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Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus

Generation and growth kinetics of SARS-CoV mutants with deletions in both nsp1 and E genes.(A) SARS-CoV genome is shown in the top, and the expanded region shows the nsp1 and E genes. White boxes represent the amino acids stretches deleted in both proteins in each virus. (B) Mutant viruses growth kinetics. Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, SARS-CoV-nsp1ΔD-ΔE and SARS-CoV-nsp1ΔD-EΔ3 at passage 1 and 10 (-p1 and -p10C) viruses at a moi of 0.001. At different times post infection, virus titers were determined by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean using data from three independent experiments.
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ppat.1005215.g013: Generation and growth kinetics of SARS-CoV mutants with deletions in both nsp1 and E genes.(A) SARS-CoV genome is shown in the top, and the expanded region shows the nsp1 and E genes. White boxes represent the amino acids stretches deleted in both proteins in each virus. (B) Mutant viruses growth kinetics. Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, SARS-CoV-nsp1ΔD-ΔE and SARS-CoV-nsp1ΔD-EΔ3 at passage 1 and 10 (-p1 and -p10C) viruses at a moi of 0.001. At different times post infection, virus titers were determined by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean using data from three independent experiments.

Mentions: In order to develop a safe vaccine candidate, mutant viruses with deletions in both nsp1 and E genes were engineered. A rSARS-CoV deleted in the nsp1 D domain and the E protein (SARS-CoV-nsp1ΔD-ΔE), and a second mutant virus with deletions of the nsp1 D domain coupled with a small deletion (E∆3) (Fig 4A) in the E protein (SARS-CoV-nsp1ΔD-EΔ3) were generated. EΔ3 deletion mutant was selected for further studies because this deletion led to a virus that grew to titers similar or higher than the SARS-CoV-∆E, in cell culture or in mice, respectively (Fig 12A and 12B). More importantly, the E∆3 virus was genetically stable after 10 passages in cell culture [53] or in vivo, maintaining its attenuated phenotype (Fig 12C), in contrast to the ∆E virus (Figs 1 and 5). This deletion was combined with another one in SARS-CoV nsp1 protein (nsp1ΔD), which was fully attenuating. The resulting virus grew to relatively high titers in vivo (Figs 9 and 13A). Viruses were rescued in Vero E6 cells, cloned and sequenced to confirm the presence of the desired mutations.


Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Generation and growth kinetics of SARS-CoV mutants with deletions in both nsp1 and E genes.(A) SARS-CoV genome is shown in the top, and the expanded region shows the nsp1 and E genes. White boxes represent the amino acids stretches deleted in both proteins in each virus. (B) Mutant viruses growth kinetics. Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, SARS-CoV-nsp1ΔD-ΔE and SARS-CoV-nsp1ΔD-EΔ3 at passage 1 and 10 (-p1 and -p10C) viruses at a moi of 0.001. At different times post infection, virus titers were determined by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean using data from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626112&req=5

ppat.1005215.g013: Generation and growth kinetics of SARS-CoV mutants with deletions in both nsp1 and E genes.(A) SARS-CoV genome is shown in the top, and the expanded region shows the nsp1 and E genes. White boxes represent the amino acids stretches deleted in both proteins in each virus. (B) Mutant viruses growth kinetics. Subconfluent monolayers of Vero E6 and DBT-mACE2 cells were infected with wt, SARS-CoV-nsp1ΔD-ΔE and SARS-CoV-nsp1ΔD-EΔ3 at passage 1 and 10 (-p1 and -p10C) viruses at a moi of 0.001. At different times post infection, virus titers were determined by plaque assay on Vero E6 cells. Error bars represent standard deviations of the mean using data from three independent experiments.
Mentions: In order to develop a safe vaccine candidate, mutant viruses with deletions in both nsp1 and E genes were engineered. A rSARS-CoV deleted in the nsp1 D domain and the E protein (SARS-CoV-nsp1ΔD-ΔE), and a second mutant virus with deletions of the nsp1 D domain coupled with a small deletion (E∆3) (Fig 4A) in the E protein (SARS-CoV-nsp1ΔD-EΔ3) were generated. EΔ3 deletion mutant was selected for further studies because this deletion led to a virus that grew to titers similar or higher than the SARS-CoV-∆E, in cell culture or in mice, respectively (Fig 12A and 12B). More importantly, the E∆3 virus was genetically stable after 10 passages in cell culture [53] or in vivo, maintaining its attenuated phenotype (Fig 12C), in contrast to the ∆E virus (Figs 1 and 5). This deletion was combined with another one in SARS-CoV nsp1 protein (nsp1ΔD), which was fully attenuating. The resulting virus grew to relatively high titers in vivo (Figs 9 and 13A). Viruses were rescued in Vero E6 cells, cloned and sequenced to confirm the presence of the desired mutations.

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus