Preclinical Evaluation Of A Split Influenza A/H7N9 Vaccine Mixed Mf59 Adjuvant

Part 1 The safety evaluation of a live attenuated recombinant influenza A/H7N9 seed strainBackgroundThe A/H7N9 influenza virus caused significant mortality and morbidity in humans during an outbreak in China in 2013.MetholdA recombinant A/H7N9 influenza seed with hemagglutinin(HA)and neuraminidase(NA)gene segments from A/Zhejiang/DTID-ZJU01/2013(H7N9)and six internal protein gene segments from A/Puerto Rico/8/34(H1N1;PR8)were generated using reverse genetics.The vaccine seed grows well in eggs.We sought to determine the genetic stability,virulence and transmissibility of this recombinant A/H7N9 vaccine seed strain to verify if it meets the requirements of an ideal vaccine seed.ResultWe successfully constructed A/H7N9 influenza vaccine seed strain A/ZJU01/PR8/2013,which has a good genetic stability and safety.The amino acid sequence was unchanged after several passages.Our results showed our recombinant A/H7N9 vaccine seed strain is safe and the virulence and transmissibility of it were decreased as compared to wild-type A/H7N9 virus,to levels comparable with PR8.We have selected this vaccine for future preclinical studies to protect animals from A/H7N9 virus infection.Part 2 Preclinical evaluation of a split influenza A/H7N9 vaccine mixed with MF59 adjuvantBackgroundDeveloping a safe and effective A/H7N9 influenza vaccine was initiated in early spring 2013,following human infections with a novel avian influenza A/H7N9 virus.MetholdIn this study,a candidate A/H7N9 vaccine seed strain is produced using reverse genetics,with HA and NA derived from a human A/H7N9 virus and the remaining genes from the PR8 backbone virus which grows well in eggs.It has a good genetic stability and safety,which meets the requirements of an ideal vaccine seed.Using the seed virus,we produced a monovalent split influenza A/H7N9 MF59-adjuvanted vaccine,which was immunogenic in mice.We sought to determine the immunogenic,protective properties,and mechanisms of a split influenza A/H7N9 vaccine mixed with MF59 adjuvant in comparison to vaccines that included other adjuvant.BALB/c mice were vaccinated with two doses of different amounts and combinations of this novel A/ZJU01/PR8/2013 split vaccine with adjuvant,then subsequently challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9)by intranasal inoculation.Furthermore,immunized BALB/c mice were followed to determine the duration of the protective immune response,monitor the dynamic changes and study possible mechanisms of the long-term immunogenicity and bioactivities.Previously vaccinated mice received an A/Zhejiang/DTID-ZJU01/2013 H7N9 challenge six months post-vaccination.Given that our A/H7N9 vaccine is selected for clinical investigation and potential human use,we performed acute toxicity,repeated dose toxicity and active systemic anaphylaxis tests to assess the safety of our A/H7N9 vaccine.ResultsWe verified that MF59 enhanced the immune response to influenza antigens.Only 1/5?1/10 HA antigen was required to achive the equial immune response reaction compared with the split vaccine,neutralizing antibody can be as high as 1024.Compared with split vaccine and aluminium adjuvant vaccine,MF59 could more potentially induce humoral immune responses and Th2 cytokine(IL-4,IL-5,IL-6,and IL-10)production after virus infection.This split A/H7N9 influenza vaccine with MF59 adjuvant could effectively induce antibody production and protect mice from A/H7N9 virus challenge.However,MF59 adjuvant and alum are poor inducers of Thl responses,NK activities and CTL activities.Long-term immunogenicity reasrech shows antibody levels decreased to between one-half and one-eighth of the peak values four months after the final dose of the vaccination.We also verified that MF59 enhanced the HI,MN,and IgG antibody titers to wide A/H7N9 virus six month after vaccination.The humoral immune response and Th2 cytokine(IL-4,IL-5,IL-6,and IL-10)production following influenza challenge was potently induced in the animals that received the split vaccine especially with the MF59 adjuvant.This vaccine could e?ectively induce antibody production and protect mice from A/H7N9 virus challenge even after six months.Toxicological experiments confirmed that the experimental animals did not exbit the vaccine related clinical symptoms such as body weight,body temperature,food intake,ophthalmic examination,blood coagulation,urine analysis and local tolerance(the injection site).Acute toxicity experiment comfirmed the clinical pathology change(spleen)and the following hematology changes:the rise of eosinophils and neutrophils,the decrease of albumin and the rise of globulin.Thoses changes were recovered in the repeated dose toxicity test.These recoverable hematologic and clinicopathologic changes occured in adjuvanted vaccines were due to the immune response to adjuvants and their adaptation to inflammation,which was acceptable.Under the conditions used in this study,the NOEAL(no obvious adverse effect level)was as high as 30 ug/0.5 ml.