title: Severe acute respiratory syndrome coronaviruses with mutations in the E protein are attenuated and promising vaccine candidates.
abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4(+) and CD8(+) T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates.
  
  
  OBJECTIVE
  Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4(+) and CD8(+) T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development.
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jose A
authors.initials: JA
authors.lastname: Regla-Nava
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jose L
authors.initials: JL
authors.lastname: Nieto-Torres
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Jimenez-Guardeño
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Raul
authors.initials: R
authors.lastname: Fernandez-Delgado
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Fett
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Castaño-Rodríguez
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Perlman
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain L.Enjuanes@cnb.csic.es.
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, Campus Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Marta L
authors.initials: ML
authors.lastname: DeDiego
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000096:biosynthesis; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D054884:Host-Pathogen Interactions; D006801:Humans; D008168:Lung / Q000276:immunology / Q000473:pathology; D008807:Mice, Inbred BALB C; D017354:Point Mutation; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D017384:Sequence Deletion; D045169:Severe Acute Respiratory Syndrome / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014765:Viral Vaccines / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology*; D000086082:Viroporin Proteins; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; C501689:E protein, SARS coronavirus; D014613:Vaccines, Attenuated; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; D000086082:Viroporin Proteins; D037521:Virulence Factors