AlShaikhahmed, K (2015) Developing Virus-Like Particles (VLPs) and Heterologous VLPs Vaccines for Epizootic Hemorrhagic Disease Virus (EHDV) Serotypes. PhD thesis, London School of Hygiene & Tropical Medicine. DOI: https://doi.org/10.17037/PUBS.02172948 Downloaded from: http://researchonline.lshtm.ac.uk/2172948/ DOI: 10.17037/PUBS.02172948 Usage Guidelines Please refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alterna- tively contact [email protected]. Available under license: http://creativecommons.org/licenses/by-nc-nd/2.5/ Developing Virus-Like Particles (VLPs) and Heterologous VLPs Vaccines for Epizootic Hemorrhagic Disease Virus (EHDV) Serotypes Kinda Alshaikhahmed Thesis submitted in accordance with the requirements for the Degree of Doctor of Philosophy University of London April 2015 Department of Pathogen Molecular Biology Faculty of Infectious and Tropical Disease LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE Funded by Boehringer Ingelheim and Private funding 1 DECLARATION OF OWN WORK All students are required to complete the following declaration when submitting their thesis. A shortened version of the School’s definition of Plagiarism and Cheating is as follows (the full definition is given in the Research Degrees Handbook): “Plagiarism is the act of presenting the ideas or discoveries of another as one’s own. To copy sentences, phrases or even striking expressions without acknowledgement in a manner which may deceive the reader as to the source is plagiarism. Where such copying or close paraphrase has occurred the mere mention of the source in a biography will not be deemed sufficient acknowledgement; in each instance, it must be referred specifically to its source. Verbatim quotations must be directly acknowledged, either in inverted commas or by indenting” (University of Kent). Plagiarism may include collusion with another student, or the unacknowledged use of a fellow student's work with or without their knowledge and consent. Similarly, the direct copying by students of their own original writings qualifies as plagiarism if the fact that the work has been or is to be presented elsewhere is not clearly stated. Cheating is similar to plagiarism, but more serious. Cheating means submitting another student's work, knowledge or ideas, while pretending that they are your own, for formal assessment or evaluation. Supervisors should be consulted if there are any doubts about what is permissible. DECLARATION BY CANDIDATE I have read and understood the School’s definition of plagiarism and cheating given in the Research Degrees Handbook. I declare that this thesis is my own work, and that I have acknowledged all results and quotations from the published or unpublished work of other people. I have read and understood the School’s definition and policy on the use of third parties (either paid or unpaid) who have contributed to the preparation of this thesis by providing copy editing and, or, proof reading services. I declare that no changes to the intellectual content or substance of this thesis were made as a result of this advice, and, that I have fully acknowledged all such contributions. I have exercised reasonable care to ensure that the work is original and does not to the best of my knowledge break any UK law or infringe any third party’s copyright or other intellectual property right. To be completed by the candidate NAME IN FULL (Block Capitals): KINDA ALSHAIKHAHMED STUDENT ID NO: LSH236219 SIGNED: Kinda AlShaikhahmed DATE: 29/04/2015 Registry Last updated – 04/07/13 2 Abstract Epizootic Hemorrhagic Disease Virus (EHDV) is an insect-transmitted pathogen of ruminants, causing periodic and significant losses in wild and captive deer populations and less frequently, a bluetongue-like disease in cattle. The serogroup of EHDV within the Orbivirus genus of the Reoviridae family consists of seven serotypes, in which emerging serotypes pose an increasing risk either regionally or globally, due to the insect vectors. To date, no vaccine against EHDV is commercially available, apart from the live-attenuated vaccine for EHDV-2 (IBAV). In this study, Virus-Like Particles (VLPs) of EHDV-1 and heterologous VLPs of EHDV-2 were generated using baculovirus multigene expression system for the synthesis of the two outer and two inner capsid proteins, essential for the formation of VLPs. The assembly of EHDV-1 recombinant structural proteins into Core-Like particles (CLPs, two proteins) and (VLPs, four proteins) was confirmed by EM analysis. The biological activity of the raised antisera to neutralise EHDV-1 was efficiently confirmed by neutralisation assay at 1:64 dilution. Cross neutralising activities were also detected against EHDV-2 and EHDV-6 serotypes at 1:8 dilution. Results presented in this study validate the potential efficacy of the VLP as a neutralising vaccine and strongly suggest its use as vaccine candidate. Additionally, an alternative approach was also initiated in this research to develop a rational vaccine against EHDV-2 using the reverse genetics system (RG). Towards this, it was first established that in vitro synthesised transcripts from purified EHDV-2 cores could generate infectious virus upon cell transfection. Note that both the generation of core transcripts and recovery of infectious virus of EHDV were not demonstrated previously. Subsequently a complete set of 10 T7 transcripts was 3 synthesised, however, it was not possible to recover any infectious virus, likely to be some unwarranted mutations. Nevertheless, these transcripts will be further investigated for future RG studies. 4 Table of contents Declaration of own work ………………………………………………………………… 2 Abstract ……………………………………………………………………………………. 3 Table of contents …………………………………………………………………………. 5 Acknowledgments ……………………………………………………………………….. 10 Abbreviations ……………………………………………………………………………... 11 List of tables and figures ……………………………………………………………….. 15 1. CHAPTER 1 ……………………………………………………………………….. 21 1.1. Reoviridae Family ………………………………………………………………... 22 1.2. Orbiviruse s ………………………………………………………………………... 25 1.3. Epizootic Hemorrhagic Disease Virus ……………………………………….. 27 1.4. Epidemiology and susceptible hosts ………………………………………... 28 1.5. Pathogenicit y …………………………………………………………………….. 31 1.6. Econ omic impact ………………………………………………………………… 34 1.7. Monitoring and control measures ……………………………………………. 35 1.7.1. Vaccination …………………………………………………………… 36 1.8. Virion morphology ………………………………………………………………. 37 1.9. The outer capsid proteins ……………………………………………………… 40 1.10. The inner core proteins …………………………………………………………. 41 1.11. Minor proteins and genome structure ……………………………………….. 43 1.12. Comparison of expression systems …………………………………………. 45 1.13. Baculovirus expression technology and its modifications ……………… 46 1.14. Baculovirus expression vector system for the production of VLPs …… 47 1.15. Immunological value of VLPs …………………………………………………. 49 1.16. BTV and other successful VLPs vaccines ………………………………….. 50 1.17. Aims of the studies ……………………………………………………………… 53 2. CHAPTER 2 ……………………………………………………………………….. 55 2.1. Baculovirus expression system ………………………………………………. 57 2.1.1. Polymerase Chain Reaction (PCR) amplification ………………... 57 2.1.2. The preparation of plasmid and Bacmid DNA ……………………. 60 2.1.3. Construction of transfer vectors for baculovirus system ………… 61 2.1.4. Preparation of chemically competent E.coli cells ………………… 66 2.1.5. Transformation of competent E.coli cells …………………………. 67 2.1.6. Electrocompetent E.coli cells ………………………………………. 68 2.1.7. Lambda red recombination and Cre recombination in E.coli …… 68 5 2.1.8. Insect cell culture …………………………………………………..... 69 2.1.9. Generation of single and multiple recombinant proteins in AcMNPV ……………………………………………………………… 70 2.1.10. Isolation and propagation of recombinant baculoviruses ……….. 71 2.1.11. Confirmation of the expression of recombinant proteins ………... 73 2.1.11.1. SDS-PAGE …………………………………………………………… 73 2.1.11.2. Western blotting ……………………………………………………… 74 2.1.12. Purification of the recombinants expressing EHDV-1 CLP ……... 75 2.1.13. Purification of EHDV-1 VLPs and heterologous VLPs of EHDV-2 ........................................................................................ 76 2.1.14. Antibodies production ……………………………………………….. 77 2.1.15. Electron Microscopy ………………………………………………… 78 2.1.16. Neutralisation assay ………………………………………………… 78 2.2. Reverse Genetics (RG) System ……………………………………………….. 79 2.2.1. Mammalian cell line ………………………………………………..... 79 2.2.2. EHDV purification, propagation and titration ……………………… 80 2.2.3.2 Purification of dsRNAs from EHDV-2 infected BSR cells ……….. 81 2.2.4. Reverse transcription-PCR (RT-PCR) ……………………………. 82 2.2.5. Construction of cDNA T7 plasmid clones ………………………… 83 2.2.6. Synthesis of EHDV-2 transcripts from cDNA plasmid clones …... 84 2.2.7. Denaturing agarose gel electrophoresis ………………………….. 85 2.2.8. Purification of EHDV-2 virus cores ………………………………… 86 2.2.9. In vitro synthesis and purification of core derived EHDV-2 mRNA ........................................................................................... 87 2.2.10. Recovery of infectious EHDV-2 by transfection of BSR cells with the core-derived or the complete set of synthetic T7 transcripts .. 88 3. CHAPTER 3 ……………………………………………………………………….. 90 3.1. In troduction ………………………………………………………………………. 91 3.2. Results …………………………………………………………………………….. 92 3.2.1. Cloning and expression of EHDV-1 structural proteins …………. 92 3.2.1.1. Cloning of EHDV-1 S2 into pAcYM1 …………………………….... 92 3.2.1.2. Expression of EHDV-1 VP2 recombinant