Rational design of respiratory syncytial virus dimeric F-subunit vaccines in protein and mRNA forms.

BACKGROUND: Respiratory syncytial virus (RSV) poses a significant public health threat, particularly to children and the elderly. Two protein-based vaccines and one mRNA vaccine have been approved, all targeting the prefusion conformation of the fusion (F) trimer. However, it has been reported that the epitope activity of the F protein gradually declines during storage, resulting in a reduction of the vaccines' immunogenicity. METHODS: In this study, we engineered a redesigned pre-F-based antigen, monomer A, derive from the RSV subtype A F protein, aiming to preserve immunodominant pre-F-specific epitopes while eliminating sub-potent ones. Following this design principle, we constructed a series of single-chain (sc) dimers and selected the one, scDimer AA, with the highest expression yield and melting temperature (T(m)). Next, we designed scDimer AB, which incorporates monomers from both subtype A and subtype B to form a heterologous sc dimer. Structural and protein characterisation analyses were conducted to verify our design. All monomeric and scDimer antigens were used to immunise rodent models. Additionally, we prepared the antigens in mRNA form and immunised BALB/c mice. Finally, we combined both antigen forms, administering intramuscular mRNA priming followed by intranasal protein delivery in mice. In all immunisation strategies, viral challenges were performed in animals to evaluate the immunologic protective effects. FINDINGS: Through rational design, we developed a monomeric and two single-chain dimeric (scDimer) proteins with the expected characteristics, including complete II, V, and Ø epitopes and a partial III epitope. The scDimers elicited stronger binding and neutralising antibody responses in rodent models compared to the monomer, and they also boosted T cell responses when combined with appropriate adjuvants. After three doses of scDimer immunisation, challenge with RSV resulted in barely detectable RSV in the tissues of immunised and challenged animals. The copies of RNA encoding N-gene were significantly reduced in the immunised groups compared to the PBS-injected control groups. We also engineered mRNA versions of the antigens and demonstrated their protective efficacy in mice. Notably, there were no significant differences between intranasal boost and intramuscular boost after one dose of intramuscular after RSV challenged, suggesting that intranasal boost provided equivalent protection to intramuscular vaccination and could reduce the risk of vaccine-enhanced disease (VED) potentially. INTERPRETATION: The scDimer-based RSV vaccines effectively protected rodents from RSV infections, highlighting their clinical potential. Our antigen design removed certain suboptimal epitope regions, enhancing the efficiency of antigen presentation and increasing the proportion of the most potent pre-F-specific neutralising antibodies. This approach provides a distinct perspective for future vaccine design. FUNDING: National Key R&D Program of China, National Science Foundation of China, Young Scientists in Basic Research, Chinese Academy of Sciences, and Special Program of China National Tobacco Corporation.