gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

Pre-clinical studies of SARS subunit vaccine candidates

Poster

  • Hui-Wen Hsiao - Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan, ROC
  • Edmond S-L. Hsieh - Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan, ROC
  • Chi-Ju Chen - Institute of Microbiology & Immunology, National Yang-Ming University, Taipei, Taiwan, ROC
  • Peggy Lian - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • Lucy Lin - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • Levent Liu - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • Leo Leng - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • Eve Ho - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • Wei-Cheng Lian - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC
  • presenting/speaker Pele C-S. Chong - Vaccine Center for R&D, National Health Research Institutes, Taipei, Taiwan, ROC

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sarsP3.01

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/sars2004/04sars108.shtml

Veröffentlicht: 26. Mai 2004

© 2004 Hsiao et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

The spike (S) protein of coronavirus (CoV) has been identified to be responsible for binding to and mediate infection of their target cells. To rapidly develop vaccine and immunotherapeutics against SARS CoV, it is important to map out the receptor binding domain(s). We have constructed and expressed recombinant fusion proteins containing human IgG Fc fragment and truncated S proteins of SARS CoV, in baculovirus. With these recombinant fusion proteins and overlapping peptides covering the entire S protein, we had identified three distinct receptor-binding domains of S protein. Using VERO E6 cells as target, we identified and mapped two separated receptor-binding domains of S protein. The low affinity binding domain (>10 μM) located within the N-terminal 333 residues and its receptor was identified to be ACE2 protein reported by W. Li et al [1], the high affinity-binding domain (~1 nM) was mapped to residues 334 to 666 and also bound strongly to ACE2 protein. When NIH/3T3, HeLa, BHK-21 and COS-7 cells as targets, we found three separated receptor-binding domains of S protein: the low affinity was mapped again to the N-terminal 333 residues, the mild binding site (1 μM) was mapped to residues 334 to 666, but the high affinity is located within residues 667 to 999.

Recombinant S fragment containing the receptor-binding domains corresponding to residues 294 to 739, rRBD2 expressed by E. coli was used as immunogens formulated in incomplete Freund's adjuvant to immunized guinea pigs and rabbits. After three immunizations, most antisera titer against rRBD2 and spike 2 (residues 334-666)-Fc had reached over 1/10,000. We observed these antisera could inhibit the ACE2 binding to rRBD2, but only partially blocking spike 2-Fc binding in ELISA. Using synthetic peptides as coating reagents in ELISA, we observed these antisera only reacted with peptides corresponding to region containing residues 404 to 533 and had no reaction with peptides from residues 524 to 618. These results were consistent with other results and further confirmed that the region around residues 524 to 618 was either incorrectly folded or non-immunogenic and most likely buried inside the spike protein like a cavity. This conformational cavity structure had to be very important for spike 2-Fc and ACE2 interaction. Macaques' immunogenicity studies using spike 1(residues 1-333)-Fc and spike 2-Fc as immunogens were performed and found similar results that most antibodies recognized regions containing residues 404 to 533. Studies of macaques' T-cell responses and virus neutralization functional assay are in progress.

This research was supported by National Science Council of Republic of China Grants # SVAC12-06 (P. Chong), SIMM07-00 and SIMM07-01 (E. Hsieh).


References

1.
W. Li et al (Nature (2003) 426:450-453