Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and AIDS patients and 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. CYP27A1, a gene that encodes a member of the cytochrome p450 family, was one of the top hits identified in the screen. It specifically encodes sterol 27-hydroxylase, which is an enzyme involved in the breakdown of cholesterol. I hypothesize that CYP27A1 is an essential gene for survival of KSHV latently infected cells, as there is evidence that cholesterol is antiviral and KSHV is known to regulate cholesterol. I have successfully cloned CRISPR guide RNAs targeting CYP27A1 into lentiviral vectors, transfected 293T cells with the vectors to make lentivirus, and transduced human tert-immortal endothelial (TIME) cells with the lentivirus to create CYP27A1 knockout cells. ICE analysis was used to confirm strong knockout of the CYP27A1 gene. Currently, we are infecting these cells with KSHV to determine if CYP27A1 is required during KSHV latent infection using cell survival and cell proliferation as readouts. I expect that knockout of CYP27A1 will result in cell death in endothelial cells latently infected with KSHV. By determining genes necessary for KSHV latency, we hope to identify potential therapeutic targets for KS tumors.

The HIV global pandemic has claimed more than 40 million lives so far, and it is an ongoing worldwide health crisis as more than 1.5 million people acquired HIV in 2021 alone. A major barrier towards a cure for HIV is the presence of replication-competent latent HIV-1 proviruses in reservoir cells, which contribute to viral persistence despite years of antiretroviral therapy. One approach to control the latent HIV reservoir is silencing HIV transcription to prevent reactivation. A comprehensive list of host factors that support latency reactivation has not been yet identified. Here, we conduct a CRISPR-Cas9-mediated gene knockout using guide RNAs packaged into budding HIV virions, serving as a readout to identify cellular factors that promote HIV latency reactivation. We used the dependency factor library (HIV-DEP) that contains hundreds of genes involved in proviral transcription. This library was transduced in bulk into two J-lat cell lines, T lymphocytes cell lines which serve as HIV latency models, and we screened for genes that, upon knock-out, prevent latency release after treating the cells with latency reversal agents (LRAs). We identified 47 genes that, when knock-out, prevented HIV latency reactivation in both J-lat models. Our findings suggest that host genes indispensable for HIV transcription also play an important role in HIV latency control. Importantly, these candidate genes could serve as targets for HIV therapeutic intervention.

HIV-2 represents approximately 1-2 million of the 38.4 million people living with HIV (PLHIV) worldwide. Existing guidelines and recommendations for the treatment of HIV-2 infection are largely derived from in vitro data and small-scale clinical studies, and are often extrapolated from studies of people living with HIV-1. Furthermore, of the two most common HIV-2 groups (A and B), only a few examples of drug resistance in group B have been reported in literature. As a result, in-depth knowledge about the mutations that arise in group B HIV-2 isolates and the roles that these mutations play in antiretroviral drug resistance is lacking. The aims of my project are (1) to develop a novel approach for creating full-length, infectious HIV-2 group B clones that encode patient-derived integrase sequences, and (2) to demonstrate that this approach can be used to characterize resistance to Integrase Inhibitors (INI), which are an important class of antiretroviral drugs. Specifically, the parental plasmid for cloning and virus expression is 7312A-JK; this plasmid contains a full-length HIV-2 genome with group B gag and pol gene sequences. To facilitate downstream steps, I have replaced the integrase-encoding portion of p7312A-JK pol with a short, synthetic linker. This cassette clone will serve as the acceptor for longer, synthetic DNA fragments that encode patient-derived group B HIV-2 integrases. My ultimate goal is to generate a panel of 10 replication-competent group B clones that can be given to members of the Gottlieb lab for culture-based drug susceptibility testing. This work is an essential step toward a more comprehensive knowledge of drug resistance in HIV-2 which, in turn, is important for evidence-based treatment of all HIV-2–infected individuals, including those infected with group B strains.

CCR5 is a co-receptor required for HIV to infect the body. Several AIDS patients were completely cured after receiving hematopoietic stem cell (HSC) transplants from donors with a mutation in their CCR5 gene making it functionally inactive. However, HSC transplantation is a very risky and expensive procedure, making it inaccessible for most AIDS patients in developing countries. Our aim was to develop a technology that introduces CCR5 gene mutations in the HSCs of AIDS patients in vivo by a single intravenous injection of a gene transfer vector. The vector delivers a genome editing enzyme (base editor) targeted to the CCR5 gene. CCR5-gene edited HSCs will give rise to CCR5-negative HIV target cells, thereby blocking HIV infection and providing life-long protection. We tested three strategies to functionally inactivate CCR5 expression: i) creating a premature stop codon, ii) eliminating the ATG start codon and iii) mutating splice acceptor sites to skip exons. We employed an advanced adenine base editor version (ABE8e) and an early version of cytidine base editor (CBE) delivered with helper-dependent adenovirus vectors (HDAd5/35++) that efficiently infect HSCs in vivo. The base editors are directed to specific target sites by single guide RNAs (sgRNAs). We screened a small library of sgRNAs and identified two species (sgSTOP2 and sgR5-1) that mediated the highest on-target editing rates and CCR5 down-regulation. HDAdAd5/35++ vectors were produced using these sequences. In a test cell line infected with these vectors, 50% of CCR5 alleles were edited. This blocked HIV infection in 40% and 95% of HDAd-ABE8e-sgSTOP2- and HDAd-CBE-sgR5-1-infected cells, respectively. We concluded that the HDAd-CBE-sgR5-1 vector is more efficient in blocking HIV infection and will further improve this vector and test it in primary human lymphocytes and HSCs in the context of HIV infection. This approach has the potential to provide a technically simple HIV/AIDS therapy.

 The Hybiske Lab studies chlamydial infections in humans, and is currently working with a National Health and Nutrition Examination Survey (NHANES) set of serum samples from the Centers for Disease Control and Prevention (CDC) to determine the seropositivity rate of chlamydia in the U.S. by testing for the presence of anti- C. trachomatis [CT] antibodies in a large multidimensional collection of patient sera. The impact of this research is highly relevant today. Chlamydia was the most reported STI in the country in 2020 and one of the leading causes of Pelvic Inflammatory Disease (PID) in females. While prior research on the prevalence of chlamydia has largely focused on specific demographic groups, this study is one of the first to research its prevalence in the general and contemporary population across the country. Using patient survey data will also help us differentiate between rectal and genital CT infection. My work centers on a novel CT peptide-based enzyme-linked immunosorbent assay (ELISA) that achieves 93.9% sensitivity and 98% specificity, far outperforming other CT detection methods including commercial assays. An additional advantage of this particular assay is that it was designed with peptide-based specificity — using 24 unique peptides that are strong B cell antigens — for the species C. trachomatis, and thus does not suffer from cross-reactivity with the closely related respiratory pathogen C. pneumoniae. We have over 3000 patient sera samples to analyze using this approach, and the 96-well assay format allows for this large number of specimens to be processed at once. I have incorporated additional analyses in my workflow, like testing for the presence of immunoglobulin species specificity across this large sample collection. Based on the data from the small percentage of the total samples collected to date, we are working with a seroprevalence rate of over 20%.

In the human immune system, there is a coevolutionary arms race occurring between pathogens and the host. Pathogens, especially viruses like HIV and SARS-CoV-2, evolve to escape the immune challenge presented by the human immune system. In return, the human immune system can re-organize and through processes that resemble Darwinian evolution, produce novel antibodies that target and neutralize the evolved pathogens. However, it is unclear who leads and who follows in this coevolutionary arms race. I introduce a bipartite, Markovian model to study a coevolving network of species and investigate causality in stochastically evolving systems. My results include novel analytical expressions for observables that discern causality, e.g., the rate of change of partial mutual information, and characterize how causal relationships change with the dimension and topology of the network. I compare the theory to simulations and describe statistical features of these processes. The tools I develop will be useful for distinguishing drivers of evolution in fitness seascapes and, more broadly, detecting causality in any type of dynamic network that undergoes nonequilibrium stochastic dynamics.