Background
The emergence of novel reassortant influenza A virus strains and zoonotic transmission to the human population is a critical factor in the development of a global pandemic, as exemplified by the 2009 swine flu pandemic and the ongoing threat of an H5N1 avian flu pandemic. Small, unconjugated synthetic peptide vaccines comprising multiple, highly conserved epitopes may provide an important strategy towards a cost-effective, easily scalable, supra-seasonal universal influenza vaccine that can target across human, swine, and avian strains, advancing pandemic preparedness.

Objective
The aim of this study was to evaluate the immunogenicity of a synthetic, single, unconjugated, multi-epitope composite peptide vaccine targeting human, swine, and avian zoonotic viruses.

Methods
Outbred mice were immunized with different doses of the single peptide vaccine LHNVD-110, and immunogenicity was evaluated using ELISA, Hemagglutinin Inhibition Assay, Microneutralization Assay, and Enzyme-linked Lectin Assay.

Results
Strong IgG1 (Th2) and IgG2a (Th1) responses were generated against HA, NA, and Matrix epitopes, and multiple strains of influenza A/H1N1pdm09, A/H3N2, A/H5N1 (HPAI), and B/Victoria viruses. Cross-reactive, neutralizing antibodies also showed inhibition of multiple viral HA and NA subtypes, demonstrating the breadth of humoral responses.

Conclusion
This single, unconjugated, multi-epitope, composite peptide vaccine generated a robust and balanced immune response inhibiting multiple HA and NA subtypes across human, swine, and avian viruses. This approach provides a cost-effective, easily scalable, seasonal, and pandemic vaccine option with the potential of a universal influenza vaccine and efficient pandemic readiness.