Oral swab analysis (OSA) is an alternative to sputum testing for tuberculosis (TB) diagnosis. Sputum is a viscous material coughed up from human airways and the most common human specimen used to test for TB. However, obtaining sputum can be difficult for some patients and occupationally hazardous for whoever is collecting the sample. Oral swabs can be collected rapidly and non-invasively, and do not require extensive processing for analysis. A larger quantity of Mycobacterium tuberculosis (Mtb) DNA can be detected using tongue swabs as compared to cheek swabs. Measurements of oral microbiota have been found to align with this trend when measured using quantitative PCR (qPCR) for bacterial DNA genes encoding conserved regions of the 16S rRNA subunit, which are common across a large variety of bacterial species and useful for comparison of relative abundance using universal bacterial qPCR. Bacterial DNA as quantified by universal qPCR was used to test whether the capacity and efficiency for biomass collection and release of alternative swab types are significantly better than previously used swabs. The qPCR protocol was identified and validated through literature review, comparing different primer sets, and testing different conditions. Tongue swab samples were collected from healthy Seattle-based volunteers, spiked with an avirulent lab-strain of Mtb (H37Ra), and processed with the QIAGEN QIAamp DNA mini kit. Alternative swab types were tested via the aforementioned qPCR protocol. The swab product that collects the most bacterial DNA are subsequently used in clinical evaluations of OSA conducted on TB patients. Data are currently being collected for analysis, and we anticipate that qPCR will determine the effectiveness of different sampling methods for the quantification of universal bacterial 16s rDNA. It is expected that this study will lead to improved OSA-based diagnostic tests for the detection of tuberculosis.

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.

Microneedles are an effective method for transdermal delivery of a variety of pharmaceutically active agents primarily because of their ability to puncture the stratum corneum. Tissue puncture using microneedles also has potential to improve drug delivery at mucosal sites such as the buccal mucosa, where topical dosing is limited by a thick epithelial layer and continuous salivary flow. However, the low tissue stiffness and wide variance in epithelial thickness present in the oral mucosa preclude direct translation of currently available transdermal microneedle application methods. Further studies of microneedle drug delivery in the oral mucosa require methods for complete and reproducible microneedle application. This project aims to address this need with a device that can apply microneedles to the buccal mucosa with reproducible penetration depth and force, metrics which are correlated with delivery efficiency. The device is designed to be tunable to accommodate microneedle arrays with various dimensions and mechanical properties. Physical parameters of the device are optimized in silico via finite element analysis simulation of tissue puncture with a microneedle array. A prototype of the device is then evaluated using a tissue phantom model to assess penetration depth and force. Performance of the lead candidate device is then validated in tissue explants. This project provides insights for future improvements to microneedle application in the oral mucosa.

Antiretroviral therapy (ART) cannot eliminate latently infected human immunodeficiency virus (HIV) reservoirs, the barrier to HIV cure. A “shock and kill” strategy has been proposed to cure HIV by using latency-reversing agents (LRAs) to reactive latent proviruses and allow for reservoir elimination. Due to the low potency and high toxicity of LRAs, none have yet been effective in reducing reservoir size in vivo. Here, we hypothesize that delivery of LRAs using nanocarriers (NCs) will improve drug solubility and safety, provide sustained drug release, and simultaneously deliver multiple drugs to reservoir tissues and cells. We developed hybrid nanocarriers to incorporate physicochemically diverse LRAs and target reservoirs in lymphatic CD4+ T cells. LRAs were formulated by physical encapsulation or covalent conjugation to the biodegradable polymer (PLGA) core. Drug combinations were evaluated in vitro using a J-Lat reporter cells and validated in CD4+ T cells from virologically suppressed patients. CD4+ T cell targeting specificity was tested ex vivo in non-human primate (NHP) peripheral blood mononuclear cells (PBMCs). Targeting and toxicity were also evaluated in vivo in mice following size optimization for increased passive drainage to lymph nodes. Optimized nanocarriers were used for identification of an LRA combination displaying synergistic latency reversal and low toxicity in vitro in model and patient cells. Long-term and specific activation of CD4+ T cells in NHP PMBCs ex vivo and in mouse lymph nodes in vivo was observed, with significant reduction in toxicity compared to free LRA delivery. This nanocarrier platform targets CD4+ T cells, successfully inducing latency reactivation in HIV reservoirs. The platform additionally enables new solutions for HIV cure with the potential to deliver anti-HIV agents, vaccines, immunomodulating agents, and gene-modifying oligonucleotide drugs.

Clouds, in general, are difficult to account for in climate and numerical weather prediction models. They represent a complex mix of radiative forcings that currently aren’t fully understood or quantifiable. Especially difficult are “mixed-phase” clouds: clouds that exist below the freezing temperature of water but are composed of both ice crystals (water in the solid phase) and supercooled liquid droplets (water in the liquid phase). Hence the name “mixed-phase”. While common, the formation of ice in these clouds remains poorly understood – further observations are vital for obtaining insight into this process. Fortunately, we have new measurements that can provide much-needed insight into their behavior. So far, I have been using measurements from the Department of Energy’s Atmospheric Radiation Measurement (ARM) Eastern North Atlantic (ENA) atmospheric observatory on Graciosa Island in the Azores archipelago which is in the northeastern Atlantic Ocean west of Portugal. The observatory is an ideal site for analyzing these clouds for several reasons, but chief among them is its Raman lidar. By measuring a phenomenon called Raman scattering and utilizing algorithms developed by a previous UW graduate student Tyler Thorsen, this instrument can detect different cloud types and aerosol sizes with high confidence up to twenty kilometers into the atmosphere. I’ve been exploring this data using the programming language Python, looking for patterns in these clouds utilizing the ground-based ENA data and verifying my findings with the lidar on the NASA satellite Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO). The poster will explore how the phase of clouds over the Azores varies with season and with temperature.