Development Of Universal H1N1 Influenza Virus Vaccine And Preparation Of Vero Cell Vaccine Strain

Influenza virus is the pathogen that caused several influenza pandemic in history and is also responsible for seasonal influenza epidemics. Influenza virus belongs to the family of Orthomyxoviridae, and could be classified into 3 genera(A, B, C). influenza A virus is the main pathogen that severe illnesses both in human and in many animal species. Each year, influenza virus causes pathogeneses or even deathes in the globe. Influenza virus evolves and mutates rapidly, leading to the failure of each year’s vaccination. So researches on universal influenza vaccines become very hot in recent years.H1N1 influenza subtype is one of the major causes of seasonal influenza. H1N1 influenza virus has the character of strain diversity and high sequence variations between sifferent strains. Traditional influenza vaccine is unable to defense our bodies against all virus strains. We have previously designed a universal HA protein sequence(CH1-1) based on “concensus sequence + conserved epitopes” strategy. We tested its ability in inducing universal immune responses in mice with DNA vaccine in the present study. After immunization, mice sera were tested for their ability to neutralize several H1N1 influenza virus strains——SC09, NC99 and FM47. And immune T cells were tested for their ability to cross-react with these virus antigens. CH1-1 induced mice sera have strong neutralizing activity against NC99 and FM47, but not against SC09. Immune T cells can cross-react with all of these viruses with high IFNγ secretions in in vitro restimulation. To obtain neutralizing antibodies against SC09, we added a plasmid that expresses HA protein of SC09 to the immunization, a combined antigen of CH1-1 and SC09-HA. After immunization, the mice sera obtained the ability to neutralize SC09 virus. The immune T cells secreted higher levels of IFNγ in in vitro restimulation with the 3 virus antigens. Moreover, the mixed plasmid vaccination protected mice against lethal challenge with SC09, NC99 and FM47 viruses, with little lung lesion 4 days post challenge, and with no viruses retained in the lung 14 days after challenge. So the mixed vaccine “p CH1-1+p SC09-HA” has the ability to induce universal immune responses against various H1N1 influenza strains. We then rescued the reassortant PR8-CH1-1 virus, and found that it is a naturally attenuated virus to mice. The PR8-CH1-1 virus was tested for its cross-reactivity with SC09 and NC99 by intranasal challenge with lethal doses of the two viruses after intranasal vaccination with PR8-CH1-1. The result showed that all vaccinated mice were protected well, with no signs of weight loss and with less lung lesion, compared with that of the PBS immunized control mice. This substantiates that PR8-CH1-1 is protective against various H1N1 influenza strains.Influenza A virus is one of the major threats to human health. Vaccination is the most effective way to prevent influenza A virus infection. We have previously obtained a Vero-cell high-yield influenza A virus vaccine donor strain through genetic mutations of the virus genes. The present study was to verify if this high-yield donor strain can support the growth of the newly emerged H7N9 influenza A virus in Vero cells to obtain a H7N9 vaccine strain. The recombinant virus, 4mut-H7N9 and PR8-H7N9, were rescued using influenza A virus reverse genetics in which the HA and NA genes of H7N9 were combined with the 6 internal genes of PR8 virus to rescue PR8-H7N9, or with the 6 internal genes of PR8-4mut to rescue 4mut-H7N9 virus. The growth of 4mut-H7N9 virus was comparied with that of PR8-H7N9 virus by growth curves and plaque morphology. The production of viral proteins from 4mut-H7N9 virus was compared with that of PR8-H7N9 by Western blot analysis and Coomassie blue staining. The PR8-H7N9 and 4mut-H7N9 viruses were successfully rescued. The virus titer of 4mut-H7N9 was about 3000 times higher than that of PR8-H7N9 at 72 hours post infection. The 4mut-H7N9 virus can form plaques of about 1mm in diameter on Vero cells, while the PR8-H7N9 virus only formed pinpoint plaques on Vero cells. The proteins levels of purified 4mut-H7N9 virus were significantly higher than those of the PR8-H7N9 virus detected by both Western blot analysis and Coomassie blue staining. Moreover, the 4mut-H7N9 virus retains the dependence of Trypsin to infect cells, ensuring its safety in vaccine production. So we got the conclusion that the reassortant 4mut-H7N9 vaccine strain rescued by reverse genetics, grows faster and better in Vero cells, which can be used as a candidate vaccine strain when facing the potential epidemic of H7N9 viruses.