The goal of this study is to characterize the activity of defensins on infection by rotavirus (RV), a double-stranded RNA virus that infects gut epithelium and is the most common cause of gastroenteritis among infants and young children. Defensins, small and abundant peptides of the innate immune response, can act on fungi, bacteria, and viruses. Although defensins are typically studied as anti-microbial, we have found that defensins both inhibit and enhance RV infection. To characterize this counterintuitive dichotomy, I first tested the activity of human and mouse alpha-defensins against a panel of human, mouse, and monkey RVs. Although we found no clear species-specific pattern, we did observe a trend in the activity of defensins produced by different cell types. In general, defensins expressed by gut epithelial cells (enteric) are enhancing, while those expressed by granulocytes (myeloid) are neutralizing. This may reflect the context of RV infection: RV will often encounter enteric defensins in the gut but seldom interact with myeloid defensins. In a competition between the effects of a neutralizing defensin and an enhancing defensin on mouse RV infection, we saw a phenotype that was the sum of their individual activities, suggesting that neutralization and enhancement are independent processes. In a time course experiment, I found that neutralizing and enhancing defensins have different kinetics: neutralization is more aligned with viral entry kinetics while enhancement persists even after virus enters the host cell. This suggests that neutralization targets viral particles while enhancement may target the host cell. This interpretation was supported by washout experiments where pretreating cells with an enhancing defensin before infection still showed enhancement of infection, but pretreating with neutralizing defensin did not have an effect on infection. Driven by the selective pressure imposed by enteric defensins, RV may have evolved to co-opt these host peptides to aid infectivity.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious agent of Kaposi’s Sarcoma—a tumor that is the most prevalent cancer found in untreated HIV-infected individuals worldwide. KSHV establishes predominantly latent infection both in cells in vitro, as well as in KS tumors. KSHV, like all viruses, lacks an independent metabolism. It dramatically alters host cellular metabolism, specifically in carbon utilization pathways. We have previously shown that, similar to cancer cells, latent KSHV-infected cells require increased glucose uptake and glycolysis for survival. Hypoxia-inducible factor 1a (HIF-1a) and HIF-2a are two homologous alpha subunits of the heterodimeric transcription factor HIF. In cancer cells, HIF induces aerobic glycolysis to enhance energy production and prevent cancer cells from damage via hypoxic stress. However, it is still unknown if latent KSHV infection requires HIF protein, and if the HIF pathway is the direct cause of the observed induction of glycolytic gene expression and downstream metabolism. I hypothesize that during latent KSHV infection, HIF-1a and/or HIF-2a are the master regulators of the altered, host cellular metabolism. I utilized an shRNA knockdown approach to determine the protein expression of glucose transporter-3 and hexokinase-1, essential glycolytic enzymes that are known to be upregulated by KSHV infection. If stabilized HIF is the master regulator of cellular metabolism (i.e. glycolysis), my expected results would show significantly lower protein expression of key glycolytic proteins during latent KSHV infection when HIF production is inhibited. This study has the potential to reveal important drug targets for future strategies to inhibit and treat latent-KSHV infection and ultimately KS tumors.

It has been 27 years since the development of the first, partially effective therapy using nucleoside analog reverse-transcriptase inhibitor, azidothymidine (AZT), against Human Immunodeficiency Virus (HIV). Upon the development of combination antiretroviral therapies (ART) interfering with the HIV life cycle, the clinical prospects of HIV infected individuals improved. However, ART cannot eliminate the virus from the body even when targeting multiple phases of the HIV life cycle. This phenomenon is due to persistence of often latent (inactive) viruses within reservoirs and compartments, which may cause reestablishment of viremia to pre-therapy levels after cessation of ART. Viral reservoirs are sites that both restrict viral replication and preserve replication-competent viral populations, and are genetically characterized as exhibiting low divergence from most recent common ancestor (MRCA) of infection and high population diversity. Virological compartments are characterized by exhibiting restricted viral gene flow. In order to develop HIV treatments that target latent viruses, we need to understand how and where the viral latency occurs in the human host. This study focuses on finding reservoir-like tissue sites in the hosts who have maintained latent HIV infection during suppressive ART. We analyzed the viral populations present in the bone marrow, brain, large intestine, liver, lung, and lymph node from one male (patient 3), and kidney, large intestine, lung, lymph node, and small intestine from another male (patient 6), both at autopsy. Viral genetic divergence and diversity were calculated based on alignments of the viral POL and ENV genes to identify possible reservoirs. We found two varient HIV population and evidence of lung being more reservoir-like compared to other tissues in patient 3, while we found no evidence that any tissue from patient 6 was more reservoir-like compared to other tissues. As lung has been implicated as potential reservoir in previous studies, we need to collect more sequences and perform the phylogenetic analysis on other cohorts.

The high mutation rate as well as the highly glycosylated surface of the human immunodeficiency virus type-1 (HIV-1) makes the generation of neutralizing antibodies (Nabs) by vaccination challenging. Previous research in the Hu lab has determined that the removal of an N-linked glycan at amino acid N197 (N7) increases sensitivity of the mutant virus to neutralizing antibodies targeting the highly variable region, V3, as well as the CD4 binding site of the HIV-1 envelope protein (Env). Based on these findings, we hypothesize that the N7 glycan modulates Nab responses, including those directed to the V3 loop of Envs from diverse HIV-1 isolates. To test this hypothesis, we immunized rabbits in a prime-boost regimen with recombinant vaccinia viruses expressing N7 glycosylated or deglycosylated versions of Env from JR-FL or PVO.4, two HIV-1 isolates that possess different biologic and antigenic properties. Serum Nab activities in immunized rabbits were determined with neutralization assays, using the reduction of infectivity of HIV-1 Env-pseudotyped viruses on a reporter cell line (TZM-bl) as the indicator of neutralization. Presence of V3-directed Nabs was detected using V3-specific peptides as competitors in neutralization assays. Results demonstrate that Nabs against HIV-1 isolates of various clades and neutralization sensitivities were generated in immunized rabbits regardless of the presence or absence of the N7 glycan on the JR-FL or PVO.4 Env. V3-directed Nabs were present in PVO.4 and JR-FL Env-immunized rabbits, accounting for >90% of the sera neutralizing activities in PVO.4 immunized rabbits against specific indicator viruses. These results suggest that the immunization regimen used does not reveal a strong effect of the N7 glycan on envelope immunogenicity. However, isolate-dependent differences in Nab response and V3 specificity were observed among immunized animals and requires further examination.

Many biological, social, and economic factors increase women's risk of contracting HIV. However, few options are available for them to protect themselves against infection. To address this problem, we are designing a female-initiated HIV prevention technology using nanocomposites made of nanofibers and drug-loaded nanoparticles. We designed nanofibers to release nanoparticles, which can penetrate the mucus layer to deliver antiviral drugs intracellularly. To better understand how nanocomposites behave during dissolution, experiments were conducted to characterize the in vitro behavior of two nanofiber polymer candidates, polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). These characterizations would inform which polymer is best suited to the conditions of vaginal drug delivery and nanoparticle loading and release. A three-day release study was performed in sink conditions to analyze the release patterns of nanoparticles from PVA fibers versus PVP fibers. Fluorescence readings and calculations showed a burst release of nanoparticles: in 30 minutes, 84% of total nanoparticle content was released from PVA, while 83% was released from PVP. Additionally, nanofiber dissolution was analyzed using agar hydrogel plates to mimic the low volume conditions in the vaginal environment. The entire PVP sample wetted out within five seconds after coming into contact with the agar, indicating that diffusion may be the mechanism of nanoparticle release from PVP. In contrast, PVA shrunk as it wetted out over the course of 60 seconds. The PVA polymer compacted in the center as it shrunk; after a minute, the area of the sample had reduced in size by 70%, suggesting that nanoparticles may be getting mechanically squeezed out. In conclusion, PVA and PVP are both able to rapidly release nanoparticles upon dissolution, although their mechanisms of nanoparticle release may differ. Ongoing research includes loading an antiviral drug into nanoparticles and conducting biodistribution studies in mice of vaginally administered nanocomposites.

The global burden of HIV exceeds 30 million individuals, who are predominantly in low resource regions. While the treatment of HIV has been dramatically improved by the advent of combination antiretroviral therapies (HAART), there is a clinical need for improved delivery systems that enable the realization of drug combinations with enhanced potency and lower toxicity that can also address the emergence of drug resistance. Delivery systems are needed to enable the combination of small molecule antiretroviral drugs (ARV), which span a wide range of physiochemical properties that precludes their simple co-delivery. Additionally, there are currently no available delivery systems for easy combination of ARV drugs and antiviral biologics such as proteins and nucleic acids. We propose the use of crosslinked lipid particles (CLPs) for the delivery of physicochemically diverse small molecule antiretroviral drugs in combination with potent antiviral neutralizing proteins against HIV-1. Due to the unique properties of lipid membranes we are able to encapsulate physicochemically diverse combinations of small molecules within separate particle compartments. This allows for finely tuned, synergistic combinations of small molecule and biologic therapeutics for simultaneous delivery. This combination delivery system will ultimately address problems that modern therapeutic strategies face with drug toxicity and limited bioavailability by maintaining a constant serum concentration of multiple ARVs simultaneously. We have demonstrated the successful synthesis of the CLP platform as well as the co-encapsulation of several ARVs. Additionally we have demonstrated the efficacy of our platform at inhibiting both cell independent and cell mediated HIV transmission, which is essential for a clinically relevant therapy or prophylactic technology. We further probed the effects that simultaneous co-delivery of multiple therapeutic agents has on drug interactions and drug synergies. Our work represents a milestone in the field of ARV delivery by allowing diverse drug combinations that were not previously realizable.

Microorganisms outnumber human cells ten to one in the average person. This microbial community interacts extensively with the human body, with both positive and negative results. My research could allow for a more targeted approach to bacterial infections rather than the commonly used general antibiotics, which eliminate both the beneficial and harmful bacteria in the body, which has been proven to cause deleterious health effects. I have been working on achieving targeted infection of the bacterium Escherichia coli by the virus M13 bacteriophage. This would allow for the virus to infect only very specific bacteria and deliver DNA encoded messages without affecting any other cells. We plan to achieve this by attaching peptides to the coat of the M13 bacteriophage and attaching a complementary protein to the target E. coli’s F-pilus, a projection on the surface of bacteria. Using a virus as a delivery mechanism allows for variability in the genetic message, such as a variety of anti-microbial proteins. The applications of this research lie in many fields, including medicine, with its applications in antibiotics and bioengineering to build more complex cellular systems.