Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody libra...Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike （S） proteins, two Fab antibodies were specific for the virus membrane （M） protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect （CPE） inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus Conclusion The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.展开更多
Background:The outbreak of Ebola virus disease(EVD)in West Africa between 2014 and 2015 was the largest EDV epidemic since the identification of Ebola virus(EBOV)in 1976,and the countries most strongly affected were S...Background:The outbreak of Ebola virus disease(EVD)in West Africa between 2014 and 2015 was the largest EDV epidemic since the identification of Ebola virus(EBOV)in 1976,and the countries most strongly affected were Sierra Leone,Guinea,and Liberia.Findings:The Sierra Leone-China Friendship Biological Safety Laboratory(SLE-CHN Biosafety Lab),a fixed Biosafety Level 3 laboratory in the capital city of Sierra Leone,was established by the Chinese government and has been active in EBOV detection since 11 March 2015.Complete management and program documents were created for the SLE-CHN Biosafety Lab,and it was divided into four zones(the green,yellow,brown,and red zones)based on the risk assessment.Different types of safe and appropriate personnel protection equipment(PPE)are used in different zones of the laboratory,and it fully meets the Biosafety Level 3 laboratory standards of the World Health Organization.Conclusion:Good preparedness,comprehensive risk assessment and operation documents,appropriate PPE,effective monitoring and intensive training,together with well-designed and reasonable laboratory sectioning are essential for guaranteeing biosafety.展开更多
基金This work was supported by Chinese National "863" R & D High Technology Programs: National SARS Key Project (2003AA208209).
文摘Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike （S） proteins, two Fab antibodies were specific for the virus membrane （M） protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect （CPE） inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus Conclusion The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.
基金supported by the China Mega-Project for Infectious Disease(2011ZX10004-101,2012ZX10004215)the Research Special Funds for Public Welfare Projects(201302006)the SKLID Development Grant(2012SKLID102).
文摘Background:The outbreak of Ebola virus disease(EVD)in West Africa between 2014 and 2015 was the largest EDV epidemic since the identification of Ebola virus(EBOV)in 1976,and the countries most strongly affected were Sierra Leone,Guinea,and Liberia.Findings:The Sierra Leone-China Friendship Biological Safety Laboratory(SLE-CHN Biosafety Lab),a fixed Biosafety Level 3 laboratory in the capital city of Sierra Leone,was established by the Chinese government and has been active in EBOV detection since 11 March 2015.Complete management and program documents were created for the SLE-CHN Biosafety Lab,and it was divided into four zones(the green,yellow,brown,and red zones)based on the risk assessment.Different types of safe and appropriate personnel protection equipment(PPE)are used in different zones of the laboratory,and it fully meets the Biosafety Level 3 laboratory standards of the World Health Organization.Conclusion:Good preparedness,comprehensive risk assessment and operation documents,appropriate PPE,effective monitoring and intensive training,together with well-designed and reasonable laboratory sectioning are essential for guaranteeing biosafety.
