| Since its first emergence in February 2013 in China,the H7N9 subtype of influenza virus has caused 1,568 infections,with a mortality rate of over 30%.Given the huge economic losses caused by the H7N9 virus to the poultry industry and the high risk posed to human health,the control and elimination of both low and highly pathogenic H7N9 viruses is imminent.Vaccination is the most effective means of preventing influenza virus infection.The current commercial H7N9 subtype of avian influenza vaccine is inactivated vaccine,which has been effective in disease prevention and control.However,traditional inactivated vaccines rely on chicken embryos for production,which has disadvantages such as insufficient supply of chicken embryos during outbreaks,environmental contamination due to the large amount of waste generated,and endogenous virus contamination.In addition,inactivated vaccines can only induce antibody response but not cellular immunity.Therefore,to meet the needs of modern poultry farming for disease prevention and control,there is a high need to develop a safe and efficient vaccine for avian influenza H7N9 subtype using new technologies.Virus like particles(VLP)are non-infectious particles self-assembled from structural proteins carrying viral antigens that mimic the natural structure of viral particles,activate cellular and humoral immune responses and induce a more complete immune protection.VLP vaccines can be produced on a large scale using the baculovirus expression system in insect suspension cells.The baculovirus expression system is widely used in clinical trials because it does not depend on chicken embryos for production and can guarantee an adequate supply of vaccine in the event of pandemic season.In addition,it has the advantages of low cost,environmental friendliness,short production cycle and high safety.Therefore,the use of baculovirus expression systems to produce influenza VLP vaccines has great potential for application.However,VLP of a certain particle size is easily recognised and cleared by the reticuloendothelial system in vivo,which to a certain extent greatly reduces the duration of action of VLP vaccines in vivo.Erythrocyte membranes have become a very promising delivery system for drugs and vaccines due to their biocompatibility,long half-life,non-immunogenicity,large surface area and as well as the fact that they do not produce toxic or harmful substances when degraded.It has been shown that nanoparticles encapsulated by red blood cell membranes can significantly enhance the circulation time of the particles in the body.In addition,splenic antigen-presenting cells have an excellent ability to target erythrocyte debris and senescent erythrocytes,making the use of erythrocyte membrane carriers as an efficient way to target antigens to the spleen.Currently,this system has attracted a lot of attention in the field of human drug delivery and vaccines,but no veterinary vaccine has been reported yet.Therefore,in this study,we propose to develop a novel vaccine based on erythrocyte membrane nanovesicles for targeted delivery of H7N9 VLP for the prevention and control of avian influenza.In this study,a nanovaccine(EM-VLP)based on erythrocyte membrane nanovesicles(EMNVs)encapsulated with self-assembled H7N9 VLP prepared through co-infecting with Sf9 cells using from three recombinant baculoviruses rBac-GD15-HA-P7,rBac-GD15-NAP8 and rBac-GD15-M1-P8.The results showed that the EM-VLP vaccine had a uniform particle size of about 259.2 nm after coating the erythrocyte membrane,with good dispersibility.Moreover,the EM-VLP vaccine significantly enhanced the humoral and cellular immune responses,providing a valuable reference for the development strategy of novel vaccines.1.Preparation and optimization of the H7N9 EM-VLP vaccineIn this section,a nano-vaccine(EM-VLP)encapsulating H7N9 subtype VLP assembled by recombinant baculovirus rBac-GD15-HA-P7,rBac-GD15-NA-P8 and rBac-GD15-M1-P8 coinfecting with Sf9 cells was successfully prepared.The morphology of VLP,EMNVs and EMVLP was observed by transmission electron microscopy.As a result,we found that the shape of VLP was in round or oval,encrusted with fibrils outside,and its diameter was about 100200 nm;EMNVs was also round,oval or in irregular morphology,and its particle size was about 100 nm;EM-VLP was round or oval particles encrusted with membranes outside,with diameter about 200 nm.The preparation methods for EM-VLP nanovaccines were also optimised,and we found that EMNVs encapsulated with VLP can either use the both ultrasound or the co-extrusion-coupled ultrasound method.Further study,the EM-VLP nanovaccines were all prepared by ultrasonication followed by co-extrusion method.The results of Malvern particle size analyser showed that the encapsulation of EMNVs improved the stability of VLP and that no damage to the main antigen and target genes of the nano vaccine occurred during the preparation of EM-VLP.2.Evaluation of the targeting effect of the H7N9 EM-VLP vaccine Studies in the previous chapter have demonstrated that EMNVs encapsulated VLP can significantly enhance the stability of VLP nanoparticles.In this chapter,we use DC2.4 and BMDC cells as cellular models to invetigate whether EM-VLP nanovaccines can be efficiently taken up by dendritic cells and further stimulate their maturation.Confocal assays showed that the EM-VLP nanovaccine could be quickely taken up by DC2.4 cells,thus stimulating the immune response level more effectively.The results of flow cytometry test indicated that incubation of EM-VLP nanovaccines with BMDC cells induced higher expression levels of CD80 and CD86 in BMDC cells.The results of IVIS spectrum imaging system showed that EM-VLP nanovaccine could be metabolized into the liver and enriched in the spleen after injected into the mouse.3.Evaluation of immune efficacy of the H7N9 EM-VLP vaccineIn the previous chapter,the EM-VLP nanovaccine was found to be effectively taken up by DC cells and stimulated the maturation of BMDC cells and was enriched in the spleen.In this chapter,mouse was used as animal models to assess the immunological efficacy of the EMVLP nanovaccine.the results of the HI antibody assay showed that the EM-VLP vaccine group induced significantly higher levels of HI antibodies compared to the VLP vaccine group.In addition,HI antibodies induced by the EM-VLP vaccine group continued to show an increasing trend after the second vaccination,suggesting that erythrocyte membrane encapsulation could prolong the humoral immune response induced by the EM-VLP vaccine.The results of the virus neutralization assay showed that the EM-VLP vaccine group induced significantly higher levels of VN antibodies compared to the VLP vaccine group.The results of ELISA showed that IgG antibodies were consistently higher in the EM-VLP vaccine group than in the VLP vaccine group at 14 d,28 d,35 d and 42 d post-immunisation and were significantly higher than that of the VLP group at day 28.The results of immune-related cytokine assay in mouse lung T cells showed that the EM-VLP vaccine group induced significantly higher IL-2 and IL-4 than the VLP vaccine group,suggesting that the cellular immune response to the VLP vaccine was significantly enhanced by encapsulating VLP with EMNVs.The results of the mouse lung inflammatory cytokine assay showed that the expression of inflammatory cytokines was significantly lower in both the EM-VLP and VLP vaccine groups than in the respective control groups on days 3 and 5 after the virus challengen,and was even lower in the EM-VLP vaccine group,but there was no significant difference between the EM-VLP and VLP vaccine groups.The results of challengen study showed that the EM-VLP nano vaccine provided 100%protection against a lethal dose of H7N9 AIV infection and significantly inhibited virus replication in the heart,liver,spleen,lung,kidney and brain organs.In addition,both the EM-VLP and VLP vaccine groups significantly inhibited virus-induced mouse lung injury,with the EM-VLP group being more effective.In conclusion,compared with the VLP vaccine group,the EM-VLP nanovaccine group produced higher levels of HI,VN and IgG antibodies,significantly enhanced the cellular immune response,and completely inhibited the virus shedding and in vivo replication of the virus after H7N9 subtype achallengen,reducing lung injury caused by virus infection. |
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