Chimeric designs of rabies virus glycoprotein G enhance baculovirus pseudotyping and immunogenicity in mice: influence of the transmembrane domain and a flexible linker

Rabies remains a lethal zoonotic disease, affecting approximately 50,000 people annually, underscoring the need for new and cost-effective vaccines capable of elicit robust immunity. Baculoviruses have emerged as promising vaccine vectors due to their ability to display antigens on their surface, making pseudotyping with rabies virus glycoprotein G (gG) a viable strategy for vaccine development. However, strategies to optimize baculovirus pseudotyping remain underexplored. In this study, we evaluated three chimeric gG designs and found that fusion to the gp64 transmembrane domain via a flexible linker significantly enhanced both baculovirus pseudotyping and the resulting immune response. The chimeras consisted of the gG ectodomain fused to the gp64 transmembrane domain either directly (gG-AN) or via a flexible linker (gG-FL), along with a variant retaining its native transmembrane domain (gG-TM). Notably, baculoviruses pseudotyped with gG-FL and gG-AN achieved the highest levels of gG incorporation into viral particles, while gG-TM impaired baculovirus replication. All pseudotyped baculoviruses elicited neutralizing antibodies against rabies virus; however, gG-FL induced the strongest immune response, as evidenced by full activation of bone marrow-derived dendritic cell markers, higher neutralizing antibody titers, and increased IFN-γ production. This enhanced immunogenicity correlated with greater protection following rabies virus challenge, with gG-FL baculovirus-immunized mice achieving the highest survival rates. Moreover, an adjuvanted gG-FL formulation showed an immunological potency of 4.65 IU/dose. These findings highlight the importance of transmembrane domain selection and flexible linker incorporation in optimizing baculovirus pseudotyping with gG and enhancing its immunogenicity against rabies virus. This strategy could inform the design of future baculovirus-based vaccines.