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This is a Quantitative Study study.

September 17, 2025Open Access

Immunogenicity and Antigenicity of the Recombinant Ectodomain of Rabies Virus Glycoprotein Containing the Human Collagen XVIII Trimerization Domain

ISIzat SmekenovAl-Farabi Kazakh National University GBG.A. BayandyAl-Farabi Kazakh National University SASanzhar AlybayevAl-Farabi Kazakh National University

Key Points

Trimeric rabies glycoprotein led to strong humoral immune responses in mice, achieving complete protection against rabies.
Mice immunized with the recombinant protein had higher ELISA titers and neutralizing activity than those with the monomeric form.
The method involved protein expression in E. coli and verification of trimer formation through size-exclusion chromatography.
This strategy for rabies vaccine development may lead to cost-effective production and simplified formulations.

Abstract

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.

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