]/HIVE in association with brain viral load. Machine learning techniques identified MAN1B1 as a key gene that distinguished HIV[+] group from HIV[+] groups with HAND. Analyses of SIV-associated RFs in brains from SIV-infected Chinese rhesus macaques with different ART regimens revealed diminished RF expression among ART-exposed SIV-infected animals although ART interruption resulted in induced expression of several RF genes including OAS3, RNASEL, MX2, and MAN1B1. Thus, the brain displays a distinct expression profile of RFs that is associated with neurological status as well as brain viral burden. Moreover, ART interruption can influence the brain’s RF profile that might contribute to disease outcomes.

Background: Immune cells, like T-cells, are modulated by several checkpoints to prevent unrestrained immune activity. Upon chronic HIV infection, these regulatory “brakes” of immune cells are co-opted to evade host-immunity. This T-cell exhaustion is due to overexpression of inhibitory immune-checkpoints (IC) that impede effector cytokine responses. As LAG3, an IC, is extensively glycosylated, the lectins, Galectin-3 and liver sinusoidal endothelial cell lectin (LSECtin), are thought to bind glycans on LAG3, engaging LAG3’s function, and thereby inhibit T-cell activation. Hence, thorough appreciation of LAG3 function, activity, regulation entails understanding its interaction with potential ligands. Here, we clarify the role of lectins, LSECtin and Galectin-3, in regulating LAG3-mediated T-cell suppression during chronic HIV infection.

Methods: We developed LAG3+ and LAG3- expressing Jurkats, a CD4+ T-cell line. A ligand-binding assay using N-glycosidase (rids glycosylation sites) will confirm the necessity of LAG3 glycosylation and test lectin-binding efficiency by comparing percent and median fluorescence intensity (MFI) of LAG3+ and LAG3- cells bound to either LSECtin/Galectin-3 by flow cytometry. Moreover, IL-2 and IFN-gamma levels within treated cells will assess differential inhibitory strengths for ligands.

Results: Percent and MFI of LSECtin/Galectin-3 binding is greater on LAG3+ cells (LSECtin: 60.4%,1187 MFI; Galectin-3: 84.1%,1345 MFI) than glycosidase-treated counterparts/LAG3- cells (LSECtin: 5.18%,838 MFI; Galectin-3: 13.0%,914 MFI), demonstrating that lectin-binding requires LAG3-glycosylation. With lectin-treatment, percent of IL-2 and IFNy production is lower (greater T-cell inhibition) in LAG3+ (23.5%;24.0%) than LAG3- cells (71.5%;84.1%) signifying that lectin-binding induces inhibitory LAG3-activity.

Significance: The created in vitro-model demonstrates lectin-binding as an overlooked mechanism instructing LAG3-activity and T-cell immunosuppression during HIV. Understanding lectin interaction in LAG3-engagement would advance optimal lectin/LAG3 antibody blockade use in relieving T-cell suppression and shock and kill alongside current immunotherapy. In deciphering and removing the “LAG3 brakes” while reversing cellular exhaustion, we can reinvigorate host-immunity and inform a functional cure for HIV/AIDS.

Background: Alterations of the intestinal barrier represent a cause for co-morbidities such as cardiovascular disease (CVD) in people living with HIV (PLWH) receiving antiretroviral therapy (ART). Such alterations coincide with the overexpression of IL-32 cytokine family, including isoforms with pleiotropic actions varying from antiviral to immunosuppressive features. We previously demonstrated that IL-32β is upregulated in the colon of ART-treated PLWH and its expression in intestinal epithelial cells (IEC) is downregulated by the Th17-hallmark cytokine IL-17A. Here, we aimed to identify new modulators of IL-32 expression in IEC, focusing on stimuli/pathways linked to HIV pathogenesis and CVD, including the Th17 cytokines IL-22 and IL-26, the gut-homing modulator retinoic acid (RA), the transcriptional repressor PPARγ, and the antiviral/immunosuppressive type I interferon (I-IFN).
Methods: The HT-29 IEC were exposed to recombinant TNF-α, IL-17A, IL-22, IL-26, and IFN-β1a the PPARγ antagonist T0070907, and all-trans retinoic acid (ATRA). IL-32β/γ/ε isoform mRNA were quantified by real-time RT-PCR.
Results: TNF-α-activated IEC upregulated predominantly IL-32β/γ, and at lower levels IL-32β mRNA expression. IFN-β1a acted in synergy with TNF-α to significantly (p<0.05; Paired t-Test, n=3 experiments) augment IL-32β/γ/ε expression (median fold change (FC): 3,05/2,05. The IL-32β/γ mRNA expression was significantly reduced by exposure to IL-17A (median FC: 0.23/0.22) and ATRA (median FC: 0.29/0.38), while IL-22 (Median FC: 1.87/1.46) and PPARγ antagonist (median FC: 1.9/3.1) exposure led to a significant increase. IL-26 did not modulate IL-32β/γ/ε expression.
Conclusions: Our results reveal the capacity of TNF-α to act in synergy with I-IFN and IL-22 to promote IL-32β/γ/ε expression in IEC and identify the PPARγ and RA pathways as new negative regulators of IL-32 expression in inflamed IEC. The identification of these novel IL-32 regulatory networks raises new questions on their potential targeting for counteracting HIV dissemination and persistent inflammation at intestinal level for preventing the occurrence of CVD in ART-treated PLWH.

Background: Limited data exist regarding the dynamics of humoral responses following COVID-19 vaccination and breakthrough SARS-CoV-2 infection in people living with HIV (PLWH) receiving suppressive antiretroviral therapy. Here, we examined the durability and specificity of neutralizing antibodies and memory B cells induced by three vaccine doses in PLWH. The impact of breakthrough infection was assessed in subset of individuals who experienced their first infection after receiving three vaccine doses.

Methods: Humoral responses against wild type (WT, Wuhan), Omicron BA.1 and BA.2.75 spike receptor binding domains (RBD) were assessed in serum of 64 PLHW up to six months post-third dose. IgG antibodies were quantified using ELISA. Neutralization activities were measured using a surrogate assay based on ACE-2 displacement. RBD-specific memory B cells were evaluated by flow cytometry.

Results: Third vaccine doses boosted all humoral measures above levels achieved after two doses, but variant-specific responses remained significantly lower compared to WT. IgG concentrations for all strains declined at a similar rate. Surrogate neutralization activities also declined over time, with those against Omicron strains falling below the limit of detection by six months in ~60% of participants. SARS-CoV-2 infection boosted IgG concentrations and neutralization activities significantly above vaccine-induced levels, though BA.2.75-specific activity remained significantly lower than BA.1. Consistent with a boost in humoral responses following a third dose, a significant increase in both WT- and Omicron-specific memory B cell responses was observed, and these responses persisted overtime.

Conclusion: A third COVID-19 vaccine dose augmented neutralizing antibody and memory B cell responses against WT and Omicron variants in PLWH receiving suppressive ART. While serum antibody levels declined over time after vaccination, the persistence of memory B cells likely contributed to robust responses observed following breakthrough infection.

Funded by the Public Health Agency of Canada (COVID-19 Immunity Task Force) and CIHR.

Background
Dysbiotic vaginal microbiota (VMB) in the female genital tract is associated with four-fold higher HIV-1 acquisition risk, while the eubiotic VMB is associated with protection. Dysbiotic VMB has been associated with significant changes in the vaginal metabolome and short chain fatty acids (SCFA) like lactic acid are known to have protective effect against HIV-1. Here, we examined the effect of a variety of SCFA found in vaginal metabolome on vaginal epithelial barrier and inflammation to help explain why women with dysbiotic VMB are more susceptible to HIV.
Methods
Clinically reported SCFA profiles of patients with BV and individuals with optimal VMB were used to create mixtures of SCFAs in concentrations that simulated dysbiotic and eubiotic conditions. These combinations of SCFA were tested on vaginal epithelial cells (VECs) grown in air liquid interface (ALI) cultures in transwells. Effect of exposure to the dysbiotic and eubiotic combinations of SCFA were examine for effect on epithelial barrier function, inflammation and HIV-1 leakage.
Results
An increase in transepithelial resistance (TER) and a decrease in FITC-dextran leakage was observed in response to treatment with eubiotic SCFA concentrations, resulting in enhancement of the integrity of the vaginal epithelial barrier and an increase in ZO-1 and desmoglein expression. VECs treated with dysbiotic SCFAs resulted in a decrease in the integrity of the vaginal epithelial barrier with an associated inflammatory cytokine response and activation of NFκB pathway. Exposing VECs to eubiotic SCFAs abrogated HIV-1 mediated decrease in TER and almost no HIV-1 leakage was observed across VECs grown in ALI conditions. In contrast, HIV-1 leakage into basolateral compartments was significantly increased when cells were treated with dysbiotic SCFAs followed by HIV-1 exposure.
Conclusion
These findings indicate the possible role of bacterial SCFA in increased in HIV-1 susceptibility seen in dysbiotic conditions and provide potential strategies for prophylaxis.

Background: Despite ART availability, HIV-infected persons develop HIV-associated neurocognitive disorders (HAND) that is associated with neuroinflammation and neuronal damage. As HIV does not infect neurons, the mechanisms underlying neuronal pathology are uncertain. Pyroptosis is an inflammatory form of regulated cell death that is driven by caspase cleavage of the gasdermin proteins. Gasdermin cleavage releases a cytotoxic N-terminal fragment that forms membrane pores to mediate lytic cell death.
Objective: To investigate the molecular mechanisms by which neurons die during HIV infection.
Methods: A multiplatform approach was used including 1) human neuronal cultures, 2) brain samples from persons with and without HAND, and 3) a nonhuman primate model of SIVmac251 infection. Western blotting, immunofluorescence, cytotoxicity assays, and ddPCR were applied to each experimental platform.
Results: Frontal cortex from persons with HIV-associated neurocognitive disorders (HAND) (n=6) showed increased neuronal gasdermin E (GSDME) expression and its cytotoxic cleavage product (N-GSDME), compared HIV-infected neurologically normal (n=8) and uninfected persons (n=10) (p<0.05). Human neurons exposed to the neurotoxic HIV-encoded viral protein R (Vpr) caused time-dependent GSDME cleavage and plasma membrane rupture (PMR) (p<0.05), measured by LDH release and Sytox uptake, which was impaired by siRNA suppression of GSDME. Pre-treatment of Vpr-exposed neurons with the caspase-1 inhibitor, VX-765, reduced cleavage of caspase-3 and GSDME resulting in less PMR (p<0.05). To validate these observations, we examined the frontal lobe tissues from SIV-infected rhesus macaques with (n=5) or without (n=6) SIV-associated neurological disease revealing increased expression of caspase-1, GSDME (p<0.05) and co-localization of active-caspase-3 with GSDME in cortical neurons from animals with brain disease.
Conclusions: The present study represents the first report of HIV-induced convergent cell death pathways in neurons through engaging both caspases-1 and -3, resulting in GSDME-mediated pyroptosis. Neuronal pyroptosis contributes to neuroinflammation and cognitive decline, which represents new therapeutic targets for HAND.

Integrase strand-transfer inhibitor (INSTI) use in sub-Saharan Africa has increased rapidly in recent years, but little is known about INSTI resistance in the non-subtype B HIV-1 that is prevalent in this region. Uganda has among the highest global burdens of HIV-1, but no studies have analyzed INSTI resistance specific to the strains of HIV-1 in this region. Here, we aim to identify novel INSTI resistance pathways specific to Ugandan HIV-1. We recently sequenced the integrase gene of 51 patients failing treatment on the INSTI raltegravir and 328 patients naïve to INSTI treatment. We identified two novel integrase mutations, I203M and I208L, which often appeared together and were strongly associated with raltegravir failure. Using our yeast-based cloning method, we generated I203M, I208L, and I203M/I208L double mutant viruses in an NL4-3 background. We then selected for high-level raltegravir resistance in these viruses, as well as in wild-type viruses, via a long-term drug-dose escalation experiment using a susceptible human cell line. We tracked the development of raltegravir resistance and the accumulation of drug-resistance mutations within integrase in each virus throughout this experiment. I203M and I208L, both alone and in combination, do not appear to increase resistance to INSTIs. However, I203M and I208L mutants developed INSTI-resistance mutations faster on average compared to wild-type. This corresponded with more rapid development of phenotypic INSTI resistance on average in the I203M and I208L mutants. Thus, we provide early evidence that the I203M and I208L mutations observed in Ugandan raltegravir-experienced patients may influence INSTI resistance by decreasing the fitness costs associated with primary drug resistance mutations. Future experiments will focus on assessing the effect of these mutations on viral fitness. These findings provide new insights on INSTI resistance in Ugandan HIV-1 and will help Ugandan practitioners better predict drug resistance profiles when considering INSTIs as a treatment option.

The HIV Envelope glycoprotein (Env) is the main target of the anti-HIV humoral response. Env can adopt different conformations: the “closed” conformation (State 1); the “partially open” conformation (State 2) and the “open” conformation (State 3). The “closed” conformation is targeted by rare neutralizing antibodies while the “open” conformation can be recognized by easily elicited non-neutralizing antibodies. Env adopts the “open” conformation upon interaction with the CD4 receptor at the surface of infected cells. However, because the viral Nef and Vpu proteins downregulate CD4 expression, Env is mainly exposed in its unliganded “closed” conformation, thus preventing its recognition by commonly elicited anti-Env antibodies.
To force Env to adopts its “open” conformations and expose vulnerable epitopes, we use small CD4 mimetic compounds (CD4mc), which imitate the interaction of CD4 with Env. We previously reported that CD4mc sensitize infected cells to antibody dependent cellular cytotoxicity (ADCC) mediated by plasma from people living with HIV (PLWH). While all plasma from PLWH responded to CD4mc, we observed a wide range of ADCC activity among them. To better understand why some HIV+ plasma are more potent at inducing ADCC, we dissected the antibody content (level and specificity) of a panel of HIV+ plasma samples obtained from a cohort of PLWH (from the FRQS AIDS network). Associations with the presence of antibodies recognizing epitopes within the ADCC potent Cluster A region were observed. Additional work related to CoRBS and CD4i mAbs are ongoing using different families of more potent CD4mc. This information will be crucial in moving forward CD4mc for eradication strategies.

Introduction: Human cohort studies associate bacterial vaginosis (BV) with adverse reproductive health outcomes like genital inflammation and increased HIV risk. We developed a bacterial challenge model to recapitulate BV-associated barrier disruption, elevated cytokine production, and increased T cell activation and recruitment. Damping of genital inflammation needs to be part of a comprehensive HIV-prevention strategy. Aspirin (ASA) is an anti-inflammatory drug that is widely available, affordable, and accepted. Human cohort studies have found that ASA modulates mucosal immunity by reducing HIV targets (CD4+CCR5+ T cells) in the FRT, but its broad impact on the vaginal mucosa is unknown. In this study, we addressed whether ASA can dampen BV-associated inflammation and epithelial barrier disruption in vivo and whether these effects can lower HIV acquisition risk.

Methods: C57BL/6 mice were intravaginally challenged with BV-associated anaerobes Mobiluncus mulieris or Gardnerella vaginalis, with daily dosing of 40mg/kg ASA. In vivo assessment of vaginal epithelial layer function using Lucifer yellow dye and immunophenotyping of vaginal T cell subsets were performed to evaluate therapeutic effects of ASA.

Results: Intravaginal challenge with Mobiluncus mulieris resulted in a 1.5-fold elevation of activated CD4+CXCR6+ T cell subsets and increased IL-17a, IL-21, and IL-22 expression signatures, whereas daily ASA treatment significantly reduced CXCR6+ T cell frequencies and Th17-associated cytokine expression. Interestingly, no significant alterations to CD4+CCR5+ T cell subsets or IFNγ expression were observed in the presence of inflammatory bacteria. Initial results also show little effect of ASA on epithelial barrier function. Intravaginal challenge with Gardnerella vaginalis revealed similar impacts on the vaginal mucosae.

Significance and Impact: Short-term ASA treatment significantly lowered key readouts of vaginal mucosal inflammation induced by BV-associated bacteria, such as the recruitment of CXCR6+ T cell subsets and pro-inflammatory cytokine production. Further assessment of ASA to lower HIV acquisition risk is warranted and currently underway.

HIV-1 is able to evade immune recognition through downregulation of major histocompatibility complex class I (MHC-I) from HIV-infected cells by using the viral protein Nef to interact with host Src family kinases (SFKs). MHC-I downregulation by Nef allows infected cells to evade immune clearance by cytotoxic T lymphocytes (CTLs). Inhibition of the Nef:SFK interaction and subsequent restoration of cell surface MHC-I expression enhances anti-HIV CTL activity. Indeed, Nef:SFK inhibitors have been investigated as adjuvants within an HIV-1 curative strategy. Previously, we developed a 2nd generation Nef:SFK inhibitor that restored surface MHC-I in vitro but displayed degradation in an in vivo murine model. We sought to improve upon the 2nd generation Nef:SFK inhibitor by developing analogs with improved in vivo activity and stability. Using organic synthesis, we have developed four 3rd generation Nef:SFK inhibitors hypothesized to have improved in vivo stability compared to their 2nd generation parental compound. We tested these inhibitors in vitro to characterize their cytotoxicity and efficacy in inhibiting Nef-induced MHC-I downregulation. Stability will be assessed in human serum by mass spectrometry and compared to progenitor compounds. Cells expressing Nef and a SFK were treated with the inhibitors for 18 hours to assess their inhibition of the Nef:SFK interaction using a luciferase-based assay. Primary human CD4+ T cells cultured with a range of concentrations of inhibitors for 24 hours showed using a luminescent assay no significant decrease in cell viability. HIV-1 NL4.3-infected primary human CD4+ T cells showed cell surface MHC-I restoration after treatment with 100 μM of H3-1A, H3-1N and H3-1T. The results of these experiments will provide us with information needed to proceed with in vivo testing and ultimately progress us closer to a potential adjuvant for anti-HIV-1 cure strategies.

Background: Approximately 50% of people living with HIV develop HIV-Associated Neurocognitive Disorders (HAND), manifested by declined behavior, motor and cognitive functions which impair their quality of life. Activation of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor known to regulate glucose/lipid metabolism can also exert anti-HIV/anti-inflammatory effects. We hypothesized that activation of PPARγ by a novel selective agonist, INT131, could suppress HIV-associated brain inflammation in vivo, in an ecotropic HIV-1 (EcoHIV) mouse model representative of HAND. The goal of this study was to examine the role of INT131 in reversing infectivity and inflammation in brain cerebellum, subcortical and cortical regions in EcoHIV infected mice.

Methods: We quantified markers of interest using qPCR analysis, including viral genes, inflammatory cytokines/chemokines, blood-brain barrier (BBB) tight junction proteins, and examined BBB permeability applying the NaF permeability assay, 21 days post intracranial injection of saline or EcoHIV (2x10^8 pg/ml) in: i) non infected mice, ii) infected EcoHIV mice, and iii) infected EcoHIV mice treated with daily oral administration of INT131 (50 mg/kg/day).

Results: Compared to saline injected controls, exposure of mice to EcoHIV, significantly increased the mRNA expression of viral genes (Vif and Tat), inflammatory markers (Tnf-α, Il-1b, Il-6, and Ifn-γ) and decreased BBB markers (Ocln, Cldn5 and Tjp-1) in brain regions. Oral administration of INT131 significantly reduced the expression of inflammatory markers and restored the expression of BBB markers to control levels. We also observed a restoration of BBB permeability, measured by NaF assay, with INT131 treatment in the EcoHIV mouse model.

Conclusion: Our in vivo work revealed that PPARγ could constitute a potential molecular target for the treatment/prevention of HIV-1 associated brain inflammation, BBB dysfunction, and potentially HAND. Future behavioural studies will investigate the efficacy of INT13l, in reversing neurocognitive deficits in the EcoHIV mouse model.

(Supported by Canadian Institutes of Health Research).

The expression of T-cell immunoglobulin and mucin 3 (TIM-3) in chronic HIV-1 infection is associated with ongoing viral replication, CD4 T-cell depletion, loss of antiviral CD8 T cell activities and progression to AIDS. It was shown recently that the HIV-1 Nef protein upregulates TIM-3 on the surface of infected cells; however, the ability of natural nef isolates to modulate TIM-3 remains to be investigated.
We assessed the ability of 131 Nef clones to upregulate TIM-3. The nef coding region was amplified from plasma HIV RNA isolated from ART-naïve individuals chronically living with HIV-1 subtype A, including 102 chronic progressors (CP) and 29 long-term survivors (LTS) and cloned. The ability of one intact, phylogenetically validated Nef clone per individual to upregulate surface TIM-3 was quantified by flow cytometry. Results were normalized to a reference clone (Nef, SF2 strain).
Our results demonstrated that participant-derived Nef clones display variable abilities to upregulate TIM-3, with a median [IQR] of 1.17 [1.06-1.29]. We found that the ability of LTS clones to upregulate TIM-3 was significantly lower (1.11 [1.01-1.24]) compared to CP (1.19 [1.06-1.31]); (p=0.01). TIM-3 upregulation activity correlated negatively with CD4 T-cell count (ρ=  0.38, p<0.0001) and positively with pVL (ρ= 0.26, p= 0.008). In addition, TIM-3 upregulation function strongly correlated with CD4 downregulation function by Nef clones (ρ= 0.61, p<0.0001), indicating that these activities may be mechanistically linked. Furthermore, TIM-3 upregulation function strongly correlated with SERINC3 (ρ= 0.30, p= 0.004) and SERINC5 (ρ= 0.52, p<0.0001) downregulation functions in the CP clones. Taken together, these findings indicate that TIM-3 upregulation activity varies among HIV-1 nef isolates and that higher ability to upregulate TIM-3 may contribute to more rapid decline of CD4 T-cell counts, enhanced viral pathogenesis and a faster disease progression.

Human Immunodeficiency Virus (HIV) persists as a global threat with over 38 million people infected. While much is known about HIV viral genomic RNA (vgRNA) packaging, vgRNA’s ability to evade the Nonsense Mediated mRNA Decay (NMD) pathway is poorly understood. Typically, NMD functions to degrade “faulty” transcripts that contain Premature Termination Codons (PTCs). Recent studies have demonstrated NMD protein UPF1 robustly and nonspecifically binds RNAs and translating ribosomes are capable of displacing UPF1. Despite the HIV RNA genome containing multiple PTCs, it is not degraded and instead is preferentially packaged into virions. The Arts laboratory has recently identified a critical packaging element termed the Genomic RNA Packaging Enhancer (GRPE) that overlaps the ribosomal frameshifting site needed for translation of the Gag-Pol polyprotein. It is hypothesized the GRPE modulates translational readthrough of the frameshift sequence allowing for UPF1 displacement and subsequent NMD evasion. We hypothesize transcripts undergoing Gag-Pol translation designates these mRNAs as genomic RNA for progeny virions. To elucidate the GRPEs involvement in packaging and NMD evasion, a modified HIV backbone with luciferase reporters was created to quantitatively measure frameshifting frequency. TaqMan qPCR will also be used to determine RNA stability and decay over time. Preliminary experiments place our constructs frameshifting frequency similar to literature values. Mutant frameshifting sequences will be created to evaluate their effects on frameshifting and RNA decay. Wildtype frameshifting frequencies will be calculated as well as in the presence of NMD inhibitors and siRNA knockdowns of NMD proteins. Understanding the mechanism behind NMD evasion could lead to the development of small molecule inhibitors to aid in antiretroviral therapy. In addition, lentiviral gene transduction systems do not harbour a GRPE and incorporation resulted in a >4-fold increase in transduction efficiency, increasing the plausibility of future clinical use.

Experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RM) has provided useful models for studying viral pathogenesis and the host immune response. Thus, viral seeding in the liver has been associated with tissue inflammation and damage and comorbidities in people living with HIV. In addition to classical CD4 T cells, Natural Killer T (NKT) cells have been reported to be infected. However, despite the role of NKT cells in hepatic immunity, little is known so far about their contribution in viral seeding. Thus, we analyzed and characterized the dynamics of hepatic CD4 and NKT cells during SIV infection.
Naïve (n=3) and SIVmac251-infected RMs (20 AID50) (n=12) were sacrificed at different time post-infection. On the day of sacrifice, liver cells were recovered and stained with specific monoclonal antibodies. Cells were analyzed by flow cytometry. Cell sorting was used to isolate cell subsets to quantify the frequency of viral DNA by qPCR.
Our results demonstrated a depletion of CD4 T cells in the liver correlating to that observed in the blood. In contrary, the percentage of NKT, characterized by the expression of CXCR6 and CD161, increased in the liver of SIV-infected RMs compared to uninfected animals. Whereas CD4 T cells from the liver displayed a T cell effector memory phenotype, NKT cells displayed a central memory phenotype. We also demonstrated that hepatic NKT cells express viral DNA.
Thus, we identified in the liver a new subset of infected T cells. These two populations represent potential viral reservoirs in hepatic tissue.