Essential Sequence Of Influenza Virus Hemagglutinin And Chimeric Hemagglutinins DNA Vaccine To Provide Protection Against Lethal Viral Infection

1.Essential Sequence of Influenza B Virus Hemagglutinin DNA to Provide Protection against Lethal Homologous Viral InfectionHemagglutinin(HA) is the main surface glycoprotein of influenza B virus.The B/Ibaraki/2/85 virus HA gene is 1758 bp in length including signal peptide sequence, HA1 sequence and HA2 sequence.We previously proved that B/Ibaraki/2/85 HA DNA induced immune response and provided effective protection in mice against challenge with homologous virus.In this study,a series of recombinant plasmids encoding truncated HA gene were constructed by PCR.BALB/c mice were immunized with the plasmids and challenged with a lethal dose of homologous virus. The essential sequence of HA DNA against influenza virus was explored by evaluation of survival rate,lung virus titer,bodyweight change and serum anti-HA antibody titer of mice.The result showed that serial deletion didn't deprive HA DNA of its protective ability until 885 nucleotides(295 amino acids) at 3'-terminal or 9 nucleotides of the signal peptide sequence at 5'-teriminal were deleted.When the signal peptide sequence was kept intact and the 5'-terminal deletion started at the beginning of the HA1 sequence,deletion of 51 nucleotides(17 amino acids) made HA DNA lose its protective ability.This suggests that the sequence nt94-876 of B/Ibaraki/2/85 virus HA DNA played an important role in protection against infection.2.Study on the Effectiveness of the Chimeric DNA Vaccine of Influenza A and B VirusWe previously proved that HA1 and essential sequences of influenza A and B virus HA DNA could provide protection against challenge with homologous virus. The HA1 or essential sequences fragments of influenza A and B virus HA genes (nt1-nt826 of A/PR/8/34 HA gene or nt1-876 of B/Ibaraki/2/85 HA gene) were cloned together into an expression vector to construct a chimeric DNA vaccine.Mice were immunized twice intramuscularly by electroporation with the constructed plasmid. One week after the booster,the mice were challenged with lethal dose of homologous type A or B virus.The effectiveness of the chimeric DNA was evaluated by the antibody response and the protective abilities,including the survival rate,lung virus titer and body weight change.The results showed that the immunized mice could be protected against not only homologous A virus but also B virus,indicating the ability of the chimeric DNA to provide cross protection.3.Essential Sequence of avain influenza HSN1 virus Hemagglutinin DNA to Provide Protection Against Lethal Homologous Viral InfectionWe construct mice adapted model of avian influenza virus A/Chi cken/JiangSu/01/ 2002(H5N1) by passage virus lung to lung several times.Firstly,we study on the effectiveness of H5 HA-DNA vaccine and inactivated vaccine or with combination with them.The results showed there was no significant difference of protection effectiveness between mice immunized with inactivated vaccine and HA-DNA.Then, a series of recombinant plasmids encoding truncated HA gene were constructed by PCR,including fragment deleted at 3'-terminal and 5'-terminal(signal peptide sequences reserved).BALB/c mice were immunized with the plasmids and challenged with a lethal dose of homologous virus.The essential sequence of HA DNA against influenza virus was explored by evaluation of survival rate,lung virus titer,bodyweight change and serum anti-HA antibody titer of mice.The serial deletion didn't deprive HA DNA of its protective ability until 849 nucleotides(283 amino acids) at 3'-terminal.When the signal peptide sequence was kept intact and the 5'-terminal deletion started at the beginning of the HA1 sequence,deletion of 81 nucleotides(27amino acids) made HA DNA lose its protective ability.This suggests that the sequence nt127-861 of H5N1 mice adapted virus HA DNA played an important role in protection against infection.