Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and survive in various settings such as soil and water. The purpose of this project is to evaluate the ability of humoral responses produced from NTM pre-exposure on the prevention of Mycobacterium tuberculosis (M.tb) disease progression in mice. Additionally, we want to determine if antibody responses from immunization with bacille Calmette-Guerin (BCG) and/or candidate tuberculosis subunit vaccines contribute to protection against M.tb. To test this, we collected serum samples from mice that received various combinations of treatments before and after infection with M.tb. Groups either received NTM in the drinking water or normal drinking water, with or without BCG vaccine, and with or without a candidate vaccine or a combination of the treatments. M. avium ATCC 25291 NTM-lysate was used as an antigen for ELISAs and it was created by growing the mycobacteria and lysing the bacterial cells by multiple freeze-thaws. NTM-specific total IgG ELISAs were performed on serum samples following different vaccine strategies. Before M.tb infection, mice vaccinated with a candidate subunit vaccine regardless of NTM exposure or BCG vaccination had a significantly higher total IgG response. After M.tb infection, mice that received immunization with BCG followed by the candidate vaccine had significantly higher NTM lysate-specific total IgG responses compared to the saline group, however, this was observed only in NTM unexposed mice. NTM-exposed mice had equivalent levels of NTM-specific total IgG in the saline group when compared with the different vaccine regimens. This suggests that NTM exposure is able to produce potent NTM-lysate total IgG antibody responses in unimmunized ‘saline’ control mice, which suggests that the mice were likely boosted following M.tb infection. Given these findings, we plan to investigate whether the NTM-specific antibody response is reducing vaccine-mediated protective responses against M.tb.
 

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.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the deadliest pathogens in human history. The aim of this project is to begin probing the metabolic reprogramming that occurs in Mtb during treatment with isoniazid (INH), a commonly used frontline drug. Unpublished work from a collaborator has identified several common metabolites that change the minimum inhibitory concentration of INH needed to kill 99% of cells in a culture. The amino acid serine -- which is well known to be involved in one-carbon metabolism through interconversion with glycine -- was found to be of particular interest due to the increase in Mtb susceptibility to INH observed in serine-supplemented culture. To further explore this effect, I have generated timecourse growth curves using an avirulent Mtb (aMtb) model while under INH treatment with and without serine. I started cultures of aMtb at log-phase growth under the following conditions: without any additives, with serine, with INH, and with both INH and serine. I then plated aliquots of defined volumes on solid media to observe colony forming units (CFU) that assess how many cells were present in each culture. I repeated CFU plating 4 and 7 days after each culture was created, which gave me sufficient data to analyze the growth patterns of aMtb under these various conditions. My findings require validation but suggest serine supplementation may play a role in lowering INH tolerance in Mtb. If this result is found to be accurate, this may implicate one-carbon metabolism as a pathway with downstream effects on Mtb tolerance to INH.

Many individuals living in refugee camps have limited access to soap. Ready access to soap gives refugees the ability to maintain their own hygiene. Proper hygiene helps to reduce the spread of diseases through proper sanitation, thereby increasing life expectancy–especially among young children and mothers. The goal of this research is to empower individuals living in refugee camps by giving them the ability to make their own soap. To achieve this goal, I first, identified methods for making soap with resources accessible to refugees, such as oils, sodium hydroxide, and protective gear like gloves to ensure their safety during the process. Second, I created and led two workshops to teach leaders within refugee camps how to make soap with these resources. I conducted pre- and post-workshop focus group questioning with 30 refugees to assess their beliefs, attitudes, and experiences with using soap and making their own soap. The results of the pre-workshop focus group with women living in refugee camps revealed that soap is important for their daily lives, particularly for those working in the farming field. They reported that having more access to soap would significantly contribute to an enhanced sense of self and promote a healthier community. Post-workshop focus group responses revealed that participants expressed positive feelings about learning to make their soap, and many expressed interest in experimenting with ingredients like milk or rose flowers for added fragrance. Participants also reported that they plan to test their handmade soap, and if satisfied with its quality and feel, intend to sell it in the local market. Empowering refugees to make their own soap has the potential to provide them with greater agency over their health, dignity, and self-sufficiency. It could also potentially create opportunities for income generation, which could significantly improve their lives.

I aim to determine if intestinal fatty acid binding protein (I-FABP) is an accurate biomarker to assess the health of Kenyan children. I-FABP is an intracellular protein whose levels rapidly elevate after intestinal damage by various illnesses. Here I will correlate I-FABP levels to pediatric health status by analyzing I-FABP concentrations in plasma from hospitalized vs healthy children in 2 Kenyan pediatric cohorts for which we have concurrent clinical and symptom data. I used R&D Quantikine Human I-FABP ELISA kits to quantify I-FABP in plasma from <5-year-old children collected at hospital discharge (n=1024) and 2-year-old healthy children (n=109). I have completed ELISAs for the hospitalized children and am currently conducting the I-FABP ELISAs for the healthy children. Median I-FABP concentration from the hospitalized children is 1380 pg/mL (IQR: 920-2027 pg/mL). Published data suggests median I-FABP levels are typically <900 in healthy children, indicating that I-FABP levels may be elevated in hospitalized vs healthy children. However, the published data from healthy children is not from a Kenyan cohort and is from an older age group, thus it is important to compare our data from hospitalized children to healthy children with similar demographics. To minimize I-FABP concentration differences due to age, I will restrict the data from the hospitalized cohort to samples from children with a median age of 2 years (range: 1-3 years) to compare to the data I am currently generating from the 2-year-old healthy children in our cohort. I expect significantly elevated I-FABP concentrations in the hospitalized children in comparison to the healthy children. I will also analyze the data according to reported intestinal diseases like diarrhea. This work will help show whether I-FABP can be used as an accurate biomarker to indicate gut health of Kenyan children after hospitalization, which can inform and guide treatment of these individuals.

Access to basic healthcare is not just a fundamental human right. It’s an urgent need that many people in Southern Ethiopia's rural communities are struggling to meet. This lack of healthcare access leads to high mortality rates among infants, children, and mothers. To address this pressing issue, an extension of clinics and health education has been proposed for the rural areas of Ethiopia, which would significantly reduce these mortality rates and increase life expectancy. The goal of our research proposal is to advocate and expand healthcare services and education across rural Southern Ethiopia, with a focus on drastically reducing mortality rates and improving life expectancy. Our research strategy involves a dual approach. Firstly, we plan to conduct a comprehensive survey to highlight the lack of primary healthcare access. This survey will include questionnaires and direct interviews with key stakeholders, including community leaders, healthcare workers, and residents from these rural communities. We aim to map out the health infrastructure, gauge the availability of medical professionals, and assess community health awareness. This data will guide us to identify critical gaps in healthcare service provision. Secondly, to understand the causes of high maternal and child mortality rates, we will review medical records and engage in discussions with healthcare providers and community members to understand the specific health challenges faced and the barriers to accessing care. Our analysis will focus on important factors such as the distance to medical facilities and the presence of healthcare personnel. By collaborating with local authorities and communities, we plan to develop targeted health education programs.Our project seeks to bridge the healthcare access gap in Ethiopia's rural areas, aiming for a holistic improvement in public health outcomes. This research represents an informed effort to make healthcare accessible to everyone, particularly those in underserved rural communities.