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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

Stability of SARS-CoV-∆E after serial passage in mice.A recombinant virus lacking the E protein (SARS-CoV-∆E) was passaged in BALB/c mice using an initial moi of 100,000 pfu. After 10 serial passages the last third of the genome was sequenced using specific primers. (A) Sequence corresponding to native 8a protein (8a) and to mutated protein generated after passage in mice (8a-dup). New incorporated amino acids are highlighted in light blue. (B) Predicted structures for 8a protein at passage 0 (8a) and 10 (8a-dup) in mice. Presence of new incorporated amino acids in 8a-dup and PBM are highlighted in light blue and red, respectively.
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ppat.1005215.g005: Stability of SARS-CoV-∆E after serial passage in mice.A recombinant virus lacking the E protein (SARS-CoV-∆E) was passaged in BALB/c mice using an initial moi of 100,000 pfu. After 10 serial passages the last third of the genome was sequenced using specific primers. (A) Sequence corresponding to native 8a protein (8a) and to mutated protein generated after passage in mice (8a-dup). New incorporated amino acids are highlighted in light blue. (B) Predicted structures for 8a protein at passage 0 (8a) and 10 (8a-dup) in mice. Presence of new incorporated amino acids in 8a-dup and PBM are highlighted in light blue and red, respectively.

Mentions: MCH-Vero and MCH-DBT exhibited an attenuated phenotype (see above). To analyze the genetic stability of recombinant SARS-CoV-∆E in BALB/c mice, virus was passaged every 48 hours by intranasal inoculation. A partial duplication of 45 nucleotides was found within 8a gene (Fig 5), leading to the incorporation of a fragment of 15 amino acids at the carboxy-terminus of 8a protein, and generating a novel 8a protein (8a-dup) with an internal PBM (CTVV) (Fig 5A and 5B). To determine the virulence of this novel virus (SARS-CoV-∆E-8a-dup), we infected a new cohort of BALB/c mice and assessed survival and weight loss, and measured virus titers. Mice infected with SARS-CoV-∆E did not lose weight and all survived. In contrast, SARS-CoV-∆E-8a-dup grew to titers similar to those of rSARS-CoV and developed profound weight loss, developing signs of illness and death by 7 dpi (Fig 6A and 6B). Histological examination of lungs from SARS-CoV-∆E-infected mice showed absence of lung damage at both dpi. In contrast, lungs of mice infected with rSARS-CoV or SARS-CoV-∆E-8a-dup showed substantial perivascular, peribronchial and interstitial cellular infiltration and edema at 2 and 4 dpi (Fig 6C). To further confirm the relevance of the partial duplication within 8a gene in the induction of virulence, a recombinant SARS-CoV-∆E with an 8a-dup gene was generated (rSARS-CoV-∆E-8a-dup). Virulence during infection with rSARS-CoV-∆E-8a-dup was evaluated as described above (Fig 6D). The engineered virus (rSARS-CoV-∆E-8a-dup) was as virulent as the SARS-CoV-∆E-8a-dup generated after SARS-CoV-∆E passage in vivo (Fig 6D). These results indicated that SARS-CoV-∆E regained virulence after serial passage in mice.


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)

Stability of SARS-CoV-∆E after serial passage in mice.A recombinant virus lacking the E protein (SARS-CoV-∆E) was passaged in BALB/c mice using an initial moi of 100,000 pfu. After 10 serial passages the last third of the genome was sequenced using specific primers. (A) Sequence corresponding to native 8a protein (8a) and to mutated protein generated after passage in mice (8a-dup). New incorporated amino acids are highlighted in light blue. (B) Predicted structures for 8a protein at passage 0 (8a) and 10 (8a-dup) in mice. Presence of new incorporated amino acids in 8a-dup and PBM are highlighted in light blue and red, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4626112&req=5

ppat.1005215.g005: Stability of SARS-CoV-∆E after serial passage in mice.A recombinant virus lacking the E protein (SARS-CoV-∆E) was passaged in BALB/c mice using an initial moi of 100,000 pfu. After 10 serial passages the last third of the genome was sequenced using specific primers. (A) Sequence corresponding to native 8a protein (8a) and to mutated protein generated after passage in mice (8a-dup). New incorporated amino acids are highlighted in light blue. (B) Predicted structures for 8a protein at passage 0 (8a) and 10 (8a-dup) in mice. Presence of new incorporated amino acids in 8a-dup and PBM are highlighted in light blue and red, respectively.
Mentions: MCH-Vero and MCH-DBT exhibited an attenuated phenotype (see above). To analyze the genetic stability of recombinant SARS-CoV-∆E in BALB/c mice, virus was passaged every 48 hours by intranasal inoculation. A partial duplication of 45 nucleotides was found within 8a gene (Fig 5), leading to the incorporation of a fragment of 15 amino acids at the carboxy-terminus of 8a protein, and generating a novel 8a protein (8a-dup) with an internal PBM (CTVV) (Fig 5A and 5B). To determine the virulence of this novel virus (SARS-CoV-∆E-8a-dup), we infected a new cohort of BALB/c mice and assessed survival and weight loss, and measured virus titers. Mice infected with SARS-CoV-∆E did not lose weight and all survived. In contrast, SARS-CoV-∆E-8a-dup grew to titers similar to those of rSARS-CoV and developed profound weight loss, developing signs of illness and death by 7 dpi (Fig 6A and 6B). Histological examination of lungs from SARS-CoV-∆E-infected mice showed absence of lung damage at both dpi. In contrast, lungs of mice infected with rSARS-CoV or SARS-CoV-∆E-8a-dup showed substantial perivascular, peribronchial and interstitial cellular infiltration and edema at 2 and 4 dpi (Fig 6C). To further confirm the relevance of the partial duplication within 8a gene in the induction of virulence, a recombinant SARS-CoV-∆E with an 8a-dup gene was generated (rSARS-CoV-∆E-8a-dup). Virulence during infection with rSARS-CoV-∆E-8a-dup was evaluated as described above (Fig 6D). The engineered virus (rSARS-CoV-∆E-8a-dup) was as virulent as the SARS-CoV-∆E-8a-dup generated after SARS-CoV-∆E passage in vivo (Fig 6D). These results indicated that SARS-CoV-∆E regained virulence after serial passage in mice.

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