National Institutes of Health (R01DK061315). Polyvalent DNA Vaccines Expressing HA Antigens of H5N1 Influenza Viruses with an Optimized Leader Sequence Elicit Cross-Protective Antibody Responses Highly pathogenic avian influenza A (HPAI) H5N1 viruses are circulating among poultry populations in parts of Asia, Africa, and the Middle East, and have caused human infections with a high mortality rate. H5 subtype hemagglutinin (HA) has evolved into phylogenetically distinct clades and subclades based on viruses isolated from various avian species. Since 1997, humans have been infected by HPAI H5N1 viruses from several clades. It is, therefore, important to develop strategies to produce protective antibody responses against H5N1 viruses from multiple clades or antigenic groups. In the current study, we optimized the signal peptide design of DNA vaccines expressing HA antigens from H5N1 viruses. Cross reactivity analysis using sera from immunized rabbits showed that antibody responses elicited by a polyvalent formulation, including HA antigens from different clades, was able to elicit broad protective antibody responses against multiple key representative H5N1 viruses across different clades. Data presented in this report support the development of a polyvalent DNA vaccine strategy against the threat of a potential H5N1 influenza pandemic. The continuous spread of highly pathogenic avian influenza Type A (HPAI) H5N1 viruses in avian species across multiple continents and frequent reports of human H5N1 infection in China and Southeast Asia highlight the threat of a potential flu pandemic in the human population. At the same time, H5N1 viruses have grown into genetically and antigentically diversified viruses. Based on phylogenetic analysis of hemagglutinin (HA) protein gene sequences, at least 10 clades of H5N1 viruses (clades 0-9) have been identified [1, 2, 3, 4, 5] . Recent studies have further assigned these viruses into four major antigenic groups (A-D) [3] . HPAI H5N1 viruses from more than one clade have caused human infection since 1997.