乙肝病毒核酸疫苗诱导H-2~b小鼠特异性免疫反应的初步研究

The humoral and cellular immune responses induced in H-2~b micee by nucleic acidvaccines encoding protein of hepatitis B virus

目的：观察并证实adr和adw亚型MHBs核酸疫苗单独应用，或与HBc核酸疫苗联合免疫诱导H-2b ，小鼠的特异性体液和细胞免疫反应。方法：C57BL/6（H-2b）小鼠随机分为4组：pJW4303组（P组，5只）；pJW4303/MHBs/adw组（W组，6只）；pJW4303/MHBs/adr组（R组，6只）；pJW4303/MHBs/adr+pJW4303/HBc组（R＋C组，6只）。免疫方法为各组小鼠每只每次注射相应的质粒DNA疫苗100μg(100μl)。免疫程序为0，2，4 w。小鼠血清抗HBs和抗HBc水平以及小鼠脾淋巴细胞培养上清中的IFN-γ和IL-4浓度用 ELISA法检测。小鼠淋巴细胞抗原特异性增殖试验采用3H-TdR掺入法，计算刺激指数（SI）。结果：W、R、R＋C各免疫组在末次免疫后4w所有小鼠均产生抗-HBs，但各免疫组间无明显差异，抗-HBs以R＋C组最早出现。R＋C组小鼠在第1次免疫后2w抗-HBc即全部阳性,P组免疫后始终未出现抗-HBs和抗-HBc。核酸疫苗免疫的w、R、R+C各组免疫小鼠HBsAg特异性脾淋巴细胞增殖指数（SI）均高于P组（P<0．01-P<o．001），R＋C（n=6）组小鼠脾淋巴细胞HBsAg特异性SI比R组显著升高（P<0．05）；R＋C免疫小鼠HBcAg特异性脾淋巴细胞增殖指数（SI）均≥2，与P组（其SI均<2）相比差异非常显著（P<0．001）。W、R、R＋c各免疫组小鼠脾淋巴细胞与HBsAg共培?

Abstract Objective:To observe and confirm the specific humoral and cellurar immune responses induced by the nucleic acid vaccinesencoding HBV PreS2+S of subtypes adw and adr and encoding HBcAg in H-2b mice. Methods: Twenty-three C57BL/6 (H-2b) mice were ran-domly divided into four groups:group pJW4303(group P); group pJW4303/MHBs/adw(group W); group pJW4303/MHBs/adr(group R) andgroup pJW4303/MHBs/adr+pJW4303/HBc(group R+C) .The imunization method is that each mouse was injected(i.m) into the quadriceps femurs muscles with the corresponding 100μg(100μl) of plasmid DNA at 0,2,4 w.ELISA determined the level of anti-HBs and anti-HBc an-tibody in each mouse sera and the concentrations of IEN-γ and IL-4 culture supernatant of spleen cells.Antigen specific proliferation of mousesplenocytes was measured by 3 H-TdR incorporating assay and the stimulation index(SI) was calculated.Results:After the last immunization,anti-HBs antibody had been detected in all mice of group W,group R and group R+C.The titers of anti-HBs were ranged from 22 329 to665.5 mU/ml.There are very significant differences between DNA vaccine groups (group W,group R,group R+C) and control group (groupP)(P<0.01),but no significant difference was observed between group W,group R and group R+C(P>0.05).Anti-HBs was first de-tected in group R+C.After the first immunization of nucleic acid vaccine of pJW4303/HBc,anti-HBc antibody appeared in all mice of groupR+C. Neither anti-HBs nor anti-HBc antibody appeared in all nice of group R+C.Neither anti-HBs nor anti-HBc was detected in group P.The stimulation index(SI) represent the capacity of antigen specific mouse proliferation of splenocytes. HBsAg-specific SIs of mouse splenocytesfrom group W,group R and group R+C is hipe than that in group P(P < 0.01 - P < 0.001) .HBsAg-specific SIs of mouse splenocytesfrom group R+C is higher than that in group R(P<0.05). HBcAg-specific SIs of mouse splenocytes from group R+C were all more than orequal to 2,but the SIs in group P were all less than 2.There was very significant difference between these two groups(P < 0.001) .By com-paring with group with group P,the levels of IFN-γ in culture supernatant of spleen cells in group W,group R and group R+C after the stimulation of HB-sAg were significant increased (P < 0.001) and the level of IL-4 in these groups were significant (P <0.001) .With the stimula-tion of HBcAg,the concentrations of IFN-γ in culture supernatant of spleen cells in group R + C is much higher than that in group P. While thelevels of IL-4 in both groups were similar and without significant difference.Conclusion:The reults in this study have revealed that nucleic ac-id vaccines encoding HBV PreS+ S of subtypes adw and adr and encoding HBcAg can induce strong humoral and cellurar immune responses,indicating the good immunogenicity of these DNA vaccines. Co-immunization with HBcAg and MHBs nucleic acid vaccines may strengthen thehumoral and cellular immune responses induced by MHBs nucleic acid vaccine. Immunizations of DNA vaccines encoding MHBs and HBcAginduce type I helper T cell responses.