Kala Hardy, Graduate Student
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
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.
An (Andy) Phan, Graduate Student
Yiping Zhu Lab
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.
Lucas Simpson, Graduate Student
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.
Chantelle White, Graduate Student
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.