Template-based coiled-coil antigens elicit neutralizing antibodies to the SARS-coronavirus

Abstract

The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.

Antibody Cross-Reactivity to Hemagglutinin Protein Antigens Demonstrates Feasibility for Development of a “Universal” Influenza A Synthetic Peptide Vaccine

“…In conclusion, we first demonstrated the usefulness of our two-stranded α-helical coiled-coil template technology in our studies to develop a synthetic peptide vaccine against Severe Acute Respiratory Syndrome (SARS) [11,12]. Here we have incorporated a 28-residue α-helical sequence from the stem domain of hemagglutinin from H1N1 and showed that we can generate polyclonal antibodies that cross-react with all H1N1 subtypes in the region 401-428.…”

Applications of immunochemistry in human health: advances in vaccinology and antibody design (IUPAC Technical Report)

“…Thus, immunogens designed by epitope transplantation have elicited structurespecific responses targeting a snake toxin, the yeast transcription factor GCN4, the severe acute respiratory syndrome coronavirus S glycoprotein, and the human immunodeficiency virus 2F5 and 4E10 epitopes [171][172][173][174]. However, the ability to graft a motif onto a scaffold is still limited by the complexity of the motif and the availability of scaffolds with suitable structures.…”

Selbstorganisation von Coiled‐Coils in der synthetischen Biologie: Inspiration und Fortschritt

“…Das Ziel dieser Studien ist die Inhibierung des Viruseintritts in Zellen. [94,95] Andere Gruppen befassen sich mit dem Entwurf von Coiled-Coil-Peptiden, die an spezifische Transkriptionsfaktoren binden, um auf diese Weise die DNA-Replikation zu modulieren. [96] Ein weiterer Aspekt ist das Umschalten der Sekundär-struktur der Peptide, die so programmiert werden können, dass sie in unterschiedlichen Umgebungen unterschiedliche Strukturen einnehmen.…”