Rg Technology H5n1 Highly Pathogenic Avian Influenza Vaccine Strain Preparation And Preliminary Evaluation

Recent outbreaks of highly pathogenic avian influenza,H5N1.(HPAI H5N1)in 15 countries and regions and associated human infections in Indonesia have ledto a heightened level of awareness and preparation for a possible influenzapandemic.WHO has predicted that a new influenza pandemic might occur in thenear future. Effective vaccines against H5N1 virus are, therefore, urgentlyneeded.Currently, HPVI type A virus(H5N1) mainly scatters in Asia, especially inIndonesia, Vietnam and China. The wild type virus isolated from human andvarious animals display the high variation by serology, pathogenicity and genestructural analysis. The viruses isolated in China belong to two clades. In this case,it is essential for mainland China to strengthen studies on the isolation specificpandemic influenza vaccine strain and development of more effective and safervaccines by novel approaches.Reverse genetics systems entirely based on cloned cDNAs of influenza viruswhose genome is negative-sense and eight-segmented have been established byNeumann, Hoffmann, et al. since 1999, and these developments have implied theperspective future of virus research. In this representation, an AIV isolate of H5N1subtype, A/xinjiang/1/2006 (H5N1), from mainland China was chosen to determinethe entire genomic sequence and characterize polygenetically. The six cDNAs fromPR8 were cloned into the bidirectional transcription/expression vector pHW2000, anda panel of reassorted viruses containing HA and NA genes from A/xinjiang/1/2006(H5N1) were generated by reverse genetics technique.Attenuated H5N1 recombinantwith modified HA genes were generated by using this technique at last, and thenassayed this reassortant as a pandemic influenza vaccine strain. The process ofrescuing pandemic influenza vaccine strain includes 3 steps,the modifying of HAgene of A/xinjiang/1/2006 (H5N1) and constructing of 8 plasmids system,rescuing the reassortant, H5N1/A/xinjiang/1/2006-RG and assaying therecombinant virus as a vaccine strain.First, the modification of HA gene of A/xinjiang/1/2006(H5N1)and constructof 8 plasmids system,The virus total RNA was extracted in BSL-3,and wasreversely transcripted into cDNA.Two full-length segments ofA/xinjiang/1/2006(H5N1)were amplified by using primer pairs with recognitionsequences for the restriction endonucleases BsmBI, BsaI or AarI. The genetic mutations were introduced into HA gene, I.e. the four basic amino acid, the sitewhich the HA gene can be splited into HA1, HA2 were deleted. Then, in our system(pHW2000), two plasmids that encode the modified HA and NA wereconstructed, together with 6 plasmids which encode the 6 gene segments of PR8,were introduced into cultured COS-1cells, resulting in the generation of newvaccine strain.The second, rescuing the reassortant, H5N1/A/xinjiang/1/2006-RG.The 2+6plasmids system which we constructed were introduced into cultured COS-1cells,resulting in the generation of new vaccine strain. During the rescued process, wepay more attention to research on the transfection condition; include the selectionof transfection agent and density of cells, the suitable time to add the TPCK andso on. Now we have found the suitable condition for rescuing pandemic influenzavirus.We detected the reassortant using RT-PCR, HA assay, IFA and electronmicroscope. The HA and NA gene sequence of reassortant and wild virusresembled each other, and the four basic amino acid in HA gene were deletedsuccessfully. The HA titer of H5N1/A/xinjiang/1/2006-RG is over 1:320.Themorphous of reassortant is normal, showing the filiform and cystic form under theelectronic microscope. Now we can rescued a pandemic virus strain in short period, andwill serve many research field about pandemic influenza virus.The third, assaying the recombinant virus (H5N1/A/xinjiang/1/2006-RG) as avaccine strain. The objective of our research is to obtain the recombinant virus toproduce the effective vaccine, so as the fellow of rescuing the reassortant, weneed to analyse the stability ,safety and immunogencity of the recombinant virus.After the passage of virus, we assayed the gene sequence, HA titer and EID50 indifferent generations. The homology of different generations virus is over 99.8%;the HA titer is over 1:320 and EID50 is about 7.All the result show that thereassortant is stabile.The CPE assay, plague assay,EID50,IVIP index were usedto detect the virulence of virus. Using this way, we can lessen the virulence, andthis kind of virus can be used to produce vaccine according to the regulation ofWHO. The diameter of plaque is 1-2mm under the TPCK-tripsin.The value ofEID50 is 7.5 and IVIP is 0.All the result display the vaccine strain which werescued can be used, and the virulence of the vaccine strain is accordant with theregulation of WHO. The immunogencity of the vaccine strain was tested byimmunizing the Balb/c mice, the HI titer is 1:40-1:640.Now we have establishedthe platform to rescue influenza vaccine strain, and this kind of vaccine strain can be used to produce the pandemic influenza vaccine.ConclusiConclusionWe have successfully rescued the recombinant virus and assayed its stability,safety and immunogencity. All the result show that the reassortant can be used toproduced the vaccine. Now we have established the key technology for thedevelopment of vaccine: reverse genetics can solve the problem of virus variationand the vaccine strain can be generated in short period; the technology in thisstudy is a better way for vaccine development of pandemic influenza.