基于梅毒核酸疫苗pcD/Tp92的不同接种策略

目的 评估基于梅毒核酸疫苗pcD/Tp92的各接种优化策略对新西兰兔梅毒螺旋体(Tp)皮肤感染的免疫保护效应.方法 核酸疫苗pcD/Tp92采用2次肌内注射免疫或肌内注射初免鼻饲加强的免疫方式结合黏膜佐剂CpG ODN及Tp92重组蛋白,分别或联合免疫新西兰兔.酶联免疫吸附法(ELISA)检测各接种策略组免疫期间(0~8周)兔特异性抗体产生及变化水平,免疫8周后兔鼻咽部、阴道黏膜SIgA产生水平及兔脾细胞IL-2、γ干扰素(IFN-γ)诱导水平,噻唑蓝法(MTT)检测兔脾淋巴细胞增殖水平.记录各组在初次免疫后第10周兔皮下接种Tp标准株感染后接种部位感染早期皮损的变化.结果 采用pcD/Tp92核酸疫苗肌内注射初免,CpG-ODN联合Tp92重组蛋白抗原鼻饲新西兰兔加强免疫的接种策略组(C2组)分别与pcD/Tp92疫苗肌注组(A2组),pcD/Tp92疫苗肌注初免,pcD/Tp92鼻饲加强免疫组(B1组)或结合黏膜佐剂CpG ODN联合免疫的接种策略组(B2组)相比,既能促进pcD/Tp92核酸疫苗在兔体内免疫期间(8周)诱生更高特异性抗体水平(C2组:1.825±0.175;A2组:1.372±0.322;B1组:0.893±0.297;B2组:1.294±0.124;P<0.05),IL-2(C2组:154.7±14.6;A2组:112.3±13.4;B1组:76.6±21.5;B2组:97.3±18.7;P<0.05)及IFN-γ(C2组:277.4±24.4;A2组:232.8±25.3;B1组:165.7±22.6;B2组:211.3±24.6;P<0.05)分泌水平,以及更高的T细胞增殖分化水平(SI C2组:3.57±0.24;A2组:3.08±0.22;B1组:2.12±0.14;B2组:2.88±0.18;P<0.05),还能刺激更高的黏膜特异性SIgA抗体,导致最低Tp感染部位皮损Tp阳性率(6.67%)及溃疡病灶形成率(6.67%)从而达到有效的保护作用.结论 pcD/Tp92核酸疫苗肌内注射初免,CpG ODN联合Tp92重组蛋白鼻饲加强免疫的接种策略能在新西兰兔体内诱导最强的黏膜免疫和免疫保护效应.

Optimization of strategies for inoculation of Treponema pallidum pcD/Tp92 DNA vaccine

Objective To evaluate immune protective effects of Treponema pallidum(Tp)pcD/Tp92 DNA vaccine delivered through different inoculation routes against Tp-induced skin infection in New Zealand rabbits. Methods A total of 108 New Zealand rabbits were randomly and equally divided into 6 groups:A1 and A2 groups treated with intramuscular injection of empty plasmids pcD and pcD/Tp92 DNA vaccine respectively for 2 sessions, B1, B2, C1 and C2 groups firstly treated with intramuscular injection of the pcD/Tp92 DNA vaccine for 1 session for primary immunization, then receiving nasogastric feeding with pcD/Tp92 DNA vaccine, pcD/Tp92 DNA vaccine+cytosine-phosphate-guanine(CpG)oligodeoxynucleotide (ODN), and recombinant Tp92 protein, and recombinant Tp92 protein+CpG ODN respectively for booster immunization. Enzyme-linked immunosorbent assay(ELISA)was conducted to detect the serum level of anti-Tp92 IgG antibody at week 0, 2, 4, 6, 8 after immunization, the SIgA level in the nasopharyngeal region and vaginal mucosa at week 8 after immunization, as well as levels of interleukin-2(IL-2)and interferon-γ (IFN-γ)in the culture supernatant of rabbit spleen cells at week 8 after immunization, and methyl thiazolyl tetrazolium(MTT)assay was performed to estimate proliferative activity of rabbit splenic lymphocytes in three rabbits from each group. At week 10 after immunization, other 15 rabbits from each group were subcutaneously inoculated with Tp standard strain, and changes of skin lesions at the inoculation site during early-stage infection were observed and recorded. Results At week 8 after immunization, the C2 group showed significantly higher serum level of anti-Tp92 IgG antibody(1.825 ± 0.175), supernatant levels of IL-2 (154.7 ± 14.6)and IFN-γ(277.4 ± 24.4), and proliferative activity of T cells(3.57 ± 0.24)compared with the A2(1.372 ± 0.322, 112.3 ± 13.4, 232.8 ± 25.3, 3.08 ± 0.22, respectively, all P<0.05), B1(0.893 ± 0.297, 76.6 ± 21.5, 165.7 ± 22.6, 2.12 ± 0.14, respectively, all P<0.05)and B2(1.294 ± 0.124, 97.3 ± 18.7, 211.3 ± 24.6, 2.88 ± 0.18, respectively, all P<0.05)groups. In addition, effective immunoprotection was achieved in the C2 group with more production of mucosa-specific SIgA antibody, as well as the lowest Tp-positive rate (6.67%) and ulcer formation rate (6.67%) in skin lesions at the inoculation sites. Conclusion The effective vaccination strategy, namely intramuscular injection of the pcD/Tp92 DNA vaccine for primary immunization followed by nasogastric feeding with mucosal adjuvant CpG ODN combined with recombinant Tp92 protein for booster immunization, can induce the strongest mucosal immune responses and immune protective effects.