SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity.
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.


CONCLUSIONS
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.
Liang
Mi-Fang
MF
State Key Laboratory for Infectious Disease Control and Prevention, Institute for Viral Disease Control and Prevention, Beijing 100052, China.
Du
Run-Lei
RL
Liu
Jing-Zhi
JZ
Li
Chuan
C
Zhang
Quan-Fu
QF
Han
Lu-Lu
LL
Yu
Jian-Shi
JS
Duan
Shu-Min
SM
Wang
Xiao-Fang
XF
Wu
Kong-Xing
KX
Xiong
Zhao-Hui
ZH
Jin
Qi
Q
Li
De-Xin
DX
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D002522:Chlorocebus aethiops; D000086862:Coronavirus M Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology*; D009500:Neutralization Tests; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000276:immunology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D045169:Severe Acute Respiratory Syndrome / Q000276:immunology* / Q000821:virology; D064370:Spike Glycoprotein, Coronavirus; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000276:immunology*
D000914:Antibodies, Viral; D000086862:Coronavirus M Proteins; C000706237:M protein, SARS-CoV; D008562:Membrane Glycoproteins; D019151:Peptide Library; D011994:Recombinant Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; C578557:spike glycoprotein, SARS-CoV