布鲁氏菌粗糙型RA343株与光滑型A19株免疫小鼠后特异性T细胞和抗体动态变化分析

Dynamic changes of specific T cells and antibodies in mice immunized with rough-type RA343 and smooth-type A19 of Brucella

摘要:

【背景】布鲁氏菌病(简称布病)严重威胁着人类健康和畜牧业的发展，使用毒力弱、免疫原性好、不干扰血清学诊断的疫苗是防控布病的有效措施。【目的】比较布鲁氏菌粗糙型RA343株与光滑型A19株免疫小鼠后机体特异性T细胞和抗体的动态变化，评价粗糙型RA343株的免疫效果。【方法】采用6周龄雌性BALB/c小鼠，共分为3组，RA343株免疫组和A19株免疫组各15只小鼠，空白对照组5只小鼠。免疫组的每只小鼠经腹股沟皮下注射0.1 mL (含菌量为1×10⁸ CFU)菌液。免疫后1、2和3周各免疫组及空白对照组分别剖杀5只小鼠，无菌取脾脏，一部分脾脏研磨分离淋巴细胞，用流式细胞术检测小鼠布鲁氏菌特异性CD4⁺T细胞与CD8⁺T细胞所占比例的变化趋势；另一部分脾脏称重后分离布鲁氏菌RA343株和A19株，评估RA343株和A19株在小鼠脾脏中的定殖情况。在剖杀小鼠的同时取外周血分离血清，用ELISA方法检测免疫后小鼠血清中IgM和IgG抗体的消长规律。采用GraphPad Prism 8.0软件作图并进行统计学分析。【结果】与A19免疫组相比，免疫后1周，RA343免疫组小鼠脾脏布鲁氏菌特异性CD4⁺IFN-γ所占比例极显著增加(P<0.01)，CD4⁺IL-2、CD8⁺IL-2和CD8⁺TNF-α所占比例显著增加(P<0.05)；免疫后2周，RA343免疫组特异性CD4⁺TNF-α所占比例极显著增加(P<0.01)，CD8⁺TNF-α所占比例显著增加(P<0.05)；免疫后3周，CD4⁺TNF-α所占比例显著增加(P<0.05)，CD8⁺IFN-γ所占比例极显著增加(P<0.01)。免疫后1–3周，RA343免疫组小鼠脾脏含菌量显著低于A19免疫组(P<0.05)，而且RA343免疫组脾脏重量低于A19免疫组。光滑型A19株免疫小鼠的IgM和IgG抗体水平在免疫后2周均显著升高；粗糙型RA343株免疫小鼠的IgM和IgG抗体采用光滑型抗原进行ELISA检测结果均为阴性。【结论】与光滑型A19株相比，布鲁氏菌粗糙型RA343株诱导的细胞免疫应答较强，而且RA343组小鼠脾脏含菌量显著低于A19组，产生抗体与光滑型布鲁氏菌抗原无交叉血清学反应，综合表明布鲁氏菌粗糙型RA343株毒力弱，免疫小鼠后可产生良好的细胞免疫反应和体液免疫反应且不干扰血清学诊断，是理想的布鲁氏菌疫苗候选株。

关键词:布鲁氏菌;流式细胞术;特异性T细胞;抗体

Abstract:

[Background] Brucellosis threatens human health and the development of animal husbandry and vaccines with low virulence, good immunogenicity, and no interference of serological diagnosis turn to be a solution. [Objective] To compare the dynamic changes of specific T cells and antibodies in mice immunized with rough-type RA343 and smooth-type A19 of Brucella, and to evaluate the immune effect of rough-type RA343. [Methods] The 6-week-old female BALB/c mice were classified into three groups, 15 in RA343 group, 15 in A19 group, and 5 in blank control group. For RA343 group and A19 group, each mouse was injected (sc) with 0.1 mL (1×10⁸ CFU) bacterial solution through into groin. A total of 5 mice in each of the three groups were killed 1, 2, and 3 weeks after immunization, respectively, and their spleens were separated. A part of the spleen was ground, and lymphocytes were separated to detect the proportions of CD4⁺T cells and CD8⁺T cells in mice (flow cytometry). The other part was weighed and then RA343 and A19 of Brucella were isolated, to evaluate the colonization of RA343 and A19 in mouse spleen. At the same time, the peripheral blood was collected and the serum was separated. For the determination of IgM and IgG in the serum of immunized mice (ELISA). GraphPad Prism 8.0 was employed for plotting and statistical analysis. [Results] The proportion of Brucella-specific CD4⁺IFN-γ in spleens (P<0.01), and the proportions of CD4⁺IL-2, CD8⁺IL-2, and CD8⁺TNF-α (P<0.05) increased in RA343 group compared with those in A19 group 1 week after immunization. Two weeks after immunization, the proportion of specific CD4⁺TNF-α (P<0.01) and the proportion of CD8⁺TNF-α (P<0.05) in RA343 group rose compared with those in A19 group. Three weeks after immunization, RA343 group registered increase of the proportion of CD4⁺TNF-α (P<0.05) and the proportion of CD8⁺IFN-γ (P<0.01) compared with A19 group. The bacterial content in spleens of RA343 group was lower than that of A19 group (P<0.05), and the spleen weight was also lower than that of A19 group 1–3 weeks after immunization. IgM and IgG levels of mice in A19 group increased significantly 2 weeks after immunization and no IgM or IgG was detected in the RA343 group. [Conclusion] Compared with A19, RA343 induced strong cellular immune response. The bacterial content in spleen of mice in RA343 group was significantly lower than that in A19 group and no serological cross-reaction occurred between antibody and A19 antigen. In summary, RA343 has low virulence, and can induce strong cellular immune response and humoral immune response in mice, with no interfere of serological diagnosis. Thus, it can be used as a candidate strain for the development of vaccine against brucellosis.

Key words:Brucella;flow cytometry;specific T cells;antibody