Helicobacter pylori is a gastric bacterial pathogen that infects more than 50% of the world's population. H. pylori colonizes and persists in the harsh, acidic environment of the human stomach yet it is not an acidophile. The ability of H. pylori to successfully colonize the stomach is dependent on several factors. This includes helical shape that is thought to enhance initial colonization, urease-dependent ammonia production which works to neutralize the pH, and chemotaxis driven motility that enables the bacteria to navigate down the acidic gradient towards the neutral gastric mucosa, its preferred niche. Studies show that flagella-driven motility plays a key role in the pathogenesis of H. pylori; motile strains display higher infection rates and colonize the stomach longer in germ-free piglets compared to non-motile strains. Although the antrum serves as the preferential colonization site due to its lacking acid producing parietal cells, H. pylori can migrate and colonize other sites. In this project, we used a soft-agar assay to characterize the spreading motility of a collection of 42 H. pylori clones isolated from a single host at two different timepoints and from different gastric regions. Our results show that there are quantitative and qualitative differences in the spreading motility of these clinical isolates. The average halo diameters measured are collectively higher in the curved rod isolates from the earlier timepoint compared to helical and rod-shaped isolates from the later timepoint. The clones also display variable chemotactic patterns in soft agar assay with most strains producing concentric rings and a few strains forming uniform halos characteristic of nonchemotactic mutants. By assessing the differences observed, our study explores the factors that impact motility, such as flagellation and cell shape, in order to provide insight into these traits that best equip H. pylori to persist in the stomach. 

 Host restriction factors are components of the innate immune system that play a vital role in inhibiting viral infection. One such example is a family of antiviral proteins, APOBEC3s, that inhibit retroviruses such as HIV by hypermutating the genome through cytidine deaminase activity. There are seven members of the APOBEC3 (A3) family found in primates, A3A-A3H. Each of these APOBEC3s vary in their protein expression levels and antiviral activity Antiviral activity of these proteins depends on expression levels and ability to be packaged in budding virions. A3C, in particular, is of interest because it is highly expressed and packaged but lacks potent antiviral activity when compared to its A3 counterparts. Moreover, when we examined A3C expression across primate evolution, we found that many primates encoded only unstable versions of A3C. Preliminary data, on the other hand, suggests an alternative role of A3C in modulating the effects of the more potent A3 proteins, A3D, A3F, and A3G. Coimmunoprecipitation assays suggest that A3C interacts with other A3 proteins, and subsequently decreases the packaging of the potent A3s into budding virions thus inhibiting their antiviral activity. Future experiments include removing endogenous A3C from cells to determine if the antiviral activity of other A3’s increases in the absence of any A3C. These data suggest that A3C has evolved to fine-tune the amount of antiviral activity of other A3 family members, thus allowing for better control of these potential mutagenic proteins.

CD8+ T-cell exhaustion during chronic human immunodeficiency virus (HIV) infection is characterized by increasing expression of exhaustion markers on the cell surface that lead to decreased function, greatly diminishing immune responses and therapeutic vaccination efficacy. Blocking the activation of one such marker (PD-1) using a monoclonal antibody (aPD-1) can help reverse exhaustion. We hypothesized that dosing with aPD-1 would boost the immune system and decrease expression of certain exhaustion markers on CD8+ T-cells throughout infection in order to improve therapeutic vaccine responses. To investigate this hypothesis, we studied how exhaustion changed over time in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques treated with a combinatorial therapeutic regimen consisting of a conserved-elements vaccine to circumvent viral mutants, GS986 to reverse latency, CCR5 gene editing to prevent viral entry, aPD-1 to reverse T-cell exhaustion, and antiretroviral treatment. We characterized exhaustion in peripheral blood mononuclear cells (PBMC) with surface staining and flow cytometry, focusing on markers of exhaustion including PD-1, CTLA-4, LAG-3, TIGIT, and TIM-3 at various timepoints throughout SHIV infection and therapeutic vaccination. We observed higher CD8+ T-cell vaccine responses in animals receiving aPD-1, compared to animals that were vaccinated but not given aPD-1, suggesting aPD-1 dosing improved vaccine responses. We did not observe any significant correlation between PD-1 signaling and expression of other exhaustion markers, suggesting that blocking PD-1 does not reduce exhaustion by decreasing the expression of other exhaustion markers. However, we found a significant negative correlation between CD8+ T-cell vaccine responses and the expression of TIGIT (Spearman r= -0.75, p=0.007). As reduced TIGIT on CD8+ T-cells correlated with increased vaccine responses, there may be a role for dual TIGIT and PD-1 blockade in future studies. Defining the role of CD8+ T-cell exhaustion in therapeutic vaccine immunogenicity and efficacy is crucial to improving combinatorial immunotherapy towards a cure for HIV.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen associated with worsening disease outcomes in cystic fibrosis (CF) patients. P. aeruginosa uses quorum sensing (QS), a cell-cell signaling system, to control expression of a variety of genes including virulence factors. In P. aeruginosa, QS is mediated in part by acyl-homoserine lactone (AHL) signals that can diffuse in and out of cells. Once AHLs accumulate, they bind to a receptor regulator that activates gene transcription. P. aeruginosa has two complete AHL QS systems, LasI-LasR and RhlI-RhlR. The two systems are arranged in a hierarchy, with the las system controlling the rhl system. QS activation in P. aeruginosa is restrained by cellular proteins that dampen the QS response. These proteins, known as “anti-activators”, attenuate QS by preventing receptor activation. Three anti-activator proteins, QscR, QslA, and QteE, have been identified in P. aeruginosa. These anti-activator proteins have additive, overlapping roles in repressing expression of QS gene products in laboratory strains but their role in the QS dynamics of CF isolates is still unclear. This project used standard molecular cloning techniques to delete or overexpress anti-activator genes in a selection of clinical isolates from CF patients. A reporter plasmid with a fluorescent marker was used to track the activity of LasR and RhlR. These experiments were used to quantify differences in QS-controlled gene activation. To test the hypothesis that anti-activators decrease the amount of LasR in the cell, Western blots were used to assess the cellular levels of QS receptors. In strains with deleted anti-activator genes, LasR levels were higher and induction was earlier. Additional tests for phenotypes controlled by QS, such as protease and pyocyanin production, were also performed. Future research should focus on evaluating these effects in additional CF isolates.

Kaposi's Sarcoma associated Herpesvirus (KSHV) is an oncogenic human herpesvirus and the etiological agent of Kaposi's Sarcoma (KS), a cancer that afflicts HIV-positive individuals worldwide and is endemic in sub-Saharan Africa. KS tumor cells, known as spindle cells, originate from latent KSHV infection of endothelial cells. We have previously used a phosphor-proteomics approach to identify changes in the phosphorylation state of proteins during KSHV infection. We identified Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) to be significantly phosphorylated during KSHV infection as compared to mock infected cells. PECAM-1 phosphorylation leads to a cascade of signaling that promotes cell adhesion, migration, and cell survival, however, the role PECAM-1 plays to aid KSHV infection is currently unknown. To determine whether PECAM-1 expression and phosphorylation is important for KS pathogenesis, I propose to assess differences in gene expression levels with RT-qPCR, validate phosphorylation levels in vitro via western blot, and generate CRISPR-lentiviral constructs to knock out PECAM-1 and express PECAM-1 isoform variants in endothelial cells. These experiments will paint a more complete picture of how PECAM-1 interacts with endothelial cellular processes during KSHV infection. Many rounds of RT-qPCR have been conducted to asses gene expression levels with highly variable results. This indicates that some uncontrolled factor in the cell culture process is affected PECAM-1 expression levels. I hypothesize that cell density is the cause behind this variability and plan to use western blotting to elucidate differences in PECAM-1 protein levels in low and highly confluent cells.

Human papillomaviruses (HPV) contribute to approximately 4.5% of cancer cases worldwide. There are currently three vaccines which protect by producing neutralizing antibodies to the structural capsid protein, L1, for each of the most oncogenic HPV types. Despite the high protective efficacy of these vaccines, not enough is known about the antibodies elicited. The aim of this project is to clone and characterize HPV-specific human monoclonal antibodies after vaccination, particularly antibodies reactive to types other than 16 and 18. Plasma cells and memory B cells were isolated from vaccinees at different time points following HPV vaccination. Antibody-encoding transcripts were amplified from pre-screened plasma cells and cloned into expression vectors - one for each antibody light and heavy chain. The expression vectors were co-transfected and the proteins were harvested and purified for one of the antibodies of interest. Antibodies reactive to HPV types 6, 11, 16, 18, 31,33 and 58 were identified. The first antibodies in the process of cloning were reactive to HPV type 11.The findings of this study will allow us to use these monoclonal antibodies as reference standards to determine the quantity of antibodies in serological assays, thus helping us identify the binding affinity and neutralizing capacity of vaccinees’ antibodies. This will aid future studies aimed at answering the question of whether the vaccine doses can be reduced to one, instead of two or three doses.