关键词: 分枝杆菌, 牛, DNA, 细菌, 佐剂, 免疫, 可重复性, 结果

Abstract: Objective  To evaluate the effectiveness and safety of an aluminum salt and BCG CpG DNA combination adjuvant system (BC-C02). Methods  Cell surface marker and intracellular cytokines were measured by FACS and ELISPOT to evaluated the immunostimulatory  effect of BCG CpG DNA adjuvant. The effects of CpG DNA adjuvant on H1N1 vaccine were evaluated by humoral immunity and protective efficacy of H1N1 vaccine. The mice were divided into 7 groups: PBS control, low dose H1N1, low dose H1N1+CpG DNA, middle dose H1N1, middle dose H1N1+CpG DNA, high dose H1N1, and high dose H1N1+CpG DNA, 20 mice per group. The effect of BC-C02 adjuvant on a new TB vaccine was evaluated by the therapeutic effect of vaccine in guinea pigs infected with M. tuberculosis, in which low dose，middle dose and high dose of vaccine (Ag85B+ BC-C02) were set, and the saline and adjuvant were used as contol, 10 guinea pigs per group. The IgE level of BC-C02 in mice and general allergic reaction of BC-C02 in guinea pigs were measured to evaluate its immunotoxicologic effect and safety.   Results  BC-C02 could stimulate B cells proliferation (CD19⁺% of CpG DNA was 41% and that of saline was 27%, t=－4.40, P<0.05), up-regulate the expression of TLR9 in B cells (CD19⁺TLR-9⁺% of CpG DNA was 2.8% and that of saline was 1.1%,  t=－5.92, P<0.05), promote the expressions of MHC Ⅱ, TLR-9 and IL-12 in macrophages (F4/80⁺I-A/I-E⁺% of CpG DNA was 82% and that of saline was 31%, F4/80⁺I-A/I-E⁺TLR-9⁺IL-12⁺% of CpG DNA was 2.1% and that of saline is 0.1%,  t_MHC-Ⅱ=－4.32,  t_{TLR-9 and IL-12}=4.80,  P<0.05). In the experiment of CpG DNA as H1N1 vaccine adjuvant, the titers of HI antibody were detected at 5 d, 7 d, 14 d and 28 d after the immunization, the antibody titers in PBS conrol group were all less than 10; the antibody titers in low dose H1N1 group < that in middle dose H1N1 group < that in high dose H1N1 group; the antibody titers in all groups of antigen with CpG DNA were higher than that in the corresponding group without CpG DNA. The protective efficacy of H1N1 vaccine showed that the survival rates of PBS, low dose H1N1, low dose H1N1+CpG DNA, high dose H1N1, high dose H1N1+CpG DNA groups were 30%，50%，90%，100%，100%, respectively, CpG DNA could promote 40% of survival rate compared with the groups without adjuvant. After the guinea pigs infected with M. tuberculosis were treated with Ag85B+ BC-C02, there were TB lesions in all of livers, lungs and spleens of saline group; there were no TB lesions in 30% of animals, and less than 30 of mean TB lesion scores (liver, lung and spleen) in 60%～70% of animals in vaccine groups. BC-C02 adjuvant could not induce the produce of antibody IgE in mice and general allergic reactions in guinea pigs. Conclusion  BC-C02 combination adjuvant system can induce cellular and humoral immune responses, improve the protective effectiveness of vaccines, and be used as a candidate adjuvant of new vaccine.

Key words: Mycobacterium bovis, DNA, Bacterial, Adjuvants, immunologic, Reproducibility of results