Tuberculosis is a major burden of disease worldwide. Although drug regimens for the treatment of tuberculosis are available, they are complex and lengthy. Efforts are being made to understand and develop novel drugs. The Parish Lab has identified a common mechanism of drug resistance in the type VII secretion system (T7SS) of Mycobacterium tuberculosis. Mutations in several components of the Esx3 T7SS system confer resistance to aminothiazole (AmT), 8-hydroxyquinoline (8HQ) and nitrothiazole salicylamide (NTS) compounds. The Esx3 T7SS is unlikely the cellular target for these compounds as four other genes had mutations, one of the four genes not being a part of the ESX-3 complex. Therefore, resistance is believed to arise from a distinct mechanism. Rv0474 is a transcriptional regulator identified to mediate resistance to AmT and 8HQ. It is a copper-responsive regulator with a copper binding motif. I am studying the Rv0474 homolog (MSMEG_0918) in Mycobacterium smegmatis, a fast-growing species used as a model organism. MSMEG_0918 lacks the copper binding motif, permitting determination of whether copper binding activity is required to confer resistance. I am using CRISPRi knock down technique to generate strains with reduced expression of MSMEG_0918, along with constructing overexpression strains. Knock down and overexpression strains allow us to determine growth, AmT/8HQ sensitivity and copper sensitivity. We propose that if copper binding activity is involved in AmT/8HQ resistance, overexpression or downregulation of MSMEG_0918 would have no effect on compound activity, because it lacks this activity. This work will determine whether MSMEG_0918 plays an equivalent role to Rv0474 in conferring resistance to AmT and 8HQ compounds. This research will provide knowledge to further global efforts in developing an adequate and effective treatment necessary for tuberculosis. 

HSV-2 is a sexually transmitted disease of global importance, with an estimated infected global population of over 500 million people, as estimated by the WHO. HSV-2 is a lifelong infection which results in painful, recurrent lesions in the genital area. Previous research has shown that high numbers of regulatory T cells (suppressive immune cells important for maintaining tolerance) are present in the mucosal tissue alongside traditional CD4+ and CD8+ T cells (T cells important for an adaptive immune response), and their role is not fully understood. The reporter strain “Nedel” is a strain of HSV-2 with an inserted mNeonGreen fluorescent marker, allowing us to visualize HSV-2 infection. Our current project is to characterize the viral kinetics of the reporter strain of HSV-2 and compare it with WT HSV-2, as well as visualize the virus in infected cells via immunofluorescence and flow cytometry. We have infected mice intravaginally with both strains of virus, collected vaginal washes for plaque assays (to determine viral load) and harvested their vaginal tracts for flow cytometry. Our first round of experiments have shown that while the general frequency of immune cells were comparable, there was significantly lower activation of these cells in the Nedel-infected mice as compared with the WT-infected mice. In ongoing experiments, we have infected the mice with a higher dose of Nedel virus in an effort to make infection more comparable to WT, and we will be harvesting vaginal tracts for flow cytometry analysis to see whether we can visualize the mNeonGreen. Our overall goal is to utilize the Nedel HSV-2 strain to investigate the role of regulatory T cells in modulating anti-viral immune response, with a broader goal of finding treatments and an effective vaccine for HSV-2.

 The herpesvirus Epstein-Barr Virus (EBV) targets B-cells and epithelial cells. The virus is transmitted by saliva and commonly associated with infectious mononucleosis. After initial infection, the virus enters a latent stage. The first oncogenic virus identified in humans, it contributes to 1.5% of all cases of human cancers worldwide, specifically cancers of B-cells and epithelial cells, and roughly 140,000 deaths/year. Currently, no treatment is available for EBV-related cancers. Due to the widespread impact of EBV on populations, including a drainage of resources in parts of the world where EBV-associated cancer rates are disproportionately high, an effective treatment will be greatly beneficial. The gH/gL complex and gB, glycoproteins necessary for virus fusion to the host cell, are conserved among herpesviruses, including EBV. Recently isolated, the monoclonal antibodies AMMO1 and AMMO5 inhibit EBV infection by preventing fusion to the host cell. AMMO1 binds to the gH/gL complex and interferes with both epithelial and B-cell infection, while AMMO5 binds to gB and can prevent epithelial cell infection. Emerging evidence suggests certain tumors express these glycoproteins, thus they may be readily targeted for immunotherapy. In this project I created Jurkat leukemic cell line T-cells which express chimeric express chimeric antigen receptors (CARs) using AMMO1 and AMMO5. This CAR should target EBV viral antigens on tumors. First, I used mutagenesis to insert DNA encoding for the AMMO1 or AMMO5 scFV, into a CAR expression plasmid and verify integration by Sanger sequencing. I then used lentiviral delivery to transduce CAR constructs into Jurkat T-cell genomic DNA. Flow cytometry was used to confirm transduction of the T-cells. Future directions with this project include increasing transduction efficiency and transducing the constructs into cytotoxic T-cell so T-cell killing assays can be used to determine the efficacy of the AMMO1 and AMMO5 CAR-T cells.

Human Immunodeficiency Virus type 1 (HIV-1) is currently a major cause of morbidity and mortality worldwide. HIV-1 isolates are classified into genetic groups M, N, O, and P. Group M is the most widespread and is further divided into at least nine genetic subtypes and over 100 circulating recombinant forms, the prevalence of which vary by geographic locations. Subtype B is the most common form in Europe and North America and as a result, receives the majority of funding and research. Because of this, most of what we know about antiretroviral drug resistance in HIV-1 is based on studies of subtype B. However, the majority of HIV-1 infections worldwide involve other “non-B” subtypes, and much less is known about drug resistance in these subtypes. Knowledge of resistance to integrase inhibitors (INIs) is particularly important, since INI-based regimens are now recommended as first-line treatment for HIV-1–infected individuals globally. My objective is to genetically alter a full-length HIV-1-encoding plasmid (HIV-1 pNL4-3) that can used as a “cassette” for cloning non-B, patient-derived integrase sequences. To do this, I have identified restriction sites flanking the integrase region that can be used to remove and replace the integrase region with a patient-derived sequence, but site-directed mutagenesis must be used to knock out additional restriction sites for the same enzymes within the plasmid to avoid undesired enzymatic activity. This will result in only the desired sites, without changing the amino acid sequence. This vector will enable us to generate recombinant viruses from patients who are failing INI-based treatment, which can be tested for resistance to INIs in cell culture. The resulting data will be used to improve prediction of drug resistance in non-B HIV-1 based on sequence information alone. These studies will help guide treatment recommendations for both individuals and populations infected with non-B-HIV-1.

Immigrant and refugee communities are disproportionately impacted by COVID-19 due to pre-existing social and health disparities. In King County, COVID-19 incidence among Black individuals is 2.8-times higher than White individuals. In addition, communities with limited English proficiency have elevated rates of hospitalization particularly in Washington state. King County testing data has not been disaggregated by racial groups, limiting our knowledge on community testing needs. To better understand these issues, our team has collaborated with the Somali Health Board (SHB) to explore the barriers to COVID-19 testing within the King County Somali Community, where approximately 30,000 Somali immigrants reside. We administered anonymous surveys to Somali and non-Somali participants aged ≥18 (n=540) at testing fairs in South King County and recruited through community outreach. Survey data were used to quantitatively determine prevalence and correlates of timely COVID-19 testing within the King County Somali Community (defined as testing within 2 days of symptom onset). We conducted in-depth interviews with healthcare workers (n=5) and policymakers (n=5) to qualitatively explore the barriers and facilitators of testing in the community. We hosted two virtual focus group discussions -- one female-identifying group (n=10) and one male identifying group (n=10) -- with members from the King County Somali community to gather personal experiences of COVID-19 testing barriers and concerns. Our preliminary results suggest that there is a trend towards longer time from symptom onset to test for Somali respondents and that Somali respondents face various barriers to testing such as distrust, misinformation, stigma, language barriers, and transportation. This work is critical to help identify Somali community barriers to COVID-19 testing and how they can be adequately mitigated to improve access and promote equity in King County’s pandemic response.

Depression is world’s leading cause of disability, yet only 1 in 23 people in low-and-middle income countries (LMICs) receive minimally adequate depression treatment. A common antidepressant is amitriptyline, which is classified as an essential medication by the World Health Organization. However, there is only limited evidence of the availability of essential psychiatric medicines in LMIC health facilities. The Demographic and Health Surveys Program conducts the Service Provision Assessment (SPA) to understand availability of services and essential medicines in LMIC health facilities. We analyzed available SPA data for Afghanistan, Bangladesh, Democratic Republic of Congo, Malawi, Nepal, Senegal and Tanzania to understand the distribution and determinants of amitriptyline availability in each country. We analyzed data from 9470 health facilities. Overall, only 12% of sampled facilities had amitriptyline on the day of assessment, suggesting that the majority do not carry amitriptyline. Multivariable regression models indicated that faith-based and private-for-profit facilities were more likely than public facilities to have amitriptyline, and that hospitals were more likely than primary care facilities to have amitriptyline. The gap between hospitals and primary facilities were wider in the same country than between them. Our results indicate a severe shortage of amitriptyline in health facilities in these eight LMICs. Efforts to narrow the mental health treatment gap by integrating mental health services into primary care will be limited without the availability of essential medicines like amitriptyline. Expanded purchasing and distribution of amitriptyline, especially to LMIC primary care facilities, is warranted.

According to the World Health Organization (WHO), more than 264 million people of all ages suffer from depression globally. Depression is associated with a variety of psychosocial and physical impairments that can each act as both a risk for and consequence of depression. In particular, sleep disturbance commonly accompanies depression and is often a target for remediation in depression treatment (e.g., CBT-I). However, some individuals are more prone to sleep problems (e.g., young adults) than others and not every individual with sleep problems or depression develops the other. Therefore, idiographic approaches that build personalized models which are sensitive to individual heterogeneity are needed to better understand day-to-day links between depression symptoms and sleep problems. In the current study, we hypothesize that the links between specific hygiene behaviors and depression symptoms from day-to-day will be different from person to person. Ambulatory assessment (AA) data was collected from young adults to test whether common sleep hygiene practices are associated with sleep quality, negative affect, anhedonia, and other depression-related phenomena uniformly across individuals. We identified the sleep hygiene behaviors that have person-specific (vs. universal) links with depression and mood across individuals. Our results will have important implications for better understanding how sleep problems exacerbate or maintain depression symptoms for each individual so that more specific aspects of behavior may be targeted in treatment (e.g., CBT-I) to improve its relevance to the individual.