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Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.

ABSTRACT: BACKGROUND: Respiratory Syncytial Virus (RSV) is an important human respiratory pathogen, particularly of infants and older adults, and despite several decades of research and development, no licensed vaccine is available. Studies have confirmed that enhancement of RSV disease does not occur after inoculation with RSV live-attenuated vaccine candidates, making such vaccines preferable. In this paper, reverse genetics was used to construct two recombinant viruses, a recombinant Long strain (rLong) and rLong-?G-EGFP; rLong-?G-EGFP is a recombinant mutant in which G was replaced with the EGFP gene, based on the Long strain of RSV. RESULTS: Both rLong and rLong-?G-EGFP were constructed successfully and recovered in Hep-2 cells, and autofluorescence was observed in rLong-?G-EGFP-infected cells during consecutive passages. Titers of rLong and rLong-?G-EGFP were ~100-fold lower than the parental strain. Although virulence was attenuated, high titers of neutralizing antibodies were induced in BALB/c mice after being inoculated with recombinant viruses in a three-dose schedule. Unexpectedly, the neutralizing antibody titer in rLong-?G-EGFP-immunized recipients did not decline significantly compared with the rLong strain. Protective efficacy of recombinant viruses in lung tissue was up to 100%, and the serum neutralizing antibody levels could stabilize at 21 days with no significant fall post-challenge. Enzyme-linked immunospot (ELISPOT) assays showed that both recombinant viruses were capable of inducing CD8+ T cell immune responses, which are crucial for virus clearance, and that rLong stimulated a higher level of IFN-? production by comparison. In terms of inducing a balanced immune response, rLong-?G-EGFP elicited slightly higher levels of IgG2a antibodies and lower levels of IgG1/IgG2a than the rLong virus. CONCLUSIONS: This study suggested that immunization with rLong and rLong-?G-EGFP were immunogenic and protected against RSV infection in the lower respiratory tract of BALB/c mice better than in the nose. Because of a relative low IgG1/IgG2a ratio, rLong-?G-EGFP was more inclined to make CD4+ T cells, shifting toward a Th1-type response, indicating that the generation of a more balanced Th1/Th2 response was desirable. This explorative study on the recombinant Long viruses also contributed to obtaining more RSV attenuated candidates by a reverse genetics approach.

SUBMITTER: Hu B

PROVIDER: S-EPMC4254404 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.

Hu Bing B Jiang Jiawei J Zhan Jianbo J Li Guoming G Jiang Yongzhong Y Guan Xuhua X Chen Yuanding Y Fang Zhizheng Z

Virology journal 20140808

<h4>Background</h4>Respiratory Syncytial Virus (RSV) is an important human respiratory pathogen, particularly of infants and older adults, and despite several decades of research and development, no licensed vaccine is available. Studies have confirmed that enhancement of RSV disease does not occur after inoculation with RSV live-attenuated vaccine candidates, making such vaccines preferable. In this paper, reverse genetics was used to construct two recombinant viruses, a recombinant Long strain ...[more]

PMID: 25107552

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Project description:Human respiratory syncytial virus (RSV) is the most important viral cause of serious pediatric respiratory illness worldwide. Currently, the most promising live-attenuated vaccine candidate is a temperature-sensitive (ts) cDNA-derived virus named rA2cp248/404/1030ΔSH, in reference to its set of attenuating mutations. In a previous clinical study, more than one-third of postvaccination nasal wash isolates exhibited partial loss of the ts phenotype. Most of this instability appeared to be due to reversion at a missense point mutation called 1030. This 1030 mutation is a single-nucleotide tyrosine-to-asparagine substitution at position 1321 (Y1321N) of the polymerase L protein that contributes to the ts and attenuation phenotypes of the vaccine candidate. The goals of the present study were to identify a reversion-resistant codon at position 1321 conferring a comparable level of attenuation and to use this to develop a genetically stable version of the vaccine virus. We modified wild-type (wt) RSV to insert each of the 20 possible amino acids at position 1321; 19 viruses were recoverable. We also investigated small deletions at or near this position, but these viruses were not recoverable. Phenotypic analysis identified alternative attenuating amino acids for position 1321. Several of these amino acids were predicted, based on the genetic code, to be refractory to deattenuation. Classical genetics, using temperature stress tests in vitro combined with nucleotide sequencing, confirmed this stability but identified a second site with a compensatory mutation at position 1313. It was possible to stabilize the 1313 site as well, providing a stable 1030 mutation. Further stress tests identified additional incidental mutations, but these did not reverse the ts/attenuation phenotype. An improved version of the vaccine candidate virus was constructed and validated in vitro by temperature stress tests and in vivo by evaluation of attenuation in seronegative chimpanzees. In addition to developing an improved version of this promising live-attenuated RSV vaccine candidate, this study demonstrated the propensity of an RNA virus to escape from attenuation but also showed that, through systematic analysis, genetics can be used to cut off the routes of escape.

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Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.

Publications

Development of a reverse genetics system for respiratory syncytial virus long strain and an immunogenicity study of the recombinant virus.

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