Rescue And Immunogenic Evaluation Of Attenuated Vaccine Candidate From A/California/07/2009

H1N1influenza virus is a type influenza virus, which can cause acute respiratoryinfectious diseases and even death. Vaccines and drugs are the main measures to controlthese diseases. Compared with the medicines, vaccines are still the primary means tocontrol the spread of influenza due to those characteristics.There are both inactivated and live attenuated influenza vaccines. Compared withinactivated influenza vaccines that only induce better humoral immunes, live attenuatedinfluenza vaccines induce not only good humoral immunes, but also better cellularimmunes and mucosal immunes. In recent years, studies have shown that cellular andmucosal immunes play important roles to prevent the spread of influenza viruses andcellular immunes enhance the vaccine protective effects. There are only inactivatedvaccines available in China. Therefore, It is an urgent problem to develop independentlylive attenuated influenza vaccines.In this study, cold-adapted, temperature-sensitive, attenuated A/California/07/2009ca(CA/AA ca) influenza virus was obtained by reverse genetic technology. Six internal genes(PB1, PB2, NP, NA, M&NS) of the virus A/Ann Arbor/6/60ca (H2N2) influenza virusand two antigenic genes (HA&NA) of the WHO recommended H1N1vaccine strainA/California/07/2009were constructed to bi-directional expression vector pAD3000andco-transfected into MDCK+COS1cells. Biological characteristics, type, phenotype ofreassortant virus strain CA/AA ca were identified by sequencing, neutralization test, IFA.Virus seed stocks were produced in the chicken and proved qualified by sterility test,mycoplasma detection, exogenous factor detection, et al. Humoral, cellular, mucosalimmunes and protective efficacy of CA/AA ca influenza virus were evaluated in mice andferrets by TCID50, ELISA, MTT and HI.The study was divided into three parts:1.Rescue and identification of candidate vaccine virus strain: CA/AA ca influenzavirus was rescued by RG, identified by RT-PCR, HI, indirect immunofluorescence, electronmicroscopy and SDS-PAGE, which proved that it was A/California/07/2009subtypes withcold-adapted, temperature-sensitive phenotype.2. Establishment, identification of seed lots and purification of this virus: Two seedbanks were established, with1:256hemagglutination titers and without bacteria, mycoplasma or exogenous factors. The virus was inoculated by105dilution into chickenembryos and incubated at33℃for72h. The virus was purified by the sucrose densitygradient centrifugation and assayed by HPLC, EM, TCID50, and so on. The CA/AA cavirus of high purification level were ultimately achieved.3. Evaluation of the immune responses induced by the vaccine: the TCID50titer of CA/AA ca virus was increased by1.25with TPCK trypsin. The titer of purified CA/AA cavirus was9.3lg TCID50/20uL, and BJ5017.5lgTCID50/20uL. Optimal dosage of CA/AA ca virus was1×106TCID50/20uL.Compared with HI titers of BALB/c mice controls that intramuscularly immunizedwith inactive A/California/07/2009virus, those of which intranasally immunized with CA/AA ca virus was lower (p<0.05) and IgA titer was higher (p<0.01), which indicated thatintranasal immunization can evoke higher mucosal immune responses, but lower humoralimmune responses. Compared with the controls, the IL-4and IFN-γ levels of live vaccinewere higher, but with IL-6level of no significance.After BALB/c mice were immunized with CA/AA ca virus, the low titers of viruswere detected in mice nose and lungs within4days. The high titers of BJ501virus in micenoses and lungs were detected after BJ501challenged, with100%mortality rate in9dayspost infection. By2weeks after the second intranasal immunization, after BJ501virus waschallenged, the BJ501virus were not detected in the mice nose, lungs, spleen, kidney andbrain, with100%survival rate.Ferrets were intranasally challenged with CA/AA ca and BJ501virus, respectively.There were no physiological changes in ferrets infected with CA/AA ca, but ferretsinfected with BJ501have clinical flu infection symptoms. The virus was detected in nose,lungs, brain at4days after challenge. Ferrets sera that immunized with CA/AA ca virusneutralized the CA/AA ca virus and BJ501, with no difference between the two viruses(p>0.05). BJ501virus was detected in the nose and lungs of CA/AA ca immunizedmice within3days after BJ501challenge.Conclusion: We successfully rescued the cold-adapted, temperature-sensitive,attenuated the CA/AA ca virus, established master and working seed lots，determinedincubation temperature, time and inoculation concentration, and purified the CA/AA cavirus by Pharmacopoeia2010requirements.Compared with A/California/07/2009virus, the results showed that CA/AA ca viruscan induce good humoral, cellular and mucosal immunes and better immune protection inmice, so do ferrets. In conclusion, CA/AA ca virus was an attenuated vaccine candidate strain.