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. 2005 May;7(5-6):882-9.
doi: 10.1016/j.micinf.2005.02.006. Epub 2005 Apr 13.

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities

Maofeng Qiu  1 , Yuling Shi, Zhaobiao Guo, Zeliang Chen, Rongqiao He, Runsheng Chen, Dongsheng Zhou, Erhei Dai, Xiaoyi Wang, Bingyin Si, Yajun Song, Jingxiang Li, Ling Yang, Jin Wang, Hongxia Wang, Xin Pang, Junhui Zhai, Zongmin Du, Ying Liu, Yong Zhang, Linhai Li, Jian Wang, Bing Sun, Ruifu Yang
Affiliations

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities

Maofeng Qiu et al. Microbes Infect. 2005 May.

Abstract

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.

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Figures

Fig. 1
Fig. 1
Structural organization and expression of the ORFs of the SARS-CoV used for this study. (A) ORFs corresponding to four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b). (B) SDS-PAGE analysis of expressed fusion proteins with Trx/6_His-tag at NH2-terminus from recombinant E. coli BL21 (DE3). Recombinant protein S3 (lane 2, 56.8 kDa), S4 (lane 3, 37.2 kDa), S5 (lane 4, 49.9 kDa), 3a (lane 5, 34.2 kDa), 3b (lane 6, 28.1 kDa), E (lane 7, 22.6 kDa), M (lane 8, 31.2 kDa), 6 (lane 9, 22.6 kDa), 7a (lane 10, 31.1 kDa), N (lane 11, 64.6 kDa), 9b (lane 12, 28.3 kDa) and plasmid pET32a vector control (lane 13) were induced by IPTG and analyzed by SDS-PAGE and stained with Coomassie brilliant blue R-250. Lane 1 shows the molecular weight markers, and arrows indicate the positions of target proteins. The polypeptides' molecular weights expressed in kDa were estimated by their relative mobility in SDS-PAGE.
Fig. 1
Fig. 1
Structural organization and expression of the ORFs of the SARS-CoV used for this study. (A) ORFs corresponding to four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b). (B) SDS-PAGE analysis of expressed fusion proteins with Trx/6_His-tag at NH2-terminus from recombinant E. coli BL21 (DE3). Recombinant protein S3 (lane 2, 56.8 kDa), S4 (lane 3, 37.2 kDa), S5 (lane 4, 49.9 kDa), 3a (lane 5, 34.2 kDa), 3b (lane 6, 28.1 kDa), E (lane 7, 22.6 kDa), M (lane 8, 31.2 kDa), 6 (lane 9, 22.6 kDa), 7a (lane 10, 31.1 kDa), N (lane 11, 64.6 kDa), 9b (lane 12, 28.3 kDa) and plasmid pET32a vector control (lane 13) were induced by IPTG and analyzed by SDS-PAGE and stained with Coomassie brilliant blue R-250. Lane 1 shows the molecular weight markers, and arrows indicate the positions of target proteins. The polypeptides' molecular weights expressed in kDa were estimated by their relative mobility in SDS-PAGE.
Fig. 2
Fig. 2
The FI values for 13 recombinant proteins to human sera by protein microarray. Thirteen recombinant proteins were applied to analyze human sera. The control and patient sera bands indicate the average FI values for each protein to the sera from 23 healthy people and 58 convalescent-phase SARS patients, respectively, and the cutoff values were calculated from the data from healthy people, cutoff = mean + 3S.D. * indicates statistically significant difference between patient sera and control.
Fig. 3
Fig. 3
Reactivity of specific IgG antibodies to individual proteins of the SARS-CoV in 58 sera from convalescent-phase SARS patients. Thirteen recombinant proteins (vertical axis) were used to analyze 58 convalescent-phase SARS patient sera (horizontal axis). The black blocks indicate positive reactions and the white ones negative.
Fig. 4
Fig. 4
Kinetics of the SARS-CoV IgG antibodies in human sera over time. The kinetics of average FI values for anti-N, anti-S3, anti-3a, anti-9b antibodies were monitored using a group of serial sera collected from people from weeks 2 to 30 after their onset of SARS. Anti-N and anti-S3 antibodies persisted until week 30, and the average FI values at week 30 were about half of those at week 4 but anti-3a and anti-9b antibodies remained at low levels all the while.
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