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High-Throughput Antigen Screening via an Invariant Chain Fusion Protein & the MHC Class II Receptor

Description

The human body’s immune system utilizes many different cell types, signaling proteins, and receptors to thwart an infectious pathogen from an individual. Adaptive immunity, particularly with CD4+ T cell lymphocytes & the MHC II receptor, was the focus of this paper by creating a custom destination vector plasmid, pFLIiP, which…

The human body’s immune system utilizes many different cell types, signaling proteins, and receptors to thwart an infectious pathogen from an individual. Adaptive immunity, particularly with CD4+ T cell lymphocytes & the MHC II receptor, was the focus of this paper by creating a custom destination vector plasmid, pFLIiP, which would contain a gateway cloning site and the nucleotides encoding the first 85 amino acids of the invariant chain protein upstream to provide a means of high-throughput antigen screening via the MHC II receptor and peptide processing pathway. The plasmid pFLIiP was successfully created and sequence verified. Both GFP and mCherry fluorescent proteins were inserted into pFLIiP via LR Clonase and successfully transfected into K562 cancer cells. Fluorescent activity read of a flow cytometer in conjunction with the differing pKa values of the two different fluorescent proteins suggested the fusion protein was in-frame and pFLIiP was successfully targeting the protein to the endosome.

ContributorsGrade, Dylan Beck (Author) / Anderson, Karen (Thesis director) / Hogue, Ian (Committee member) / Knappenberger, Mark (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2020-05

The Impact of IL-36γ Treatment on HSV-2 Replication and Immune Cell Recruitment in the Female Reproductive Tract

Description

Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections (STI), affecting over 267 million women worldwide. HSV-2 causes a chronic, latent infection that increases the risk for acquisition with other STI, including HIV. Currently, there is no vaccine against HSV-2 and novel anti-viral treatments are…

Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections (STI), affecting over 267 million women worldwide. HSV-2 causes a chronic, latent infection that increases the risk for acquisition with other STI, including HIV. Currently, there is no vaccine against HSV-2 and novel anti-viral treatments are needed. IL-36γ is a newly characterized cytokine that has been shown to play a role in inflammation and be upregulated in response to microbial infection and tissue damage. We have shown that IL-36γ is expressed in the female reproductive tract (FRT) and is upregulated by HSV-2 infection in vitro and in vivo. IL-36γ in turn induces production of proinflammatory cytokines and chemokines in human vaginal epithelial cells (VEC) that can aid in immune cell recruitment. We hypothesize that IL-36γ is a key regulator of mucosal inflammation in the FRT and functions to limit HSV-2 infection. We have demonstrated that IL-36γ treatment prior to infection protects against HSV-2 replication, disease severity, and promotes survival in a lethal mouse model. Thus, the objective of this study is to understand the mechanisms whereby IL-36γ inhibits HSV-2 replication. To understand the impact of IL-36γ on the HSV-2 lifecycle, we pretreated VEC with IL-36γ and evaluated viral titer during virus attachment and entry, replication, and cell-to-cell spread by plaque assay. Pretreatment with IL-36γ 4h prior to infection did not significantly reduce viral titers in VEC monolayers relative to untreated groups. This suggesting that IL-36γ may play a more significant role in immune cell recruitment during HSV-2 infection. To test this, FRT tissue samples from HSV-2 infected IL-36γ -/- and WT mice were analyzed by histochemistry to characterize immune cell recruitment. No clear pattern was determined for tissue samples in which cell clusters were observed and cell type within recruited clusters was unable to be identified at the current magnification. As these projects continue, the data will aid in elucidating the mechanism and level to which IL-36γ impacts HSV-2 infection in human VEC and FRT models.

ContributorsAlexander, Thessaly E (Author) / Herbst-Kralovetz, Melissa (Thesis director) / Capco, David (Committee member) / Hogue, Ian (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)

Created2019-05