The Study Of Seasonal Trivalent Live Attenuated Influenza Vaccine Based On Vero Cell

Vaccination is the main effective method to combat influenza,and there are three different categories of influenza vaccine using in the world,as these:split,subunit and live attenuated influenza vaccine(LAIV).LAIVcan induce a variety of adaptive and long lasting immune responses by intranasal spray,including serum antibodies,mucosal immunity and cytotoxic T lymphocytes targeted to conserved virus epitopes.Nowadays,LAIV,registered for use in human,are produced by chick embryos only in Russia and America.Chick-embryo-based vaccines have disadvantages,such as ovalbumin allergy and long preparation period.Meanwhile,they have the risks of spreading vir?lence factors derived from chicken po?ltry for there is no inactivated process in LAIV production.The WHO recommended the Vero cells to produce human vaccines(e.g.rabies and polio)in 1995.Additional,the advantage of Vero cell based influenza vaccine is that it can be large-scale c?ltivation through the fermentation tank and avoids the variation of influenza virus surface antigen.But most influenza viruses can't grow in Vero cells.It is an important obstacle to the development of influenza vaccines based on Vero cell.So far,no Vero cell-based LAIV is reported worldwild.It is critical to c?lture cold adapted influenza viruses for the development of Vero cell-based LAIV.There was just only one cold adaptation strain in Vero cell(A/Singapore/1/57ca(H2N2)reported in Singapore,and no cold-adapted influenza B virus in vero cell was reported.Fortunately,over the past decade,we developed two Vero cell-adapted(Va)and cold adapted(ca)influenza virus strains by gradient cooling temperature,named as following:A/Yunnan/1/2005Vca(H3N2Vca)and B/Yunnan/1/2011 Vca(BVca).These two strains owing triple feature(ca,Va and attenuation(att)characteristic),which had be confirmed by serious experiment data,co?ld be high-yield in Vero cells at 25 ?.Firstly,the eight gene fragments(PB2,PB1,PA,HA,NP,NA,M and NS)of H3N2Vca and BVca influenza virus were inserted into the bidirectional expression plasmid(PHW2000)and select plasmids that co?ld work togerher.That was the donor plasmid bank.Then we cloned the surface genes(HA and NA)of the vaccine strains(A/ChristChurch/16/2010?A/Texas/50/2012and B/Massachusetts/2/2012)recommended by WHO in the 2013-2014 flu season in the northern hemisphere using the same methodSecondly,six recombinant plasmid(PB2,PB1,PA,NP,M and NS)from donor plasmid bank and two recombinant plasmid(HA and NA)from parental plasmid bank were used to generate new LAIV vaccine strains.These new strains were named as:A/ChristChurch/16/2010Vca,A/Texas/50/2012,and B/Massachusetts/2/2012.All of these new strains had triple features(ca,Va and att),which were evident by dozens of passage.Complete normal morphology were observed by electron microscopy,with f?ll caps?le.Genome composition of new strains fragments were consistent with previous designed.Furthermore,we explored the serum-free medium to c?lture Vero cells and established the SFM-Vero cell bank;Vero cells were passaged at ratio of 1:2.36hours later,the seed reassortment influenza viruses were added with 0.1 MOI and the pH of medium,containing 0.8?g/ml TPCK trypsin,was 7.2-7.4.This was the most suiTab.c?lture conditions.After a series of large-scale c?ltivation and purification processes(including?ltrafiltration concentrated,sucrose density gradient centrifugation),we harvested the original monovalent vaccine solution.Furthermore,trivalent LAIV was made according to their Infectious droplets degrees.Then we evaluated the immunogenicity of the trivalent LAIV using mice and ferrets animal model.28 days after nasal administration,the serum HI titer against H1N1?H3N2 and BY influenza virus in mice was 1:234.67,1:248.3 and 1:239.3 respectively.After boost once at the 28th day,the HI titer co?ld be higher,as 1:490.667 against H1N1,1:606.7against H3N2 and 1:485.3 against BY,respectively.Additionally,the secretory IgA(sIgA)in lung lavage fluid were distributed as 2217.6ng/ml and 3329.5ng/ml after one and twice inoc?lation at 14th day post-vaccination.In the ferrets experiment,our trivalent LAIV co?ld induce HI titer as high as 1:2000.And the cytokine(IL-4,IL-6)levels were significantly increasing compared with the negative control.In addition,by the challenge experiment res?lts,our trivalent LAIV had 100%protection efficiency in the challenge experiment res?lts.The genomic libraries of attenuated influenza A and B virus based on Vero cells were constructed and used for the preparation of trivalent(tetravalent)seasonal LAIV virus seed.Subsequent preparation of trivalent Vero cell seasonal seasonal LAIV had better safety,immunogenicity and protective effecicy.