SEA为载体蛋白的A/C群脑膜炎奈瑟菌结合物初步免疫效果评价

Preliminary evaluation on immune effect from immunization group A/C meningococcal polysaccharide conjugates by using SEA as protein carrier

目的对以金黄色葡萄球菌肠毒素A(staphylococcal enterotoxin A,SEA)为载体蛋白的A/C群脑膜炎奈瑟菌结合物的免疫效果进行初步评价。方法采用1-氰基-4-二甲氨基-砒啶四氟硼酸(1-cyano-dimethylamino pyridiniumtetrafluoroborate,CDAP)活化法,将SEA、破伤风类毒素(tetanus toxin,TT)分别与A群脑膜炎奈瑟菌荚膜多糖(group A N.meningitidis capsular polysaccharide,GAMP)、C群脑膜炎奈瑟菌荚膜多糖(group C N.meningitidis cap-sular polysaccharide,GCMP)结合制备结合物;将结合物分别免疫BALB/c小鼠,腹部皮下免疫3次,于第1针免后第9天、第19天、第27天眼眶采血,分离血清备用;检测体液免疫及细胞免疫反应水平。结果 SEA与GAMP结合后能增强抗-GAMP Ig G抗体水平,第3次免后GAMP-SEA组多糖抗体水平达到1∶12 800,高于GAMP组1∶400,但GCMP-SEA组结果不理想。SEA与GAMP结合后均能激发细胞免疫反应,IFN-γ和IL-4的SFC与GAMP组比较均升高,且IFN-γ高于IL-4。SEA与GAMP、GCMP结合后Th1/Th2细胞亚群比值分别达到23.48和22.19,高于GAMP组(14.09)和GCMP组(16.73),差异有统计学意义(P<0.05),提示SEA与GAMP、GCMP结合后可激发细胞免疫。结论 SEA与GAMP、GCMP结合后既能增强GAMP、GCMP的免疫原性,提高体液免疫反应,又能激发细胞免疫反应,提示SEA具备作为A/C群脑膜炎奈瑟菌结合疫苗载体蛋白的可行性。

Objective Preliminary evaluation was carried out on cellular and humoral immune effect by immunization of group A and group C meningococcal polysaccharide （GAMP, GCMP） conjugates by using recombinant staphylococcal enterotoxin A （SEA） as a protein carrier. Methods SEA protein and tetanus toxoid （TT） were conjugated to group A and group C meningococcal capsular polysaccharide, separately, activated by CDAP （1-cyano-dimethylamino pyridinium tetrafluoroborate）. Each conjugate was injected BALB/c mice subcutaneously on day 0, 10, 20. The serum samples were collected on day 9, 19, 27 after first immunization, and stored at -20 ℃ for detecting their humoral and cellular immune responses. Results The recombinant SEA can increase the level of anti-GAMP antibodies after it conjugated with GAMP, with an anti-GAMP IgG 1 ： 12 800 after the third immunization, in camparison it was higher than 1 ： 400 in GAMP group. However, the results of the GCMP-SEA group were not disirable. SEA could stimulate cellular response, compared with CAMP group SFC （ spot forming cell） in IFN-γ and IL-4 group were higher, and IFN-γ level was higher than IL-4. The ratios of Th1/Th2 cells reached 23.48 and 22.19, respectively, after SEA combined with GAMP and GCMP, higher than the GAMP （14.09） and GCMP group （16.73）, there was a statistical meaning （P 〈 0.05 ）, it suggested that SEA could stimulate cellular response. Conclusion The SEA was able to increase both of humoral response and cellular response in immunized mice after conjugated with GAMP and GCMP, this indicated that the SEA had a potential to use as a protein carrier in preparation of group A/C meningococcal polysaecharide conjugate vaccine.