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Journal of Virology, March 2005, p. 3289-3296, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3289-3296.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Choong-Tat Keng,1,{dagger} Aihua Zhang,2,{dagger} Shuo Shen,1* Kuo-Ming Lip,1 Burtram C. Fielding,1 Timothy H. P. Tan,1 Chih-Fong Chou,1 Chay Boon Loh,1 Sifang Wang,1 Jianlin Fu,1 Xiaoming Yang,2 Seng Gee Lim,1 Wanjin Hong,1 and Yee-Joo Tan1

Institute of Molecular and Cell Biology, Singapore,1 Wuhan Institute of Biological Products, Wuhan, People's Republic of China2

Received 6 September 2004/ Accepted 2 November 2004

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-S{Delta}10 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.

* Corresponding author. Mailing address: Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Dr., Singapore 138673. Phone: 65 6586 9622. Fax: 65 67791117. E-mail: shenshuo{at}imcb.a-star.edu.sg.

{dagger} C.-T.K. and A.Z. contributed equally to this article.

Journal of Virology, March 2005, p. 3289-3296, Vol. 79, No. 6
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.6.3289-3296.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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