Desige And Development Of Pandemic Influenza Vaccine Using Reverse Genetics

Recent outbreaks of highly pathogenic avian influenza(HPAI H5N1) in 15 countries and regions and associated human infections in Indonesia have led to a heightened level of awareness and preparation for a possible influenza pandemic.WHO has predicted that a new influenza pandemic might occur in the near future. Effective vaccines against H5N1 virus are, therefore, urgently needed.Vaccination is considered one of the most-effective preventive measures for the control of influenza pandemics.However,Vrius variation,egg-based production and intramuscular vaccination with needles are barriers for production of vaccine.With the recommendation of WHO,we need find the effective strategies for developing vaccine against influenza pandemic.Currently,HPVI type A virus(H5N1) mainly scatter in Asia,especially in Indonesia,Vietnam and China.The wild type virus isolated from human and various animals display the high variation by serology,pathogenicity and gene structural analysis. The viruses isolated in China belong to two clades.So we need to develop the prototype vaccine,now,include the inactive whole virion vaccine,the split vaccine,the live attenuated vaccine and the subunit vaccine.Among of the total,the split vaccine has the rare and good quality.It can be used in different old person,including the old and the children below 12 year old.it is more safe than live attenuated vaccine and can induced more strong immne response than subunit vaccine. Now,influenza split vaccines have been prepared and are undergoing clinical evaluation in several countries.In China,we have develop the influenza split vaccine for the first time,to solve the problem of meeting an emergency and nought.We also develop the new strategies for vaccine,include reverse genetics for generation vaccine strain,Vero cell instead of eggs to production and alternative routes of administration. R&D of Pandemic Influenza Split VaccineWe purchased the virus vaccine strain-VNH5N1-PR8/CDC-RG(H5N1)which was recombinated from A/Viet Nam/1203/2004 (HA,NA) and PR8 from CDC in American.We have successfully established the seed lots for the vaccine strain including the master seed lot and the working seed lot.The HA titer, identity, EID50, Sterility test, mycoplasma and extraneous agents detection were carried out seperately,and prove that the virus seed lots are safe and stabile, accordant with the regulations of WHO.Fellow the establishment of seed lots,we have selected the best condition for virus culture,harvest,inactivation,split and purification.The tempreature and time for the virus culture is 33℃for 52h.To use 1:4000 formaldehyde to inactive virus for 72h, and select the Sepharose 4 Fast Flow to purify the virus and 0.5% Triton X-100 to split virus about 2h.At last,we prepared the stock solution of vaccine.One approach for increasing the number of available split vaccine doses without a compensatory increase in egg production is to enhance the immunogenicity of the antigen; addition of an effective, safe adjuvant could result in a more immunogenic vaccines and also enable injection of a smaller quantity of antigen to elicit an effective immune response,so we select 1.2mg/ml AL as adjuvant and 3.75,7.5,15.0,30.0,45.0,60.0, 75.0,90.0μg/0.5ml dose form to intramuscular vaccinate the mice and guinea pigs one dose and two dose.The HI test showed that 7.5,15.0,30.0μg/0.5ml dose form will be selected into the clinical trial,and immunizd two dose.The safety and stability of the pandemic vaccine are also important. Allergy test,single dose toxicity test,abnormal toxicity test,acute-term toxicity test were carried out seperately to test the safety of vaccine.We placed the vaccine in different conditon, 2～8℃,22～25℃,37℃to detect the HI titer,HA content,pH value and appearance to determine the vaccine stability.Our split vaccin can kept in 2～8℃for two years by the vaccine accelerate test.The vaccine prepared with this virus was assessed in a double-blind Phase I clinical trial, the results of which showed that, although antibody responses indicative of immune protection were achieved by administration of the vaccine with AL adjuvant.R&D of Key Technology for Pandemic influenza vaccineThe technology for pandemic influenza vaccine include generation the virus vaccine strain,selection of cultural medium and alternative routes of administration.RG(reverse genetics): New vaccines that exploit reverse genetics technology are being developed,and the knowledge that the pathogenicity of avian influenza viruses is primarily determined by HA variation.The virus vaccine strain can be rescued by this technique in time when the influenza pandemic break out.There is no vaccine strain reference laboratory in China,we want to establish it using the 8 plasmids system. Briefly, in our system (pHW2000), six plasmids that encode exact copies of the six individual RNA segments of the PR8, together with two plasmids that express the two viral proteins (HA,NA) of A/Anhui/1/2005 (H5N1), are introduced into cultured COS-1cells, resulting in the generation of new vaccine strain.RT-PCR,HA test,electron microscope,IFA were carried out to detect the recombinant virus.The result showed that the gene sequence,HA titer(1:320),virus morphous and virulence accordant with the virus vaccine strain.Cell-culture-based vaccine:Given that chicken embryonated eggs, which are currently used for inactivated-vaccine production, would be in a short supply during a pandemic, the development of Vero cell-culture-based H5 vaccines is an attractive alternative approach. Modification of the Vero cell that that can be easily infected by influenza virus or modification of vaccine strain that grow well in cell cultures are two useful strategies.We constructed pCDNA3.1-ST3GalⅢexpressing vector,transfected Vero cell,got the new Vero cell line which can express the 2-3 type receptor efficiently.The vaccine strain, PR8/A/Anhui/1/2005 can replicated in modified Vero cell,the TCID50 is 106.5-7,is higher than usual Vero cell.We exchanged NS gene of PR8 into NS gene of Eng53/v-a to get the modified virus,but we did not obtain the reasonable result.Alternative routes of administration: We investigated the aerosolized vaccine for influenza pandemic which can reduced the antigen quantity and be used widely. Split vaccines combined with adjuvants-such as AL as well as outer membrane proteins of Neisseria meningitidis, referred to as proteosomes have been delivered directly to the nasal mucosa in animal models.We detected the secreting type IgA in saliva and lung lavage,and we also detedted the antibody in serum.The IgA titer in lung lavage is 1:640.All the results displayed the the aerosolized vaccine with protosome is the good selection for pandemic vaccine. ConclusionIn order to respond to the influenza pandemic in China,we have finished the influenza split prototype vaccine to meet an emergeny outbreak of influenza.We also established the key technology for developing of vaccine:reverse genetics can slove the problem of virus variation and the vaccine strain can be generated in short of period; the development of Vero cell-culture-based vaccine can provide the stabile system to overcome the obstacle of a short supply of eggs during a pandemic; aerosolized vaccine for influenza pandemic can induced the systemic immunity and mucosal immunity at the same time,and also induced cross immunity among different virus strain.These findings indicate that the technology in this study is a better way for vaccine development of pandemic influenza.