A Multi-Epitope Chimeric Hemagglutinin Vaccine Elicits Cross-Protective Immunity against Avian Influenza H5N8 and H9N2 Subtypes in Poultry

Bozorgkhoo, Zahra; Hemati, Behzad; Taghizadeh, Morteza; Tebianian, Majid; Ghaderi, Mostafa

doi:10.22092/ari.2025.368770.3564

A Multi-Epitope Chimeric Hemagglutinin Vaccine Elicits Cross-Protective Immunity against Avian Influenza H5N8 and H9N2 Subtypes in Poultry

Articles in Press

Document Type : Original Articles

Authors

¹ Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran

² Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

10.22092/ari.2025.368770.3564

Abstract

Avian influenza viruses, notably H5N8 (HPAI) and H9N2 (LPAI), threaten public health due to zoonotic potential and genetic adaptability. Avian influenza viruses circulate in poultry and can sometimes directly infect humans after contact with infected birds. Some existing vaccines have proven useful in controlling these severe infections. Traditional vaccines targeting variable hemagglutinin (HA) head domains necessitate frequent updates. This study designed a chimeric HA (cHA H9/H5) vaccine targeting conserved epitopes from H5N8 and H9N2 HA proteins to elicit broad immunity.
A multi-epitope construct, integrating B-cell and cytotoxic T lymphocyte epitopes linked via stabilizing sequences (KK, AAY, GPGPG), was a codon-optimized construct, expressed in E. coli BL21(DE3), and purified (>95% purity, 22 mg/L yield). Specific pathogen-free chickens (n=20/group) received two doses of cHA H9/H5 formulated with Alum or freund’s adjuvants, compared to commercial H5N8/H9N2 vaccines. Humoral responses were assessed via hemagglutination inhibition (HI) assays.
The cHA H9/H5 vaccine induced robust HI titers against homologous H9N2 (log₂ GMT 10–12) and hetero-subtypic H5N8 (log₂ GMT 6–8), surpassing commercial vaccines. While H9N2 vaccines lacked cross-reactive H5N8 antibodies, cHA H9/H5 elicited neutralizing titers (20–80) against H5N8. Freund’s adjuvant enhanced immunogenicity significantly, with sustained post-boost antibody levels.
These results highlight the cHA H9/H5 vaccine’s ability to overcome strain-specific limitations by targeting conserved epitopes, inducing cross-reactive immunity critical for pandemic preparedness. Adjuvant selection proved pivotal in optimizing responses, aligning with prior chimeric HA vaccine research. This study advances the development of universal influenza vaccines, offering a promising strategy to mitigate risks posed by different evolving avian influenza variants.

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