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Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral Messenger RNA Cap Methyltransferase

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Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral Messenger RNA Cap Methyltransferase Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yu Zhang The Graduate Program in Food Science and Technology The Ohio State University 2014 Dissertation Committee: Dr. Jianrong Li, advisor Dr. Melvin Pascall Dr. Stefan Niewiesk Dr. Tracey Papenfuss Copyrighted by Yu Zhang 2014 Abstract Human metapneumovirus (hMPV) is a newly discovered paramyxovirus, first identified in 2001 in the Netherlands in infants and children with acute respiratory tract infections. Soon after its discovery, hMPV was recognized as a globally prevalent pathogen. Epidemiological studies suggest that 5 to 15% of all respiratory tract infections in infants and young children are caused by hMPV, a proportion second only to that of human respiratory syncytial virus (hRSV). Despite major efforts, there are no therapeutics or vaccines available for hMPV. In the last decade, approaches to generate vaccines employing viral proteins or inactivated vaccines have failed either due to a lack of immunogenicity or the potential for causing enhanced pulmonary disease upon natural infection with the same virus. In contrast to inactivated vaccines, enhanced lung diseases have not been observed for candidate live attenuated hMPV vaccines. Thus, a living attenuated vaccine is the most promising vaccine candidate for hMPV. However, it has been a challenge to identify an hMPV vaccine strain that has an optimal balance between attenuation and immunogenicity. In addition, hMPV grows poorly in cell culture and the growth is trypsin-dependent. To evaluate the safety and efficacy of a vaccine candidate, a robust small animal model is required. ii To enhance the growth of hMPV, the cleavage site (99RQSR102 motif) of fusion (F) protein was mutated to 99RRRR102 in an infectious clone of hMPV. The resultant recombinant hMPV (rhMPV) displayed trypsin-independent growth phenotype and promoted earlier cytopathic effects (CPE) in cell culture. Interestingly, rhMPV formed clear viral plaques in a number of mammalian cell lines (such as Vero-E6 and LLC-MK2 cells) in an agarose overlay plaque assay, which allows for plaque purification of the viruses. Thus, the trypsin-independent rhMPV is an improved backbone virus for development of live attenuated vaccines. To identify a small animal model for hMPV, we compared the replication and pathogenesis of rhMPV in BALB/c mice, Syrian golden hamsters, and cotton rats. It was found that BALB/c mice are not permissive for rhMPV infection. In hamsters, rhMPV had an efficient replication in nasal turbinate but was restricted in lungs. In contrast, hMPV replicated efficiently in both nasal turbinate and lung when intranasally administered with three doses (104, 105 and 106 PFU) in cotton rats. Lungs of cotton rats infected by rhMPV developed histological changes including interstitial pneumonia, mononuclear cells infiltrates, and increased lumen exudates. Immunohistochemistry examination found that viral antigens were expressed at the luminal surfaces of the bronchial epithelium cells in lungs. Thus, we conclude that that cotton rat is a robust small animal model for rhMPV infection. We hypothesize that viral messenger RNA (mRNA) cap methyltransferase iii (MTase) is a novel target to identify a live attenuated hMPV strain that has a proper balance between attenuation and immunogenicity. The rationale for this hypothesis is that mRNA cap methylation is essential for viral gene expression and, subsequently, viral replication. To test this hypothesis, we performed a mutagenesis analysis in the putative mRNA cap MTase catalytic site and S-adenosyl methionine (SAM) binding site located in the conserved region VI (CR-VI) of large (L) polymerase protein. Alanine substitutions to the amino acid residues involved in MTase catalysis and SAM binding diminished reporter gene expression in a minigenome replication assay. Using a reverse genetic system, we recovered three recombinant hMPVs carrying mutations in the SAM binding site. Trans-methylation assay showed that these rhMPV mutants were specifically defective in ribose 2’-O, but not guanine N-7 (G-N-7) methylation. These MTase-defective rhMPVs showed delayed growth kinetics, reduced viral genome replication and mRNA synthesis, and formed smaller plaques in cell culture compared to wildtype rhMPV. Therefore, rhMPVs lacking 2’-O methylation were highly attenuated in cell culture. To determine whether MTase-defective rhMPVs can be used as live attenuated vaccine candidates, recombinants rhMPV-G1696A, G1700A, and D1755A were inoculated intranasally into cotton rats. It was found that MTase-defective rhMPVs were highly attenuated in viral replication in upper and lower respiratory tracts in cotton rats. In addition, these recombinant viruses caused minimal lung histological changes and viral antigen expression in bronchial epithelium cells. Importantly, cotton iv rats vaccinated with these MTase-defective rhMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with wildtype rhMPV. In conclusion, we found that mRNA cap MTase is a novel target to rationally design live attenuated vaccines for hMPV. These MTase-defective rhMPVs were sufficiently attenuated but retained high immunogenicity in cotton rats. Hence, MTase-defective rhMPVs are excellent vaccine candidates for hMPV. v Dedication This dissertation is dedicated to my parents Pei-Xin Zhang and Yun-Zhu Wang for their love and support. vi Acknowledgement First and foremost, I would like express my deepest gratitude to my advisor Dr. Jianrong Li for his guidance and support during my time in his lab. I am always impressed and inspired by his devotion and enthusiasm to science. Without him, I would not have achieved my dissertation. I also want to thank my committee members Dr. Stefan Niewiesk, Dr. Melvin Pascall, and Dr. Tracey Papenfuss for the mentorship and the intellectual environment they provided. All members in Dr. Li’s group: Dr. Yuanmei Ma, Fangfei Lou, Erin DiCaprio, Dr. Yongwei Wei, Dr. Hui Cai, Brie Coyle, Jing Sun, Xueya Liang, Dr. Xiaodong Zhang, Yue Duan, Dr. Junan Li, Kurtis Feng, Ashley Predmore, Elbashir Araud, Jiawei Yeap, Anastasia Purgianto, Dr. Ran Zhao, Dr. Xiangjie Yao, and Yang Zhu, Rongzhang Wang, and Dr. Xianjun Dai, I really appreciate and thank you all for the help and friendship over the years. I would like to thank all my friends from our collaborate labs, Devra Huey, Dr. Dhohyung Kim, Michelle (Gia) Green, Dr. Miyoung Kim, Yaoling Shu, Dr. Supranee Chaiwatpongsakorn, Dr. Heather Costello, Sara Johnson, Dr. Gabriel Sanglay, Dr. Lizanel Feliciano; thank you for your help all these years. I also would like to thank the entire faculty, staff and students in Department of Food Science. vii Vita June 2002…………………………………… Huzhou High School June 2006…………………………………… B.S. Biotechnology, East China University of Science and Technology, Shanghai, China September 2008 –present…………………… Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Publications Zhang Y, Li J, Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase (Manuscript in preparation) Wei Y, Zhang Y, Cai H, Mirza MA, Iorio MR, Peeples EM, Niewiesk S, Li J, Roles of the putative integrin-binding motif of the human metapneumovirus fusion (F) protein in viii cell-cell fusion, viral infectivity, and pathogenesis, J Viro., Manuscript Submitted in November, 2013 Kim MY, Ma Y, Zhang Y, Li J, Shu Y, Oglesbee M., hsp70-dependent antiviral immunity against cytopathic neuronal infection by vesicular stomatitis virus. J Virol. 2013 Oct;87(19):10668-78 Ma Y, Wei Y, Zhang X, Zhang Y, Cai H, Zhu Y, Shilo K, Oglesbee M, Krakowka S, Whelan S, Li J, Messenger RNA cap methylation influences pathogenesis of vesicular stomatitis virus in vivo. Manuscript accepted in December, 2013 Zhang Y, Wei Y, Li J, Li J, Development and optimization of a direct plaque assay for human and avian metapneumoviruses, J Virol Methods. 2012 Oct;185(1):61-8. PATENTS Jianrong Li, Yongwei Wei, Yu Zhang. Novel live attenuated vaccines for human metapneumovirus. Patent filed Jun 2012 Jianrong Li, Yuanmei MA, Yu Zhang. World Patent Application WO 2012170997 A1, Paramyxovirus immunogens and related materials and methods, Patent publication date: Dec 13, 2012 BOOK CHAPTERS Li J, Zhang Y. Messenger RNA Cap Methylation in Vesicular Stomatitis Virus, a Prototype of Non‐Segmented Negative‐Sense RNA Virus, Methylation - From DNA, RNA and Histones to Diseases and Treatment, Prof. Anica Dricu (Ed.), ISBN: 978-953-51-0881-8, InTech, DOI: 10.5772/54598. November 28, 2012 ix Fields of Study Major Field: Food Science and Technology Minor Field: Molecular Virology x Table of Contents Abstract……………… ..................................................................................................ii Dedication…………… ................................................................................................. xi Acknowledgement .......................................................................................................vii Vita……………….. ................................................................................................... viii List of Tables……………… ......................................................................................
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