Matching Items (5)
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022
Description
Sepsis is a deadly and debilitating condition resulting from a hyperinflammatory response to infection. Most organ systems are severely impacted, including the neurological complications for survivors of sepsis. Sepsis associated encephalopathy (SAE) is characterized by dysregulated molecular pathways of the immune response impinging upon normal central nervous system (CNS) function and ultimately resulting in lasting cognitive and behavioral impairments. Sepsis predominantly occurs in a few neonates but mostly elderly individuals where they are at high risk of sepsis-induced delirium and other neurological implications that may have overlap with neurodegenerative diseases. This study seeks to identify gene candidates that exhibit altered transcriptional expression in tissues between pigs injected with saline control vs lipopolysaccharide (LPS) to model the early inflammatory aspects of the septic response. Specifically, brain frontal cortex was examined to see which genes and pathways are altered at these early stages and could be targeted for further investigation to alter the cognitive/behavioral decline seen in sepsis survivors. This experiment uses a bulk RNA-seq approach on Yorkshire pigs to identify the variance in gene expression profile. Data analysis showed several gene candidates that were downregulated in the brain in response to LPS that point to early endothelial cell disruption, including OCLN (occludin), SLC19A3 (thiamine transporter), and SLC52A3 (riboflavin transporter). Genes that were upregulated in LPS brain samples implicate endothelial cell dysfunction as well as immune/inflammatory alterations, possibly due to alterations in microglia, the primary immune cell of the brain. Several studies are now underway to understand the cellular origin of these transcriptional changes, as well as analyzing the molecular signatures altered in response to sepsis in whole blood and kidney using bulk RNAseq. In conclusion, specific gene candidates were identified as early changes in the septic brain that could be targets to prevent long-term cognitive and behavioral changes in future studies, establishing a baseline panel to interrogate in animal models with the goal of advancing treatments for human patients who experience sepsis.
ContributorsNeill, Ryan (Author) / Fryer, John D (Thesis advisor) / Hogue, Ian (Thesis advisor) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2021
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 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
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 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
Description
Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection.
ContributorsKoyuncu, Orkide O. (Author) / MacGibeny, Margaret A. (Author) / Hogue, Ian (Author) / Enquist, Lynn W. (Author) / College of Liberal Arts and Sciences (Contributor)
Created2017-10-26