Comparative Evaluation of Three Nanoparticle Vaccines Targeting the Prefusion F Protein of Respiratory Syncytial Virus: Immunogenicity and Protective Efficacy

Jie Jiang, Hongqiao Hu, Lei Cao, Naiying Mao, Zhen Zhu, Na Wang, Yuqing Shi, Hai Li, Yan Zhang National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Disease, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, People’s Republic of ChinaCorrespondence: Yan Zhang; Hai Li, Email [email protected]; [email protected]: To evaluate the immunogenic potential of three different nanoparticle (NP) platforms for respiratory syncytial virus (RSV) prefusion (pre-F) protein vaccines.Methods: Three NP platforms— 24-mer ferritin (Fe), 60-mer lumazine synthase (LuS), and 120-subunit I53-50—were engineered to display RSV pre-F trimers (DS2) via SpyTag-SpyCatcher (ST-SC) conjugation (DS2-Fe, DS2-LuS) or direct genetic fusion (DS2-I53-50). The assembled particles were characterized using size-exclusion chromatography (SEC), SDS-PAGE, electron microscopy (EM), and dynamic light scattering (DLS). Antigenicity was evaluated using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) with prefusion-specific neutralizing antibodies. Immunogenicity and protective efficacy were evaluated in BALB/c mice following a prime-boost immunization, with analyses of humoral and cellular immune responses as well as post-challenge protection.Results: All three NP platforms successfully displayed the DS2 antigen while preserving its prefusion conformation. Notably, DS2-I53-50 demonstrated superior assembly quality and particle homogeneity relative to DS2-Fe and DS2-LuS. Compared to soluble DS2, all three DS2-NPs exhibited enhanced binding affinity (7- to 12-fold increase) to prefusion-specific antibodies (D25, AM14). In vivo, all DS2-NPs elicited higher levels of RSV-specific neutralizing antibodies and induced a more balanced Th1/Th2 immune response, with DS2-I53-50 generating significantly greater neutralizing antibody titers (1.7- to 2.4-fold increase) against both prototype RSV strains (LONG, 18537) and circulating genotypes (ON1, BA9). Immune cell profiling further revealed that all three DS2-NPs enhanced germinal center formation, facilitated follicular dendritic cell recruitment, and expanded memory T cell populations. Following RSV challenge, all DS2-NPs vaccines conferred significant protection, evidenced by accelerated weight recovery, reduced lung viral loads, and mitigated pulmonary pathology. Among them, DS2-I53-50 provided the most robust protection, achieving a 3.7-log reduction in viral titers and minimal lung pathology.Conclusion: NP platforms significantly enhanced the immunogenicity of RSV DS2 antigens, with DS2-I53-50 eliciting the strongest immune responses and protective efficacy. These findings underscore the potential of rationally designed NP-based vaccines for RSV. Keywords: respiratory syncytial virus, RSV, nanoparticle vaccine, ferritin, lumazine synthase, I53-50, prefusion F protein, immunogenicity, protective efficacy