We previously demonstrated that Th17-polarized CD4+ T-cells are enriched in viral reservoirs (VR) in people with HIV-1 (PWH) receiving antiretroviral therapy (ART), and that the Th17 master regulator RORC2 acts as positive regulator of HIV replication/outgrowth. REV-ERBalpha/beta acts as transcriptional repressors of RORC2. Thus, we hypothesized that REV-ERBalpha/beta inhibits HIV-1 transcription/replication via mechanisms involving the repression of RORC2.
Memory CD4+ T-cells of HIV-uninfected individuals were exposed to replication-competent and single-round HIV-1 constructs upon CD3/CD28 triggering in vitro. Early and late reverse transcripts, and integrated HIV-DNA were quantified by nested real-time PCR. HIV-1 replication was quantified by FACS and ELISA. A quantitative viral outgrowth assay (VOA) was performed using memory CD4+ T-cells of ART-treated PWH. The REV-ERBalpha/beta agonist SR9011 and the antagonist SR8278 were used to modulate REV-ERBalpha/beta activity. RNA-Sequencing was performed using the Illumina technology. Gene expression was quantified by real-time RT-PCR and western blotting.
SR9011 decreased RORC2 and IL-17A expression, and reduced HIV-1 replication in vitro and viral outgrowth in T-cells of ART-treated PWH. SR8278 reduced HIV replication in vitro and viral outgrowth, in part via decreasing the expression of the HIV-1 co-receptor CCR5. In a single-round infection model, the REV-ERBalpha/beta drugs increased the intracellular expression of HIV-p24 but decreased the release of viral particles. These effects coincided with an increased SAMHD1 phosphorylation, indicative of efficient reverse transcription. Surface expression of BST2 was increased by SR9011 but not SR8278. RNA-Seq analysis revealed an increase of APOBEC3G mRNA in the presence of SR8278.
These results provide evidence that REV-ERB modulates Th17 effector functions by repressing RORC2 and interferes with HIV replication at multiple levels including entry (CCR5), reverse transcription (SAMHD1), integration, translation, virion release (BST2) and infectivity (APOBEC3G), with the REV-ERB blockade reverting HIV latency, while limiting viral propagation Thus, REV-ERB pharmacological targeting may be used in HIV cure/remission strategies.

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV-1 transcription in colon-infiltrating CD4+ T-cells, and decreased systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin.

Experiments were performed with CD4+ T-cells isolated by negative selection using magnetic beads from HIV-uninfected participants, infected with wild type CCR5-tropic HIV-1 NL4.3BaL (HIVNL4.3BaL) or single round VSV-G-pseudotyped HIV-1 (HIVVSVG) in vitro, as well as T-cells of ART-treated PWH ex vivo (n=8/group/experiment). HIV-1 replication was measured by real-time nested PCR (early/late reverse transcripts, integrated HIV-DNA), ELISA (soluble HIV-p24) and flow cytometry (intracellular HIV-p24). A viral outgrowth assay (VOA) was used to measure replication-competent HIV-1 reservoirs. Cell-associated HIV-RNA was measured by real-time RT-PCR. The HIV-1 RNA/DNA ratio was used as a surrogate marker of HIV-1 transcription. Surface BST2/Tetherin and intra-nuclear BCL2 expression, as well as binding of broadly neutralizing (bNAbs; 2G12, PGT121, PGT126, PGT151, 3BNC117, 101074, VRC03) and non neutralizing (nnAbs; F240, 17b, A32) HIV-1 Env Abs, were measured by flow cytometry.

In the VOA performed with CD4+ T-cells from ART-treated PWH, and upon infection in vitro with HIVNL4.3BaL and HIVVSVG, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T-cells by the PGT126 bnAbs, recognizing the “closed” conformation of HIV 1 Env. This improved recognition could potentially translate in killing of infected T-cells by ADCC.

Thus, metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Tissues harboring HIV-1 are potential key reservoirs that are established early during infection and represent a major barrier for an HIV-1 cure. An essential and permanent step in the HIV life cycle is the integration of its viral genome into its host’s genome. This integration site targeting is not random and plays a critical role in the expression and long-term survival of the integrated provirus. HIV-1 integration site profiles have been extensively studied in peripheral blood from infected individuals; however, less is known about integration site distribution in clinically derived anatomical tissues. To better understand the heterogeneity of tissue HIV reservoirs, we characterized the genomic environment surrounding integrated HIV-1 proviruses from a unique biobank of cryopreserved biopsy samples of esophagus, peripheral blood leukocytes/peripheral blood mononuclear cells (PBL/PBMC), stomach, duodenum, colon and brain tissue from HIV-1 subtype B infected individuals between the years of 1993 and 2010. Integration site profiles with respect to common genomic features were comparable in all tissues except brain, which exhibited significantly reduced integration frequency in genes and increased integration in short interspersed nuclear elements (SINEs) and DNaseI hypersensitivity sites. Strikingly, we observed differential targeting of non-canonical B-form DNA (non-B DNA) with distinct tissue-specific preferences. In addition, we identified remarkable integration site overlap between tissues and between individuals with integration hotspots enriched in non-B DNA. Lastly, we identified several genes that were highly targeted for integration in all tissues that have been linked to disease. Our data offers fresh insights into the genomic landscape surrounding integration sites of HIV-1 in various tissues of infected individuals.

One of the biggest obstacles in curing HIV-1 infection is the elimination of infected cells that remain in a dormant state and do not actively replicate, a state referred to as latency. One important facet of HIV-1 latency involves the ability of the HIV-1 genome to integrate into regions of the human genome that promote latency. Previously, we showed that HIV-1 specifically targets non-canonical B-form DNA (non-B DNA) features, particularly guanine quadruplex (G4), for integration. Non-B DNA structures are alternative DNA structures that differ from the regular B-DNA double helix conformation and assume different shapes and structures, akin to a genomic form of Braille. We showed that integration near one form of non-B DNA, G-quadruplex (G4) DNA, increases the pool of latently infected cells. To better understand how the HIV-1 pre-integration complex (PIC) targets G4 DNA, we compared the integration site profiles of HIV-1 containing subtype A, B or D variants of integrase. We showed that the subtype D integrase variant significantly enhanced integration site targeting of G4 DNA compared to subtypes A and B. Co-immunoprecipitation of the different HIV-1 PICs, followed by comparative liquid chromatography mass spectrometry analysis of the PIC proteins, identified Nucleolin, Nucleophosmin, and Heterogenous nuclear ribonucleoprotein A1 as G4 DNA binding candidates that were enriched in subtype D PICs. As such, this data identifies novel human protein candidates that may direct the PIC to host G4 DNA during integration and influence integration patterns. Likewise, our findings also shed light on potential mechanisms behind HIV-1 latency and introduces promising avenues for antiviral drug therapies targeted to eradicating latent HIV-1 reservoirs.

Background: Dolutegravir (DTG) is a well-tolerated drug used in first-line combination antiretroviral therapy. However, DTG is associated with oxidative stress in clinical and in vitro studies, a pathway that may contribute to the initial small increase in rates of neural tube defects (NTD) reported in 2018. We investigated the effect of DTG on oxygen-dependent enzymes before and after neural tube closure in a murine pregnancy model.

Methods: C57BL/6 mice were mated and randomized 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. Embryos and placentas were collected at gestational days 9.5 and 11.5. RT-PCR and western blot assessed gene and protein expression of relevant enzymes with HPRT, and β-actin used as housekeeping genes/proteins. Statistical analyses were performed using one-way ANOVA with Tukey’s post-test or Kruskal-Wallis with Dunn’s post-test.

Results: Embryo mRNA levels of superoxide dismutase 1 (SOD1) and SOD2 were similar between groups at GD9.5 (pre-neural tube closure). Post neural tube closure (GD11.5), embryo SOD1 mRNA levels were significantly lower, while protein levels were higher in both DTG groups versus control (1xDTG p= <0.0001, 5xDTG p=0.0079 for SOD1 mRNA; 1xDTG p= 0.0036, 5xDTG p=0.0136 for protein). SOD2 mRNA expression was significantly lower in embryos exposed to the therapeutic dose of DTG versus controls (p<0.0001), but no differences were observed at the protein level. Placental SOD1 protein expression was significantly higher in GD9.5 DTG-treated dams, but significantly lower in GD11.5 DTG-treated dams (p<0.001).

Conclusion: DTG was associated with a lower embryonic mRNA and higher protein levels of the anti-oxidant enzyme SOD1 in the post-neural tube closure window, suggesting that DTG may dysregulate anti-oxidant pathways that may result in oxidative stress common to many pathways leading to congenital defects.

Background: Integrase inhibitors (INSTI) are the most recommended antiretrovirals around the world. However, some people experience weight gain upon initiating INSTIs, and this might implicate metabolic off-target effects. Mitochondria play an important role in metabolism, and mitochondrial DNA (mtDNA) content is a biomarker for mitochondrial dysfunction. Here, we aimed to measure mtDNA content changes in women with HIV who have switched to an INSTI-based regimen, compared to women who remained on their protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) containing regimens.

Methods: This was a retrospective study. All women enrolled in the CARMA and/or BCC3 study who had blood specimens available before and after switching to INSTIs were included. Each woman was matched 1:1 for age, ethnicity, PI/NNRTI pre-switch, and smoking status with women who remained on their PI/NNRTI-containing regimens. Blood mtDNA was determined using monochrome multiplex qPCR and % change per year compared between groups by Mann Whitney U Test.

Results: Fifty-six women (40 PI-, 16 NNRTI-regimen) were matched with 56 controls (Table 1). While older age was associated with lower mtDNA content (p=0.0045), no significant differences in the change in mtDNA/year were detected between women who switched to INSTI and those who continued their regimens (p=0.77). Mitochondrial DNA content changes were not associated with ethnicity, smoking status, viral load or CD4 count.

Conclusions: Switching to INSTI showed no association with mtDNA content, a reassuring finding. However, given the large variance observed, associations between changes in mtDNA and experiences of adverse effects should be further investigated.

Background:
The HIV viral reservoir persists in latently infected CD4+ cells and rebounds upon treatment interruption, hindering a sterilizing cure. Understanding the mechanisms of virus re-emergence may inform cure treatment strategies. Potential factors include HIV escape from CTL responses or inadequate HIV-specific CTL responses. It is unknown whether mutational escape from HIV-specific CTL responses occurs during viral rebound in early-treated individuals (<6 months).

Methods:
To determine if rebound HIV displays mutational escape from pre-cART T cell responses, subject immune responses were epitope mapped and plasma virus was sequenced prior to treatment and during analytical treatment interruption (ATI). Participants (n=4) were treated during the early phase of infection and later participated in the placebo group for an HIV therapeutic vaccine trial in which ATI was a component (NCT01859325). We mapped subject-specific anti-HIV epitopes using IFN-γ ELISpot assays with peptides spanning Gag, Pol, Env, and Nef (NIH reagents program). Plasma virus was sequenced pre-cART and post-peak viral rebound during ATI and mutational escape was assessed by comparing amino acid sequences of CTL-eliciting epitopes from both timepoints.

Results:
By ELISpot, only Gag epitopes elicited responses in all subjects. Furthermore, we determined that amino acid sequences from plasma HIV RNA harvested prior to cART initiation and following peak viral rebound during ATI showed no instances of mutational escape in confirmed CTL-eliciting epitopes. When comparing pre and post ATI sequences, only one mutation was observed in an individual during ATI which putatively corresponds to Env escape from the antibody response.

Conclusions:
Our results suggest that immune epitope escape to pre-existing T cell responses is not a major factor in viral rebound of HIV during treatment interruption for early-subjects patients. Other immunological factors such as T Cell exhaustion, or reservoir resistance to killing by CTL should be investigated to understand viral rebound dynamics during ATI.

A significant roadblock to the generation of an HIV-1 vaccine has been the inability to elicit cross-clade neutralizing antibody responses capable of protecting against the exceptionally high level of HIV-1 genetic diversity circulating in human populations. By targeting HIV-1 variants with high transmission fitness for our vaccine development, we hope to exploit the phenotypic characteristics that allow theses viruses to establish new infections. By then eliciting antibodies capable of neutralizing these transmitted/founder(T/F) viruses we hypothesize that we will induce an enhanced protective response as compared to immunogens generated from lab adapted HIV-1 variants. To that end, we used a Mann lab bioinformatics tool to screen thousands (n=7705) of HIV-1 subtype B sequences within the Los Alamos database, to identify viruses which exhibit characteristics for enhanced transmission fitness. Using our in-silico approach we have identified three highly transmissible subtype B viruses to form our baseline immunogens. Additionally, we have engineered a series of structural and N-linked glycan mutations into the glycan shield of our HIV immunogens to expose specific sites known to be highly vulnerable to neutralizing antibody mediated attack. The resulting mutant viruses were shown to have increased vulnerability to neutralization by a variety of cross-clade broadly neutralizing antibodies (BnAbs). Preliminary data evaluating the antigenicity of our HIV Immunogens in BnAb expressing murine B-cells show our most vulnerable immunogens were also highly antigenic. We are in the process of generating virus-like particles (VLPs) of our most promising vaccine candidates using our established protocols. Our VLPs are genome-less assemblies of HIV structural proteins which are morphologically indistinguishable from HIV-1. As vaccine delivery platforms, we have published data showing our VLP immunogens possess in-vitro CD4 T cell immune priming and recall capabilities. By generating cross-clade neutralizing antibodies through vaccination, this research will have overcome the most significant roadblocks to HIV vaccine development.

The HIV reservoir is the major barrier to cure, yet the factors that influence reservoir size during ART remain incompletely characterized. We investigated sociodemographic, clinical, and immunogenetic correlates of HIV reservoir size in 105 ART-treated PLWH.
HIV reservoir size was measured in genomic DNA from CD4+ T-cells using the Intact Proviral DNA Assay, where primers/probes were adapted to within-host HIV polymorphism where required. HLA class I types were characterized by sequence-based typing.
Participants were 90% male and median 51 (interquartile range [IQR] 38-59) years old. Participants had a median recent CD4+ T-cell count of 740 (IQR 508-953) cells/mm³, and a median nadir CD4+ T-cell count of 260 (IQR 110-480) cells/mm³. They had been receiving ART for a median 8.7 (IQR 4.4-13.2) years, and 65% were receiving an INSTI-based regimen at time of sampling. Most (92%) HIV infections were subtype B.
The median intact proviral load (i.e. reservoir size) was 79 (IQR 31-194) HIV copies/million CD4+ T-cells. The median total proviral load (i.e. intact and defective) was 984 (IQR 509-2143) HIV copies/million CD4+ T-cells. These measures correlated strongly (Spearman ρ=0.72, p<3e-16). HLA-B*07:02 carriage was associated with both larger reservoirs and total proviral load, while HLA-A*02:01 was associated with larger total proviral load (all p<0.02, q<0.18). In univariable analyses, lower nadir and recent CD4+ T-cell counts, and receiving a non-INSTI-based regimen, were associated with larger reservoirs (p<0.03). Older age, lower nadir CD4+ T-cell count, longer time on ART, and receiving a non-INSTI-based regimen were associated with higher total proviral load (all p<0.002), but only older age and lower nadir CD4+ T-cell count remained significant after multivariable analysis (both p<0.05).
We identified older age and lower nadir CD4+ T-cell count as correlates of higher total proviral loads during ART. The mechanisms underlying HLA associations with reservoir size require further investigation.

The HIV envelope glycoprotein (Env) is critical for infection, as it is the key protein mediating viral attachment and entry. Moreover, it is the only viral protein present on the HIV surface, making it a central target in vaccine development. However, Env is notoriously difficult to target because it is present in low amounts on the virus surface, has high genetic variability, and a high level of glycosylation. These features present key challenges to studying the basic biology of HIV Env using conventional techniques, and have limited knowledge discovery. Herein, we demonstrate the utility of a novel approach to study HIV Env, using Flow Virometry (FV), an emerging technique that can sensitively probe virion surfaces in a high-throughput, single-particle manner.

We performed FV on HIVIIIB produced in the H9 T cell line, using indirect staining techniques with a panel of 85 monoclonal anti-Env antibodies. A subset of antibody stains were performed in the presence/absence of soluble CD4, to detect differences in Env conformations. We also assessed the accessibility and quantity of Env in the context of different virus models, comparing antibody binding to the same HIV isolate (HIVBaL) produced in different cell types (293T, T cell lines, and PBMC).

We observed that many anti-Env antibodies can be used in indirect staining techniques to study HIV Env with FV, with the highest quantitative staining results using anti-V3 loop antibodies. Env staining with FV was influenced by the virus model used, with highly divergent staining observed on identical viral isolates produced in different cell types. We also observed that some antibodies perform differently when tested in parallel across different assays (FV, neutralization, and virus capture) applied to identical viral stocks. By staining viruses in the presence/absence of soluble CD4, we showed that FV can sensitively assess differences in the HIV trimer conformation.

Vaccination has been critical in stemming the COVID-19 pandemic; however, the effectiveness of vaccination in immunocompromised populations remains an important area of research. Studies have shown end-stage renal disease (ESRD) patients have muted memory B cell formation and reduced humoral responses to COVID-19 vaccination; however, early innate immune responses have yet to be characterized. To address this, we collected blood before (BD1) and 1-4 days post dose 1 (PD1) of BNT162b2 vaccination in ESRD patients (n= 39 BD1, 35 PD1) and healthy controls (34 BD1, 15 PD1) for quantification of 20 plasma cytokines. Detailed enrolment and follow-up questionnaires capturing demographic, medical and COVID-19 infection information was collected from participants. Samples were also collected for RNA sequencing and cellular and antibody responses at multiple timepoints post vaccination. We observed ESRD patients to have an elevated (p<0.01) baseline (BD1) inflammatory cytokine profile (IFN-γ, IL-1β, IL-6, TNF-α, eotaxin, IP-10, MCP-1, MIP1-α, MIP1-β) compared to healthy controls. When stratifying by sex, female ESRD patients had a higher concentration of key inflammatory cytokines than males at baseline which appears to drive the inflammatory profile in the full sample. This trend is also found when considering clinical biomarkers (MIP1-α, MIP1-β) for ESRD, with a higher concentration found in female ESRD patients. Despite baseline inflammation, ESRD patients were able to mount cytokine responses to vaccination similar to those of healthy controls, with females in both populations more reactogenic to vaccination than males. Specifically, IL-2, IL-10, and IP-10 were more significant in ESRD females (0.001p<0.0001) compared to ESRD males (0.01

Despite advances in antiretroviral therapy, human immunodeficiency virus (HIV) persists in lymphoid organs. HIV specifically infects T lymphocytes coexpressing the CD4 and the CCR5 or CXCR4 coreceptors. Unlike its counterpart HIV, CXCR4 is not used by the simian immunodeficiency virus (SIV). In vitro, it has been suggested that SIV uses alternative coreceptors such as GPR15. However, little is known about the dynamics of CD4 T cells expressing GPR15 compared to those expressing CCR5. Here, we analyzed the expression level and the dynamics of CCR5- and GPR15-expressing CD4 T cells in rhesus macaque (RM).

RM infected with SIVmac251 (20 AID50) were sacrificed at different times post-infection. Blood and lymphoid organs were recovered, and cells were stained with specific antibodies and then analyzed by flow cytometry.

Firstly, we observed that naïve cells CD4 T cells express low levels of coreceptors whereas effector memory and terminal differentiated CD4 T cells express CCR5. T cells expressing CCR5 do not express GPR15 suggesting distinct T cell subsets. Thus, our results indicated that central memory CD4 T cells represent the main population expressing GPR15. In the blood and peripheral lymph nodes, we observed the early depletion of CD4 T lymphocytes expressing the CCR5 and GPR15 receptors. Of interest and consistent with immune activation, we found that CD4 T cells expressing CCR5 are increased at eight weeks post-infection. Similarly, we found that peripheral blood CD4 T cells expressing GPR15 are also increased. Antiretroviral administration prevented such early depletion.
The depletion of GPR15-expressing CD4 T cells like those expressing CCR5 cells strongly suggest the use of GPR15 as a coreceptor for SIV infection in RMs.

HIV-1 is the causative agent of a global pandemic infecting tens of millions of people and resulting in the death of hundreds of thousands each year. Lenacapavir (GS-6207) is a novel antiretroviral recently approved by the FDA and the first drug to target the HIV-1 capsid. Lenacapavir binds to hexameric units within the capsid and causes hyperstabilization, inhibiting proper virion assembly and uncoating. The M66I mutation in the capsid protein was previously shown to be the primary cause of resistance against Lenacapavir in clinical and laboratory settings. However, M66I mutants also have significantly lower fitness than wild-type viruses in the absence of Lenacapavir. The mechanism by which the M66I mutation confers resistance to Lenacapavir while maintaining the viability of the virus is unknown. We hypothesize that treatment with Lenacapavir selects for HIV-1 virions possessing a mixture of M66I and wildtype capsid proteins produced during co-infection events. We predict that this viral trans-encapsidation confers sufficient resistance to Lenacapavir without over-compromising the function of the viral capsid. To determine the effect of M66I/wildtype trans-encapsidation on Lenacapavir resistance, HEK293T cells were cotransfected by plasmids encoding wild-type and mutant HIV-1 fused with EGFP in the presence or absence of Lenacapavir. Viral titres were determined by assaying for reverse transcriptase activity or EGFP fluorescence. We found that in the presence of Lenacapavir, M66I-mutant viruses were produced at higher titres than in its absence. Further research will investigate the effect of M66I/wild-type trans-encapsidation on capsid stability and uncoating in the presence of Lenacapavir. Additionally, dose response analysis will be used to further investigate how production of M66I viruses is enhanced by Lenacapavir. Finally, mass spectrometry will be used to quantify the M66I:wild-type capsid ratio in the fittest virions following Lenacapavir selection.

Background: Despite the success of ART, people living with HIV (PLWH) suffer from inflammatory comorbidities such as coronary artery disease (CAD). Tryptophan (Trp) catabolism into Kynurenine (Kyn) via the IFNg-inducible enzyme indolamine2,3-dioxygenase (IDO), is associated with HIV disease progression and atherosclerosis. Herein, we assessed the Trp metabolism in PLWH with subclinical CAD.

Materials and Methods: Plasma specimens from HIV+CAD+ (n=34), HIV+CAD- (n=32), HIV-CAD+ (n=17) and (4) HIV-CAD- (n=23) participants were obtained form the Canadian HIV Aging Cohort Study. CAD was assessed by cardiac computed tomography angiography. Plasma levels of Tryptophan metabolites were measured by solid-phase-extraction liquid chromatography–tandem mass spectrometry. Soluble inflammatory mediators were measured by Luminex or ELISA.

Results: IDO activity defined by Kyn/Trp ratio, was significantly increased in HIV+CAD+ compared to HIV-CAD- (p=0.0023) and HIV-CAD+ (p=0.0018). Accordingly, plasma IFNg levels were highest in HIV+CAD+ individuals within study groups and correlated positively with Kyn/Trp ratio (p=0.02, r=0.435). Positive correlation of Kyn/Trp ratio with plasma IP-10 levels in both HIV+ and HIV- groups were observed, while independent of the CAD status. Significant increases in the Trp metabolites kynurenic acid, althranilic acid were observed in PLWH, while the levels of xanthurenic acid were decreased in PLHW independent of their CAD status. HIV+CAD+ individuals also exhibited with the highest plasma levels of the markers of gut mucosal damage REG-3α and IFABP within study groups, but only IFABP was associated with the CAD status. Increase in the levels of other inflammatory mediators sTNFR-II and IL-6 were associated to the presence of HIV, but independent of CAD.

Conclusions: IDO activity and Trp/Kyn ratios were increased in HIV+CAD+ individuals compared to both HIV negative groups, along with their higher levels of the markers of the gut mucosal damage and IFNg.

The major barrier to a cure for HIV-1 is the reservoir of latently infected cells primarily within memory CD4+ T cells. Thus, understanding the regulation of HIV-1 expression is critical for developing curative therapies for HIV-1, by affecting either provirus transcription or processing of viral RNA. Our lab determined that SR kinases, known regulators of RNA splicing, can modulate HIV-1 gene expression and latency. Specifically, we found that CLK1 suppresses transcription initiation from the HIV-1 promoter, while CLK2 affects post-initiation events essential for the accumulation of viral mRNA. Our investigations have shown that depletion of CLK1 also increases the percentage of cells responding to latency-reversing agents (LRAs), indicating that CLK1 plays a role in regulating HIV-1 latency. To further investigate these mechanisms, we have examined the impact of depleting specific CLKs on HIV-1 promoter activity. Consistent with our current model, reducing CLK1 levels enhances HIV-1 promoter function, while depletion of CLK2 or CLK3 has no effect. In experiments investigating the effects of CLK2 depletion on nascent HIV-1 RNA expression, we observed a significant decrease in completely spliced viral RNA abundance but other viral RNAs were minimally affected, indicating that CLK2 is essential for the removal of the last intron. Parallel ChIP assays revealed that all CLKs selectively associate with the HIV-1 promoter, consistent with the hypothesis that CLKs act in a local fashion to regulate various steps of HIV-1 gene expression. Additionally, we have demonstrated that the depletion of CLKs to levels that affect HIV-1 gene expression has limited impact on expression of most host genes, at the level of RNA abundance, alternative splicing, or polyadenylation. Collectively, these findings highlight the sensitivity of HIV-1 gene expression to small changes in CLK activity, indicating that CLKs are promising targets for development of curative strategies for HIV.

The HIV/AIDS pandemic, originating from spillovers of SIV in chimpanzees and sooty mangabeys, has surpassed COVID-19 in casualties — AIDS killed more than five times more people than COVID-19. In elite controllers (0.5% of HIV infected individuals), viral replication remains undetectable despite the presence of a replication-competent viral reservoir. Integration site disparities between elite controllers and long-term antiretroviral therapy patients shed light on viral persistence mechanisms. Distinct reservoir configurations prompt questions about HIV integration site selection preferences: are there differences in integration site selection between the two types of HIV? Largely confined to the West of Africa, HIV-2 receives less attention due to its slower progression compared to the globally dominant HIV-1. The genomic sites of HIV-2 integration require deeper exploration. Strategies to determine viral integration sites include bulk sequencing that can be cost challenging. The recent mapping approaches are very promising techniques, sensitivity scalable down to single-cell level. However, low throughput and high cost remains an issue. Practically, the limitations do not work in favor of clinical study. Here, we propose the use of a novel high throughput sequencing mapping technique based on linear amplification and A-tailing that will address some of the limitations of previous mapping strategies to identify the site and frequency of HIV-1 and HIV-2/SIV integration in cell models. The protocol is optimized using HIV-1 infected cell lines. We have obtained the first dataset, and the high-resolution maps provide in-depth understanding of insertion hotspots. This technology has the potential to characterize highly complex samples with multiple target sequences and has sufficient sensitivity, specificity, and robustness to detect and sequence even single cell insertion events via an initial linear amplification step. The long-term plan is to apply this new mapping approach on clinical samples.

Title: Investigational Studies to Understand the Decreases in Lymphocytes Seen Clinically with Islatravir (ISL) and Enabling the Initiation of New Clinical Trials
Purpose:
ISL is an inhibitor of HIV-1 replication. Its active form (ISL-triphosphate; ISL-TP) preferentially accumulates in lymphocytes. During Phase 2/3 clinical trials, ISL-related decreases in lymphocytes were observed at doses (e.g., ≥0.75 mg QD) corresponding to ISL-TP concentrations ≥35 µM, but not at doses associated with an ISL-TP concentration of 9µM). Studies were conducted to investigate possible mechanisms for these lymphocyte decreases.
Method:
In vitro effects of ISL and several HIV NRTIs on TK6 cells (lymphoblast cell-line) population doubling and mitochondrial DNA content were evaluated. In vitro effects of ISL on human PBMC cytotoxicity, activation, and function were also assessed. ISL TP was evaluated for its ability to inhibit human DNA polymerases α, β, and γ. Additionally, ISL was evaluated in a 10-week immunophenotyping study in mice.
Results:
In TK6 cells, ISL and most NRTIs resulted in population growth inhibition at similar triphosphate concentrations. ISL-TP concentrations that resulted in cytotoxicity in TK6 cells and human PBMCs were comparable. ISL-TP weakly inhibited DNA polymerase α. Consistent with the lack of inhibition of polymerase γ, ISL did not impact mitochondrial DNA content. In mice, decreased lymphocyte counts were observed at ISL-TP PBMC levels associated with lymphocyte decreases in humans. Additionally, effects on B and/or T cell activation in splenocytes from mice dosed with ISL were observed at ISL-TP levels that greatly exceed those achieved at the clinical dose without lymphocyte effects.
Conclusions:
Preferential accumulation of high ISL-TP concentrations in lymphocytes can lead to cytotoxicity, possibly through DNA polymerase α inhibition as a contributing factor. These effects are shared with several marketed NRTIs, at similarly high triphosphate levels. Mitochondrial toxicity is not a contributing mechanism. These investigations, together with clinical and modelling data, identified an ISL-TP threshold (9µM), and subsequently ISL QD and QW clinical doses) below which decreases in lymphocytes are not expected.

Due to the existence of a latently infected immune cell population, termed the latent reservoir, Human Immunodeficiency Virus Type 1 (HIV-1) persists in people living with HIV-1 (PLWH) on antiretroviral treatment (ART). Recent studies have shown that the ability of the HIV-1 accessory protein Nef to downregulate Major Histocompatibility Complex Class I (MHC-I) positively correlates with the size of the latent reservoir in PLWH on ART. Most research on the latent reservoir involves high-income countries dominated by the Subtype B strains. As such, little is known of the impact non-Subtype B Nef proteins have on the latent reservoirs of PLWH in lower-middle-income countries.
To address this, the MHC-I downregulation ability of non-Subtype B Nef proteins from ART-treated PLWH in Uganda was characterized and demonstrated a positive correlation on latent reservoir size. However, it is unknown if Nef’s other functions affect latent reservoir size. SERINC5 – a cell surface host restriction factor – incorporates into the host-derived membrane during egress and reduces progeny virion infectivity. To counteract this, Nef downregulates SERINC5 from the cell surface to restore infectivity.
As SERINC5 downregulation is important for enhancing infectivity, we hypothesize that the ability of non-Subtype B Nef derived from PLWH in Uganda to downregulate SERINC5 will also positively correlate with the associated latent reservoir size. To test this, primary nef sequences will be cloned into pN1 expression vectors to produce a Nef protein C-terminally linked to the eGFP fluorophore upon cellular expression. The clones will be expressed in CD4+ HeLa cells along with plasmids encoding SERINC5, with cell surface levels being determined via flow cytometry. Additionally, we will carry out luciferase assays to determine how SERINC5 downregulation impacts virion infectivity.
Overall, we aim to characterize non-subtype B Nef’s ability to downregulate SERINC5, and its relationship with latent reservoir size in PLWH on ART.

Background: HIV remains a major global health issue, with over 39 million people living with the virus and 1.3 million new infections in 2022 alone. During infection, HIV specifically targets the CD4 molecule found on T helper cells (Th cells) to mediate its viral entry. There are five well-characterized subsets of CD4 T cells: Th1, Th2, Th17, regulatory T cells, and follicular helper T cells. The Th subsets are particularly important to the immune system due to their specialized cytokine productions, enabling them to target specific classes of pathogens. Moreover, different Th subsets can exhibit varying levels of susceptibility to HIV infection. To investigate these HIV target cells, we are developing a flow cytometric assay to detect Th subsets and measure their expression of biomarkers associated with increased HIV risk.

Methods: The development of this assay involved utilizing peripheral blood mononuclear cells (PBMCs) from healthy Winnipeg donors (n=4). Following PBMC isolation, cells were pre-stained for chemokine receptors to identify specific Th subsets: Th1 (CCR6-CXCR3+); Th2 (CCR6-CXCR3-CCR4+); Th17(CXCR3-CCR6+CD161+); Th1/17 (CCR6+CXCR3+). Following pre-staining, the cells were subjected to ex vivo staining for T cell markers (CD3, CD4, and CD8 T cells) and activation markers (CD38, HLA-DR, and CD69).

Results: This assay has been optimized for clone interactions, fluorescence compensation, voltage titration, and antibody titration. Upon implementation, this assay can measure the % frequency of the following Th subsets in PBMCs: Th1 (% mean ± SD: 12.10± 1.54), Th2 (12.60 ± 8.37), Th17 (6.38 ± 3.36), Th1/Th17 (6.53 ± 2.52). Moreover, the assay can also measure activation markers associated with increased HIV risk (CD38, HLA-DR, CD69, CCR5) in each Th subset, resulting in 24 potential readouts.

Conclusion: Moving forward, this assay will be employed to characterize HIV susceptibility in PBMCs collected from sex workers in Nairobi, Kenya.

Heavily glycosylated, the envelope of human immunodeficiency virus 1 (HIV-1) plays a key role in its transmission. During transmission, a single HIV-1 clone, the transmitter/founder (T/F) virus, establishes infection within the new host. Previous work done by our group has focused on characterizing the traits of T/F HIV-1 that allow for its successful transmission and identified glycosylation as a selection factor for transmission in human genital tissue. During ex vivo experiments when genital tissues were saturated with lectins that prevented glycans on the HIV-1 envelope from binding, the virus was better able to enter the tissue and establish infection. Thus, the role of HIV-1 envelope glycosylation and its influence on the establishment of infection is a central focus of our group’s research.

We believe that the type and amount of glycans on the HIV-1 envelope may be a selection factor for efficient transmission. However, TF HIV-1 propagated in vitro has the specific glycan patterns of the cell lines used for propagation. These glycan patterns are distinct from those present on the TF HIV-1 from an infected individual's blood or vaginal tract. We propose to instead, systematically modify the glycans on HIV-1 to determine the best glycan composition for the most efficient heterosexual transmission. Glycans will be modified by treating virions with glycosidases and propagating viruses in cell lines with inhibited transferases created using CRISPR/Cas9. These glycan-modified HIV-1 virions will then be tested for transmission fitness in human genital tissues to determine the best glycan composition for efficient transmission. HIV-1 envelope glycosylation continues to be a major challenge in vaccine development. Through this project, we aim to identify the most optimal glycan composition for an HIV-1 vaccine design and to expand the scientific understanding of glycans and their role in HIV-1 transmission.

Over 38 million people live with HIV infection, of whom 25% will experience neurological disability. HIV infection activates the brain’s innate immune cells causing inflammation and neuronal death that underpins the neurodegenerative disorder, HIV-associated neurocognitive disorder (HAND). Inflammasome activation triggers the proteolytic cleavage and release of specific cytokines (IL-1beta and -18) and a type of inflammatory programmed cell death termed pyroptosis. Pyroptosis is mediated by a group of proteins called the gasdermins that undergo proteolytic cleavage mediated by caspases and other proteases; the cleaved gasdermins cause plasma membrane pore formation with ensuing cell lysis and death. We investigated that the primate-specific pyroptosis-associated protein, gasdermin B (GSDMB). In the brain tissues from patients with HAND, GSDMB was highly expressed based on the RNA-seq analyses of frontal cortex that was confirmed by RT-PCR. Immunofluorescence studies revealed increased GSDMB expression in glial cells, which was verified by GSDMB western blotting of neural cells showing increased expression in astrocytes. RNA-seq showed increased caspase-1 transcripts in brain samples from HIV-infected persons. Cleavage of GSDMB by caspase-1 resulted in a N-terminus moiety (N-GSDMB). Immunodetection revealed GSDMB to be expressed in oligodendrocytes and astrocytes of HIV [+] brains and antiretroviral therapy (ART) treatment in patients with HIV decreased GSDMB expression versus non treated patients. Expression of N-GSDMB in transfected astrocytes caused pyroptosis, evidenced by increased LDH release, in contrast to the transfected full-length or C-terminus of GSDMB. In a model of SIV infection of nonhuman primates, we observed increased GSDMB immunoreactivity in the brains of animals with encephalitis including the N-GSDMB moiety. In conclusion, GSDMB was principally expressed in astrocytes and oligodendrocytes within human brain of HAND patients while ART supressed GSDMB expression. N-GSDMB generation caused pyroptosis in astrocytes. The GSDMB cleavage-pyroptosis pathway could be considered a druggable target that contributes to neurodegeneration in HIV infection.

Background: COVID-19 vaccines have significantly reduced the public health impacts of SARS-CoV-2. However, an increase in the incidence of other respiratory infections has been seen following peaks of SARS-CoV-2 infection in Canada and in other settings. This observation raises concerns that SARS-CoV-2 infection may dampen existing immunity against other pathogens. Here, we examined the impact of SARS-CoV-2 infection on immune responses to unrelated viruses among a cohort of vaccinated individuals.

Methods: Study participants included 38 adults who remained SARS-CoV-2 naive until at least one-month after receiving their third dose of COVID-19 vaccine. Of these, 19 participants subsequently experienced SARS-CoV-2 infection (hybrid group) and 19 participants remained naive (control group). IgG antibody responses against common respiratory pathogens (RSV, Influenza A and Influenza B) were quantified using Multi-plex ELISA at one month (pre-infection for all) and six months (post-infection for hybrid group) following COVID-19 vaccination. Similarly, CD4+ and CD8+ T-cell responses against CMV (C), Epstein-Barr virus (E) and Influenza virus (F) were assessed using CEF peptide pools with an activation induced marker assay.

Results: IgG responses to RSV, Influenza A and Influenza B were similar at both time points in controls and in hybrid individuals pre- and post-infection. In contrast, a significant decline in CEF-specific CD8+ T cells was observed among hybrid individuals post-infection (p=0.006), whereas these responses remained unchanged among control individuals.

Conclusion: Our results suggest that CD8+ T cell responses are impaired following SARS-CoV-infection, despite prior COVID-19 vaccination. We observed no change in IgG responses against RSV or Influenza viruses post-infection or in CD4+ T cell responses to CEF peptides, indicating that immune dysfunction due to SARS-CoV-2 may be selective. Further analyses are warranted to dissect the specific responses that may be impacted by SARS-CoV-2 infection.

Funded by Michael Smith Health Research BC and the CIHR Canadian HIV Trials Network.

Background: COVID-19 vaccines have significantly reduced the public health impacts of SARS-CoV-2. However, limited data exist regarding the dynamics of memory B cell responses following COVID-19 vaccination in people living with HIV (PLWH) receiving suppressive antiretroviral therapy. Here, we examined the durability and specificity of memory B cells induced by three vaccine doses in PLWH.

Methods: Memory B cells specific for SARS-CoV-2 spike receptor binding domains (RBD) derived from wild type (WT, Wuhan) and Omicron BA.1 strains were quantified in 66 PLHW at one and six months post-third vaccine dose using flow cytometry.

Results: WT-, Omicron- and dual/cross-reactive memory B cells were observed in all individuals after three vaccine doses and these responses persisted over time. Nevertheless, Omicron-specific responses were significantly lower compared to WT (p<0.0001). Interestingly, individuals who subsequently experienced SARS-CoV-2 breakthrough infection displayed a lower frequency of WT- and dual-specific B cells at one month post-third dose compared to those who remained naïve (p<0.03).

Conclusion: Our results demonstrate that three doses of COVID-19 vaccine elicited memory B cell responses against WT and Omicron variants in PLWH receiving suppressive ART. The persistence of memory B cells likely contributes to robust responses observed following breakthrough infection and may contribute to an individual’s susceptibility to infection. Overall, our results underscore the immune benefits of COVID-19 vaccines among PLWH.

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

BACKGROUND/AIMS
An understanding of HIV diversity in the latent pool and the impact of this recrudescent virus population upon latency reversal is poorly understood. This study investigates the dynamics of HIV-1 proviruses (latent and non-infectious) in subtype B and subtype D infected individuals under long-term treated with cART (up to 20 years) and upon latency reversal.
METHODS
Five subtype B and 16 subtype D HIV-1 infected individuals receiving cART after 10 to 20 years were analyzed for this study. Nested-PCR and RT/nested-PCR were used to amplify nearly full-length HIV proviruses from extracted DNA samples from HIV-positive donors PBMC on cART and virus released from the same samples following latency reversal. We measured proviral load by qPCR, and viral diversity by next-generation sequencing using both Illumina MiSeq and Oxford Nanopore.
RESULTS
Ten nearly full-length proviruses could be amplified from the HIV-1 subtype D samples (of 16) and three from HIV-1 subtype B infected participants (of 5), all on long-term stable cART. The Gag and Pol genes were less diverse than the Env genes. Although the proviral load decreased with the time length of stable cART, the overall genetic diversity of the latent HIV population did not decrease, nor did the fraction of predicted replication-competent to incompetent proviral integrants in this CD4 T cell population. Latent HIV that activated and released following latency reversal clustered with proviral DNA in these CD4+ cells of individuals on long-term cART. However, an identical match could not be found in the provirus compared to the released, replicating virus clones sequenced following latency reversal from the same CD4 T cell sample of the same individuals on long-term cART.
CONCLUSION
Our results demonstrate a highly divergent pattern of intra-host viral diversity in both subtype B and subtype D HIV-1 proviruses in individuals on long-term cART.

People with non-subtype B HIV are underrepresented in studies of reservoir-origin viremia, defined as persistent low-level viremia (PLLV) during ART and post-ART rebound viremia. We characterized PLLV in an individual with HIV subtype A ("P1"), and HIV rebounding in plasma following ART interruption in an individual who experienced HIV superinfection with a unique recombinant form (URF) ("P2").

P1, diagnosed in 2003, initiated ART in Fall 2006. Viremia was largely suppressed until early 2020, after which a prolonged PLLV episode (50-709 copies/ml) occurred. During this time, plasma ARV levels were confirmed by mass spectrometry, and longitudinal plasma genotyping identified a clonal sequence lacking drug resistance mutations. This was consistent with a reservoir origin, so we conducted full-genome proviral sequencing to locate the source. P2, diagnosed in 2010 with HIV subtype B, subsequently experienced superinfection with a subtype G/CRF02_AG URF before initiating ART in Spring 2012. P2 interrupted ART in 2018, allowing us to characterize rebound viruses in context of on-ART proviral diversity.

P1's PLLV sequence harboured a 3-base deletion in HIV's major splice donor (MSD) site, which is predicted to yield non-infectious virus. Proviral sequencing identified 12 clones identical to the PLLV, consistent with a clonally-expanded source. Preliminary virus culture experiments using molecular clones supported the PLLV as non-infectious; restoring the deleted bases rescued function. For P2, on-ART proviral sequencing revealed 20% subtype B and 80% URF sequences, with replication-competent proviruses of both subtypes identified by viral outgrowth. After ART interruption, rebound HIV was genetically diverse and comprised 9% B and 91% URF, with no novel within-host recombinants detected. Notably, more recently-archived lineages emerged in plasma first.

In non-B HIV subtypes, clonally-expanded reservoir cells harbouring MSD-defective HIV can drive non-infectious PLLV. During ART interruption, diverse HIV lineages reactivate sequentially. These findings deepen our understanding of HIV reservoir dynamics in non-B subtypes.

Background: Dried blood spots (DBS) have been increasingly adopted due to their ease of use and stability over fresh blood. The Roche HI2QCAP is a qualitative assay that detects HIV-1 total nucleic acid from DBS samples to assist with HIV diagnosis. However, the assay’s performance among HIV viral controllers is unknown. We sought to evaluate the utility of DBS specimens tested with HI2QCAP among a cohort of individuals with HIV-1 viral control phenotypes.

Methods: Patients actively followed at a tertiary hospital-based HIV clinic were reviewed. Adults aged eighteen and older who were serologically diagnosed with HIV-1 and maintained viremic control without any anti-retroviral therapy were included. Viremic control was defined as plasma HIV-1 RNA viral loads of ≤400 copies/mL over the most recent twelve-month period or for ≥90% of all viral load measurements. Samples were collected for parallel testing of DBS with the HI2QCAP assay and whole blood with a gold-standard HIV reference lab-developed pro-viral assay targeting HIV-1 pol and LTR-gag regions. Descriptive statistics were used and results were compared for categorical agreement.

Results: Seven individuals met the inclusion criteria and underwent parallel testing. Participants’ median age was 48 years old and four were female. DBS testing achieved categorical agreement with reference testing in all but one patient who resulted DBS negative, reference assay positive for pol and LTR-gag, and had a plasma HIV-1 RNA viral load of 197 copies/mL. Additionally, DBS testing correctly identified a patient as positive who was pol positive but LTR-gag negative by the reference assay.

Conclusions: DBS-based testing demonstrated reliable and robust performance for the qualitative HIV-1 assay HI2QCAP among HIV viral controllers. These initial results support the current approach of orthogonal testing algorithms employed by HIV reference laboratories and encourage future studies with larger cohorts to validate DBS testing among unique HIV immuno-phenotypes.

The major obstacle to HIV cure is the persistence, despite antiretroviral therapy (ART), of a pool of latently infected cells. Most infected CD4+ T cells persisting under ART display a central (TCM) or effector memory (TEM) phenotype. Considering the differences between them, we hypothesized that the mechanisms underlying the persistence of infected TCM and TEM cells may differ.

We studied longitudinal samples (3-24 years of ART) from 10 ART-treated participants, using a modified HIV-Flow approach to analyze the phenotype of cells harboring inducible proviruses. CD4+ T cells were stimulated for 24h with anti-CD3/anti-CD28 antibodies, which minimally affected the expression of cellular markers and allowed us to compare the phenotype of p24+ and p24- cells.

p24+ cells expressed lower levels of HLA-ABC than p24- cells (p=0.026, fold decrease 1.4), likely reflecting the downregulation of class I molecules by Nef and Vpu. The immune checkpoint molecules PD-1 and TIGIT, which were previously shown to favor latency maintenance, were both expressed at higher levels in p24+ cells (p<0.0001, fold increase 1.8 and p<0.0001, fold increase 3.4, respectively). Similarly, CD127, the receptor of IL-7, was expressed at higher levels in p24+ cells (p<0.0001, fold increase 2.4). Furthermore, p24+ cells more frequently expressed the anti-apoptotic protein Bcl-2 (p=0.006, fold increase 1.1) as well as Granzymes A and B (p<0.0001, fold increase 6.2 and p=0.02, fold increase 6.8). The only notable difference between TCM and TEM in p24+ cells was a stronger downregulation of HLA-ABC in TEM compared to TCM (p=0.025, fold decrease = 2.7). A longitudinal analysis of our limited number of samples, revealed increased expressions of granzymes A and B over time during suppressive ART (p=0.009, r=0.55 and p=0.005, r=0.59, respectively).

Our data suggest that persistently infected cells have increased capacity for cytotoxicity, survival and viral silencing compared to non-infected cells during ART.

HIV viral load (VL) is variable between individuals, with host genetics and viral fitness contributing to this variability. For example, a locus on chromosome 1, near CHD1L, is associated with control of HIV replication. Recently we showed that this region is also linked to amino acid (AA) variation in HIV reverse transcriptase (RT) and gag in a South African cohort primarily infected with HIV subtype C. To assess the reproducibility of these findings, across diverse populations and viral subtypes, we acquired plasma for 33 PLWH from Kenya. HIV sequences for RT, protease (PR), and integrase (IN) were acquired via Sanger sequencing and genotypes for 4 host variants near CHD1L were acquired by qPCR. Of 33 individuals, we were able acquire RT, PR, and IN sequences for 27 individuals primarily infected with subtypes A and D. Genotyping was successful for all 27 individuals and 5 individuals were heterozygous for the minor allele of the 4 variants. Logistic regression was used to test the association between HIV AA variants in RT, PR, and IN with host alleles near CHD1L. Due to the small sample number, we were unable to test for an association at RT codon 248, as a majority of sequences did not cover RT up to codon 248. However, adding to our previous findings, we observed significant associations between the CHD1L variant rs7519713 and codons 60 (p=1.3x10-2) and 216 (p=3.3x10-2) of IN suggesting a mechanism of HIV restriction by CHD1L at integration. Here, we report on the success of HIV sequencing and host genotyping from plasma samples >15 years old and we show evidence of selective pressure by variants near CHD1L on IN. Our findings provide further insight into how genetic variability, near the CHD1L locus, contributes to viral evolution and host control in a population highly affected by HIV.

While antiretroviral drugs cannot cure HIV-1 infections, they allow millions of HIV-1 patients globally to live essentially normal lives. However, the development of drug resistance against antiretroviral drugs presents a major barrier. Drug resistance has been documented against all classes of antiretroviral drugs, and resistant strains can be transmitted to newly infected, treatment-naïve patients. Pretreatment drug resistance is a major problem in low-to-middle income counties, where alternative drug availability may be limited. Uganda is a geographically diverse country with distinct rural and urban regions, and it has among the highest global burden of HIV-1. Here, we aim to determine the frequency of pretreatment drug resistance regionally and over time in Uganda. We collected >3000 protease/reverse transcriptase sequences from treatment-naïve Ugandan patients between 1997 and 2017. This represents by far the largest collection of pretreatment sequences from Uganda and the only sequence set divided by region to our knowledge. We predicted drug resistance against the most commonly used antiretroviral drugs using the Stanford DRM penalty scoring system. We note a low frequency (<2%) of resistance against the WHO-recommended nucleoside reverse transcriptase inhibitors, especially in central Uganda. Similarly, resistance against protease inhibitors is low (<2%) over time and in all regions of Uganda. However, resistance against non-nucleoside reverse transcriptase inhibitors is alarmingly high (>15% in central Uganda) and appears to be increasing over time. Thus, non-nucleoside reverse transcriptase inhibitors should not be recommended for widespread use in patients initiating antiretroviral therapy in Uganda. Similarly, this work serves as a warning to other countries which still recommend the use of non-nucleoside reverse transcriptase inhibitors in initial therapies in their national guidelines.

Background/Objectives: The HIV-1 LTR regulates the expression of viral genes, and hence, HIV replication. Interferon regulatory factors (IRF-1, IRF-8) have been shown to regulate the transactivation and repression of the HIV-1 LTR, via binding to the ISRE and forming a complex with NF-κB. This study examined the hypothesis that mutation(s) at the NF-kB and/or ISRE of the HIV-1 LTR may impact HIV fitness and hence, the pathogenesis observed in HIV-infected patients.
Methods: HIV-1 sequences of 5’ LTR from published HIV database (n=411, Los Alamos National Laboratory) and 97 HIV-infected patients of Kenyan Pumwani Sex-Worker Cohort were analyzed using ClustalW. Longitudinal CD4 counts of antiretroviral-treatment-naïve patients enrolled in the study were used as a measure of disease progression.
Results: In all HIV-1 sequences analyzed, the LTR NF-κB binding motif was highly conserved and essentially identical in all samples, including subtypes A, A1, B, C, and D. Similarly, the LTR ISRE motif was also highly conserved, with >99% identity. However, some LTR sequences contained two-tandem ISRE motifs. The majority of these 2-ISRE sequences were found in subtypes A1, A1-recombinants, G, and U. Longitudinal study of HIV provirus from HIV-infected patients (n=6) showed conservation of LTR sequences over time (4-9 years). When HIV-infected patients were grouped into 1-ISRE and 2-ISRE, based on HIV-1 provirus sequences, the 2-ISRE patients had a relatively faster drop in CD4 counts, suggesting a more rapid disease progression.
Conclusions: The NF-κB motif and ISRE of HIV-1 LTR is likely highly critical to viral fitness, as the sequences are conserved over time and between different subtypes examined. Furthermore, duplication of ISRE in the HIV-1 LTR may contribute to increased viral replication and more rapid disease progression.

Human and simian immunodeficiency viruses (HIV/SIV) are widely known to deplete CD4 T lymphocytes, a key component of the immune system. The CD4 T cells are implicated in humoral responses by helping B cells in B cell follicles, a T-dependent humoral response that is distinct from extrafollicular B cell activation, which is T-independent. During the acute phase, a drastic T cell depletion is reported that may impact memory B cell immune response such as vaccine.

We assessed in rhesus macaque (RM) the impact of SIVmac251 infection on a vaccine response developed beforehand. Thus, a HIV/SIV vaccine candidate was administrated in RMs. B cell differentiation and dynamics were monitored throughout the vaccination protocol and after infection. Blood samples, as well as peripheral lymph nodes, were collected and analyzed by flow cytometry.

Firstly, we analyzed B cells given their crucial role in the development of humoral response leading to neutralizing antibodies. Following a prime and a boost, we noticed a differentiation of B cells as well as an increase in the expression of CD95, a marker of activated germinal center reaction, but also the expression of PD-1 as compared to the placebo. Whereas an increase in PD-1 expression is reported to be a marker of naïve B cell activation, our results highlighted higher expression of PD-1 on activated memory and resting memory B cells following vaccination. This population may represent regulatory B cells such as B-1 cells. Studies are in progress to better define their specificities. Intriguingly, these populations are increased following SIV infection in both vaccinees and controls. Additional works are in progress using alternative vaccines such as Influenza for determining the specificities of the B cells dynamics and the role of this cell subset.

This work will draw a preliminary portrait of the interactions between vaccine responses and SIV infection.

Background: The cytotoxic immunity of memory CD8 T-cells (Mem) depends on their produced ATP levels. Although autophagy inducers improve ATP-dependent cytotoxic Mem function in PLWH, it is however ineffective in helping their exhausted PD-1+CD39+subset. Since heat shock protein 60 (HSP60) can also regulate ATP production in Mem, we investigated if exhausted cells found in PLWH display defective HSP60 energetic support, and if so, could we rescue this defect with dimethyl alpha-ketoglutarate (DMKG); indeed, DMKG is a diester AKG analog, which can supplement mitochondrial metabolism.

Methods: We assessed the HSP60-regulated ATP production in CD8 Mem including anti-HIV-1 cells in PLWH and HIVneg donors; For that, CD8 Mem were T-cell activated [TcR engagement, and HIV-1 peptide stimulation], with or without (w/wo) anti-PD-1 blocking antibodies (Abs), autophagy inducer AICAR, and dimethyl alpha-ketoglutarate (DMKG). At 16 hours post-activation, we assessed in both total, non-exhausted and PD-1/CD39 dual-stained CD8 Mem, HSP60 expression, autophagy activity, mitochondrial ATP production, and cytotoxic functions [defined as the percentages of perforin/Granzyme-B dual-stained cells]. Of note, we determined both the whole and FAO-mediated mitochondrial ATP-linked respiration levels, using Seahorse flux analyzer.

Results: Our data confirmed lowest HSP60 expression in exhausted CD8 Mem of PLWH after T-cell activation when compared to other cells, despite similar rescuable autophagy activity with AICAR. Blocking PD-1 signaling in exhausted Mem enhanced their HSP60-dependent ATP-linked respiration. Although induction of autophagy alone was confirmed ineffective in rescuing ATP-dependent cytotoxic function of exhausted Mem in PLWH, our metabolic investigation further revealed that DMKG supplementation led to enhanced percentages of cytotoxic cells, with levels that were comparable to HIVneg donor.

Conclusions: Stimulating mitochondrial energy metabolism with metabolic drugs for better effector CD8 T-cell immunity is an enticing concept for PLWH. In this context, our data have revealed that DMKG may be considered in rescuing defective immunity in exhausted HSP60low cells.

From the seasonal influenza to the ever enduring crisis of HIV, the viruses’ ability to rapidly adapt and resist current therapeutics has become increasingly evident. The existing arsenal of antiviral medications, often targeted to viral proteins such as polymerase or protease in HIV, or neuraminidase for influenza to prevent new viral particles release, have struggled to keep up with the rapid evolutionary adaptation of viruses. Both HIV and Influenza genomes are compact, coding for multiple proteins whose expression relies on alternative RNA splicing for their replication. This requirement for a cellular process presents an opportunity to approach antiviral strategies from a host perspective by targeting RNA processing steps. We hypothesize that modulation of key splicing machineries and their regulators can hinder virus replication. SR (Serine-Arginine) proteins and Heterogeneous nuclear ribonucleoproteins (hnRNPs) are conserved host proteins involved in both host and viral RNA processing. We curated a chemical library of SR kinase inhibitors and compounds that modulate HNRNPs’ binding and expression to test our hypothesis. Screening of this library identified several compounds that are able to inhibit both HIV and influenza replication. Here, we present various evidence in the context of HIV and influenza on the capacity of select compounds to reduce viral RNA and protein, accumulation at the level of RNA synthesis, stability or changes in alternative splicing site usage that correlate with changes in SR protein phosphorylation patterns. These studies set the stage for the future exploration of inhibitors that target the RNA processing steps as antivirals, potentially leading to a therapeutic breakthrough in the ongoing battle against viral infections.

Human Immunodeficiency Virus Type 1 (HIV-1) is a global burden, with an estimated 36.9 million people currently living with HIV-1 (PLWH). HIV-1 evades the immune system using its accessory proteins to interact with host cellular proteins. One of those accessory proteins, Nef, decreases the levels of cell surface molecules such as cluster of differentiation 4 (CD4), major histocompatibility complex class I (MHC-I), and Serine Incorporator 5 (SERINC5) to accelerate T cell decline in untreated PLWH. As we have previously measured the levels of CD4 and SERINC5 levels in PLWH, which may be related to associated disease progression, we hypothesized that the ability of Nef to downregulate surface molecules could affect the overall pathogenesis of HIV-1.

We wish to investigate our hypothesis using a recently discovered hypervirulent variant of Subtype B HIV-1 in the Netherlands, which led to higher viral load and quicker T cell decline in PLWH harbouring this variant. To uncover the relationship between Nef functions and HIV-1 virulence, we aim to measure other surface molecule downregulation activities mediated by Nef in the hypervirulent HIV-1 subtype B variant during infection. Furthermore, although the common pathway of Nef-mediated SERINC5 and CD4 downregulation is characterized, we have identified a polymorphic motif in Nef that only influenced SERINC5 downregulation. We will explore how this motif affects Nef functions and potential novel pathways.

This research will provide insight into Nef functions that could enhance HIV-1 subtype B virulence and explore Nef motifs involved in modulating surface molecule downregulation. Overall, this research may shed light on the development of new therapeutic interventions to control HIV-1 pathogenicity during an infection.

CD4+ T cells are essential for helping B cells and cytotoxic CD8 T cells. Notably, a subset of CD4+ T cells exhibits cytotoxic activity, known as CD4 CTL, that has been previously described in the blood and to participle in the control of viral infections. They express molecules related to CD8 T cells like granzyme B and perforin. However little is known about the distribution of these effector cells in the tissues. Thus, we investigated the dynamics of these cytotoxic CD4 T cells during SIV infection in rhesus macaques.
Over forty rhesus macaques (RMs) were studied, including healthy, SIVmac251-infected RMs, ART treated and RMs subjected to ART interruption. After sacrifice, lymphoid tissues, including the spleen, mesenteric and axillary/inguinal lymph nodes, as well as blood were recovered. Dynamics and phenotype of CD4 T cells expressing cytotoxic molecules (perforin and granzyme B) were analyzed by flow cytometry. Bulk RNA-seq transcriptomic experiments were performed on distinct CD4+ T cell subsets to analyze more in deep transcriptional gene profiles.
Our results demonstrated that cytotoxic CD4 T cells are predominantly present in the blood and spleen of healthy RMs highlighting the compartmentalization of these cells in different anatomical tissues. Interestingly, we observed that the percentages of this population decrease during SIV-infection. Additionally, we observed that differentiated CD4 T cells exhibit a prevalent expression of cytotoxic molecules reflecting the full differentiation of these cells although associated with short-half life. Early ART transiently prevents the depletion of this population in the blood that nevertheless decreases upon ART discontinuation.
Thus, their depletion in the blood and spleen, early after infection, may contribute to the absence of effector cytotoxic CD4 T cells associated with the absence of viral control.