Of the estimated 38 million HIV infections globally, approximately 1-2 million are people living with HIV-2 (PLHIV-2). Infection with HIV-2, which is endemic in West Africa, is characterized by lower viral loads (VL) and slower disease progression to AIDS than HIV-1. However, many PLHIV-2 eventually progress to AIDS and death if left untreated. Antiretroviral therapy (ART) for HIV-2 is complicated by fewer effective drugs and significant challenges in drug resistance testing to identify appropriate therapy. This study aims to validate a novel method of HIV-2 drug resistance testing using nucleic acid from dried blood spots (DBS). One hundred and fifty DBS have been collected as part of two clinical studies of ART for HIV-2 in Senegal. HIV viral nucleic acid (DNA/RNA) is extracted and the regions encoding protease (PR) and reverse transcriptase (RT), which are the most common drug targets, are amplified by PCR, then sequenced. Sequence data are examined for evidence of drug resistance-associated mutations. To date, we have tested 115 samples with a median viral load of 84 (range: 0-24,000) and obtained drug resistance data from 43 (37.3%). Testing was most commonly successful from samples with higher viral loads; samples with viral loads >250 copies/ml were successful 75.7% of the time. We observed several drug resistance mutations, including PR V47A (5 patients), I50V (3 patients), and L90M (2 patients), and RT K65R (4 patients), Q151M (2 patients), and M184V (10 patients). Eight patients had no known resistance mutations. Once DBS testing is complete, we will select a subset of sequences to compare to genotypic resistance testing from corresponding plasma samples to evaluate the sensitivity of DBS-based testing vs. standard methods. DBS-based resistance testing has the potential to revolutionize ART for HIV-2 by allowing faster, easier, and cheaper assessment of drug resistance to optimize second-line therapy for HIV-2-infected patients.

Immunological memory prevents reinfection by a pathogen. This protection is accomplished by memory T cells expressing T cell receptors (TCR) specific for previously encountered pathogen-derived peptides (antigens). Conventionally, memory T cells are thought to be inert during novel infections because there is no interaction between their TCRs with their specific antigens. Despite this, we and others have demonstrated that these T cells (here termed “bystanders”) can be activated by inflammatory signals alone and gain cytotoxic effector function in the absence of TCR-antigen interaction. This study aims to determine how inflammation regulates and attenuates bystander responses and how we can leverage these cells therapeutically. Using in vitro cell stimulations, we found that the inhibitory receptor, programmed cell death protein 1 (PD-1), is strongly upregulated by bystanders after exposure to certain inflammatory cytokines. This finding is unique because the current paradigm is that PD-1 expression is caused by TCR stimulation and PD-1 represents a target to manipulate bystander responses. Further, in mouse models of vaccination, we found that bystander-mediated killing can limit vaccine antigen. We believe that interfering with bystander T cell effector functionality could be targeted to improve antigen-specific vaccine responses. Through understanding the mechanisms that dictate bystander function, we may better modulate bystander T cells function during infection, vaccination, and cancer to improve patient outcomes.

Early initiation of antiretroviral therapy (ART) among people living with HIV significantly improves health outcomes and survival rates. Based on these findings, the World Health Organization recommended provision of ART to all people living with HIV regardless of disease status. In 2019, the Government of Tanzania initiated task-sharing to nurse-initiated HIV care as a way to mitigate health systems challenges associated with increasing numbers of patients eligible for ART. As health providers take on new responsibilities, it is critical to assess the quality of HIV care. We are presenting observational data collected during a technical assistance program across 5 regions in Tanzania. Supportive supervision teams visited 20 health care facilities to observe provision of HIV care. During baseline observations, teams used a 41-point checklist to assess individual health care providers administering HIV care. Domains within the checklist included visit type, introduction, adherence counseling, consultation, and communication and support. Following the observations, the teams reviewed individual results with the providers and offered feedback. A repeat evaluation was performed after 3-months. Data has been summarized as counts, proportions, means, and standard deviations. Chi-squared tests of proportions have been used to compare pre/post data. Data analysis has yet to be completed. Findings from our analysis will measure differences in quality of HIV care over time in the context of HIV task-sharing and will provide specific information about domains of quality of HIV care. Results will be shared with the Government of Tanzania to inform future program directions. Quality assessment is important in providing HIV treatment in accordance with national guidelines. Our data analysis will inform future clinical training and health systems monitoring to ensure efficient and effective HIV care in Tanzania.

Malaria devastates communities around the world, and researchers are striving to solve this enormous global health problem. Vaccine candidates could control malaria in these areas. Unfortunately, producing a single dose of these vaccines requires millions of malaria parasites from thousands of mosquitoes. Currently mosquitoes are dissected by hand, and the parasites are extracted from the dissected material with a tiny pestle. This work is time-consuming, expensive, and produces low parasite counts. With current techniques, vaccines will be difficult and expensive to produce, preventing them from protecting malaria-endemic populations. My project is to overcome the technical challenges described above by developing a system that will automatically dissect mosquitoes and extract malaria parasites. Our team has built a mosquito dissection robot and an automatic grinder. By hand, an experienced technician can dissect 150 mosquitoes per hour, but we are designing our robot to dissect over 1200 in the same time. By automating this process we improve yields and save time. In the future we will manufacture and distribute these tools to other labs. To confirm the quality of the parasites our robots extract, we are conducting extensive biological testing.

Malaria, a disease caused by Plasmodium parasites, is an enormous public health burden, especially in resource-poor areas of the world. After the female Anopheles mosquito deposits the Plasmodium sporozoite stage to the host through its saliva during blood feeding, the sporozoite quickly makes its way to the liver where it selectively invades a hepatocyte. The parasite replicates within the host cell, eventually re-entering the blood stream where it causes symptomatic infection. My project focuses on the liver stage of malaria, where complex hepatocyte signaling pathways contribute to parasite development and replication. Previously, our lab demonstrated that host signaling pathways that control lipid peroxidation are crucial to regulating liver stage infection during the first 24 hours. Inhibiting SLC7a11, a protein associated with the regulation of lipid peroxidation, led to increased peroxidated lipids within the infected cell and reduced Plasmodium liver stage parasite infection. Interestingly, lipid peroxides were localized to the infected hepatocyte, leading us to question the kinetic and spatial distribution of peroxidated lipids within the infected hepatocyte. I cultured Hepa 1-6 cells and infected them with Plasmodium yoelii sporozoites. After infection, I treated the cultures with drugs that promote or inhibit SLC7a11. Lipid peroxide levels and localization were observed by fluorescent microscopy at six hours post-infection and mean fluorescent intensity was quantified. At six hours post-infection, I observed no significant difference in lipid peroxidation when comparing infected and uninfected cells, suggesting that lipid peroxidation in infected cells occurs at some point between 6 and 24 hours. This project will allow us to gain a better understanding of how the lipid peroxidation pathway contributes to limiting Plasmodium infection within the liver and how it might be targeted by therapeutics to selectively kill parasites without harming the host.

Human Immunodeficiency Virus (HIV) remains on the forefront of research due to the ongoing global epidemic. HIV is comprised of two genetically different types, HIV-1 and HIV-2. HIV-2 is inherently resistant to some classes of antiretroviral drugs, and many HIV-2 patients develop drug resistance to first-line and subsequent regimens. HIV-2 can further be divided into two distinct genetic groups: A and B. While both are endemic to West Africa, group A accounts for the majority of infections and remains the most studied of the two groups. In-depth knowledge of drug resistance in HIV-2 group B is lacking, as only a few patients with drug-resistant virus are described in the literature and there have been no systematic efforts to characterize the drug resistance patterns of HIV-2 group B isolates in cell culture. My project's goal is to build drug resistance mutations, documented in literature, for HIV-2 group A into a full-length HIV-2 group B infectious molecular clone. Those results are used to compare the relative drug resistance conferred by those mutations to the phenotypes observed for equivalent mutants of HIV-2 group A. More specifically, common drug resistance mutations are introduced into the pol gene of a group B clone, individual mutant clones are isolated, and these are used to transfect replication-competent cells for virus production and drug susceptibility testing. Inhibitors targeting the reverse transcriptase, protease and integrase targets of HIV-2 are evaluated. The resultant drug resistance profiles are then compared to those found in published datasets for HIV-2 group A to determine how HIV-2 group A and group B mutants differ in terms of the magnitude and/or scope of drug resistance. These data are essential for developing evidence-based treatment guidelines for HIV-2–infected patients that harbor drug-resistant group B strains.

Human immunodeficiency virus (HIV) infection is a significant global health issue, with approximately 75 million infections, and over 35 million deaths, since the beginning of the AIDS pandemic. The majority of these are attributable to HIV type 1 (HIV-1). A second form of HIV – HIV type 2 (HIV-2) – is endemic in West Africa and has spread to other areas with socioeconomic ties to the region. Historically, regimens for first-line treatment of HIV-2 have differed from those used in HIV-1-infected patients due to the intrinsic resistance of HIV-2 to nonnucleoside reverse transcriptase inhibitors. This distinction is coming to an end, as countries throughout West Africa are implementing a new WHO-recommended treatment regimen for first-line treatment of all HIV-infected patients, including those with HIV-2. The regimen, known as TLD, is comprised of the nucleoside reverse transcriptase inhibitors tenofovir and lamivudine and the integrase inhibitor, dolutegravir that has potent activity against both HIV-1 and HIV-2. Although treatment-emergent drug resistance has been well characterized for HIV-2 patients receiving tenofovir and lamivudine, there are few data regarding resistance mechanisms in patients receiving the third component of TLD, dolutegravir. The two objectives of my project are: (1) to construct a system for generating recombinant HIV-2 clones that encode and express integrase sequences from TLD-treated HIV-2 patients, and (2) to determine the in vitro susceptibility of viruses produced from the patient-derived clones to the integrase inhibitor dolutegravir. Specifically, I am engineering a plasmid vector into which patient-derived integrase sequences can be ligated for virus production and drug resistance testing in culture. The plasmid vector produced in this study will be used to characterize novel genetic pathways to dolutegravir resistance in HIV-2 and will help identify patients who are failing TLD treatment due to drug resistance. This information is crucial for improving treatment outcomes in HIV-2-infected individuals worldwide.