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Development of Diagnostic Assays for Melioidosis, Tularemia, Plague And

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Development of Diagnostic Assays for Melioidosis, Tularemia, Plague And University of Nevada, Reno Development of Diagnostic Assays for Melioidosis, Tularemia, Plague and COVID19 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Biology By Derrick Hau Dr. David P. AuCoin / Dissertation Advisor December 2020 THE GRADUATE• SCHOOL We recommend that the dissertation prepared under our supervision by entitled be accepted in partial fulfillment of the requirements for the degree of Advisor Committee Member Committee Member Committee Member Graduate School Representative David W. Zeh, Ph.D., Dean Graduate School December 2020 i Abstract Infectious diseases are caused by pathogenic organisms which can be spread throughout communities by direct and indirect contact. Burkholderia pseudomallei, Francisella tularenisis, and Yersinia pestis are the causative agents of melioidosis, tularemia and plague, respectively. These bacteria pertain to the United States of America Federal Select Agent Program as they are associated with high mortality rates, lack of medical interventions and are potential agents of bioterrorism. The novel coronavirus disease (COVID-19) has resulted in a global pandemic due to the highly infectious nature and elevated virulence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Proper diagnosis of these infections is warranted to administer appropriate medical care and minimize further spreading. Current practices of diagnosing melioidosis, tularemia, plague and COVID-19 are inadequate due to limited resources and the untimely nature of the techniques. Commonly, diagnosing an infectious disease is by the direct detection of the causative agent. Isolation by bacterial culture is the gold standard for melioidosis, tularemia and plague infections; detection of SAR-CoV-2 nucleic acid by real-time polymerase chain reaction (RT-PCR) is the gold standard for diagnosing COVID-19. These techniques, however, can often be time consuming and require laboratory equipment and trained individuals not readily available in low-technology settings. The present dissertation outlines the development of alternative diagnostic assays for melioidosis, tularemia, plague, and COVID-19 as three sections: (I) Diagnostic Target Identification, (II) Immunoassay Development, (III) Evaluation of Immunoassays ii First, the identifications of circulating B. pseudomallei and F. tularensis proteins in clinical specimens were performed using a multi-armed approach to determine putative biomarkers of melioidosis and tularemia. The approach consisted of three methods for identifying circulating bacterial proteins resulting in a comprehensive methodology for determining potential biomarkers. The three methods were (i) In vivo Microbial Antigen Discovery (InMAD), (ii) patient serological markers and (iii) protein profiling by mass spectrometry. Converging results from the three analyses yielded putative targets to evaluate as biomarker of melioidosis or tularemia. The B. pseudomallei target list consisted of seven proteins: BPSS1531, BPSL2298, BPSL1504, BPSS0311, BPSL3092, BPSL1445, and BPSL3319. The F. tularensis target list consisted of five proteins: FTT1357/1712, FTT0308, FTT0928c, FTT0954c and FTT1349/1704. Upon validation, protein targets can be used to develop alternative assays for the diagnosing melioidosis and tularemia. Second, immunoassays were developed for the detection of two Y. pestis proteins suggested as biomarkers of plague: low-calcium response V (LcrV) and capsular fraction-1 (F1). A total of twenty-two high affinity monoclonal antibodies (mAbs) were isolated from BALB/c mice immunized with recombinant LcrV, F1 and LcrV-F1 fusion protein via hybridoma technology. mAbs were characterized by Western blots, enzyme- linked immunosorbent assays (ELISA), and surface plasmon resonance. Antigen- capture ELISAs and lateral flow immunoassays (LFI) were developed using the mAbs and optimized for analytical sensitivity. Prototype LFIs were evaluated to detect LcrV and F1 in surrogate clinical specimens. A multiplexed LFI detecting both LcrV and F1 was assessed against a panel of Y. pestis isolates, clinical near neighbors and other bacterial Select Agents indicating high assay specificity. The immunoassays developed can be iii used to evaluate clinical samples, further determining the diagnostic power of the LcrV and F1 proteins in clinical samples. Third, the capsular polysaccharide (CPS) of B. pseudomallei has been identified as a biomarker of melioidosis. Previous studies indicate CPS is filtered through the kidneys and excreted in urine during an infection. The Active Melioidosis Detect Plus (AMD+TM) is an updated version of the point-of-care, rapid diagnostic tool developed through a collaboration between the Diagnostics Discovery Laboratory and InBios International Inc (Seattle, WA). The AMD+TM LFI was used to evaluate twenty melioidosis urine samples which includes temporal sets collected from two patients receiving treatment. CPS was detected in 80% of the melioidosis urine samples by the AMD+TM LFI. Additionally, three isolates of B. pseudomallei (K96243, 1026b, Bp82) were assessed to determine concentrations of CPS in cultures grown in vitro. Results suggest a large concentration of CPS is produced in vitro, with quantifiable amounts by ELISA within hours of inoculation. This suggests the detection of CPS in bacterial culture may be an alternative method for diagnosing melioidosis with the sensitivity of bacterial culture, and specificity and timeliness of an immunoassay. Lastly, the COVID-19 pandemic has led to over 46 million infections and 1.2 million deaths worldwide. SARS-CoV-2 is highly infectious and more virulent than other known coronaviruses. Diagnoses by RT-PCR and contact tracing have been essential for minimizing the spread of infection, however additional countermeasures including vaccines and therapeutics are warranted. Viral neutralization is associated with blocking the receptor-binding domain of the spike protein (RBD). A cohort study examining antibody titers against RBD in individuals who have recovered from acute COVID-19 iv suggest low and waning titers within months post-recovery. As accounts of reinfections have been documented, low titers may further indicate minimal protective immunity for those who have previously been infected. Additionally, two individuals had 4-fold to 8- fold increases in IgG response ninety days after enrollment and may suggest reinfection. Further evaluation of patient history will elucidate the possibilities of re-exposure and assess IgG response to RBD as a retroactive method of diagnosing COVID-19 reinfections. v Acknowledgements I would like to express my gratitude to my advisor, Dr. David AuCoin. I am grateful for the opportunity to pursue a graduate degree in his laboratory as well as for the mentorship I have acquired over the past several years. I would also like to acknowledge my committee members: Dr. Thomas Kozel, Dr. Cyprian Rossetto, Dr. Paul Brett, and Dr. David Quilici. I am thankful for your knowledge and support throughout the course of this work. I would like to share my appreciate for all past and present members of the Diagnostic Discovery Laboratory and the Molecular Microbiology and Immunology department. I am thankful for everyone’s contributions to my academic growth and for keeping a vibrant work environment. I would like to thank my family and friends for their love and endless support. And lastly, a special shoutout to Spam, the best pup who has stuck by my side throughout the entire process. vi Table of Contents PAGE Abstract...............................................................................................................................i Acknowledgements……….................................................................................................v Table of Contents………………….....................................................................................vi List of Tables..…………...…………………........................................................................ix List of Figures.…………...…………………........................................................................xi Chapter 1: Introduction......................................................................................................1 1.1 Overview 1.2 Melioidosis 1.3 Tularemia 1.4 Plague 1.5 COVID19 Chapter 2: Multi-armed Approach for Identifying Circulating Bacterial Proteins in Melioidosis and Tularemia Patient Samples....................................................................12 2.1 Abstract 2.2 Background 2.3 Methods 2.4 Results vii 2.5 Discussion 2.6 Figures and Tables Chapter 3. Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis………………………….…………………………………………………………………68 3.1 Abstract 3.2 Background 3.3 Methods 3.4 Results 3.5 Discussion 3.6 Figures and Tables Chapter 4. Detection and Quantitation of Capsular Polysaccharide (CPS) in Clinical Melioidosis Samples and Laboratory-grown Burkholderia pseudomallei Isolates……..112 4.1 Abstract 4.2 Introduction 4.3 Materials and Methods 4.4 Results 4.5 Discussion 4.6 Figures and Tables Chapter 5. Temporal Profile of Immunoglobulin G Titers against SARS-CoV-2 RBD in Recovered Patients of Northern Nevada…………………………………………………...135 4.1 Abstract 4.2 Introduction viii 4.3 Methods 4.4 Results 4.5 Discussion 4.6 Figures Chapter 6: Conclusion…………………….............…………………………………………143 Literature Cited……………………………………………………………………………......146 ix List of Tables Chapter 2: Multi-armed Approach
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