| In 2013,the first human case of avian influenza virus(AIV)was reported in China,and as of June 2022,the H7N9 influenza virus has infected 1,568 people and caused 616 deaths,with a high mortality rate(http://www.fao.org/ag/againfo/programmes/en/empres/H7N9/situation_update.html).H7N9 AIV is not only a serious threat to the poultry industry,but also to human health.A highly safe and effective vaccine is therefore needed to prevent a potential pandemic of H7N9 subtype AIV.Vaccination has been the most important way to prevent influenza,and polyvalent inactivated vaccine is currently the most common method of vaccination against influenza virus-related diseases for human use.The main drawbacks of inactivated vaccines are the safety issues associated with allergic reactions and the difficulty in providing durable immune protection.Virus-like Particles(VLP)contain a high density of antigenic proteins and are able to enter cells as effectively as natural viruses,offering good immunogenicity and safety.In addition,the ability to quickly produce VLPs and at low cost is an advantage in the event of a potential pandemic.Glycosylation is a common post-translational modification that plays an important role in protein biology.Common glycosylation modifications are an important way for pathogens to obscure their own epitopes and engage in immune evasion.Therefore,altering the immunogenicity of vaccines by changing antigenic motifs,adding or removing potential glycosylation modifications to develop novel vaccines has become a new tool in vaccine research.In this study,we evaluated the immune protective efficacy of the H7N9 VLP vaccine in BALB/c mice using an insect cell-baculovirus expression system;three strains of recombinant baculovirus with altered HA glycosylation modification sites were constructed and the VLP was prepared to evaluate the immune protective efficacy of different combinations of HA glycosylation modifications in SPF chickens and BALB/c mice.The results showed that the VLP vaccine provided 100%clinical protection against the highly pathogenic H7N9 AIV in both immunized SPF chickens and B ALB/c mice.To be noted,the double 133 and 158 HA glycosylation modifications significantly enhanced the humoral immune response in experimental animals,attenuated pathological changes in the mouse lung,and significantly reduced the secretion of inflammatory cytokines in the lungs of mice,providing a reference for the design of novel H7N9 influenza vaccines.1.Evaluation of the immune effect of H7N9 subtype avian influenza virus-like particles vaccine in miceIn this study,H7N9 VLP vaccines were prepared by co-infecting Sf9 cells with recombinant baculoviruses rBac-GD15-HA,rBac-GD15-NA and rBac-GD15-M1 expressing the H7N9 subtype HA,NA and M1 genes which previously constructed in our laboratory.And the vaccines were purified to enhance immunogenicity.Four-weekold BALB/c mice were immunized with purified and unpurified H7N9 VLP vaccine.The immunization procedure was a second booster three weeks after the first immunization;the immunization dose was a single 10 μg;and the immunization route was via intramuscular injection.At the two weeks after the booster immunization,the vaccine groups all induced high hemagglutination-inhibiting(HI),virus-neutralizing(VN)and IgG antibodies in the hosts.Using the S8/H7N9 mouse-adapted strain as challenge virus,mice in the PBS group showed significant weight loss and all died within 8 days;both vaccines provided 100%clinical protection and completely inhibited virus replication in mouse tissues and organs.The VLP vaccine significantly increased the expression of IL-2,IL-4 and IFN-y,and significantly inhibited the expression of IFN-1β,IL-6,TNF-α,MIP-1α,IFN-γ,RANTES,IP-10 and other cytokines in mouse lungs.Moreover,the purified VLP was more effective than the unpurified VLP in these respects.The results of histopathological sections of lung showed that all vaccine groups effectively attenuated the lung injury after virus infection in immunized mice.Therefore,the H7N9 VLP vaccine prepared in this study provided complete clinical protection in mice,significantly reduced tissue damage in the lungs,thus provided a basis for the subsequent construction of an H7N9 VLP vaccine with an altered glycosylation modification pattern.2.Effect of HA glycosylation modification at positions 38,133,158 on the immunogenicity of H7N9 subtype of avian influenza VLP vaccineTo investigate the effect of glycosylation modifications on the immunogenicity of the H7N9 subtype of avian influenza VLP vaccine,three influenza virus HA sequences with altered motifs associated with glycosylation modifications were constructed and the recombinant baculovirus were successfully rescued,namely rBac-GD15-HA-38(-),rBac-GD15-HA-133(+)158(+)and rBac-GD15-HA-133(+)158(-).Three mutant baculoviruses were used as HA protein donors and the corresponding VLP was prepared and named VLP-38(-),VLP-133(+)158(+)and VLP-133(+)158(-),respectively.The results showed that the 133(+)158(+)double glycosylation modifications increased the antigen production.Challenge experiments using GD15 virus revealed no clinical signs and no mortality in SPF chickens in each vaccine group during the 14-day observation period,while chickens in the PBS control group showed clear clinical symptoms of influenza and all died within 7 days.Both VLP vaccines provided 100%clinical protection against H7N9 HPAIV in SPF chickens.Results of tissue viral load assays showed that all groups of VLP vaccines completely inhibited virus replication in tissue organs.Mouse experiments showed that after two immunizations,the vaccine in the VLP-133(+)158(+)group induced significantly higher production of HI and VN antibodies in mice than in the WT group.Using the S8/H7N9 murine adapted strain as the challenge virus,mice in the PBS group showed significant weight loss,and all died within 8 days,while all vaccine group provided 100%clinical protection and completely inhibited virus replication in mouse organs.The results of T cell immunerelated cytokines showed that the VLP-133(+)158(+)group significantly elevated the expression of IL-2 and IL-4.In addition,all the VLP vaccine significantly inhibited the expression of inflammatory cytokines in the lungs,including IFN-1β,IL-6,TNF-α,MIP-lα,IFN-γ,RANTES,and IP-10.,and the VLP-133(+)158(+)group showed a promising advantages in inhibiting TNF-α and MIP-1α and RANTES.In this study,we constructed three HA glycosylation modification site-altered recombinant baculoviruses and successfully assembling the corresponding VLP.The VLP-133(+)158(+)double glycosylation mutation group produced significantly higher amount of antigen than that of the wild-type VLP.In SPF chickens and BALB/c mice experiments,the VLP-133(+)158(+)double glycosylation mutation group produced significantly higher humoral immune responses than the other groups,significantly attenuated pathological changes in the lungs of SPF chickens,and significantly reduced the secretion of inflammatory cytokines in the mouse lung.These results suggest that glycosylation modifications have considerate potential for the design of novel avian and mammalian influenza vaccines. |
