Selection, Expression and Vaccination with Recombinant Rhodnius
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SELECTION, EXPRESSION AND VACCINATION WITH RECOMBINANT RHODNIUS PROLIXUS ANTIGENS by ALICE CHARLOTTE SUTCLIFFE (Under the Direction of Donald E. Champagne) ABSTRACT Triatomine (Hemiptera:Reduviidae) control is an important aspect of reducing the spread of Trypanosma cruzi, the causative agent of Chagas disease, for which no vaccine currently exists. The development of a triatomine vaccine would increase the number of prevention and control methods available. Here, I describe the selection of twelve Rhodnius prolixus proteins, their recombinant expression and use in vaccine trials in mice as well as the investigation of function of one of these protein targets. Overall, my results do not establish biological function for this protein target and they indicate that induction of an antibody responses can occur in mice vaccinated with recombinant R prolixus proteins but this was not a sufficient protective response. INDEX WORDS: Rhodnius prolixus, ectoparasite vaccine, complement inhibitor, recombinant protein SELECTION, EXPRESSION AND VACCINATION WITH RECOMBINANT RHODNIUS PROLIXUS ANTIGENS by ALICE CHARLOTTE SUTCLIFFE B.Sc, University of Guelph, 2007 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2016 © 2016 Alice Charlotte Sutcliffe All Rights Reserved SELECTION, EXPRESSION AND VACCINATION WITH RECOMBINANT RHODNIUS PROLIXUS ANTIGENS by ALICE CHARLOTTE SUTCLIFFE Major Professor: Donald E. Champagne Committee: Mark R. Brown Rick L. Tarleton Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia May 2016 DEDICATION For my dad iv ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Donald Champagne and the rest of my committee members, Dr. Mark Brown and Dr. Rick Tarleton for providing me with guidance throughout this process. Thank you to Dr. Ellen Dotson, Dr. Robert Wirtz, Dr. Bill Hawley and Paul Howell for supporting my research and thank you to past and present members of the Tarleton Lab: Phil Yao, Gretchen Cooley and Britton Davis for all of your help and expertise. Thank you to my friends and CDC lab mates, especially Dan, Gena and Jessica for all of your advice and encouragement. Thank you doesn’t begin to describe my gratitude towards my mom, dad, Russell, Zev and Julia. You all kept me going in your own ways, no matter what, and understood the time that I needed to dedicate to this. For that, I am eternally appreciative. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION .........................................................................................................1 General Introduction ................................................................................................1 Transmission of Chagas Disease .............................................................................1 Disease Course, Symptoms and Treatment .............................................................2 Ecology ....................................................................................................................3 Control and Prevention ............................................................................................5 Vaccines against Vectors .........................................................................................7 Statement of the Problem .........................................................................................7 Research Goals.........................................................................................................8 2 LITERATURE REVIEW: ARTHROPOD ECTOPARASITE VACCINES ..............10 Introduction ............................................................................................................10 Ectoparasite Vaccines ............................................................................................14 Vaccine Targets .....................................................................................................18 Pan-Arthropod Vaccines ........................................................................................29 Conclusions ............................................................................................................32 vi 3 SELECTION, EXPRESSION AND EVALUATION OF VACCINE TARGETS IN RHODNIUS PROLIXUS ..............................................................................................34 Introduction ............................................................................................................34 Objectives ..............................................................................................................38 Materials and Methods ...........................................................................................38 Results and Discussion ..........................................................................................57 Conclusions ...........................................................................................................69 4 INVESTIGATION OF VACCINE TARGET RPRC010285 FROM RHODNIUS PROLIXUS ...................................................................................................................87 Introduction ............................................................................................................87 Objectives ..............................................................................................................90 Materials and Methods ...........................................................................................90 Results and Discussion ..........................................................................................98 Conclusions .........................................................................................................102 REFERENCES ................................................................................................................113 APPENDIX I ...................................................................................................................137 vii LIST OF TABLES Page Table 3.1: Summary of primers used, expected PCR product length and theoretical protein product size ........................................................................................................................73 Table 3.2: Summary of selected protein targets.............................................................................74 Table 3.3: Summary of VectorBase tBLASTn results...................................................................75 Table 3.4: Summary of Rhodnius prolixus feed data over three feeding trials..............................76 Table 3.5: Hazard ratio estimates from Cox proportional hazard analysis ....................................77 Table 4.1: Plate layout summary of complement inhibition assays conducted ...........................105 Table 4.2: Summary of Rhodnius prolixus double-stranded RNA (dsRNA) feed data over three trials ..............................................................................................................................106 Table 4.3: Hazard ratio estimates from Cox proportional hazard analysis ..................................107 viii LIST OF FIGURES Page Figure 3.1: Serine protease amino acid comparison ......................................................................78 Figure 3.2: Initial PCR amplification of RPRC010285 and RPRC002329 ...................................79 Figure 3.3: SDS-PAGE analysis of purified protein expressed by Escherichia coli BL21(DE3)pLysS ..............................................................................................................80 Figure 3.4: Bioplex results of mice vaccinated with recombinant proteins .................................81 Figure 3.5: Kaplan-Meier one-minus survival curves of Rhodnius prolixus N1s fed on mice vaccinated with RPTMP04372, RPTMP12451 and RPRC010285 and naïve mice ..........83 Figure 3.6: Western blot of Rhodnius prolixus anterior midgut tissues exposed to serum from group 2 vaccinated mice ....................................................................................................84 Figure 3.7: Immunohistochemistry micrographs of Rhodnius prolixus anterior midgut tissues ...85 Figure 3.8: Flowchart summarizing the methods and approach used in Chapter 3 .......................86 Figure 4.1: Kaplan-Meier one-minus survival curves comparing cumulative molting ...............108 Figure 4.2: Gel containing dsRNA extracted from Escherichia coli HT115(DE3) culture ........110 Figure 4.3: Percent inhibition ±SD from complement inhibition assays .....................................111 ix CHAPTER 1 INTRODUCTION General Introduction Chagas disease (American trypanosomiasis), caused by the flagellate protozoan parasite, Trypanosoma cruzi, is transmitted by infected triatomine (Hemiptera: Reduviidae) bugs to humans and animals, mostly in Latin America. Trypanosoma cruzi is estimated to infect 6-7 million people worldwide (WHO 2015) and up to 90 million people in endemic regions are at risk (Coura 2007). The annual global burden is estimated to be