Preparation And Preliminary Evaluation Of Vero Derived Highly Pathogenic Human Avain Influenza Vaccine Strain

Influenza is an acute respiratory disease caused by influenza virus, which has been spread to all over the world. There were three worldwide flu pandemics in 20th century caused by H1N1, H2N2, H3N2, respectively. Especially in the 1918'Spanish flu', more than 20 million people died which brought serious threat to human society. Recently, since the first outbreak of the highly pathogenic avian influenza virus of the H5N1 subtype was reported in humans in Hong Kong in 1997, it has been spread to many areas of the world. From January 2003 to June 2nd 2009, 429 laboratory-confirmed H5N1-infected human cases were reported to the World Health Organization, 262 dead, and the mortality was above 60%. Because there was a harmful effect on human health and world economy, WHO claims all the countries should take measures to deal with pandemic influenza.Vaccination is an effective method for preventing and controlling avian influenza. At present, the main material for producing influenza vaccine is embryonated chicken egg. However, this is an extremely cumbersome system with many disadvantages with respect to time-consuming generation of vaccine strain, lack of reliable supplies of high quality eggs and so on. The source of eggs has become the key point for producing sufficient influenza vaccines once the outbreak of pandemic influenza, which might result in the failure of controlling and preventing pandemic influenza in time. As a result, it has been a great item to carry out the research of material for producing human influenza vaccine. Continuous cell line is a good material for producing vaccines. There were two kinds of cells which have been approved to produce human influenza vaccine, including Vero cell lines and MDCK cell lines. The former cell lines have been licensed in the world, and the later have been allowed to be applied in Europe, too. Unfortunately, poor adaptability and low yield for influenza virus in Vero cells become the capital problems to solve. So, it is an urgent need to carry out the research of Vero high-adaptation.The development of influenza reverse genetics provides a powerful implement for generating vaccine strain. The first step for producing human influenza vaccine is just to generate vaccine strain. Compared with traditional method of obtaining vaccine strain from co-transfecting eggs with PR8 and circulating virus, it is more reasonable and faster to use reverse genetics. At the same time, this method will reduce the time of producing candidate strain, which will be allowed to deal with avian influenza in time. The NS gene is the nonstructural gene of influenza virus, including NS1 and NS2 gene segments and coding the NS1, NS2 proteins. There are many studies have demonstrated the NS1 protein play a significant role in translation and replication of viral RNA. The second protein encoded by the NS gene segment is the NS2 protein (nuclear export protein, NEP). Although little is known about the function of NEP, this polypeptide interacts with nucleoprotein and mediates the export of viral ribonucleoproteins (RNP). In addition, the nuclear export signal (NES) motif of NS2 protein had been shown to be crucial for the production of live vaccine viruses. These studies have shown that the NS gene play an important role in replication of influenza virus.Vero cells were approved to produce human vaccines. Earlier studies indicated that influenza viruses did not replicate productively in Vero cells. In order to prepare influenza vaccine in large scale from Vero cells, we take two measures: on one hand, a reassortant influenza virus was generated by reverse genetics from Vero cells, and the rescued virus contained the surface glycoprotein genes from circulating virus and was allowed to propagate to high titers in Vero cells; on the other hand, Vero cells were screened to acquire more sensitive cell strain for generating more influenza virus. These measures could pave the way for producing safety, effective human influenza vaccine in large scale with Vero cells, which provided technique possibly useful for preventing and controlling outbreak of influenza.The aim of this study was to generate Vero high-yielding H5N1 influenza vaccine by reverse genetics. With the NS gene mutation it was modified to Vero-adapted. HA and NA genes were derived from the circulating strain and the other genes from PR8. A reassortant virus was generated from Vero cells according to 1+2+5 virus reassortant mode by using reverse genetics.The results of this study can be divided into three parts:1. Recovery of Vero high-yielding influenza vaccine strain: firstly, the HA and NA genes of A/Anhui/01/2005 (H5N1) (attenuated strain) were amplified by RT-PCR and be identified by sequencing. Secondly, the NS gene of PR8 was modified to Vero-adapted and the NS gene was synthesized to construct plasmid pHW2000-HA(A),pHW2000-NA(A),pHW2000-NS, respectively. Changes in NS gene: site-specific mutagenesis at 21, 58, 60, 127, 174, 189 amino acids in NS1, and its 231-238 amino acids were deleted; for NS2 gene: site-specific mutagenesis at 16, 31, 86, 107 amino acids, and its 231-238 amino acids were deleted. With the correct sequence verification, 7+1 mode cotransfection was performed to identify their function. Thirdly, with other five inner genes of the PR8 eight-plasmid rescue system sever as the backbone, virus was rescued according to 1+2+5 mode by co-transfecting Vero cells. The rescued virus was identified as the aim strain by sequencing, and it was in line with typical influenza morphologic characteristics. These results demonstrated we had rescued influenza virus successfully.2. Identification of biology characteristic for the rescued virus: the rescued virus contained the HA and NA genes of H5N1-A/Anhui/01/2005, and the rescued virus was identified as H5N1 subtype by HI antigenic analysis; In addition, the modified NS gene played an important role for its replication. In presence of TPCK-trypsin, Vero monolayer was infected equal amount of rescued virus and its parental virus, respectively, incubated at 37℃, 5%CO2. The CPE on Vero was observed with microscope and the time for CPE was recorded. These results demonstrated that the half times to peak yield for the rgAnhui/NS reassortant virus was shorter than that of its parental virus (18h, 25h). Furthermore, comparison of growth kinetics for rgAnhui/NS and rgAnhui/PR8 in Vero cells was performed to show the effect of the NS gene on subsequent virus amplification in Vero cells. Any improvement from an increase in yield would be negated by changes in the antigenicity of a vaccine. Vaccine production procedures must allow the produced virus to retain the antigenic properties of the parent viruses. The rescued virus was continually passaged ten times in Vero cells to determine its genetic stability. The HA gene segment was sequenced and compared with the parental virus at 2nd, 4th, 6th, 8th, 10th passages, respectively. 99.9% identity of the HA sequence showed that the replace of NS gene could not change the antigenicity of the recovery virus, which provided guarantee for vaccine production.3. Tests of the safety and immunogenicity for the rescued virus: the rescued virus was amplified in Vero cells and harvested from supernatant fluid. The viruses were purified by sucrose density gradient centrifugation (SDGC). Purified viruses were titered with EID50 and TCID50. The main antigen of rescued virus was determined by SDS-PAGE. The pathogenicity of the rescued virus was determined in eggs and mice model by intranasal immunization. There were no any dead cases caused by the rescued virus, which showed that the rescued virus was safe. We tested the immunogenicity of the rescued virus by subcutaneously inoculating the mice. And then, the purified virus was made into inactivate vaccine, and add Al(OH)3 as adjuvant. Balb/c mice were immunized through intramuscular, 15μg HA/mice. Mice were immunized secondly after 14 days. The serum of mice was harvested through vena caudalis on 7th, 14th, 21st, 28th day for determining IgG titers and HI titers. The results showed that the rescued virus had similar immunogenicity with its parental virus.Conclusion: In this study a reassortant virus strain was rescued successfully in Vero cells by reverse genetics, H5N1-A/Anhui/01/2005-NS, abbreviated rgAnhui/NS. The rescued vaccine strain contained the modified NS gene and was able to grow faster and higher titers in Vero cells than its parental virus did. In addition, the rescued virus contained the surface protein genes from reassortant H5N1-A/Anhui/01/2005 strain. The results of HI assay showed that the rescued virus was H5N1 subtype, accorded expectation. We found that there were no any changes in HA and NA segments by sequencing, which suggested its antigenicity remained stabilization. There were no any dead cases caused by the rescued virus, which indicated that the rescued virus was safe. Test of immunogenicity for the rescued virus in mice indicated that it could induce efficient immune response. And there was no significant difference between antibody titers induced by the rescued virus and its parent virus. And these results provided guarantee for producing influenza vaccine with cells.