Construction And Immunological Study On Zika Virus Recombinant Rabies Viruses And Bacterial-like Particles

Zika virus(ZIKV)disease,caused by ZIKV,is a zoonotic infectious disease.It spread worldwide in 2015,resulting in a pandemic associated with Congenital Zika Syndrome in newborns and and Guillain Barrésyndrome in adults.On December 1st,2015,the World Health Organization(WHO)and the Pan American Health Organization declared a global warning on ZIKV disease.In November 2016,the WHO announced that ZIKV was no longer a public health emergency of international concern,but it is still a significant and sustained public health problem.Moreover,ZIKV was listed in the“Research and Development(R&D)Blueprint for Action to Prevent Epidemics”.In February 2018,the WHO listed ZIKV disease as one of the priority study diseases,and it is one of the imported zoonoses in China in recent years.Because of the possibility of repeated outbreaks and the lack of specific drugs and vaccines,research on the development of a safe and efficient vaccine is important for responding to the outbreak and ensuring public health.In this paper,based on rabies virus(RABV)and gram positive enhancer matrix(GEM)platform,ZIKV recombinant RABVs and bacterial-like particles were prepared and evaluated in order to provide technical and product support for disease prevention.Based on the reverse genetic operating system of RABV SRV9 strain,three recombinant RABVs carrying ZIKV pr M-E gene were constructed,named ZI-D,ZI-E and ZI-F respectively:ZI-D:pr M-E gene;ZI-E:transmembrane(TM)domain of pr M-E replaced with the TM-cytoplasmic domain(TM-CD)of SRV9;ZI-F:signal peptide and TM domain of pr M-E replaced with the region of SRV9.Confocal analysis showed that RABV G protein and ZIKV E protein could all be detected in ZI-D and ZI-E infected cells,and a large number of ZIKV E protein could be co-located with RABV G protein on the surface of ZI-E infected cells.In ZI-F infected cells,only RABV G protein could be detected.The immunogold electron microscopy results showed that ZIKV E protein labeled gold particles were around the surface of ZI-D and ZI-E.The purified viruses were identified by Western-blot(WB).The protein band of ZIKV E could be detected in ZI-D and ZI-E,and the expression of ZI-E was more than that of ZI-D;there was no target band in ZI-F.Moreover,there were no significant differences in growth abilities among recombinant viruses(ZI-D and ZI-E)and the parental virus.After 25 successive passages of ZI-D and ZI-E,PCR detection were positive.In conclusion,two recombinant RABVs,named ZI-D and ZI-E,were successfully obtained.The genome passages were stable;the expression of foreign protein did not affect the growth kinetics of recombinant viruses,and the virus titer could reach 10^8.63 TCID₅₀/m L.After inactivated by?-propiolactone and purified,ZI-D and ZI-E were mixed with ISA 201 VG&poly(I:C)adjuvant.Cellular and humoral immune responses were detected after immunization in mice.Vaccines achieved appreciable ZIKV specific Ig G responses with Th2 type.On the 6^th week after immunization,the average neutralizing antibody against ZIKV of two groups were 1:19.8 and 1:25.6respectively,which were both above the protective level line reported in the literature(PRNT₅₀ value>1:10).In addition,the titers of RABV neutralizing antibodies in immune groups were all above 23.38 IU/m L,which were above the protective level(0.5 IU/m L).On the 3^rd and 6^th day of the first immunization,immune groups could significantly activate B lymphocytes in inguinal lymph nodes of mice.One week after the third immunization,the splenocytes in ZI-D and ZI-E group proliferated efficiently.The capacity of splenocytes secreting IFN-?and IL-4 increased significantly,and the capacity of ZI-E group was higher than ZI-D group.The cytokines secreted into the supernatant were increased in varying degrees including Th1 cytokines(IL-2,IFN-?and TNF-?)and Th2 cytokines(IL-4,IL-5,IL-6 and IL-10).In addition,compared with ZI-D group and control group,the activation degrees of CD4⁺T cells,CD8⁺T cells and B cells(CD4⁺/CD8⁺/CD19⁺&CD69⁺)in ZI-E group were significantly increased,and the proportions of central memory T cells of CD4⁺and CD8⁺T cells(CD4⁺/CD8⁺&CD44⁺&CD62L⁺)were also significantly increased.In conclusion,ZI-E could induce specific humoral and cellular immune responses against ZIKV,and could also produce high-level neutralizing antibodies against RABV.Based on GEM display platform,we prepared ZIKV bacterial-like particles.Firstly,we constructed recombinant baculoviruses that expressed ZIKV pr M-E and PA3 fusion proteins.The results showed that the fusion proteins without stem-transmembrane(ST-TM)were secreted into the culture medium supernatant and successfully bound to the surface of GEM.The fusion proteins containing pr M-E ST-TM were hardly secreted into the supernatant and could be detected only in the sonicated supernatant with uneven band size.Moreover,the fusion proteins with the ST-TM region could not bind to the surface of GEM effectively.Therefore,three kinds of secreted fusion proteins(signal peptides were derived from ZIKV pr M-E,Japanese encephalitis virus pr M-E and baculovirus GP67 protein,respectively)were selected and combined with GEM to prepare bacterial-like particles.The results of immunofluorescence and WB were positive.In conclusion,three kinds of ZIKV bacterial-like particles that could display ZIKV E protein on the surface of GEM were obtained,named ZI-?-PA-GEM,ZI-JE-?-PA-GEM and ZI-GP-?-PA-GEM,respectively.ZIKV bacterial-like particles were mixed with ISA 201 VG&poly(I:C)adjuvant and evaluated immumogenicity in mice.Anti-ZIKV Ig G could be detected in the serum with Th2 immunity.Six weeks after the first immunization,the average value of neutralizing antibodies were more than 1:24.9.The results of cellular immune response showed that on the 3^rd and 6^th days after the first immunization,the immune groups showed significantly activated B lymphocytes in the inguinal lymph nodes.On the 6^th and 9^th days,dendritic cells(DC)were significantly activated.One week after the third immunization,splenocytes in immune groups proliferated efficiently,and ZI-?-PA-GEM group showed the greatest increase in proliferation.ELISpot detection of IFN-?and IL-4 showed that there were significant differences between immune groups and the control group,and the levels of ZI-?-PA-GEM group were the highest.The cytokines in the supernatant(including IFN-?,IL-4,IL-6 and IL-10)also increased in varying degrees.The activation of CD4⁺T cells,CD8⁺T cells and B cells(CD4⁺/CD8⁺/CD19⁺&CD69⁺)in ZI-?-PA-GEM group increased,and the proportion of central memory T cells in CD4⁺T and CD8⁺T cells(CD4⁺/CD8⁺&CD44⁺&CD62L⁺)also increased significantly.In conclusion,the bacterial-like particle ZI-?-PA-GEM could not only induce effective humoral immune response,but also promote the proliferation of splenocytes,the secretion of various cytokines,the activation of T/B cells and the proportion increase of central memory T cells.In conclusion,ZIKV recombinant RABV ZI-E and bacterial-like particle ZI-?-PA-GEM were prepared and screened in this study.They could effectively induce humoral and cellular immune responses.In addition,ZI-E can potentially be used as the ZIKV-RABV binary vaccine.This study laid new ideas and technical foundation for the development of Zika vaccine.