Trainees

 
Shahad Ahmed, Graduate Student
Serra-Moreno Laboratory
Email Shahad

My main research focus is on the cellular defense protein SERINC5, an antiviral restriction factor that inhibits HIV entry by altering the conformation of the viral envelope, thereby impairing the ability of nascent virions to infect target cells. In addition to its role in blocking HIV entry, recent reports demonstrated that SERINC5 exerts multipronged actions in host defense, restricting a broad range of viruses through multiple mechanisms. In line with these findings, our lab has identified a novel role of SERINC5 by which it inhibits HIV gene expression and consequently impairs virion production. Our subsequent studies revealed that SERINC5 achieves this by impairing HIV transcription. Consistent with these findings, we observed that SERINC5 similarly downregulates plasmid gene expression, suggesting that SERINC5 is involved in an innate host defense pathway that senses and silences invading genetic elements. Therefore, there is a critical need to elucidate the mechanism by which SERINC5 senses and impairs the transcription of non-self-DNA. Without this knowledge, the therapeutic potential of SERINC5 against invading genetic materials will remain unexplored.

 
Zachary Boodoo, Graduate Student
Singh Laboratory
Email Shahad

Even on combined antiretroviral therapy (cART), people living with HIV (PLWH) experience a low level of viral replication that engenders a chronic inflammatory state and increases their risk to develop atherosclerosis (AS). Previous work by our lab has demonstrated platelet-monocyte complexes (PMCs) occur with greater frequency in PLWH due to aberrant platelet activation, and these complexes are associated with higher expression of monocyte activation markers and increased numbers pro-inflammatory CD16+ intermediate and non-classical monocytes (NCM). It is currently unknown if platelet-monocyte interactions are more consequential and have differential effects on monocyte behavior depending on an individual’s HIV and AS status. In addition, the fate of PMCs in the vascular disease microenvironment, and the platelet-induced transcriptional changes that underlie monocyte dysfunction, are not well characterized.

Considering these unknowns, we have implemented a study cohort to investigate the effects of chronic HIV infection on atherosclerosis. Participants were age-, sex-, and Reynolds risk score-matched and divided into four subgroups based on their HIV and AS status (HIV-AS-, HIV+AS-, HIV-AS+, and HIV+AS+). Single-cell RNA sequencing and unbiased clustering of patient monocytes identified eight cell subsets that were conserved across donor groups, one of which was classified as PMCs based on significant abundance of platelet-associated gene signatures. TGFβ and downstream IL-10 signaling was upregulated in the PMC cluster compared to non-PMC subsets and enriched in the HIV+AS+ subgroup, identifying an additive effect of the two disease conditions on this pathway in PMCs. Bulk RNA sequencing and ELISA analysis of platelet-monocyte cocultures recapitulated these results. Simulation of AS and chronic HIV infection conditions in vitro with oxLDL and LPS, respectively, showed that PMC-derived macrophages had a pro-suppressive immune phenotype that was exacerbated by treatments. Our results support the hypothesis that PMC phenotypes are uniquely influenced by HIV and AS conditions and provide a rationale for antiplatelet therapy as a therapeutic intervention to reduce AS risk and severity in PLWH. Ongoing experiments include determining if PMC-defining gene signatures are relevant to monocyte expression patterns, and how PMCs and PMC-derived macrophages influence the vascular microenvironment by designing, optimizing, and utilizing a 3D hydrogel model of the human intima.

 Kala Hardy, Graduate Student
Takimoto Lab
Email Kala

I study viral host shutoff proteins, which are expressed by viruses to reduce host protein expression. Nsp1 fills this role in coronaviruses. The shutoff mechanism of nsp1 from SARS-CoV-2 has been characterized, but the mechanism used by seasonal human coronaviruses has not been investigated. My research focuses on how nsp1 from seasonal human coronaviruses causes host shutoff.

 Vania Lopez-Ruiz, Graduate Student
Robert Lab
Email Vania

Tuberculosis (TB) infections remain a significant burden on the developing world, as they continue to be a leading cause of death worldwide. Drug resistance to TB is on the rise and understanding the mode of the pathogenesis of the disease is of utmost importance in the search for novel immunotherapies targeting TB. Studying TB in a laboratory setting is difficult due to its slow growth and high pathogenicity level. Mycobacterium marinum (Mm) is a reliable model organism to study M. tuberculosis (Mtb), which causes TB, with a faster doubling time and lower pathogenicity in humans. Mm causes TB-like disease in the African clawed frog, Xenopus laevis. X. laevis is an invaluable animal model that can help us study how infection with Mm drives responses from major histocompatibility complex (MHC)-I independent immune pathways because X. laevis tadpoles do not express classical polymorphic MHC class I molecules until after metamorphosis. Instead, the tadpoles are dependent on interactions between innate T cells (iT) and non-polymorphic MHC-I-like molecules. XNC4 is a novel non-polymorphic MHC-I-like molecule found in X. laevis. Since MHC-I-like molecules are not polymorphic they are attractive targets for immunotherapies in individuals irrespective of their genetic heterogeneity. Notably, our lab showed that transgenic tadpoles deficient in XNC4 are significantly more susceptible to Mm infection than wild-type controls. However, the mechanism underlying this susceptibility remains unknown. Therefore, the objective of the present research is to elucidate the mechanism by which MHC class I-like XNC4 is expressed at the surface of antigen-presenting cells (APCs) such as macrophages to activate and control host resistance activation against Mm pathogens.

This research will contribute to a better understanding of the intricate interactions of Mm and other mycobacteria with their host. Indeed, MHC-I-like genes are widespread across vertebrates including humans. Defining the requirements for XNC4 cell surface expression will provide insight into the mechanism involved in binding putative ligands derived from mycobacteria and in anti-Mm iT cell activation. Canonical stabilization of classical MHC-I requires dimerization with b2-macroglobulin (b2m) and peptide loading in the endoplasmic reticulum (ER) enabled by transporters TAP1 & 2; and the ER aminopeptidase ERAAP1 for presentation at the cell surface. However, some MHC-I-like molecules function independently of TAP and ERAAP and do not require b2m for cell surface expression. Our unpublished data indicate that XNC4 binds unusually long peptides, which is reminiscent of HLA-F in humans and MHC-II which are TAP and ERAAP independent. Based on our published findings and preliminary evidence, we hypothesize that the surface expression of XNC4 is independent of peptide loading via TAP and ERAAP or dimerization with b2m. If XNC4 is independent of b2m and peptide loading, we must understand how Mm infections drive a specific response.

 
Liam Peterson, Graduate Student
Brewer/Beck Laboratories
Email Liam

We have shown that S. aureus enhances viral susceptibility of keratinocytes (KC). However, little is known about the effect of fungi on KC viral susceptibility. We found that exposure of KC to Candida (C.) albicans (104 colony forming units [CFU]) or C. parapsilosis (103 CFU) diminished viral susceptibility, whereas Saccharomyces cerevisiae or Malassezia sympodialis exposure had little or no effect, respectively. To extend these findings to additional Candida species (spp) we observed that KC exposed to either C. tropicalis (102 CFU) or C. glabrata (104 CFU) reduced vaccina virus-induced cytopathic effect (CPE) (65% decrease, p=0.066; 32% decrease, p<0.05, respectively). Collectively, these findings suggest that Candida spp diminish KC viral susceptibility. To understand the molecular changes occurring in Candida-exposed KC that would promote an antiviral response, we quantified gene transcripts associated with antiviral activity from KC following C. parapsilosis or C. albicans exposure (104 CFU). We observed an increase in genes encoding inflammasome (NLRP3, 6.2-fold increase, p<0.05; IL1B, 21.6-fold increase, p<0.0001), antimicrobial peptides (RNASE7, 6.2-fold increase, p=0.051; DEFB3, 102.3-fold increase, p<0.05) and type 1 interferons (IFNB, 1.5-fold increase, p<0.05; IFNA1, 2.1-fold increase, p<0.05). Only two genes were significantly upregulated following C. albicans exposure; IL1B (11.9-fold increase, p<0.01) and RNASE7 (3.37-fold increase, p<0.05). Furthermore, 6 hours post-viral infection, IL1B (2.52-fold increase), NLRP3 (3.08-fold increase), DEFB3 (35.4-fold increase), and RNASE7 (3.92-fold increase) transcripts remained at higher levels compared to C. parapsilosis-unexposed KC. To test whether enhanced gene expression following Candida exposure leads to secretion of antiviral proteins from KC that act in an autocrine fashion, we generated conditioned supernatants from yeast alone and yeast-KC co-cultures and exposed KC to these cell-free supernatants prior to viral infection. These supernatants did not diminish viral susceptibility, suggesting yeast-KC contact is likely critical for Candida spp anti-viral effect. This research demonstrates the contrasting effects of different microbes (Candida spp and S. aureus) on the viral susceptibility of human skin. Ongoing work includes understanding the antiviral pathway conferring resistance of KC colonized with fungi to viral infection. This is being done through transcriptomics of yeast exposed KC to look at concurrent host pathways stimulated across different yeast conditions as well as CRISPR/Cas9 gene knockout of selected targets.

 An (Andy) Phan, Graduate Student
Yiping Zhu Lab
Email Andy

My research focus is on identifying novel host factors responsible for the regulation of HIV-1 gene expression. The pathogen targets and depletes immune cells, leading to an incurable infection and a damaged immune system. Infected cells may establish a latent state where viral gene expression is transcriptionally silenced through epigenetic mechanisms, and thus infected cells are unable to be cleared by the immune system or through antiretroviral drugs. My goal is to identify host factors responsible for the silencing of viral DNA and the mechanisms by which they regulate viral gene expression. This is important in the context of providing insight into the potential development of antiretroviral therapy based on the implications for HIV-1 immune escape and gene expression mechanisms.

 
Keegan Proctor, Graduate Student
Varble Laboratory
Email Keegan

Staphylococcus aureus is a leading cause of bacterial infections. However, it also permanently colonizes ~30% of the human population without symptoms, acting as a commensal member of the human microbiome. Individual strains of S. aureus can have significantly different genetic compositions from each other, and the presence of virulence factors and antibiotic resistance genes within a given strain plays a pivotal role in determining the severity and frequency of infections from an individual strain. These genetic factors are commonly found on mobile genetic elements that can quickly spread between different bacteria, making the treatment of S. aureus infections difficult. Horizontal gene transfer (HGT) of these mobile elements is therefore a driver of S. aureus pathogenicity, but it remains unclear what factors determine how well individual mobile elements are able to move between strains. To identify and characterize these unknown factors, we have developed a pipeline to screen a large collection of clinically isolated S. aureus for individual genetic factors which impact the acquisition of foreign DNA. As part of this work, we have also developed an experimental system to deliver transposons to previously intractable clinical strains of S. aureus, expanding the scope of our experiments. Further understanding of bacterial factors involved in HGT could aid in predicting or potentially controlling the spread of antibiotic resistance as well as rational design of mobile genetic elements as tools.

 Lucas Simpson, Graduate Student
Munger Lab
Email Lucas

Human cytomegalovirus (HCMV) is a highly adapted betaherpes virus that is the leading cause of congenital infections in the world. Further, infection of immunocompromised individuals, such as AIDS patients, transplant recipients, and cancer patients receiving immunosuppressive therapies, leads to severe and sometimes fatal disease. HCMV modulates various facets of host cell metabolism and several cellular metabolic activities have been identified that are important for HCMV infection. However, many virally-induced metabolic activities have never been assessed for their contributions to infection. Further, for those activities previously found to be important for infection, additional questions remain about their broad applicability to infection of different cell types, their infection under different physiological conditions, and how those metabolic activities affect different programs of infection, e.g., lytic versus latent infection. To address these questions, we are developing a high-throughput pipeline to assess HCMV’s reliance on various cellular metabolic activities in different conditions. We will screen a library of metabolic inhibitors to assess HCMV’s metabolic vulnerabilities in multiple cell types, as well as with more physiologically-relevant media. It is expected that this pipeline will identify metabolic activities that are broadly important for HCMV infection, thereby highlighting those that require further study with respect to the mechanisms of their activation and their contributions to HCMV infection.

Nicole Waild Graduate Student
Munger Lab
Email Nicole

Human cytomegalovirus (HCMV) is a herpesvirus that infects approximately 50-60% of people in the United States before they turn 40, with most infections occurring in children under the age of five. HCMV causes severe disease in immunocompromised individuals and is a leading cause of birth defects. However, the mechanisms through which HCMV gene products affect cellular functions and support infection are not fully understood. The UL26 viral protein has been shown to be necessary to block anti-viral gene expression and sufficient to attenuate NF-κB signaling, and virus lacking a functional UL26 ORF replicates to decreased viral titers. The discrete mechanisms through which these phenotypes are achieved are unclear. The host transcription factor JUNB, which was identified in a UL26-BioID proximity screen, was found to be a putative anti-viral factor through a targeted CRISPR screen. Preliminary validation using CRISPRi demonstrated that the loss of JUNB enhances ∆UL26 viral replication. Additionally, JUNB inactivation blocks the accumulation of anti-viral genes, specifically Interferon-stimulated gene 15 (ISG15), a known JUNB target, and an anti-viral gene strongly induced during ∆UL26 infection. Lastly, immunofluorescent studies indicate that UL26 is necessary to prevent JUNB nuclear puncta that are associated with JUNB activation. The interplay between UL26 and JUNB’s transcriptional activity will be further characterized by performing RNA-seq, and the putative interaction between these proteins will be assessed using bimolecular fluorescent complementation and co-immunoprecipitation.

 Chantelle White, Graduate Student
Sant Lab
Email Chantelle

A hallmark of SARS-CoV-2 infection is the degree of variability in disease presentation, ranging from asymptomatic to a severe, pneumonia-like illness, often ending in fatality. One key factor to consider as relevant to the course of the disease is T cell memory to endemic CoV (sHCoVs). There is considerable sequence conservation between sHCoV and SARS-CoV-2, suggesting the potential recruitment of sHCoV-reactive memory CD4 T cells into the immune response to SARS-CoV-2. To evaluate this issue, CD4 T cells isolated from the PBMC of 48 healthy human donors, ranging from 20 -80 years old, were stimulated with peptides representing the entire translated region of the Spike (S), S1 and S2 domains and Nucleocapsid (N) proteins from each sHCoV. The magnitude and functional potential of CD4 T cell responses were quantified by cytokine EliSpot. Th1-like responses were characterized by the antigen-specific production of IFNγ and IL-2. Similarly, ThCTL-like responses were characterized by the production of GrzB and GrzA. These studies revealed that the magnitude and specificity of CD4 T cell responses to sHCoV S and N varied across individuals, with a significant decline in the magnitude of the response with age. There was a notable bias towards the S2 domain of S. Our previous studies, assessing the primary response to SARS-CoV-2 S in a naïve mouse model, revealed that epitopes targeted for CD4 T cell recognition were dispersed throughout the entirety of the S protein. When investigating these human responses in the context of a lifetime of repeat infections, it is important to note that the S2 domain is enriched for conserved epitopes, relative to the S1. This suggests that prior exposures to these conserved epitopes may drive the preferential expansion of these S2-specific CD4 T cell subsets, increasing their likelihood of recall into the SARS-CoV-2 responses. CD4 T cells elicited by each virus demonstrate distinct functional potential, particularly NL63 and HKU1-reactive CD4 T cells that exhibited robust cytotoxic potential. Furthermore, there was a significant age-associated decline in S and N-reactive, IFNγ and IL-2 producers but HKU1 S-specific GrzB producers did not demonstrate a significant decline with respect to age. These studies reveal the variability in both the magnitude and cytotoxic potential of sHCoV-reactive CD4 T cells across individuals and with age. These results suggest that the recruitment of these populations into the immune response to SARS-CoV-2 will be equally varied, differentially impacting disease outcomes. Currently, with the University’s acquisition of the Cytek Aurora, we have been able to design and are now optimizing a 33-color, flow cytometry panel that will be utilized to determine the multipotency, transcription factor expression patterns and homing potential of these HCoV-reactive CD4 T cells.