Immune Effects Of CVB3 VP1 Protein Given In Different Injection Sites And Doses And Combined With Various Adjuvant

Objective: Coxsackievirus is a member of Piconravirus entervirus. Coxsackievirus Group B Type 3(CVB3) is a major pathogen of human viral myocarditis, which is the leading cause of neonate and adolescent sudden death. Until now, there is no virus-specific preventive vaccine available for human .The VP1 is a major capsid protein of CVB3, which include many epitopes of B and T cells. It can elicit the production of effective neutralizing antibody in mice.Gene engineering subunit vaccine can be extracted from the pathogens directly or produced by prokaryotic and eukaryotic expressing system which have been created to express the target protiens. Subunit vaccine has been proved to be the strongest immunogen that can elicit a higher level production of antibody. These vaccines showed no side reaction caused by nonantigenic elements, and was more safe than other types of vaccnine in use. In previous study, a subunit vaccine, CVB3 VP1 was prepared from prokaryotic expressing system. It was shown that the VP1 vaccine could induce a higher titres of antibody and cell mediated immune response. But the titers of antibody produced were usually too weak to provide the host effective immune protection. Therefore, it is an important strategy for us to improve the immunological potency of VP1 protein and induce an effective CVB3-specific immune responses, as well as enhance the protective efficacy.The immune response elicited by antigens is affected by many factors: which include the animal species to be vaccinated, route of administration, the dose of antigen inoculated, the times and interval of immunization, and the immunological adjuvant compounds used and so on. In this study, a subunit vaccine, CVB3 VP1 protein prepared from prokaryotic expressing system and purified. The VP1 were inoculated in three frequently used injection site, with three doses of antigen and combined with different adjuvants. The immune response was then evaluated by testing the humoral immune and cell mediated immune response and protection of mice from CVB3 challenge.Methods:1 The prokaryotic expression plasmid pET-His/VP1 was transformed into E.coli BL21(DE3). The transformed E.coli. BL21(DE3) were cultured and induced to express VP1 protein with IPTG. The product expressed was analyzed by SDS-PAGE electrophoresis and verified in Western blotting by monoclonal antibody specific for the 6×histidine tag.2 The transformed bacteria were lysed by supersonic wave treatment. The supernatant and inclusion pellet were checked by SDS-PAGE to analysis the solubility of the expressed product. The result was that the protein was expressed in the form of inclusion body. The inclusion bodies were solubilized in urea and the fusion protein was purified with Ni affinity chromatography column under denaturing condition. The purified protein was concentrated by PEG8000.3 Animal experiments were performed in two stages. The first stage: BALB/c mice aged 6-8 weeks were divided into four groups, with twelve mice in each. The VP1 protein was diluted in PBS, and injected in peritoneal cavity,quadriceps,and subcutaneously in the nape. The mice were immunized three times at a three week interval with 100μg of protein. The primary immunizations were performed with FCA, and boost immunization with FIA. Fourteen days after every injection, sera were collected and tittered for CVB3-specific neutralizing antibodies by virus neutralizing test and for the IgG by ELISA. The optimal injection site was determined by comparison of the results.4 In the second stage of the experiments: BALB/c mice aged 6-8 weeks were divided into six groups,with eighteen mice in each. The mice were immunized three times at a three weeks'interval. The VP1 protein is injected in optimal injection site with different dosesâ‘ Low-dose group (50μg protein)â‘¡Middle-dose group (100μg protein )â‘¢High-dose group (150μg protein). In the groups ofâ‘ ,â‘¡,andâ‘¢, the primary immunizations were carried out with VP1 combined with FCA, and the boost with FIA(1:1). In optimal injection site and dose(100μg protein), the mice were immunized with VP1 protein in combination with different adjuvants: in the groupâ‘£montanide ISA720 group⑤Al(OH)3 groupâ‘¥PBS group. Fourteen days after every injection, sera were collected and tested for CVB3-specific neutralizing antibodies and specific IgG antibody by ELISA. Three weeks after the third immunization, splenocytes obtained from three mice of each group were stimulated by inactivated CVB3 and ConA. The lymphocytic proliferative activities and specific CTL cytotoxic activities were tested by CCK-8 assay; In the same time, the other twelve mice were challenged with 4 LD₅₀ of CVB3 and the numbers of surviving animals was monitored for twenty-one days post infection; Furthermore, the rest mice of each group were challenged with 3 LD₅₀ of CVB3 and sacrificed seven days later to evaluate the virus titers in the blood.Results:1 VP1 protein was expressed in prokaryotic expression system successfully and the protein was expressed in a inclusion form. The protein was verified by Western blot analysis with His antibody.2 Titers of neutralizing antibody in different administration route groups after every immunization were: intraperitoneal group: 1:7.08,1:28.18,1:42.66; subcutaneous group: 1:7.08,1:11.22,1:35.48; intramuscular group: 1: 8.91,1:47.86,1:70.79. The mean titers of specific IgG antibody were : intraperitoneal group: 1:155,1: 7600,1:11800; subcutaneously group :1:240, 1:8300,1:17000 ;Intramuscular group: 1:425,1:22400,1:38400. The statistical analysis showed that the differences of neutralizing antibody titers and the mean titers of specific IgG of every group were significant between diferent times of immunization (P<0.05). After the third immunization the differences between different groups were significant (P<0.01). The Intramuscular group were elicited higher titers of antibody than intraperitoneal group, and the titers of antibody clicited in intraperitoneal group were higher titers than those in subcutaneously group.3 The mean titers of neutralizing antibody after every immunization in different dose groups were: low dose group: 1:6.31, 1:42.66, 1:47.86; middle dose group: 1:7.08, 1:44.67, 1:63.10; high dose group: 1:7.08, 1:50.12,1:70.79 ;The mean titers of specific IgG antibody were: low dose group: 1:200, 1:6400, 1:19200; middle dose group: 1:398, 1:21000, 1:36300; high dose group :1:425, 1:23040, 1:51200. The statistical analysis showed that excepting PBS group, the differences of neutralizing antibody titers and the mean titers of specific IgG antibody of every group were significant after every times of immunization (P<0.05).After the third immunization the difference was significant (P<0.05). high dose group elicited higher titers than middle dose group , middle dose group elicited higher titers than low dose group.4 The mean titers of neutralizing antibody after every immunization in different adjuvant groups were: Adjuvant AL(OH)3 group: 1:5.62, 1:12.59, 1:26.92; Adjuvant FA group:1:7.08, 1:44.67, 1:63.10; Adjuvant ISA720: 1:8.91, 1: 44.67, 1: 85.11; The mean titers of specific IgG antibody were : Adjuvant AL(OH)₃ group: 1:150, 1:1600,1:11800; Adjuvant FA group: 1:398, 1:21000, 1:36300; Adjuvant ISA720 group: 1:280, 1: 22300, 1: 38400; The statistical analysis showed that excepting PBS group, the differences of neutralizing antibody titers and the mean titers of specific IgG antibody after every times of immunization of every group were significant (P<0.05). After the third immunization the difference was significant (P<0.05). neutralizing antibody: Adjuvant ISA720 group elicited higher titers of antibody than adjuvant FA group, adjuvant FA group elicited higher titers of antibody than adjuvant AL(OH)3 group, IgG antibody: Adjuvant ISA720 group and adjuvant FA group elicited higher titers of IgG than adjuvant AL(OH)3 group.5 Splenocytes from the vaccinated mice showed different proliferative activity. When stimulated by CVB3 or ConA,the splenocytes from all groups showed significantly higher proliferative activity than that of PBS group (P<0.05). High dose group showed stronger proliferative activity than middle dose group and low dose group(P<0.05). Adjuvant ISA720 group showed stronger proliferative activity than adjuvant FA group and adjuvant AL(OH)3 group (P<0.05). When stimulated by ConA , the differences were not significant (P<0.05). 6 The splenocytes from all groups showed significantly stronger specific lymphocytic CTL cytotoxic activity than that of PBS group . High dose group showed stronger CTL cytotoxic activity than middle dose group and low dose group (P<0.05). Adjuvant ISA720 group showed stronger CTL cytotoxic activity than adjuvant FA group and adjuvant AL(OH)3 group (P<0.05).7 Up to the 21th day after CVB3 challenge, the survival rates of PBS group, low dose group, middle dose group, high dose group, adjuvant AL(OH)3 group and Adjuvant ISA720 group were 0, 66.67%,66.67%;50%,33.33%,50 %, respectively. Chi-square test indicated that differences among the groups were not significant. It indicated that the survival time of low dose group and middle dose group were better than high dose group (P<0.05) and that of Adjuvant FA group was better than adjuvant AL(OH)3 group (P<0.05).8 After 3LD₅₀ CVB3 challenge, the virus titers in blood of all groups were significantly lower than that of PBS group (P<0.05). The titers in high dose group was lower than in middle dose group and low dose group(P<0.05). the titer in Adjuvant ISA720 group was lower than adjuvant AL(OH)3 group (P<0.05).Conclusion:1 VP1 protein was expressed in E. coli successfully and the fusion protein was purified.2 Different route of administration to BALB/c mice neutralizing antibody and IgG antibody increased after every times of immunization. It was showed intramuscular group elicited stronger humoral immune responses.3 With different injected dose ,neutralizing antibody and IgG antibody increased along with injeccted dose. But it indicated that the survival time of low dose group is best .4 VP1 protein was immunized mice in combination with different adjuvants. Adjuvant FA and ISA720 has induced better immune response in comparison to AL(OH)3. Since FCA is not approved for human use,we conclude that ISA720 is an effective adjuvant can be used for further studies.5 In conclusion, the results suggested that 50μg-100μg protein combined with adjuvant FA and ISA720 given by intramuscular injection might induce an improved immune responses.