UTMB Health SHARED (Univ. of Texas Medical Branch at Galveston)
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Vaccine Development Strategies for Crimean-Congo Hemorrhagic Fever Virus and its Tick Vector
No full textCrimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus with an extensive geographic range and complex ecology. Ticks within the Hyalomma genera are the primary vector and reservoir of the virus and maintain the virus through a cryptic tick-vertebrate-tick cycle in nature. However, infection of humans with CCHFV can result in the severe disease known as Crimean-Congo hemorrhagic fever (CCHF). As humans are the only species that display clinical signs of disease from infection with CCHFV, vaccine development has focused on preventing human disease. Despite extensive research, no vaccine candidates have advanced to clinical trials. This dissertation aimed to utilize different vaccine platform technologies to target unique points in the CCHFV transmission cycle as new strategies for vaccine development for CCHFV. Vaccine candidates were developed against ticks for the preferred hosts of immature and adult H. marginatum ticks, and against the virus for prevention of human CCHF. The two anti-tick vaccine candidates utilized viral-vectored vaccine technology encoding the concealed tick antigen Subolesin or a chimeric Subolesin and rabies virus glycoprotein antigen. Neither vaccine candidate induced stronger humoral immunity than the conventional approach of purified protein in adjuvant. These studies showed that the vaccine type, specific viral vector used and the generation of a chimeric Subolesin antigen are key considerations for developing future anti-tick vaccines. For prevention of human CCHF, a candidate multi-epitope antigen for CCHFV was developed using bioinformatics and a plasmid-based DNA vaccine was evaluated in vitro and in vivo. Although the multi-epitope antigen was not immunogenic, these studies provide information about predicted immunogenic regions of the CCHFV glycoprotein precursor that should be evaluated for inclusion in rational vaccine development. Overall, this dissertation has evaluated three different vaccine development strategies, and provides information for future tick and CCHFV vaccine development research
The Effect of Integration of TeamSTEPPS® Strategies Within an Orientation Program on New Graduate Nurse Team Attitudes
No full textThe problem this research addressed was identification of potential relationships between a TeamSTEPPS® based orientation program and NGN Teamwork Attitudes. The purpose of this study was to determine if integration of TeamSTEPPS® into the NGN orientation program increased measurements of Teamwork Attitudes (TA). Specific Aim 1: In the NGN, determine TA pre-TeamSTEPPS® training, as measured by the TeamSTEPPS® Teamwork Attitude Questionnaire (T-TAQ). Specific Aim 2: In the NGN, determine TA post-TeamSTEPPS® training, as measured by the T-TAQ. Specific Aim 3: In the NGN, compare TA pre/post-TeamSTEPPS® training, as measured by the T-TAQ. The theoretical framework guiding the study was Gibson’s Theory of Affordances. The methodology for this study was a quasi-experimental pre/post-test study design. The significance of the study was knowledge about TeamSTEPPS® increased measured teamwork attitudes and facilitated integration of NGNs into the interprofessional team
The development and use of cutting edge next generation sequencing methodologies to study RNA viruses
No full textWith new next generation sequencing technologies and methodologies being published often, our ability to sequence viruses and host transcriptomes (in response to viral infection) has expanded – driving virology forward. Here we show the development and use of novel methodologies to study RNA viruses from their impact on the host, to variations in viral genomes, to how context of an infection may alter disease outcomes. We have published methods such as Tiled Click-Seq (TCS) and poly-A Click-Seq (PAC-Seq) with corresponding pipelines (Virus Recombination Mapper and Differential Poly-A Cluster [DPAC]) to study variation in viral genomes and transcriptomic changes respectively. We have also used single nuclei RNA sequencing (snRNA-Seq) for a more granular look at transcriptomic changes using the package Seurat in R.
As the 2015-2016 Zika virus (ZIKV) outbreak in S. America was associated with development of microcephaly in infants born to expectant mothers infected early in pregnancy we wanted to study the transfer of ZIKV from mother to fetus. As this involves placental infection, we extracted total cellular RNA from ZIKV infected (or mock-infected) human placental (JEG3) cells and used it to construct PAC-Seq libraries. Subsequent DPAC analysis provided data on differential gene expression, alternative poly-adenylation (APA), and use of alternative terminal exons. We found that up-regulated poly-A sites (PASs) lacked the sequences for canonical poly-adenylation (AAUAAA ~20 nts upstream of the PAS or a GU region just downstream) that were found in down-regulated PASs. Here we present a potential mechanism for the large-scale APA occurring in response to ZIKV infection in JEG3 cells.
Microcephaly, and other CNS issues, can be symptoms of ZIKV infection we wanted to look at the brain as well. As opioid overdose deaths have increased in recently in the U.S. (coinciding with the COVID-19 pandemic) we investigated the potential impact of opioid use on severity of neurological disease from RNA virus infection, looking at expression of genes involved in SARS-CoV-2 or ZIKV infection in the mesolimbic pathway, using both snRNA-Seq and PAC-Seq. Our results suggest that opioid use may exacerbate symptoms of these infections by up-regulating inflammation and down-regulating anti-viral pathways in this brain region
Evaluation of a gold nanoparticle platform as highly immunogenic and protective therapy against Burkholderia mallei, B. pseudomallei, and Enterohemorrhagic Escherichia coli O157:H7
No full textBurkholderia mallei (Bm) and B. pseudomallei (Bpm) are facultative intracellular pathogens and the causative agents of glanders and melioidosis. At present, effective vaccines for the prevention of glanders, or melioidosis in humans, are not available. However, renewed attention has been directed toward developing Burkholderia vaccines because of the pathogens' seemingly ideal characteristics for malicious use as a biothreat agent. Additionally, a vaccine will also have significant value for the immunization of at-risk populations in melioidosis/glanders endemic areas of the world. Therefore, the following studies' long-term goal is to develop a platform that allows for the efficient generation of a multicomponent vaccine that can protect against both glanders and melioidosis. In this study, glycoconjugates coupled to gold nanoparticles (AuNPs) were tested for their protective properties in clinically relevant infection models. The flexible NP platform allow us to incorporate novel antigens identified previously by a reverse vaccinology model. In these studies, we have established an optimal immunization procedure to test the efficacies of several protein-polysaccharide NPs when delivered alone or in a combination containing various proteins in a clinically relevant and highly controlled aerosol murine model of infection. We have demonstrated that several glycoconjugates offer protection against lethality when delivered intranasally, with increased protection afforded by a refined formulating containing the most immunogenic proteins. Also, we have shown that antigen-specific humoral responses play a significant role in the protection induced by various AuNP-coupled glycoconjugate formulations. Similarly, we have shown a protective effect of AuNP-coupled proteins in a colonization model of Enterohemorrhagic Escherichia coli (EHEC). Furthermore, we showed that vaccination with AuNP-coupled to EHEC-specific antigens elicits strong systemic and mucosal response associated with protection against EHEC colonization. This work proposes to bridge the properties of subunit vaccination with those of synthetic nanomaterials to enhance immune responses to vaccines against pathogenic bacteria
CELF1, PTBP1, and RBFOX2-mediated alternative splicing regulation in cardiovascular diseases
No full textAlternative splicing (AS) is dysregulated in Type 1 diabetic (T1D) and hypoplastic left heart syndrome (HLHS) patient hearts but the mechanisms responsible are unclear. Here, we provide evidence that in these patient’s hearts that dysregulation of the RNA binding proteins (RBPs) CELF1, PTBP1, and RBFOX2 contribute to AS changes. Utilizing genome-wide approaches, we identified extensive changes in AS patterns in T1D mouse hearts. We discovered that many aberrantly spliced genes in T1D hearts have CELF1 and PTBP1 binding sites. CELF1-regulated AS affects key genes within signaling pathways relevant to diabetes pathogenesis. Disruption of CELF1 binding sites impairs AS regulation by CELF1. We show that a spliced variant of PTBP1 that is highly expressed in normal newborn mouse hearts is aberrantly expressed in adult T1D mouse hearts. We also demonstrated that inducible expression of diabetes-induced PTBP1 spliced variant has less repressive splicing function. Notably, PTBP1 antagonizes RBFOX2-mediated AS of selected mRNA targets in this context. In summary, our results indicate that CELF1 and PTBP1 target RNAs are aberrantly spliced in the T1D heart, leading to abnormal gene expression.
RBFOX2 is significantly associated with HLHS. In HLHS, three damaging de novo RBFOX2 mutations (nonsense, frameshift, and splice site) have been identified. Here we provide evidence that the nonsense and frameshift RBFOX2 mutants are unable to promote proper splicing of their target genes despite normal subcellular localization of these mutants. Further, we show that the nonsense mutant interacts with a subset of proteins that wildtype RBFOX2 does not interact with, indicating that this nonsense mutant may constitute a gain of function. These discoveries pave the way for targeting RBPs and their RNA networks as novel therapies for cardiac complications of diabetes and HLHS
Bloodmeal determinants of Zika virus infection of Aedes (Stegomyia) aegypti mosquitoes
No full textThe following dissertation aims to ascertain how factors in the blood of viremic mammals influence the infection of Aedes (Stegomyia) spp. mosquitoes with the arboviruses Zika (ZIKV). It has been repeatedly observed in both flaviviruses and alphaviruses that bloodmeals taken from viremic animals are more infectious than artificial bloodmeals. Although this finding has been demonstrated to be at least in part mediated by clotting factors facilitating increased viral contact with the mosquito midgut epithelium following a natural bloodmeal, a comprehensive and mechanistic exploration of this observation has not been undertaken, a gap the proposed project seeks to address. I hypothesize that there are endocrine, inflammatory, and chemical factors of in vivo mammalian blood, resulting from viral infection, that increase the oral infection by ZIKV in Aedes aegypti mosquitoes. Utilizing multiple mosquito species (Aedes aegypti, Ae. albopictus, Culex quinquefasciatus, and Sabethes cyaneus) I first determined the general basal levels of vector competence these species demonstrated for various strains of ZIKV. Subsequently experiments directly comparing artificial bloodmeals derived from murine and non-human primates to ZIKV viremic animals were conducted to ascertain what, if any, soluble factors were found in association with any instances of increased infectivity in Ae. aegypti. Finally, manipulation of the constituents of bloodmeals were conducted by means of artificial supplementation of bloodmeals (glycation of erythrocytes, addition of TGF-β) as well as via utilization of genetically modified mice (LEPRdb/db; displaying a type II diabetic/metabolic syndrome-like phenotype). Corresponding to previous reports, I found that viremic animals largely proved more infectious than artificial bloodmeals, although in some experimental paradigms (NHPs) this was not found to be statistically significant. Strikingly, in murine bloodmeals the use of “semi-artificial” bloodmeals (whole blood mixed 1:1 with virus) as opposed to washed erythrocytes mixed with virus increased infectivity of ZIKV in Ae. aegypti mosquitoes. Furthermore, artificially glycated erythrocytes, or inclusion of TGF-β in bloodmeals substantially increased infectivity of ZIKV in Ae. aegypti suggesting that elements of blood lost in some artificial bloodmeal formulations may play a role in the observed loss of infectivity. These findings are incorporated into recommendations for vector competence study designs settings of both high and low resources
Male-specific mechanisms in a murine model of nociplastic pain
No full textIn order to address how acute nociceptive pain can transition to nociplastic pain, we developed a murine model in which acute injury-induced mechanical hypersensitivity was prolonged beyond its normal resolution time following postinjury stimulation at normally innocuous intensity. This model utilized intraplantar capsaicin injection or plantar incision as an acute injury, and vibration or warm water immersion for postinjury stimulation. The prolonged mechanical hypersensitivity in both males and females lasted at least 21 days in the absence of peripheral inflammation, indicating the nociplastic nature of this hypersensitivity. The persistent mechanical hypersensitivity was attenuated by morphine or gabapentin in both sexes but was maintained by sex-specific mechanisms: specifically, by ongoing peripheral afferent activity at the initial injury site in females and by reactive spinal microglia in males. Further investigation into the male-specific mechanisms underlying the nociplastic pain state revealed that activation of spinal microglia drives the postinjury vibration stimulation-triggered transition to a nociplastic pain state, but that microglia activation was not mediated by the BDNF-TrkB pathway, unlike other chronic pain models. After an acute peripheral injury, GABAergic disinhibition was required for postinjury vibration stimulation to trigger the spinal microglia-driven transition to a nociplastic pain state. Even in the absence of an inciting peripheral injury, vibration stimulation could trigger the transition to a spinal microglia-mediated nociplastic pain state in males following direct spinal GABAergic disinhibition by intrathecal injection of the GABAA receptor antagonist bicuculline or the GABAB receptor antagonist CGP 52432. In females, spinal GABAB receptor inhibition, but not GABAA receptor inhibition, followed by vibration stimulation was able to trigger a transition to the nociplastic pain state. However, this pain state in females was not mediated by spinal microglia. Proinflammatory cytokines, but not prostaglandins, at the spinal level contributed to the maintenance of nociplastic pain state in males. Overall, these findings provide key insights for understanding the sex-specific mechanisms underlying the transition to and maintenance of the nociplastic pain state, indicating that spinal microglia are potential therapeutic targets to prevent and treat nociplastic pain in males
Exploiting the Conflict Within: Examining Tripartite Interactions for Development of Next Generation Mosquito Microbiome Vector Control Strategies.
No full textMosquitoes harbor various microbes that profoundly influence many aspects of their biology, including vector competence. Given their intimate association, these microbes have established a wide range of strategies aiding them in their transmission, either horizontally or vertically, making them highly attractive for applied vector control approaches to prevent the spread of arthropod-borne disease. The mosquito microbiome responds to environmental and host cues as well as microbial interactions and diet. This body of work focuses on a number of aspects import for the development of a robust microbial driven control stratagem. Next generation sequencing of the 16S ribosomal RNA gene was utilized to examine the microbiome in each of the objectives presented here. The first objective explored how different sugar types influence the microbiome of Aedes aegypti resulted in two main conclusions. First, mosquitoes that are reared in separarte environments had distinct microbiomes. The second finding was that, although sugar type only impatcted the overall microbial community structure in the New Orleans mosquito line from Liverpool, the Galveston mosquito line from Galveston and the NO line from Liverpool both experienced altered responses to each sugar by specific bacterial taxa. The second objective characterized the relationship between the microbiome and Zika virus (ZIKV) in both lab reared and field collected Ae. aegypti mosquitoes. Here bacterial representatives of the Acetobacteraceae and Enterobacteriaceae families were correlated to ZIKV infection. The influence of these bacteria was found to be independent of mosquito immunity. Additionally, mosquitoes exposed to ZIKV had increased levels of these Acetobacteraceae and Enterobacteriaceae bacteria. These results suggest that ZIKV infection were both mosquito and viral strain specific. The third objective, which examines host small RNA interplay between mosquitoes and ZIKV infection, found that infection lead to dramatic increases in short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). Additionally, 17 host miRNAs had altered levels across multiple time points. Finally, the mosquito RNAi response to ZIKV targeted the NS5 region, while ZIKV in response produced virus-derived piRNA-like small RNAs (vipRNAs). Together, these results establish the foundations for developing a microbial based control strategy, in which bacteria could be engineered to deliver RNAi-stimulating RNAs in mosquito hosts to prevent the spread of arboviruses like ZIKV
Characterization of Candidate Mutations for Use in a Live Attenuated West Nile Virus Vaccine
No full textWest Nile virus (WNV) is a mosquito-borne flavivirus that causes neurological disease and fatalities annually in the United States and Europe, but no human WNV vaccines have been licensed. Studies of WNV vaccine candidates and of vaccines for related flaviviruses indicate that a live attenuated vaccine based on the full-length WNV genome will likely induce the most robust protective immune response compared to other vaccination strategies. This dissertation describes investigation of potentially attenuating mutations in WNV to aid in candidate vaccine development. Single gene mutations were investigated in the envelope (E), NS4B, and NS5 proteins. Additionally, multigenic mutants were investigated that combined mutations in the NS1 protein with mutations in the E or NS4B proteins, as a vaccine with multigenic mutations should have fewer chances of reversion to virulence. The E mutation investigated, E138K, did not have a mouse attenuated phenotype, and virulence was associated with reversion to wild-type genotype. Of seven NS4B mutations investigated, only P54A and P54G mutants were significantly attenuated in mice. While the P54G mutant was genotypically stable, P54A was capable of reversion. In the NS5 protein, two single mutants were studied independently (K61A and E218A) and in combination as a double mutant. The NS5 single mutants were attenuated in mice, however, the double mutant had reduced attenuation associated with reversion at both mutated residues. For the multigenic mutants, strongly attenuating NS1 glycosylation site mutations (NS1mut) were combined with NS4B-C102S, NS4B-W103Y, or E-E138K. Although NS4B-C102S and NS4B-W103Y mutations did not independently attenuate WNV mouse virulence, the multigenic mutants retained the attenuated phenotype of the NS1mut. In comparison, the NS1mut+E-E138K mutant was not attenuated, and instead, the genotype showed evidence of reversion. Overall, NS4B-P54G and NS5-K61A mutations were the most strongly attenuated and stable mutations investigated, and thus, they should be considered during WNV vaccine development. Additionally, the NS1mut glycosylation site mutations should be stabilized with additional amino acid substitutions to continue to use this mutant in vaccine design. Overall, this dissertation describes the attenuating phenotype and stability of mutations in diverse regions of the WNV genome and provides insight into WNV rational vaccine development strategies
Data Mining for Signal Detection of Targeted Therapy Related Drug Toxicity in Breast Cancer Patients
No full textApplication of signal detection methods using claims data can improve post-marketing drug surveillance. The aim of this study is to compare two routinely used approaches, the proportional reporting ratio (PRR) and Gamma Poisson Shrinker (GPS) with a tree-based scan statistic (TBSS). Using data from the Texas Cancer Registry and Surveillance, Epidemiology and End Results linked to Medicare from 2010-2014 we identified 8,949 patients with breast cancer treated with chemotherapy and 2,542 patients treated with trastuzumab in addition to chemotherapy. Inpatient and outpatient visits up to 1 year from start of therapy were used to identify adverse events (AEs). For each method two signaling thresholds were evaluated. Across all methods we found a total of 34 signals associated with use of trastuzumab. Clinical review determined that most identified signals represented known AEs or confounding. GPS on the highest signaling threshold failed to detect a well-established AE when time of follow-up was less than 6 months. Overall there was considerable agreement between methods with GPS being the most conservative. PRR and TBSS may be more appropriate in exploratory drug safety studies using this dataset