Human immunodeficiency virus type 2 (HIV-2) is endemic in West Africa and is also found in areas with socioeconomic ties to the region. Historically, advancements in the treatment of HIV-2 infection have been slow compared to HIV-1, and there is an urgent need to identify effective antiretrovirals (ARVs) for people with HIV-2 (PWH2) who harbor drug-resistant virus. Lenacapavir (SUNLENCA®) may help fill this gap in the HIV-2 treatment landscape. Lenacapavir is a long-acting, injectable ARV that targets the HIV-1 capsid protein and is approved for use in adults with multidrug-resistant HIV-1 infection. We recently showed that lenacapavir is highly active against HIV-2 isolates in culture, albeit with a 11- to 14-fold lower potency relative to HIV-1. These data suggest that lenacapavir binds to HIV-2 capsid cores in a manner similar to that observed in HIV-1. To further test this hypothesis, I am using site-directed mutagenesis (SDM) to introduce specific amino acid changes into the putative lenacapavir-binding region of the HIV-2 capsid protein. These changes fall into two categories: 1) mutations that convert the targeted residue in HIV-2 to the corresponding amino acid residue found in HIV-1; and 2) mutations that are associated with the development of lenacapavir resistance in patients with HIV-1 infection. To date, I have constructed a 5.5-kilobase plasmid clone that contains the capsid-encoding region of HIV-2ST (plus downstream flanking sequences) in a pBluescript plasmid vector. I introduced the desired mutations by SDM, and verified that the plasmids are correct via automated Sanger sequencing. My next step is to clone the mutated capsid sequences into a full-length clone of the HIV-2 ST genome for virus production and culture-based drug susceptibility testing. These studies have direct implications for improving HIV-related care and public health in West Africa and other areas where significant numbers of PWH2 reside.

To form Human Immunodeficiency Virus type 1 (HIV-1) virions, the HIV-1 Gag polyprotein multimerizes, traffics to the cell membrane, assembles into virions, and buds as viral particles. HIV uses the infected cell proteins to support viral assembly and export from the cell. Although some cellular proteins have been identified that participate in viral assembly and budding, the spatial and temporal "cellular proteome" is not known. Insights as to the order of proteins involved in facilitating assembly and budding provide information on viral infection and potential therapy targets. I am utilizing a split-APEX2-mediated proximity labeling method to identify which host factors interact with Gag during assembly. The basis of the method is splitting APEX2 into two segments encoded into separate Gag sequences so that the APEX2 segments rejoin for complete enzymatic function when Gag dimerizes and multimerizes. Once the APEX2 enzyme reconstitutes, we activate it to biotinylate cellular host proteins within ~20 nm of Gag during the processes of virion formation. I aim to deliver this system to primary human CD4+ T-cells and macrophages via two lentiviral vectors containing transgenes for either AP-Gag-P2A-EGFP or EX-Gag-P2A-mCherry. Cells express the dual fluorescence markers EGFP and mCherry if both Split-APEX2 domains are present, and we enrich these cells by cell sorting. APEX2 is restored and enzymatically active, allowing proximity-dependent biotinylation of Gag-host cell proteins for SILAC-based quantitative proteomic mapping during virion assembly. We are using this method to quantitatively map the Gag-host protein "interactome" in the infected cellular microenvironment in subtypes of human primary CD4+ T-cells and macrophages. Therefore, this research studies Gag interactions of potential HIV-1 host dependency factors in CD4+ T-cells and macrophages, the natural cell populations for HIV-1 infection. Previously, we conducted a similar study in HEK293Ts, and we anticipate elucidating a similar Gag-host cell protein analysis with this study.

Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and is a prevalent AIDS-associated malignancy. Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS. While all herpesviruses are capable of both lytic and latent replication programs, KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell - a cell expressing markers of the endothelium. There is limited viral gene expression during latency so it is difficult to target the virus directly. Therefore, our approach is to target host cellular requirements for KSHV latent infection. Previously, the Lagunoff Lab performed a genome wide CRISPR-Cas9 screen targeting over 18,000 human genes to identify cellular genes essential only to cells latently infected with KSHV. ACADS and CHD1 are two genes identified as some of the top hits from the screen. ACADS encodes a tetrameric mitochondrial flavoprotein, which catalyzes the first step of mitochondrial beta-oxidation. CHD1, or chromodomain helicase DNA binding protein 1 alters gene expression by chromatin modification. I hypothesize that ACADS and CHD1 are required for survival of latently infected KSHV cells but not uninfected cells. To test this hypothesis, I created knockout tert-immortalized microvascular endothelial (TIME) cells of each gene with CRISPR-Cas9 and plasmids containing guide RNAs used in the original screen. Then, I infected control and knockout cells with either KSHV or mock, and conducted trypan blue assay at 72 hours post infection to measure percent of live cells. Preliminary data suggests an increase in cell death for KSHV infected ACADS knockout cells compared to the control cells. In future experiments, I expect a significant decrease in the percentage of live cells in the ACADs and CHD1 knockout cells compared to the control and uninfected cells.

Infection from Mycobacterium tuberculosis is the leading cause of death due to infectious disease worldwide, with rates of tuberculosis infection greatest in low and middle-income countries (LMICs). Tuberculous meningitis (TBM) is the most severe form of M. tuberculosis disease with nearly half of all cases resulting in death or neurological consequences. Recent studies in our lab have found that single-nucleotide polymorphisms (SNPs) in MUC5AC, a secretory lung mucin, are associated with increased TBM susceptibility, morbidity, and mortality. The purpose of my study is to identify the functional MUC5AC SNP. Four candidate SNPs were selected within the MUC5AC promoter region based on high linkage-disequilibrium scores across multiple global populations with a SNP in the MUC5AC promoter, rs28737416. I utilized molecular cloning techniques to combine a luciferase-expressing plasmid with isolated regions of the human MUC5AC promoter containing the SNPs of interest and subsequently transformed this recombinant plasmid into competent cells. Next, I performed site-directed mutagenesis at the SNPs of interest and am currently transfecting mutants into HEK293T cells to investigate how genotypic variation in each candidate SNP influences promoter function by measuring luciferase expression. I anticipate variants in at least one SNP of interest will reduce gene expression (measured by luciferase expression), indicating functionality. Characterization of this genetic mutation will provide insight into TBM susceptibility across populations and could inform studies of novel therapeutics to treat TBM.


The tumor microenvironment (TME) exhibits several obstacles to immunotherapies including suppressive signals and a lack of costimulatory ligands. The TME causes T cell exhaustion and reprogramming of immune cells (e.g. macrophages) from an anti- to pro-tumor phenotype. Therapeutic approaches such as agonist CD40 monoclonal antibodies stimulate macrophages to induce anti-tumor programming. However, enthusiasm for monoclonal antibodies has been dampened by dose-limiting toxicities and inadequate pharmacokinetics. We are developing novel adoptive cell therapies by armoring tumor-targeted T cells with fusion proteins that provide costimulatory signals that are absent in the TME. Dual Costimulatory Receptors (DCR) are composed of the CD40L ectodomain combined with a costimulatory receptor endodomain. CD40L-DCRs are engineered into CD8 T cells to provide positive signals: CD40L interacts with TME macrophages, while simultaneously stimulating CD8 T cells via the costimulatory endodomain. I hypothesize that CD40L-DCR T cells are reprogramming macrophages from pro- to anti-tumor programming, providing greater therapeutic efficacy against tumors. Our in vivo studies showed improved survival using different CD40L-DCR candidates in a murine model of acute myeloid leukemia. In the CD40L DCR-T cell-treated mice, macrophages exhibited elevated levels of costimulatory molecules, suggesting an anti-tumor phenotype. To understand how CD40L-DCR T cells induce macrophage reprogramming, I am conducting in vitro experiments, co-culturing bone marrow-derived macrophages (BMDM), tumor cells, and CD40L-DCR T cells. I predict that CD40L-DCR T cells will induce BMDM to reprogram into an antitumor phenotype and increase phagocytosis of tumor cells. I will use IncuCyte and flow cytometry technologies to determine how BMDM programming is altered and how BMDM phagocytosis of tumor cells changes over time. These studies will support the translational potential of CD40L DCR-T cell therapy, opening doors to novel therapeutic interventions against a range of cancers.