| Influenza virus belongs to Orthomyxoviridae,and is divided into four types(A,B,C,D).It can cause infections and diseases in human,poultry,pigs,horses,bats and other animals.Avian influenza is an infectious disease caused by influenza A virus,which can not only spread rapidly among birds and poultry,but also infect humans.The H5N1 subtype is a highly pathogenic avian influenza virus,which was first found to be capable of infecting humans in 1997.So far,there have been nearly 1,000 cases of H5N1 infection with a mortality rate of more than 50%,leading to huge impact and losses on human health and economic.At present,influenza vaccination is still the most effective and economical meansto prevent influenza,but most of the flu vaccines on the market today are inactivated vaccines derived from chicken embryo.The traditional technique of these vaccines has many limitations,such as the preparation period of chicken embryo is long and the supply of chicken embryo is insufficient,especially during a pandemic,which may affect the timely availability of influenza vaccines.Therefore,the establishment of a rapid influenza vaccine research and production platform is crucial to the prevention and control of influenza.Virus-like particles(VLPs)are hollow particles containing viral structural proteins.Without virus genome,VLPs have higher safety,and the particle structures are similar to those of whole virus vaccines,which can induce stronger immune effect in the body.VLPs do not depend on the production system of chicken embryo and adopts in vitro cell culture.Vaccinia virus Tiantan strain is a vaccine strain used for the prevention of smallpox,and is also very suitable for the expression of exogenous proteins.It has a large genome,many non-essential genes,so it can contain long exogenous gene fragments.The host range is wide and can be amplified in many different cell lines.Vaccinia virus Tiantan replicates in cytoplasm and is of high safety,with a long history of human applications.In this study,vaccinia virus Tiantan strain was used as the expression vector to express VLPs in mammalian cells,and its immunogenicity was analyzed by vaccination in mice.Firstly,the recombinant vaccinia virus shuttle plasmid pSCCK-HA/NA was designed and constructed.Then the shuttle plasmid was co-transfected with vaccinia virus Tiantan strain into Vero cells.After several rounds of fluorescence screening,recombinant virus rVTT-HA/NA expressing the HA and NA proteins of H5N1 avian influenza virus was successfully obtained.The expression of HA and NA proteins was confirmed by Western blot.Virus-like particles were obtained by sucrose density gradient centrifugation and purification,particles similar in shape and size to H5N1 can be seen under an electron microscope.The total protein content was 93.49?g/mL measured by Lowry method and HA protein content was24.58?g/mL measured by SRID.The mice were divided into three groups(1,2,3),and each group was vaccinated with VLPs doses of 1?g,5?g,and 10?g each mouse respectively.At the same time,H5N1 whole virus inactivated vaccine(1?g/mouse)was used as positive control.The results of humoral immunity showed that the neutralization activity of serum antibody was greater than 1:40 in the 2 and 3 groups.The titer of the third group reached to 1:180,and was comparable to that of the whole virus inactivated vaccine group at week 8.The titer of IgG detected by ELISA was significantly increased in all three groups after enhanced immunization,and the titer remained stable,with no significant decrease until the eighth week.The results of cellular immunity showed that the proportions of spleen CD8~+T cells induced by virus-like particles in three groups were higher than that of the positive control,and the proportion of lymphocytes secreting cytokine IFN in the spleen was also higher than the positive control,which proved that virus-like particles induced stronger cellular immunity.This study can provide a foundation for the development of new virus-like particles influenza vaccine. |
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