High epitope density in a single recombinant protein molecule of the extracellular domain of influenza A virus M2 protein significantly enhances protective immunity

The degree of epitope density has been shown to be a critical factor influencing the magnitude of epitope-specific responses. However, whether high epitope density in just a single protein molecule can still enhance the humoral response or, more importantly, the protective immunity, has not been determined. To test this, five glutathione-S-transferase fusion proteins bearing various numbers of copies of the M2e epitope on M2 protein of influenza virus (1, 2, 4, 8 and 16 copies) were prepared, and used to immunize mice and rabbits. Our data show clearly that M2e-specific humoral response was enhanced with increasing epitope density. By lethal challenge assay in mice, it was observed that recombinant proteins with higher M2e epitope densities resulted in higher survival rates and slower weight losses. The survival rate was directly related to the degree of epitope density in the single recombinant protein: 100% in the case of 16 M2e copies; 50% with 4 M2e epitopes; and 0% with one.