Molecular engineering of respiratory syncytial virus immunogen for rational redesign of prophylactic peptide vaccines against pediatric viral pneumonia

The immunogen FFL_001 of respiratory syncytial virus (RSV), a widely spread virus that infects the lungs and breathing passages, is a three-helix bundle protein of 115 amino acids long that consists of three helical arms H1, H2 and H3. Here, we attempted to perform molecular engineering of the immunogen, aiming to considerably reduce the protein scaffold of FFL_001 with only moderate activity loss. Structural analysis and molecular dynamics (MD) simulations revealed that two helices H2 and H3 of the FFL_001 three-helix bundle can directly interact with its cognate monoclonal antibody Motavizumab, while the remaining helix H1 plays a crucial role in stabilisation of the three-helix bundle conformation. Binding of the two split peptide segments separately representing FFL_001 two-helix bundle H2-H3 and Motavizumab-binding site to the antibody would incur considerable entropy penalty as compared to binding of the intact FFL_001, suggesting that peptide segments are highly flexible that exhibit a strong intrinsic disorder in solution. In this respect, a scheme was proposed to rationally redesign the immunogen protein scaffold by truncation and cyclisation of the three-helix bundle. The cyclisation was conducted on the spatially vicinal residue pairs in H2 and H3 helical arms by mutating the residue to cysteine and introducing a disulphide bond across them. Consequently, we obtained three cyclic peptides that were theoretically predicted to have strong binding potency towards Motavizumab. In order to substantiate the computational finding, binding affinity of the designed cyclic peptide and its linear counterpart was determined. Consistently, no binding can be found between the linear peptide and Motavizumab (K_d = n.d.), while a moderately high affinity was observed for cyclic peptide (K_d = 16.2 nM).