Virus-like And Bacterium-like Particles Of Tick-borne Encephalitis Virus Preparation For Immunization Research

Tick-borne encephalitis（TBE）,also known as forest encephalitis,is a natural foci of nervous system disease caused by tick-borne encephalitis virus（TBEV）.It causes fatal encephalitis in humans and animals with long-term sequelae,seriously threatening human and public health.With the global climate changing,ticks,the vector that transmits the virus,have been expanding in recent years.As a result,TBE has exhibited new epidemic characteristics,re-emerging and prevalent in old foci,and appeared new foci constantly.Moreover,new virus subtypes have emerged.It is worth noting that there is no specific treatment for the disease at present,so vaccine immunization is essential for preventing and controlling tick-borne encephalitis.Inactivated vaccines are the currently approved tick-borne encephalitis vaccines due to the advantage of high safety.However,they often require multiple immunizations to stimulate the body to produce effective protection,which results in high costs and a short duration of immunization.Therefore,it is of great significance to develop new genetically engineered vaccines with high safety and strong immunogenicity for preventing and controlling tick-borne encephalitis.New vaccines based on virus-like particles（VLPs）and bacterium-like particles（BLPs）not only have good safety,but also stimulate the body to produce both cellular and humoral immunity.Therefore,they are the main directions for the current TBE vaccine research.In this study,the baculovirus-insect cell expression system was used to express TBEV-prM-E protein and E-PA3 fusion protein,to prepare TBEV VLPs and TBEV BLPs,respectively.At the same time,immunization research on mice was conducted to evaluate their safety and immunogenicity.An indirect ELISA method for the detection was established based on E-DⅢprotein expressed in prokaryotic.They lay the foundation for the research of new vaccines for tick-borne encephalitis.Specific research contents include:（1）Expression of tick-borne encephalitis virus E-DⅢand development of indirect ELISA for antibody detection.The gene encoding DomainⅢof TBEV E protein was amplified and the recombinant plasmid p ET-30a（+）-TBEV-E-DⅢwas constructed and transformed to competent E.coli BL21（DE3）for expression under the induction of IPTG.The expressed product was purified by Ni-NTA chromatography.An indirect ELISA was established to detect antibodies against TBEV using TBEV E-DⅢprotein as coating antigen.The results showed that TBEV E-DⅢprotein was expressed and aggregated to form inclusion bodies in E.coli,and the optimal induction condition was 0.6 mmol/L IPTG induction for 6 hours at 30℃.The optimal concentration of coating antigen was1 mg/L.The ELISA was specific to detect the antibody against TBEV at positive serum,and no cross reaction to the positive sera of Japanese encephalitis virus（JEV）,Zika virus（ZIKV）,or West Nile Virus（WNV）.The coefficients of variation for the inter-and intra-batch assays were less than 10%.In summary,the recombinant protein TBEV E-DⅢwas successfully expressed and purified.Indirect ELISA using TBEV E-DⅢas coating antigen was successfully established to detect antibodies against TBEV with high specificity,sensitivity and repeatability.It could be used to detect TBEV-specific antibodies in serum.（2）Virus-like particles of tick-borne encephalitis virus preparation for immunization research.The recombinant baculovirus r BV-prM-E-prM-E expressing the TBEV prM-E protein was constructed by the baculovirus-insect cell expression system.The identification results of indirect immunofluorescence,Western Blot and genomic PCR showed that the recombinant baculovirus r BV-prM-E-prM-E could successfully express the TBEV prM-E protein after infecting Sf9 cells.TBEV VLPs were prepared by inoculating recombinant baculovirus in suspension-cultured Sf9 cells,and then purified by density gradient centrifugation.The resultant TBEV VLPs were mixed with complex adjuvant Poly（I:C）＆Montanide ISA 201VG to prepare the VLPs vaccine,and evaluated the immune effect by immunizing BALB/c mice.Activation of DC cells and B cells was detected in inguinal lymph node cells one week after the first immunization of mice with TBEV VLPs;the activation of T cells and the production of memory T cells were detected in the spleen one week after the third immunization;a significant increase in the number of splenocytes with secreting IFN-γand IL-4.The production of TBEV-specific IgG（1:10）was detected in the serum of mice 1 week after the second immunization,and the level of TBEV IgG antibody increased significantly1 week after the third immunization,up to 1:10^5.5.The IgG antibody level remained at1:10^4.3 3 months after the third immunization.In summary,TBEV VLPs could stimulate the body to produce cellular immunity and humoral immunity.The third immunization could produce immune memory and induce mice to produce high and long-lasting specific IgG antibodies.（3）Bacterium-like particles of tick-borne encephalitis virus preparation for immunization research.The recombinant baculovirus r BV-E-PA3-E-PA3 expressing the E-PA3 fusion protein was constructed using the baculovirus-insect cell expression system.The identified recombinant baculovirus r BV-E-PA3-E-PA3 was inoculated into suspension-cultured Sf9 cells and the corresponding culture supernatant was combined with Gram positive enhancer matrix（GEM）particles to prepare TBEV BLPs.The results of Western blot and transmission electron microscopy showed that the E-PA3 fusion proteins successfully anchored on the surface of GEM particles.BLPs vaccines were prepared by mixing with complex adjuvant Poly（I:C）＆Montanide ISA 201VG.BALB/c mice were immunized by intramuscular injection to evaluate the immune effect.The results suggested that the production of TBEV-specific IgG(1:10^1.7)was detected in the serum of mice 1 week after the first immunization,and the level of TBEV IgG antibody increased significantly 1 week after the second immunization,up to 1:10^6.9.The IgG antibody level remained at 1:10~6 5 months after the second immunization.Activation of DCs and B cells could be detected in inguinal lymph node cells one week after the first immunization of mice with TBEV BLPs;the activation of T cells and the production of memory T cells were detected in the spleen one week after the third immunization;a significant increase in the number of splenocytes with secreting IFN-γand IL-4.The secretion of Th1-type cytokine TNF-αand Th2-type cytokines IL-5,IL-6,IL-10,IL-1βin splenocytes increased significantly.In conclusion,TBEV BLPs could stimulate the body to produce strong cellular and humoral immunity.The second immunizations could produce immune memory and induce mice to produce excellent high and long-lasting specific IgG antibodies.The TBEV VLPs and TBEV BLPs prepared in this study can stimulate the body to produce specific cellular and humoral immunity to varying degrees,and TBEV BLPs have higher and longer-lasting immune effects.They could be used as TBEV vaccine candidates.