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Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase

Zhang, Yu
http://rave.ohiolink.edu/etdc/view?acc_num=osu1388027781

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Food Science and Technology.
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. 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 (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 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.
Jianrong Li (Advisor)
Melvin Pascall (Committee Member)
Stefan Niewiesk (Committee Member)
Tracey Papenfuss (Committee Member)
190 p.
Biochemistry; Biology; Immunology; Virology
human metapneumovirus; hMPV; methyltransferase; MTase; cotton rat; Sigmodon hispidus; vaccine; animal model; large polymerase protein L

Recommended Citations

Citations

  • Zhang, Y. (2014). Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1388027781

    APA Style (7th edition)

  • Zhang, Yu. Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1388027781.

    MLA Style (8th edition)

  • Zhang, Yu. "Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral messenger RNA cap methyltransferase." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1388027781

    Chicago Manual of Style (17th edition)

osu1388027781
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© 2014, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.