Strategies for Designing Peptide Immunogens To Elicit α-Helical Conformation-Specific Antibodies Reactive with Native Proteins

摘要

Synthetic peptide vaccines against epitopes from the native proteins of pathogenic organisms have the potential to replace traditional vaccines if they can mimic the structure of the epitope found in the native protein target. We have developed a robust technology to elicit antibodies that recognize α-helical sequences of native proteins using a peptide template that consists of a parallel, two-stranded, α-helical coiled-coil. The surface-exposed residues from a helical sequence of interest are inserted into the template to elicit conformation-specific antibodies that recognize the same sequence in the native protein. This strategy was used to develop a vaccine candidate for the pathogen responsible for a 2003 outbreak of severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV). The SARS-CoV Spike (S) glycoprotein is a class I viral fusion protein that possesses regions containing hydrophobic heptad repeats (HR) at the C-terminus (HRC) and at the N-terminus (HRN) of the fusion domain of S protein. The coiled-coil structures formed by the HRC and HRN regions undergo a series of conformational changes that ultimately mediate membrane fusion during virus entry and virus transmission between host cells. Three different peptides were designed to display the HRC region as a one-stranded peptide, templated two-stranded coiled-coil or peptide scaffold-three stranded coiled-coil and conjugated to keyhole limpet haemocyanin (KLH) to form the immunogens used to elicit antibodies in rabbits. We prepared three additional constructs to evaluate these antibodies, a stabilized HRC trimer (GCN4-HRC-GCN4 construct to mimic the prefusion conformation of S protein), a less stable and more flexible trimer of HRC (HRC-GCN4) and a third construct comprised of HRC and HRN peptides to form the six-helix bundle of the postfusion conformation of S protein. Even though all three peptide immunogens contained the same HRC sequence, their corresponding antibodies demonstrated a wide range of affinities to the GCN4 constructs, BSA-peptide conjugates and the native S protein and had different virus neutralizing activities. The templated two-stranded HRC peptide had the highest helical content and was the most thermally stable peptide immunogen of the three peptide immunogens examined here. Importantly, the antibody elicited against this peptide was the only antibody capable of binding the prefusion state of the native S protein, preventing virus entry and inhibiting S protein mediated cell-cell fusion. This antibody exhibited the strongest binding to the GCN4 constructs (HRC-GCN4 and GCN4-HRC-GCN4) and had the weakest affinity for the postfusion conformation of HRC (six-helix bundle construct). Our conformation-stabilized two-stranded coiled-coil template acts as an excellent platform to elicit a-helix-specific antibodies against native proteins and can be exploited to develop vaccine candidates against a wide variety of viral pathogens where a-helical regions are important for viral entry. Here, we review techniques to generate effective synthetic peptide immunogens to elicit antibodies that recognize native proteins and present our work targeting SARS-CoV.