|Heers Dack, Kristen|
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2004
Publication Date: 6/1/2004
Citation: Elliott, M.B., Pryharski, K.S., Yu, Q., Parks, C.L., Laughlin, T.S., Gupta, K., Lerch, R.A., Randolph, V.B., Lapierre, N.A., Heers Dack, K.M., Hancock, G.E. 2004. Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro. Journal of Virology. 2004 Jun:78(11):5773-83. Interpretive Summary: Human respiratory syncytial virus (RSV) is a leading viral cause of severe lower respiratory tract disease, including bronchiolitis and pneumonia, in infants and young children worldwide. It causes annual epidemics and is responsible for about 100,000 hospitalizations and 4,500 deaths among infants and young children in the United States per year. In addition, RSV infection can cause severe respiratory illness in elderly and immunodeficient adults. At present there are no licensed vaccines and effective drugs available for routine use to prevent the disease caused by RSV. Therefore, there is a great need for vaccine development to prevent the disease. On the surface of RSV, there are two major glycoproteins, F and G, which are thought to play important roles in stimulating human immuno-response. Recently it was shown that RSV completely deleted of G protein do not grow very well in animal models and human, implying the existence of an attenuation site in G protein that may be manipulated for vaccine development. In the study, we sought to identify this site. By using a genetic engineering technology, we constructed several recombinant RSVs that have G protein partially deleted in the C-terminal regions. We demonstrate for the first time that the C-terminal domain of G protein is required for efficient replication in both mouse and human cells. Furthermore, the data suggest that the C terminal region plays a critical role in maturation of RSV from the cell. Importantly, truncation at the C-terminus did not appear to impair the immunogenic properties of G protein. This new information is important for design of future attenuated vaccines for RSV.
Technical Abstract: The design of live, attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. The prototype vaccines were not suitable for newborn infants primarily because of insufficient attenuation. However, genetic instability and reversion to a less attenuated phenotype also caused concern. In this study we sought to construct novel mutants through genetic mutation of the attachment G protein. Complete deletion of G protein previously yielded RSV strains that were attenuated for replication in vivo. Using reverse genetics, recombinant (r) RSV strains were engineered with truncations from the C-terminus of G protein. The resultant rRSV strains were designated rA2dG118, rA2cpdG174, rA2cpdG193, and rA2cpdG213 with truncations at amino acids 118, 174, 193, and 213 respectively. All rA2cpdG strains were attenuated for growth in vitro and in the respiratory tract of BALB/c mice, but not restricted for growth at 370C. None of the mutations significantly affected nascent rRSV strain genome synthesis in human lung epithelial (A549) cell lines, but infectious virus titers in the culture medium were dramatically diminished. Hence, the data suggest that the C-terminal 85 amino acids of G protein contain a site important for efficient genome packaging or virus budding from the infected cell. When used to immunize mice, the rA2cpdG strains generated immune responses that were efficacious and similar to those elicited by the temperature sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that rRSV strains with truncated G protein are immunogenic, less likely to revert to less attenuated phenotype, and thus potential candidates for development of attenuated vaccines against RSV.