| Vaccination is one of the most effective ways to combat influenza. In China, split, inactivated influenza vaccines and subunit vaccines are used. Live attenuated influenza vaccines (LAIVs) have considerable advantages over inactivated vaccines because of intranasal delivery, inducing mucosal and cellular immunity besides humoral immunity. While live attenuated influenza vaccines are produced exclusively in Russia and America, and live attenuated influenza virus strains are A/Ann Arbor/6/60(H2N2) and B/Ann Arbor/1/66, A/Leningrad/134/17/57(H2N2) and B/USSR/60/69, respectively. LAIV was first licensed for use in the US in2003, named as Flumist(?). It was available only for healthy individuals aged5-49years in2003and approved for use in children2years and older since2008. However, LAIVs were produced in embryonated chicken eggs. Egg-based vaccines have risks of egg contamination and spread of contagious disease of poultry. Meanwhile, the limited capacity of the egg-dependent vaccine supply could be problematic in terms of securing enough doses when facing a pandemic situation, such as occurred in2009, or in the event of a pandemic originating from a highly pathogenic avian virus, such as the H5N1virus. Given all this, it is recommended to substitute continuous mammalian cell lines for eggs to produce influenza vaccines. African green monkey Vero cells are approved by the World Health Organization as a safe substrate for vaccine production for human use. Most influenza virus strains grew poorly in Vero cells. So far, there are only there Vero cell-based influenza vaccines produced in a bio safety level3facility. Vepacel(?)(Baxter) is a pre-pandemic influenza vaccine against the H5N1subtype of influenza A (A/Vietnam1203/2004) and was approved in all European Union Member States. Celvapan(?)(Baxter) is a whole-virion, Vero cell-derived, inactivated pandemic influenza vaccine containing A(H1N1)pdm09virus and was approved in Europe. Preflucel(?)(Baxter) is a Vero cell-derived, seasonal trivalent split, inactivated vaccine, the phase3trial of which was performed in healthy adults (age18-49years) in the US. Vero cell-based LAIVs are not available yet. It is critical to culture cold adapted virus strains in Vero cells for the development of Vero cell-based LAIVs. Only one cold adapted virus strain in Vero cells, A/Singapore/1/57ca, was reported. Fortunately, on the basis of the Vero cell-adapted strain A/Yunnan/1/2005Va(H3N2) in our lab, one cold adapted virus strain in Vero cells was developed by gradient cooling and cultured for50serial passage at25℃. In this study, the strain A/Yunnan/1/2005Vca(H3N2)P50was cultured for another22serial passage at25℃. As a result,50%Tissue Culture Infectious Dose (TCID50) per milliliter was from10"’to10-7.5, and log10TCID50/mL at25℃,33℃,39℃were7.5,7.7,3.7, indicating cold adaptation (ca) and temperature sensitivity (ts) phenotypes of the strain. Ferrets were inoculated i.n. with10’TCIDso Vca, and logio TCID50/g tissue of turbinates and lungs were2.6±0.3and1.4±0.1, which demonstrated that the cold adapted virus strain in Vero cells exhibited attenuation (att) phenotype. In hemagglutination inhibition test, the HI titer with H3N2anti-serum was1:1280without cross-reactive titers with H1N1、Bv and By anti-serum. Nucleotide alignment betweenVcaP50and VcaP70, showed that there is no missense mutations.It is crucial to determine the genes related to cold adaptation for development of cold adapted virus strain in Vero cells and LAIVs. It was reported mostly that segments PB2, PB1and PA were related to cold adaptation. In the study, nucleotide and protein alignments between Va and Vca strains were performed, and then3D structures of them were analyzed, which indicated that PB1was the possible segment. PB1plasmids were exchanged to rescue virus Vca/VaPB1and Va/VcaPB1by reverse genetic technology. Rescued viruses were cultured in Vero cells for4serial passages. The log10TCID50/mL of Vca/VaPBl at25℃and33℃were7.1and3.8, while that of Va/VcaPBl were7.4and6.5. It was demonstrated that the gene PB1was related to cold adaptation in Vero cells.It is widely believed that the emergence of a new influenza pandemic caused by avian strains is only a matter of time and that a safe, effective and easily manufactured vaccine is required. The Virus stain A/Yunnan/1/2005Vca (H3N2), as a donor strain, may be reassortment with highly pathogenic avian influenza (HPAI) viruses to produce LAIV seeds for influenza pandemic. There existed biosafety risks to use live viruses on evaluating immunological effects. It may be preferable to substitute pseudotyped viral particles (pp) for live viruses. In the study, pp of A(H1N1)pdm09(A/California/7/2009) and HPAI H5N1(A/Anhui/1/2005) were generated. Pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. The difference between pp and the corresponding viruses, was not statistically significant. Moreover, the positive rate of H5N1antibody was2.1%in population sera detection, and GMT (95%CI) was17.82(8.72,36.43), and the positive rate of H1N1antibody was 53.5%and GMT (95%CI)25.78(22.09,30.08). In conclusion, pp was reliable in neutralizing antibody detection. |
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