Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.
Identifieur interne : 000D47 ( PubMed/Corpus ); précédent : 000D46; suivant : 000D48Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.
Auteurs : Jose M. Jimenez-Guarde O ; Jose A. Regla-Nava ; Jose L. Nieto-Torres ; Marta L. Dediego ; Carlos Casta O-Rodriguez ; Raul Fernandez-Delgado ; Stanley Perlman ; Luis EnjuanesSource :
- PLoS pathogens [ 1553-7374 ] ; 2015.
English descriptors
- KwdEn :
- MESH :
- chemical , immunology : Vaccines, Attenuated, Vaccines, Synthetic, Viral Vaccines.
- growth & development : SARS Virus.
- immunology : SARS Virus.
- pathogenicity : SARS Virus.
- Animals, Cells, Cultured, Female, Mice, Mice, Inbred BALB C, Virulence.
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.
DOI: 10.1371/journal.ppat.1005215
PubMed: 26513244
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Regla-Nava</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Nieto Torres, Jose L" sort="Nieto Torres, Jose L" uniqKey="Nieto Torres J" first="Jose L" last="Nieto-Torres">Jose L. Nieto-Torres</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Dediego, Marta L" sort="Dediego, Marta L" uniqKey="Dediego M" first="Marta L" last="Dediego">Marta L. 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Jimenez-Guarde O</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Regla Nava, Jose A" sort="Regla Nava, Jose A" uniqKey="Regla Nava J" first="Jose A" last="Regla-Nava">Jose A. Regla-Nava</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Nieto Torres, Jose L" sort="Nieto Torres, Jose L" uniqKey="Nieto Torres J" first="Jose L" last="Nieto-Torres">Jose L. Nieto-Torres</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Dediego, Marta L" sort="Dediego, Marta L" uniqKey="Dediego M" first="Marta L" last="Dediego">Marta L. Dediego</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Casta O Rodriguez, Carlos" sort="Casta O Rodriguez, Carlos" uniqKey="Casta O Rodriguez C" first="Carlos" last="Casta O-Rodriguez">Carlos Casta O-Rodriguez</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Fernandez Delgado, Raul" sort="Fernandez Delgado, Raul" uniqKey="Fernandez Delgado R" first="Raul" last="Fernandez-Delgado">Raul Fernandez-Delgado</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author><author><name sortKey="Perlman, Stanley" sort="Perlman, Stanley" uniqKey="Perlman S" first="Stanley" last="Perlman">Stanley Perlman</name><affiliation><nlm:affiliation>Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.</nlm:affiliation></affiliation></author><author><name sortKey="Enjuanes, Luis" sort="Enjuanes, Luis" uniqKey="Enjuanes L" first="Luis" last="Enjuanes">Luis Enjuanes</name><affiliation><nlm:affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</nlm:affiliation></affiliation></author></analytic><series><title level="j">PLoS pathogens</title><idno type="eISSN">1553-7374</idno><imprint><date when="2015" type="published">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term><term>Cells, Cultured</term><term>Female</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>SARS Virus (growth & development)</term><term>SARS Virus (immunology)</term><term>SARS Virus (pathogenicity)</term><term>Vaccines, Attenuated (immunology)</term><term>Vaccines, Synthetic (immunology)</term><term>Viral Vaccines (immunology)</term><term>Virulence</term></keywords><keywords scheme="MESH" type="chemical" qualifier="immunology" xml:lang="en"><term>Vaccines, Attenuated</term><term>Vaccines, Synthetic</term><term>Viral Vaccines</term></keywords><keywords scheme="MESH" qualifier="growth & development" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="immunology" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" qualifier="pathogenicity" xml:lang="en"><term>SARS Virus</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Animals</term><term>Cells, Cultured</term><term>Female</term><term>Mice</term><term>Mice, Inbred BALB C</term><term>Virulence</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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. </div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">26513244</PMID><DateCompleted><Year>2016</Year><Month>04</Month><Day>11</Day></DateCompleted><DateRevised><Year>2019</Year><Month>02</Month><Day>02</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Electronic">1553-7374</ISSN><JournalIssue CitedMedium="Internet"><Volume>11</Volume><Issue>10</Issue><PubDate><Year>2015</Year><Month>Oct</Month></PubDate></JournalIssue><Title>PLoS pathogens</Title><ISOAbbreviation>PLoS Pathog.</ISOAbbreviation></Journal><ArticleTitle>Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.</ArticleTitle><Pagination><MedlinePgn>e1005215</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1371/journal.ppat.1005215</ELocationID><Abstract><AbstractText>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. </AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Jimenez-Guardeño</LastName><ForeName>Jose M</ForeName><Initials>JM</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Regla-Nava</LastName><ForeName>Jose A</ForeName><Initials>JA</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Nieto-Torres</LastName><ForeName>Jose L</ForeName><Initials>JL</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>DeDiego</LastName><ForeName>Marta L</ForeName><Initials>ML</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Castaño-Rodriguez</LastName><ForeName>Carlos</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Fernandez-Delgado</LastName><ForeName>Raul</ForeName><Initials>R</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Perlman</LastName><ForeName>Stanley</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Enjuanes</LastName><ForeName>Luis</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>P01 AI060699</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant><Grant><GrantID>5P01AI060699</GrantID><Acronym>AI</Acronym><Agency>NIAID NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D052061">Research Support, N.I.H., Extramural</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2015</Year><Month>10</Month><Day>29</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>PLoS Pathog</MedlineTA><NlmUniqueID>101238921</NlmUniqueID><ISSNLinking>1553-7366</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014613">Vaccines, Attenuated</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014614">Vaccines, Synthetic</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D014765">Viral Vaccines</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D002478" MajorTopicYN="N">Cells, Cultured</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D005260" MajorTopicYN="N">Female</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D051379" MajorTopicYN="N">Mice</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D008807" MajorTopicYN="N">Mice, Inbred BALB C</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D045473" MajorTopicYN="N">SARS Virus</DescriptorName><QualifierName UI="Q000254" MajorTopicYN="N">growth & development</QualifierName><QualifierName UI="Q000276" MajorTopicYN="Y">immunology</QualifierName><QualifierName UI="Q000472" MajorTopicYN="N">pathogenicity</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014613" MajorTopicYN="N">Vaccines, Attenuated</DescriptorName><QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014614" MajorTopicYN="N">Vaccines, Synthetic</DescriptorName><QualifierName UI="Q000276" MajorTopicYN="N">immunology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014765" MajorTopicYN="N">Viral Vaccines</DescriptorName><QualifierName UI="Q000276" MajorTopicYN="Y">immunology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D014774" MajorTopicYN="N">Virulence</DescriptorName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2015</Year><Month>06</Month><Day>05</Day></PubMedPubDate><PubMedPubDate 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