Background: Rabies remains a fatal zoonotic disease, necessitating effective and affordable vaccines. While current vaccines are effective, they require multiple doses and may not induce long-lasting immunity in all settings. The rabies virus glycoprotein (RABV-G) is the principal antigen responsible for eliciting virus-neutralizing antibodies, but its recombinant monomeric forms often suffer from poor immunogenicity due to misfolding and aggregation. Methods: A recombinant trimeric RABV-G ectodomain (rRABV-G-XVIII) was engineered by fusing it to a human collagen XVIII-derived trimerization domain. The protein was expressed in E. coli, purified under denaturing conditions, and refolded. Trimer formation was verified using size-exclusion chromatography. Mice were immunized with rRABV-G-XVIII, with or without adjuvant, and compared to a monomeric form (rRABV-GE). Antigen-specific antibody responses were measured by ELISA, neutralizing activity was assessed, and protective efficacy was evaluated via intracerebral challenge with the CVS-27 rabies strain. Results: rRABV-G-XVIII formed stable trimers and induced strong humoral immune responses, with high ELISA titers and virus-neutralizing activity comparable to an inactivated rabies vaccine. Mice immunized with rRABV-GE showed lower antibody responses and partial protection, which improved with adjuvant. All rRABV-G-XVIII-immunized mice were fully protected against rabies challenge, independent of adjuvant use. Conclusions: Stabilization of RABV-G in its native trimeric conformation markedly improves immunogenicity and protective efficacy. This approach offers a promising strategy for the development of rabies subunit vac-cines with simplified formulations and potential for cost-effective production in bacterial systems.

Rabies remains a serious global public health concern. The envelope glycoprotein of the rabies virus (RABV-G) is the principal viral antigen and harbors critical neutralizing epitopes, making it a promising target for subunit vaccine development and diagnostic applications. Nevertheless, the efficient production of recombinant RABV-G with preserved antigenic and immunogenic properties remains technically demanding. In this work, we developed an optimized strategy for the expression and purification of recombinant RABV-G and assessed its antigenicity and immunogenicity. The rabies virus glycoprotein ectodomain (rRABV-GE) was successfully expressed in Escherichia coli Rosetta2(DE3)pLysS and purified in a biologically relevant form. The rRABV-GE gene was codon-optimized and cloned into the pET-28c(+) expression vector, resulting in recombinant protein expression largely in an insoluble state. Consequently, the protein was subjected to refolding and purification procedures. To improve protein solubility, hydrophobic loop regions were substituted with flexible linker sequences. Protein purification was performed under denaturing conditions using urea, followed by affinity chromatography. The identity and antigenicity of the purified protein were confirmed by Western blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). ELISA testing was performed using rabbit sera collected from animals immunized with the commercial Purified Vero Cell Rabies Vaccine (RHABDOVAC-1). Collectively, these findings indicate that the optimized bacterial expression platform enables the production of biologically relevant rRABV-GE and provides a cost-effective approach for the development of rabies subunit vaccines and diagnostic reagents. In summary, the recombinant rRABV-G protein produced in this study appears to be a suitable diagnostic antigen for ELISA-based and other rabies virus immunodiagnostic uses. Keywords: Rabies virus, glycoprotein ectodomain, antibody, ELISA, Rosetta2(DE3)pLysS, vaccine.