The main obstacle towards Human Immunodeficiency Virus’s (HIV) eradication is latency. A better understanding of HIV gene expression is required to fight latency. Our laboratory identified the Pre-Initiation Complex of HIV (PICH), a cellular protein complex that binds HIV’s promoter. The identification of PICH proteins followed by their knockdown showed that they have a positive impact of HIV gene expression. We are studying the role of each PICH protein on HIV transcription to evaluate their potential as drug targets. My project focuses on PICH115, a protein that interacts with Tat.

First, Chromatin ImmunoPrecipitation assays indicated that PICH115 can be found on HIV proximal promoter.
Then, we mapped the interaction site between Tat and PICH115. CoImmunoPrecipitation experiments, performed by transfecting plasmids coding for Flag-Tat and and different domains of His-PICH115 in HEK293T cells, showed that the N-acetyltransferase and the tRNA-Binding domains of PICH115 are required to interact with Tat in cellulo.
To verify if this interaction was direct, we carried out GST-Pulldown with recombinant proteins of fragments of GST-Tat and HA-PICH115. We observed a direct interaction between the core domain of Tat and the N-acetyltransferase and tRNA-Binding domains of PICH115 in vitro.
We finally demonstrated that this interaction has consequences on Tat interaction with TAR RNA, an essential step for HIV transcription. Indeed, in ElectroMobility Shift Assays, when a fluorescent TAR RNA probe is incubated with recombinant Tat, the presence of recombinant PICH115 increases the binding of Tat to TAR.

To conclude, we showed that PICH115 binds HIV’s proximal promoter, we mapped Tat and PICH115 interaction site and highlighted its impact on Tat-TAR interaction. Eventually, we will explore the role of the N-acetyltransferase function of PICH115 on HIV gene expression. These data will refine our understanding of HIV gene expression to develop new Shock and Kill or Block and Lock strategies.

Historically, persistent low-level viremia (pLLV) during ART had been attributed to incomplete antiretroviral adherence/absorption or drug resistance, but new evidence indicates it can originate from the reservoir. We are investigating the potential reservoir origin of plasma virus in an individual with pLLV.

Longitudinal plasma and a recent PBMC sample are being analyzed using sequencing, phylogenetics, and the intact proviral DNA assay (IPDA) to elucidate the reservoir origin of pLLV.

The participant, who has a subtype A or CRF01_AE infection, was diagnosed with HIV in 2003 and initiated ART in Oct 2006. With the exception of two “blips”, pVL was suppressed until February 2020, after which 30 consecutive pVL were detectable (between 50-709 copies/ml) despite receiving 4 active drug classes. No adherence concerns were noted and the presence of drug in plasma was confirmed by mass spectrometry. Drug resistance genotyping of 12 plasma samples during the pLLV revealed an essentially clonal sequence with no resistance mutations, that was distinct from prior genotypes. Bulk sequencing of 5' and gp41 regions confirmed that the pLLV was clonal with occasional limited diversity. To date, 47 proviruses have been single-genome-amplified from PBMC. Of these, two were genetically intact and the remaining 45 were predominantly 3’-defective. One of the intact proviruses matched the pLLV sequence across all query regions. Moreover, it (and the pLLV) shares a unique deletion in the 5' IPDA probe site, allowing sequence-specific quantification of its abundance in the reservoir. We plan to reconstruct within-host HIV evolutionary history from pre-ART plasma, allowing us to infer pLLV age and lineage origin.

Results to date suggest that the reservoir cell harbouring this intact provirus could be a member of a clonally-expanded population that is fueling pLLV. Combining detailed single-genome-sequencing with customized IPDA and within-host HIV dynamics analyses can help elucidate the reservoir origins of pLLV.

Antiretroviral therapy (ART) lowers plasma viral load to undetectable levels. However, in individuals under ART, integrated proviruses remain transcriptionally silent but replication competent, in multiple cellular compartments, named viral reservoirs. Here, we investigate the possible role of HIV accessory viral protein R (Vpr) in limiting the establishment of transcriptionally silent proviruses in repressive regions of the chromatin, a condition prone to the establishment of viral reservoirs. Previous studies have shown that Vpr hijacks the Cullin4-DDB1DCAF1 E3 ubiquitin ligase complex to target DNA repair factors (e.g., HLTF, UNG2) and epigenetic modulators (e.g., HUSH and NuRD complexes). Such events lead to G2-M arrest and apoptosis in dividing cells. In addition, Vpr enhances viral replication in terminally differentiated myeloid cells. Our study assesses for the first time the role of Vpr in the establishment of productive infection and viral latency during infection of Bone-Marrow-Liver-Thymus (BLT) humanized mice. NOD/SCID/IL2rγnull mice were engrafted with human fetal liver hematopoietic stem cells (CD34+) and autologous thymus, promoting maturation and education of T-cell progenitors. BLT mice were infected with a wild-type (WT) or a Vpr-defective HIV-1 strain for 4 or 11 weeks. Preliminary results suggest that absence of Vpr reduces the overall infection efficiency in BLT mice as well as during ex-vivo infection of splenic hCD4+ T isolated from uninfected mice. In contrast, as previously reported, no difference was observed upon infection of primary human CD4+ T cells or T-cell lines. Furthermore, in 2 out of 6 infected mice, Vpr-defective virus reverted to a WT sequence and displayed a WT phenotype, indicating that there is a selective replication advantage for maintaining Vpr function in BLT mice. Current efforts on understanding the cellular and molecular mechanisms underlying Vpr-mediated enhancement of HIV infection in vivo is likely to shed light on novel host restrictions that HIV-1 must overcome to persist.

BACKGROUND/AIMS
The persistence of a latent HIV reservoir within the population of CD4 T cells fails combined antiretroviral therapy. The elimination of this latent viral reservoir is a crucial aspect of current strategies for an HIV-1 cure. Our previous non-infectious virus-like particle (VLP) specifically reactivated the latent viral pool in HIV-specific CD4+ T cells of individuals treated with antiretroviral therapy during acute HIV infection. In this study, we propose that this VLP can also reactivate the latent viral pool during chronic HIV infection, regardless of subtype B or D.
METHODS
This study examined 15 subtype B and 17 subtype D HIV-1 infected individuals receiving cART after 6 to 20 years. We used our previous VLP called ACT-VEC to induce transcriptional activity in provirus. Ex vivo co-culture experiments were used to assess its efficacy in patients treated in the chronic stage of infection. We measured antigenicity, latency reversal, and virus diversity using IFN-g ELISpot, qRT-PCR, and deep sequencing respectively.
RESULTS
ACT-VEC was highly effective in reactivating latent HIV-1 in CD4 T cells of chronic patients on cART, releasing 100 to 1000-fold more HIV-1 into culture supernatant than other stimulations with common recall antigens, HDAC inhibitors, and PKC agonists. Even after 6-20 years of cART, ACT-VEC reactivated 1000-fold more HIV-1 latent virus than in patients treated during acute infection. The amount of HIV-1 released with ACT-VEC correlated directly with the infectious virus produced from the same patients' activated PBMCs. A high proportion of latent infectious viruses with genetic diversity were released by ACT-VEC latency reversal as determined by Nanopore and Illumina sequencing of viral genomic RNA.
CONCLUSION
ACT-VEC effectively reactivates latent HIV-1 in patients with chronic infection, targeting HIV-specific CD4+ T cells. Even after 20 years of cART, ACT-VEC reactivated the large proportion of HIV-1 reservoir in both Ugandans and Canadians.

Although antiretroviral therapy (ART) controls viral replication, HIV persists as an integrated provirus in latently infected CD4+ T cells. Clonal expansion of infected cells has been shown to contribute to the maintenance of HIV reservoirs during ART. However, the antigen specificities of the majority of these T cells clones are unknown. Since HIV infection induces the translocation of bacterial products from the gut to the blood, we hypothesized that microbiota derived antigens could be responsible for the proliferation of latently infected CD4+ T cells.

We developed a sensitive and specific flow-cytometry based assay to identify bacteria-specific CD4+ T cells in the blood. PBMCs isolated from 10 people living with HIV under ART were stimulated for 18h with 12 bacterial lysates from gut microbiota species (including E. coli L. Acidophilus and R. Intestinalis) and 3 pathogenic species (M. Tuberculosis, S. Typhimurium, and C. Difficile). We observed that the combined expression of CD40L, CD69 and 4-1BB could discriminate microbiota-specific CD4+ T from the LPS induced signal and that activation of bacteria-specific cells was abolished when HLA-I was blocked. All participants had detectable microbiota-specific CD4+ T cells with a mean of 0.07% of circulating CD4+ T cells. The highest frequencies of bacteria specific cells were detected against C. Difficile, R. Intestinalis and S. Typhimurium (mean of 0.14%). We are using this activation induced marker approach to sort bacteria-specific CD4+ T cells and perform HIV genome near full length amplification to measure the frequency of microbiota-specific CD4+ T cells harbouring HIV proviruses.

Our results indicate that microbiota-reactive CD4+ T cells are frequently detected in the blood of people living with HIV. Preliminary data indicate that microbiota-specific CD4+ T cells harbour HIV proviruses. Using next generation sequencing, we will then determine if bacteria-specific CD4+ T cells are enriched in intact HIV proviruses.

Despite key advancements in combating HIV-1, a functional cure has yet to be realized. This is primarily driven by the HIV accessory protein, Nef, and its interactions with host proteins that collectively promote HIV immune evasion and contribute to its pathogenicity. Through its interaction with Src family tyrosine kinases (SFK), Nef initiates and misdirects a signalling cascade that results in major histocompatibility complex class I (MHC-I) cell surface down-regulation and sequestration at the trans-Golgi network. By extension, this reduces the presentation of HIV-derived epitopes in the context of MHC-I to limit immune recognition and represents a major impediment towards the development of HIV cure strategies. We therefore performed an in silico docking screen and identified a small molecule inhibitor (H3-1) of the Nef-SFK interaction. Indeed, H3-1 counteracted Nef-dependent MHC-I down-regulation in preliminary studies to improve cell surface MHC-I expression in primary human CD4+ T cells without associated cytotoxicity. We then assessed H3-1 as a Nef inhibitor in a transgenic mouse model of a HIV/AIDS-like phenotype. Following addition of H3-1 to ex vivo culture, transgenic mouse CD4+ splenocytes displayed marked enhancement of (i) total cell surface MHC-I and (ii) cell surface MHC-I complexed to a model epitope. Mass spectrometry revealed that H3-1 was rapidly cleared in vivo after oral and intraperitoneal administration. This in vivo instability is likely due to the peptidic nature of H3-1 and therefore, we have used organic synthesis to generate a peptidomimetic panel of H3-1 analogues with improved biostability. H3-1 analogues displayed improved ex vivo plasma stability and future studies will assess in vivo plasma stability in transgenic mice. The synthesis of biostable H3-1 analogues capable of resisting Nef-mediated MHC-I down-regulation represents an important step forward in evaluating the role of Nef-SFK inhibitors as adjuvants within a HIV/AIDS functional cure.

The “kick and kill” approach of eradicating HIV requires the reactivation of virus-infected long-lived cells harboring integrated latent HIV proviruses known as “viral reservoir (VR)”. Latency reversing agents (LRAs) can reactivate latent viruses to enhance their immune recognition but are inefficient at sensitizing reactivated cells to death. Recent evidence reveals that latently HIV-infected cells have an intrinsic resistance to cell death responses modulated by the BCL-2 pro-survival protein. Previously, we showed that a novel bivalent SMAC mimetic (SM) reactivates latently infected CD4+ T cells by engaging the non-canonical NF-ĸB pathway and induces a modest reduction of VR in humanized mice. In this study, we examined if a novel BCL-2 inhibitor alone or in combination with the SM could augment the sensitivity of latently-infected cells to cell death. Using HIV latency models of CD4+ T cells, our in vitro results reveal that the BCL-2 inhibitor alone possesses selective toxicity towards latently-infected cells (2D10, JLat 10.6 CD4+ T cell models of latency) but is not sufficient to reactivate efficiently integrated proviruses. Sequential treatment of the SM and BCL-2 inhibitor can additively reduce the frequency of latently-infected cells via the activation of caspase-3. In primary CD4+T cells infected with a dual reporter virus, the combination of BCL-2 inhibitor with SM reduces the frequency of latently-infected cells by ~30%. In a study with humanized CCST mice (Colas et al, Stem Cell Reports, in press), combined treatment of the Bcl-2 inhibitor and SM significantly reduces viral load and shows a trend towards a reduction of p24+CD4+T cells in the spleen and liver. Overall, these results provide the rationale to evaluate the therapeutic potential of combining LRAs with Bcl-2 antagonists in the context of VR elimination strategies in preclinical humanized mouse models.

Introduction: The main challenge in finding an HIV cure is the HIV reservoir of latently infected CD4+ T-cells and persistently infected macrophages. These cells are phenotypically indistinguishable from their healthy counterparts and thus difficult to target. We have demonstrated that latently/persistently HIV infected cells are susceptible to killing by the interferon sensitive oncolytic rhabdovirus virus MG1. The efficiency of MG1 killing can be potentially increased by using small pro-apoptotic molecules called second mitochondria derived activators of caspases (SMAC) mimetics. SMAC mimetics have been shown to increase oncolytic virus killing in several different cancer models and have also been shown to selectively kill HIV infected cells.
Methodology: First, latently HIV infected J1.1 cells and OM10.1 cells were infected with MG1-eGFP in low volume for 2 hours, and then treated with SMAC mimetics. In subsequent experiments, cells were treated with SMAC mimetics for 24 hours and then infected with MG1. Flow cytometry was done 24 and/or 48 hours post infection to assess cell viability via PI staining, and caspase 3/7 activity via FLICA staining.
Results: For latently infected J1.1 cells, either concurrent SMAC mimetic treatment and MG1-eGFP infection or 24-hour pre-treatment with SMAC mimetic followed by MG1 infection resulted in significantly greater killing than with MG1 alone. This increased killing was accompanied by increased casp3/7 activation. For the latently infected OM10.1 cells, only SMAC mimetic treatment followed by MG1 infection resulted in increased cell death and increased caspase3/7 activation.
Conclusion: This project identifies a potential novel cure strategy by employing combination therapy with oncolytic viruses and SMAC mimetics to eradicate the viral HIV reservoir and demonstrates the importance of cell type in evaluating such strategies.