A keystone bacterium involved in the pathogenesis of chronic periodontitis, commonly known as gum disease, is Porphyromonas gingivalis. This pathogen produces multiple structures on the surface of its cells categorized as virulence factors, including lipopolysaccharides (LPS) and outer membrane vesicles (OMV). Lipopolysaccharides are anchored to the outer membrane of P. gingivalis by Lipid A structures. Previous studies in our lab have found that the abundance and cargo selection of OMVs released from the cell’s surface can be modulated by the structure of Lipid A on P. gingivalis. The virulence factors investigated assist P. gingivalis in the colonization of the host at a cellular level – such as the formation of biofilm, or aggregated bacteria on a surface. OMVs released from the cell's surface operate as a delivery system for the various structures found on the outer membrane of the bacterial cell. We hypothesize that the biofilm density and morphology formed by P. gingivalis are influenced by the changes in OMV abundance and content derived from the modulations in the Lipid A structure. To test this, we utilize biofilm assays, where live cultures from various strains with differing Lipid A structures can grow and aggregate on a glass coverslip for 48-72 hours. Morphology differences are revealed from the analysis of the biofilms and pixel intensity is quantified and compared among strains. Various assays are used to compare the activity and concentrations of protein and lipid cargoes within the OMVs to understand how they are connected to biofilm morphology. The biofilms formed by P. gingivalis contribute to its pathogenesis, therefore it is important to understand the impact that secreted outer membrane vesicles have on its structure and integrity.

My research project aims to combat inflammation and fibrosis caused by biomaterials and implants. Nondegradable biomaterials can provide long-term stability in the body but can elicit a foreign body response, such as inflammation. The project focuses on finding ways to repair and replace damaged tissue at the location of the implant.The project involves engineered M1 cells which were created and published by the Giachelli Lab within the Department of Bioengineering at UW. We have two groups of mice: one control group that has been injected with engineered TLR4 (Toll-Like Receptor 4) cells but not given the CID (Chemically Induced Dimerizers) drug, and another group that has been injected with both the engineered TLR4 cells and given the CID drug. The inside of the cell was isolated in both groups of mice and the extracellular domain was removed. For the group with CID, the inside domain of the cell was bound with CID which is thought to result in dimerization and activation of the M1 phenotype.At this stage in the project, we have tissue samples from both these groups, and I will analyze these samples using H&E staining thus allowing me to measure healing parameters, like how dense the collagen structure is around the material and analyze the effect of CID on the state of the tissue. For expected results, I believe the group with CID will have a denser collagen structure around the material than the group without CID.Conducting the analysis on these tissue samples will help address the effect of CID on healing parameters, such as inflammation. Furthermore, this work will help us develop a better understanding of the roles that M1 and M2 phenotypes play in the healing process which can lead to new therapies that reduce the inflammatory response elicited by biomedical devices. 

Rotaviruses (RVs) are non-enveloped, fecal-oral pathogens of the Reoviridae family that cause acute severe gastroenteritis. RVs encounter various host immune defenses, including antimicrobial peptides like alpha-defensins, which are constitutively secreted in the small intestine. Defensins are small, amphipathic, cationic peptides with broad antimicrobial activity. Our lab has recently shown defensins can modulate RV infection; some RV strains are neutralized by alpha-defensins, while other strains are resistant to or enhanced by alpha-defensins. For instance, mouse rotavirus (EDIM) infection is enhanced by rhesus macaque myeloid alpha-defensin (RMAD1), while rhesus rotavirus (RRV) is neutralized by it. Using viral genetics, we identified viral protein 4 (VP4), the spike protein, as the determinant for both defensin-mediated neutralization and enhancement. In the intestine, VP4 is cleaved by trypsin into two subdomains, VP5* and VP8*. This cleavage is necessary to make the virus infectious. VP5* and VP8* have distinct functions, membrane penetration and cell binding, respectively. The goal of my project is to uncover the crucial subdomain of VP4 that determines the defensin-mediated phenotypes. To accomplish this, I designed and created plasmids encoding chimeric VP4 proteins consisting of combinations of VP5* and VP8* from a defensin-enhanced RV (EDIM) and defensin-neutralized RVs (RRV and SA11). I used a newly described RV reverse genetics system to generate infectious RVs containing these chimeric VP4 proteins. With my rescued chimeric viruses, I am assessing how defensins modulate the infectivity of chimeric viruses compared to the parental, wildtype RVs. My current data suggests that the determinant domain is not solely VP5* or VP8*, but a combination of both. By identifying the defensin determinant, we will gain important insight into the mechanism of defensin-mediated neutralization and enhancement.

An effective peptide-based cancer vaccine requires efficient intracellular delivery of antigen peptides to activate tumor-killing immune responses. However, the localization of peptide antigens during endosomal release to optimize the immune response remains under-investigated. The Pun Lab has developed the self-assembling Virus-Inspired Polymers for Endosomal Release (VIPER) that can induce endosomal escape of peptide antigens. The current VIPER formulation employs reducible disulfide bonds for antigen conjugation. We hypothesize that the controlled antigen peptides release mediated by endosomal proteases can result in more effective antigen presentation that leads to more potent tumor-killing cell responses. In this project, I replace VIPER’s antigen conjugation strategy with the pentafluorobenzyl (PFB) moiety (VIPER-NR) and introduce an enzyme-labile linker in the antigen sequence to determine the optimal peptide release kinetics and localization for optimal tumor-killing cell responses. I synthesized a library of enzyme-labile linkers for the ovalbumin antigen peptide of VIPER-NR. I utilized a dendritic cell cross-presentation assay to screen for linkers associated with that efficient antigen presentation in vitro. Subsequently, I conducted enzyme-mediated drug release assay and red blood cell lysis assay in vitro to evaluate the cleaved antigen profile and endosomal escape induction capacity of VIPER-NR with cleavable linkers compared to VIPER-NR. We expect early release of antigens during endocytosis with these modified antigen sequences in VIPER-NR in vitro and significantly elevated cytotoxic T-cell response against the model antigen in vivo. This project could improve the effectiveness of the VIPER system as a peptide vaccine delivery platform. It would also provide further understanding of optimal antigen release profiles for a better tumor-killing cell response in cancer vaccine applications.