Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents.
The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
Keng
Choong-Tat
CT
Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Dr., Singapore 138673.
Zhang
Aihua
A
Shen
Shuo
S
Lip
Kuo-Ming
KM
Fielding
Burtram C
BC
Tan
Timothy H P
TH
Chou
Chih-Fong
CF
Loh
Chay Boon
CB
Wang
Sifang
S
Fu
Jianlin
J
Yang
Xiaoming
X
Lim
Seng Gee
SG
Hong
Wanjin
W
Tan
Yee-Joo
YJ
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D015153:Blotting, Western; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000502:physiology; D009500:Neutralization Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology* / Q000502:physiology
D000914:Antibodies, Viral; D000939:Epitopes; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus
Virology. 2001 Jan 20;279(2):371-4
11162792
J Virol. 1997 Jul;71(7):5251-8
9188593
N Engl J Med. 2003 May 15;348(20):1967-76
12690091
N Engl J Med. 2003 May 15;348(20):1953-66
12690092
Nature. 2003 May 15;423(6937):240
12748632
Lancet. 2003 May 24;361(9371):1779-85
12781537
J Virol. 2003 Aug;77(16):8801-11
12885899
Biochem Biophys Res Commun. 2003 Dec 26;312(4):1159-64
14651994
Nature. 2003 Nov 27;426(6965):450-4
14647384
Lancet. 2003 Dec 6;362(9399):1895-6
14667748
J Biol Chem. 2004 Jan 30;279(5):3197-201
14670965
Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2536-41
14983044
Clin Diagn Lab Immunol. 2004 Mar;11(2):362-71
15013989
J Virol. 2004 Apr;78(7):3572-7
15016880
Lancet. 2004 Mar 20;363(9413):938-47
15043961
Nature. 2004 Apr 1;428(6982):561-4
15024391
J Virol. 2004 May;78(9):4552-60
15078936
Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6641-6
15096611
J Biol Chem. 2004 May 14;279(20):20836-49
14996844
Biochem Biophys Res Commun. 2004 Jun 18;319(1):283-8
15158473
J Virol. 2004 Jun;78(12):6134-42
15163706
Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8455-60
15150417
Biochem Biophys Res Commun. 2004 Jul 2;319(3):746-52
15184046
Proc Natl Acad Sci U S A. 2004 Jun 8;101(23):8709-14
15161975
J Virol. 2004 Jul;78(13):6938-45
15194770
J Virol. 2004 Jul;78(13):7217-26
15194798
Lancet. 2004 Jun 26;363(9427):2122-7
15220033
Lancet. 2004 Jun 26;363(9427):2139-41
15220038
Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9804-9
15210961
J Clin Virol. 2004 Sep;31(1):66-8
15288616
J Gen Virol. 1988 Dec;69 ( Pt 12):2939-52
3058868
J Virol. 1990 Jan;64(1):339-46
2403441
J Gen Virol. 1990 Feb;71 ( Pt 2):263-70
2155283
J Virol. 1990 Dec;64(12):6270-3
1700835
Lancet. 2003 Apr 19;361(9366):1319-25
12711465