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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

Protection conferred by immunization with SARS-CoV-double mutants.BALB/c mice (16-week-old) were mock-immunized or immunized with 6000 pfu of SARS-CoV-nsp1ΔD-EΔ3 viruses at passage 1 and 10 (-p1, -p10C and -p10M), and challenged at day 21 post-immunization with 100,000 pfu of wt virus (5 mice per group). Weight loss (A) and survival (B) were recorded daily.
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ppat.1005215.g016: Protection conferred by immunization with SARS-CoV-double mutants.BALB/c mice (16-week-old) were mock-immunized or immunized with 6000 pfu of SARS-CoV-nsp1ΔD-EΔ3 viruses at passage 1 and 10 (-p1, -p10C and -p10M), and challenged at day 21 post-immunization with 100,000 pfu of wt virus (5 mice per group). Weight loss (A) and survival (B) were recorded daily.

Mentions: To determine whether SARS-CoV-nsp1-ΔD-EΔ3 confers protection against challenge with rSARS-CoV, BALB/c mice were either immunized with SARS-CoV-nsp1ΔD-EΔ3-p1, -p10C and -p10M or non-immunized, as a control. At 21 days postimmunization, mice were challenged with rSARS-CoV administered by the same route. Non-immunized mice lost weight and all died on day 6 after the challenge (Fig 16A and 16B). In contrast, vaccination with the attenuated mutant viruses completely protected mice against challenge with rSARS-CoV (Fig 16A and 16B), indicating that the double mutant virus is a promising vaccine candidate.


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)

Protection conferred by immunization with SARS-CoV-double mutants.BALB/c mice (16-week-old) were mock-immunized or immunized with 6000 pfu of SARS-CoV-nsp1ΔD-EΔ3 viruses at passage 1 and 10 (-p1, -p10C and -p10M), and challenged at day 21 post-immunization with 100,000 pfu of wt virus (5 mice per group). Weight loss (A) and survival (B) were recorded daily.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005215.g016: Protection conferred by immunization with SARS-CoV-double mutants.BALB/c mice (16-week-old) were mock-immunized or immunized with 6000 pfu of SARS-CoV-nsp1ΔD-EΔ3 viruses at passage 1 and 10 (-p1, -p10C and -p10M), and challenged at day 21 post-immunization with 100,000 pfu of wt virus (5 mice per group). Weight loss (A) and survival (B) were recorded daily.
Mentions: To determine whether SARS-CoV-nsp1-ΔD-EΔ3 confers protection against challenge with rSARS-CoV, BALB/c mice were either immunized with SARS-CoV-nsp1ΔD-EΔ3-p1, -p10C and -p10M or non-immunized, as a control. At 21 days postimmunization, mice were challenged with rSARS-CoV administered by the same route. Non-immunized mice lost weight and all died on day 6 after the challenge (Fig 16A and 16B). In contrast, vaccination with the attenuated mutant viruses completely protected mice against challenge with rSARS-CoV (Fig 16A and 16B), indicating that the double mutant virus is a promising vaccine candidate.

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