Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice

Fan, C.; Cohen, A. A.; Dam, K.-M. A.; Rorick, A. V.; Priso Fils, A.-C. I.; Yang, Z.; Gnanapragasam, P. N.; Segovia, L. N.; Huey-Tubman, K. E.; Moon, W. J.; Lin, P. J.; Bjorkman, P. J.; Hoffmann, M. A.

doi:10.1101/2025.08.18.670983

bioRxiv · 10.1101/2025.08.18.670983

Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice

Fan, C.·Cohen, A. A.·Dam, K.-M. A.·Rorick, A. V.·Priso Fils, A.-C. I.·Yang, Z.·Gnanapragasam, P. N.·Segovia, L. N.·Huey-Tubman, K. E.·Moon, W. J.·Lin, P. J.·Bjorkman, P. J.·Hoffmann, M. A.

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Abstract

mRNA vaccines emerged as a leading vaccine technology during the COVID-19 pandemic. However, their sustained protective efficacies were limited by relatively short-lived antibody responses and the emergence of SARS-CoV-2 variants, necessitating frequent and variant-updated boosters. We recently developed the ESCRT- and ALIX-binding region (EABR) mRNA vaccine platform, which encodes engineered immunogens that induce budding of enveloped virus-like particles (eVLPs) from the plasma membrane, thereby resulting in presentation of immunogens on cell surfaces and eVLPs. Prior studies showed that spike (S)-EABR mRNA-LNP immunizations elicited enhanced neutralizing antibody responses against ancestral and variant SARS-CoV-2 compared with conventional S mRNA-LNP in naive mice, but the effectiveness of S-EABR mRNA-LNP boosters in the context of pre-existing immunity has not been investigated. Here, we evaluated monovalent Wuhan-Hu-1 (Wu1) and bivalent (Wu1/BA.5) S-EABR mRNA-LNP boosters in mice pre-vaccinated with conventional Wu1 S mRNA-LNP. Compared to conventional S mRNA-LNP boosters, the EABR approach enhanced monovalent and bivalent mRNA-LNP booster-induced neutralizing responses against Omicron subvariants BA.1, BA.5, BQ.1.1, and XBB.1, with bivalent S-EABR mRNA-LNP consistently eliciting the highest titers. Epitope mapping of polyclonal antisera by deep mutational scanning revealed that bivalent S-EABR mRNA-LNP boosted diverse "polyclass" anti-receptor-binding domain (RBD) responses, suggesting balanced targeting of multiple RBD epitope classes. In contrast, monovalent S, bivalent S, and monovalent S-EABR mRNA-LNP boosters elicited less diverse polyclonal serum responses primarily targeting immunodominant RBD epitopes. Cryo-EM structures demonstrated that bivalent mRNA immunizations promote S heterotrimer formation, potentially enhancing bivalent S-EABR mRNA-LNP booster-induced antibody breadth and polyclass epitope targeting by activating cross-reactive B cells through intra-S crosslinking. These findings support the future design of bivalent or multivalent S-EABR mRNA-LNP boosters as a promising strategy to confer broader, and therefore potentially more durable, protection against emerging SARS-CoV-2 variants and other rapidly evolving viruses.

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Fan, C., Cohen, A. A., Dam, K.-M. A., Rorick, A. V., Priso Fils, A.-C. I., Yang, Z., Gnanapragasam, P. N., Segovia, L. N., Huey-Tubman, K. E., Moon, W. J., Lin, P. J., Bjorkman, P. J., Hoffmann, M. A.. 2025-08-19. Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice. https://doi.org/10.1101/2025.08.18.670983

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