u Ottawa L'Universite canadierme Canada's university ITTTT FACULTE DES ETUDES SUPERIEURES 1^=1 FACULTY OF GRADUATE AND ET POSTOCTORALES U Ottawa POSDOCTORAL STUDIES L'UmversitC canadierino Canada's university Karen Rowlandson "A0tIu"RDElATHESE7AUtWRO?"THl¥S~ Ph.D. (Microbiology and Immunology) GRADE/DEGREE Department of Biochemistry, Microbiology and Immunology PAC.QJE^ Production and Evaluation of Plant-Derived Vaccines for Cytomegalovirus Using Guinea Pig as an Animal Model TITRE DE LA THESE / TITLE OF THESIS Eilleen Tackaberry DIRECTEUR (DIRECTRICE) DE LA THESE / THESIS SUPERVISOR CO-DIRECTEUR (CO-DIRECTRICE) DE LA THESE/THESIS CO-SUPERVISOR EXAMINATEURS (EXAMINATRICES) DE LA THESE/THESIS EXAMINERS Wangxue Chen (National Research Council) Lionel Filion Sean Li Martin Pelchat Gary W. Slater Le Doyen de la Faculte des etudes superieures et postdoctorales / Dean of the Faculty of Graduate and Postdoctoral Studies Production and evaluation of plant-derived vaccines for cytomegalovirus using guinea pig as an animal model Karen Rowlandson Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the PhD degree in Microbiology & Immunology Department of Biochemistry, Microbiology & Immunology Faculty of Medicine University of Ottawa © Karen Rowlandson, Ottawa, Canada, 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre r6f6rence ISBN: 978-0-494-61256-9 Our file Notre reference ISBN: 978-0-494-61256-9 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lntemet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada Abstract Human cytomegalovirus (HCMV) infection is usually asymptomatic in healthy individuals. However, severe disease and/or death can occur in immunocompromised patients. The overall objective of this project was to develop a plant-derived CMV subunit vaccine and determine its effectiveness. Since CMV is species-specific, guinea pig CMV (GPCMV) was used as a model for HCMV. Four gene constructs were engineered to contain either the rice glutelin-1 promoter (Gtl) or the maize ubiquitin promoter, and the gene for either glycoprotein B (gB) or phosphoprotein 65 (pp65) of GPCMV. Agrobacterium tumefaciens were transformed with the Gtl-gB construct and subsequently used to transform Oryza sativa (rice) or Arabidopsis thaliana. Due to technical difficulties associated with rice transformations, experimental work was continued with only A. thaliana. PCR analysis of plant tissue confirmed maintenance of the gB transgene for seven generations, and Western blots of selected seed extracts revealed a gB-specific band not found in non-transformed A. thaliana extracts. Guinea pigs (4 per group) were subcutaneously immunized with 20 mg of seed protein extract from GPCMV gB A. thaliana plants (estimated to contain approximately 50 ug of gB), an equal amount of total protein from non-transformed A. thaliana seeds (negative control), or baculovirus-derived GPCMV gB (positive control). Two animals immunized with seed-derived gB demonstrated GPCMV-specific antibody responses when analysed by ELIS A. Western blot analysis confirmed that this response was specific for GPCMV gB. Neutralization assays were performed using serum obtained 14 days after the last immunization. Three of the 4 animals immunized with the gB seed extract showed viral neutralizing responses, with titres ranging from 40 to 3240. These results indicate that GPCMV gB expressed in the seeds of A. i thaliana can generate a GPCMV-specific immune response. Based on data from GPCMV- specific ELISAs, Western blots, and viral neutralization assays using serum from immunized animals, I concluded that gB was authentically expressed in A. thaliana seeds with at least some epitopes similar to those found on native GPCMV gB that were able to neutralize viral infectivity. ii Acknowledgments I first wish to thank Dr. Eilleen Tackaberry for providing me with support but at the same time allowing me the independence to grow as a graduate student, for encouraging me when the results did not, and for giving me a project that kept me on my toes over the years. Many thanks to my thesis advisory committee, Dr. Bill Casley, Dr. Ken Dimock, Dr. Anil Dudani, Dr. Hans Schernthaner, and Dr. Kathie Wright for their help and encouragement throughout the many years. I am also indebted to my lab mates for their help over the years, even after they left lab! Special thanks to Diana George, whose help was unwavering, even on days when it only involved watering plant after plant. Many thanks to everyone within the Centre of Biologies Research at Health Canada, who graciously helped me throughout my years as a student. I would also like to thank Health Canada for financial support. Thanks to the laboratory of Dr. Mark Schleiss, who made me feel welcome in Minneapolis and for supplying me with a tremendous amount of help. I want to thank my family for all of their encouragement and support over the years. In particular to my sister Tracy, who kept me "company" during my travels. Thank-you Kelly and Matt for your friendship and support throughout this project. I wish to also thank Mr. Savage, my high school math teacher, who engrained in me the notion of "intestinal fortitude". Finally, but not least, I wish to thank Errol, the second half of team Karen/Errol. This thesis would not exist without his help, insight, patience, and support. iii Table of Contents Abstract i Acknowledgements iii Table of Contents iv List of Abbreviations x List of Figures xii List of Tables xiv 1.0 Introduction 1 1.1 General overview of project 1 1.2 Human cytomegalovirus 1 1.2.1 A major health concern 1 1.2.2 Molecular biology 2 1.2.3 HCMV strains 3 1.2.4 Viral replication, life cycle, and expression of proteins 4 1.2.5 CMV latency and immune evasion 7 1.2.6 Transmission of HCMV 9 1.2.7 HCMV in vulnerable populations 10 1.3 Treatments for HCMV 13 1.3.1 Antiviral drugs 13 1.3.2 Immunoprophylaxis 15 1.3.3 Live viral vaccines 16 1.3.4 Dense bodies as vaccines 20 1.3.5 Subunit vaccines 21 1.3.5.1 Purified recombinant gB 22 1.3.5.2 Viral vectors 24 1.3.5.1 DNAplasmids 26 1.4 Animal models of CMV 28 1.4.1 Primate models 29 1.4.2 Guinea pig model 30 iv 1.5 Plants as expression systems 32 1.5.1 Why use plants? 33 1.5.2 Expression of therapeutic proteins in plants 34 1.5.3 Expression of subunit vaccines in plants 35 1.5.4 Plant-derived vaccines for mucosal immunization 36 1.5.5 Challenges with plant-derived vaccines 38 2.0 Rationale, Hypotheses, and Objectives 42 2.1 Rationale 42 2.2 Hypotheses 42 2.3 Objectives 42 3.0 Materials and Methods 44 3.1 Engineering of gene constructs 44 3.1.1 Plasmids 44 3.1.2 Polymerase chain reaction 44 3.1.3 Quantification and purification of DNA by agarose gel electrophoresis 47 3.1.4 Ligation reactions 47 3.1.5 Bacterial transformations 47 3.1.6 Extraction of plasmid DNA from bacterial cultures 48 3.1.7 Restriction enzyme digests 49 3.1.8 Enzymatic generation of blunt ends 49 3.2 Transformation of Agrobacterium tumefaciens 49 3.3 Transformation, selection, and regeneration of rice 51 3.4 Transformation, selection, and regeneration of Arabidopsis thaliana 51 3.4.1 General cultivation of A thaliana 51 3.4.2 Nomenclature for generations of transformed A. thaliana 52 3.4.3 Floral dip method of transformation 52 3.4.4 Selection of transformed seeds by antibiotic resistance 54 3.4.5 Regeneration of antibiotic-resistant plantlets 54 3.5 Testing for expression of P-glucuronidase (GUS) in transformed tissues 55 3.6 Screening for presence of transgene in transformed plants 55 v 3.6.1 Extraction and quantitation of plant genomic DNA 55 3.6.2 Screening for the presence of the transgene by PCR 56 3.7 Protein extraction from seeds 56 3.8 Quantitation of proteins 57 3.9 Enzyme-linked immunosorbant assay (ELISA) 57 3.10 Western blots 57 3.11 Immunization of guinea pigs 58 3.11.1 Experimental animals 58 3.11.2 Immunizations 59 3.11.3 Collection of serum 59 3.12 Neutralization of GPCMV infectivity 59 3.12.1 Culturing of guinea pig lung fibroblasts 59 3.12.2 Generation of viral stock 60 3.12.3 Determination of viral titre 60 3.12.4 Viral neutralization assay 61 4.0 Results 63 4.1 Engineering of plants expression vectors 63 4.1.1 Construction of pCAMBIA1301/Gtl/ss/gB/NOS 63 4.1.2 Construction of pCAMBIA1301/Gtl/pp65/NOS 68 4.1.3 Construction of pCAMBIA1301/Ubi/gB/NOS 72 4.1.4 Construction of pCAMBIA1301/Ubi/pp65/NOS 76 4.2 Screening of transformed A.