EP_073. Design and expression of a multiepitope recombinant respiratory syncytial virus in Ogataea polymorpha for mucosal vaccine development

Nor Aziyah Mat-Rahim^1*, Khairul Nizam Mohd Khalid², Mohd Ishtiaq Anasir¹, Siti Nur Zawani Rosli³,Helmi Husaini Zainal Fithri⁴, Ernie Zuraida Ali²

1. Virology Unit, Infectious Diseases Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Shah Alam, Selangor, Malaysia

2. Inborn Error of Metabolism and Genetic Unit, Nutrition, Metabolism and Cardiovascular Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Shah Alam, Selangor, Malaysia

3. Bacteriology Unit, Infectious Diseases Research Centre, Institute for Medical Research, National Institutes of Health, Ministry of Health Malaysia, 40170 Shah Alam, Selangor, Malaysia

4. Research Unit for Bioinformatics and Computational Biology (RUBIC), Kuliyyah of Science, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia

*Corresponding author: Nor Aziyah Mat-Rahim. This email address is being protected from spambots. You need JavaScript enabled to view it.

ABSTRACT

INTRODUCTION: Respiratory syncytial virus (RSV) is a leading cause of paediatric respiratory infections and poses significant risks to elder adults, especially those with underlying medical conditions. Vaccines for the virus are available, however, current vaccines are administered parenterally (intramuscularly). As the virus infects via the mucosal route, a mucosal vaccine will provide broader and more effective protection against the infection. Here, we leverage the yeast expression system that offers a scalable and cost-effective platform to produce a multiepitope gene for mucosal vaccine development.

OBJECTIVE(S): This study aimed to (1) design a multiepitope antigen targeting conserved domains of the RSV glycoprotein (G), and (2) express it in Ogataea polymorpha (O. polymorpha) using a methanol-inducible promoter.

MATERIALS & METHODS: The multiepitope genes were identified using reverse vaccinology and validated for antigenicity using VaxiJen 2.0. The structural stability was assessed via molecular modelling, docking, and molecular dynamics simulations. The designed gene was codon-optimised for O. polymorpha and cloned into the pHIPH4 vector (methanol-inducible) and transformed into O. polymorpha. Transformants were selected via hygromycin resistance, and protein expression was confirmed by SDS-PAGE and immunostaining analysis.

RESULTS: The multiepitope gene encoding three antigenic domains, connected by linkers, was successfully designed and transformed into O. polymorpha. The expression of multiepitope RSV protein in O. polymorpha under methanol induction has been demonstrated and the binding of the multiepitope recombinant protein towards specific anti-RSV antibody confirms its antigenic potential.

CONCLUSION: We have demonstrated the potential of yeast-expressed multiepitope antigen as a promising candidate for an effective mucosal RSV vaccine. O. polymorpha offers unique advantages over conventional yeast systems (Saccharomyces cerevisiae and Komagataella phaffii), including high protein yield, stable genomic integration, and favourable post-translational modifications. Future work will focus on the formulation, immunogenicity testing, and preclinical efficacy studies. 

KEYWORDS: Respiratory syncytial virus, Mucosal vaccine, Ogataea polymorpha, Multiepitope, Thermostability