Ma Jinzhu, Yu Liquan, Song Baifen, Yu Yongzhong, Zhang Shaoduo, Wei Yuhua, Wu Zhijun, Yao Di, Yu Wei, Zhu Zhanbo, Cui Yudong
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China.
College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China.
J Med Microbiol. 2018 Mar;67(3):432-440. doi: 10.1099/jmm.0.000666. Epub 2018 Jan 17.
In this study, we prepared GapC1-150-IsdB126-361-TRAP (GIT) proteins plus heat-labile enterotoxin B (LTB) as an intra-molecular adjuvant, together with CpG to further enhance its immunogenicity.
Initially, the target genes were acquired and inserted into pET-32a (+) vectors to express LTB-GIT protein. LTB-GIT expression was confirmed by Western blotting and its immunocompetence was estimated through ELISA. Further, we immunized BALB/c mice with the LTB-GIT plus CpG adjuvant. After the second immunization, the antigen-specific CD4 cell responses for IFN-γ, IL-2, IL-4 and IL-10 were monitored by intracellular cytokine staining (ICS) assay. After the third immunization, the level of IgG antibodies in the serum from immunized groups was assessed by ELISA, and the protective immune response was appraised by Staphylococcus aureus and Streptococcus dysgalactiae challenge.
The ELISA results showed that the OD450nm value of the LTB-GIT group was significantly higher than that of the BSA group. The group immunized with LTB-GIT plus CpG exhibited significantly stronger CD4 T cell responses for IFN-γ, IL-2, IL-4 and IL-10 compared to the group immunized with LTB-GIT, GIT alone orLTB-GIT plus CpG. In addition, the group immunized with LTB-GIT plus CpG generated the highest level of IgG antibodies against GIT among all of the groups, and our results also showed that LTB-GIT plus CpG markedly improved the survival percentage of mice compared to other groups.
We confirmed that the novel double adjuvants, LTB and CpG, are able to significantly improve GIT-induced immune responses. This formula could be a promising strategy for enhancing the immune efficacy of multi-subunit vaccines against Staphylococcus aureus and streptococcal infection.
在本研究中，我们制备了GapC1-150-IsdB126-361-TRAP（GIT）蛋白，并将热不稳定肠毒素B（LTB）作为分子内佐剂，同时加入CpG以进一步增强其免疫原性。
首先，获取目标基因并将其插入pET-32a（+）载体中以表达LTB-GIT蛋白。通过蛋白质印迹法确认LTB-GIT的表达，并通过酶联免疫吸附测定法评估其免疫活性。此外，我们用LTB-GIT加CpG佐剂免疫BALB/c小鼠。第二次免疫后，通过细胞内细胞因子染色（ICS）测定法监测针对IFN-γ、IL-2、IL-4和IL-10的抗原特异性CD4细胞反应。第三次免疫后，通过酶联免疫吸附测定法评估免疫组血清中IgG抗体的水平，并通过金黄色葡萄球菌和停乳链球菌攻击来评估保护性免疫反应。
酶联免疫吸附测定结果显示，LTB-GIT组的OD450nm值显著高于牛血清白蛋白组。与用LTB-GIT、单独的GIT或LTB-GIT加CpG免疫的组相比，用LTB-GIT加CpG免疫的组对IFN-γ、IL-2、IL-4和IL-10表现出明显更强的CD4 T细胞反应。此外，在所有组中，用LTB-GIT加CpG免疫的组产生了最高水平的针对GIT的IgG抗体，并且我们的结果还表明，与其他组相比，LTB-GIT加CpG显著提高了小鼠存活率。
我们证实新型双重佐剂LTB和CpG能够显著改善GIT诱导的免疫反应。该配方可能是增强多亚基疫苗针对金黄色葡萄球菌和链球菌感染免疫效力的一种有前景的策略。
