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

Abstract

BackgroundRespiratory 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) glycoprotein. However, it has been reported that the F protein transitions to the post-fusion state during storage, resulting in a reduction of the vaccines immunogenicity. MethodsIn this study, we engineered novel pre-F-based antigens to preserve pre-F-specific immunodominant epitopes while eliminating sub-potent ones. Based on this, we constructed a series of single-chain dimers and selected the one with the highest expression yield and melting temperature (Tm). Next, we created a heterodimer, scDimer AB. Structural and protein characterization analyses were conducted to verify our design. All monomeric and scDimer antigens were used to immunize rodent models. Additionally, we prepared the antigens in mRNA form and immunized BALB/c mice. Finally, we combined both antigen forms, administering intramuscular mRNA priming followed by intranasal protein delivery in mice. In all immunization strategies, viral challenges were performed in animals to evaluate the immunologic protective effects. FindingsThrough rational design, we developed a monomeric and two single-chain dimeric (scDimer) proteins with the expected characteristics, including complete II, V, and O epitopes and a partial III epitope. The scDimers elicited stronger binding and neutralizing 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 immunization, live RSV was barely detectable in the tissues of infected animals. The copies of RNA encoding N-gene were significantly reduced in the immunized groups compared to the PBS-injected control groups. We also engineered mRNA versions of the antigens and verified their protective efficacy in mice. Notably, there were no significant differences between intranasal boost and intramuscular two doses after RSV challenged, suggesting that intranasal boost provided equivalent protection to intramuscular vaccination and could reduce the risk of vaccine-enhanced disease (VED) potentially. InterpretationThe 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 neutralizing antibodies. This approach provides a novel perspective for future vaccine design. FundingNational 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. RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSThe licensed respiratory syncytial virus (RSV) vaccines were based on RSV pre-F structure. We conducted a PubMed search for articles published in English from databases inception to March 31, 2025, using the search terms "RSV" (interchanged with "respiratory syncytial virus"), "pre-F" (interchanged with "prefusion F") and "vaccine". Our search revealed that most of the articles focus on combining pre-F with RSV G protein or other proteins to form viral-like particles (VLPs) or nanoparticles. The second-largest category of articles examined the antibodies level elicited by the pre-F antigens in animal model or some special cohorts. Other studies addressed topics such as optimization of injection dosage and immunization strategies, methodologies for detecting pre-F-specific antibodies, and the development of mRNA-LNP vaccines. Notably, only 4 articles described novel designs of RSV pre-F antigens, all of which were trimeric and based exclusively on the RSV A subtype F protein sequence. Added value of this studyTo our knowledge, our design is the first single-chain dimer version of RSV pre-F antigen. The engineered antigens demonstrated high yield, thermostability, and immunogenicity comparable to that of DS-Cav1. Additionally, we developed the first chimeric RSV-antigen covering both subtype A and B, thereby broadening the potential protective spectrum. We further validated the immunogenicity of our designs in mRNA form and as a protein-mRNA combination. This research emphasizes the potential of constructing and combining minimized, stable antigen modules, offering a novel approach for future vaccine development. Implications of all the available evidenceIn this study, we present a novel approach for constructing and combining minimized, stable antigen modules for future vaccine design. The strong immunogenicity of our antigens underscores their potential for clinical application. Furthermore, given the structural similarities between the RSV antigen and those of other Pneumoviruses, such as human metapneumovirus (hMPV), our design strategy could be extended to the development of multivalent vaccines.