Shi Chang-hong, Zhang Ting-fen, Zhu De-sheng, Zhang Hai, Bai Bing, Zhao Yong, Yue Chen-li, Zhao Lei, Liu Jian-li
Laboratory Animal Centre, Fourth Military Medical University, Xi'an, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2009 Aug;32(8):603-7.
To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide.
BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test.
The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3.21 +/- 0.21) were higher than the synthetic peptide + IFA group (2.40 +/- 0.15). The IFN-gamma levels induced among the 3 experiment groups [(182 +/- 6), (194 +/- 9) and (179 +/- 8) mg/L] were lower than the BCG group [(275 +/- 10) mg/L], but higher than the saline group [(71 +/- 3) mg/L]. The IFN-gamma level induced among the 3 experiment groups did not show any marked difference. Although the protection induced by HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA all showed resistance against MTB H(37)Rv infection in the spleens or lungs (the bacterial logarithmic loads of spleen: 6.74 +/- 0.14, 6.60 +/- 0.13 and 6.81 +/- 0.28; the bacterial logarithmic loads of lung: 5.81 +/- 0.21, 5.74 +/- 0.27 and 6.65 +/- 0.32), none of them was better than the conventional BCG (the bacterial logarithmic loads of spleen and lung: 5.95 +/- 0.17 and 5.62 +/- 0.23).
Both HSP16.3 and its synthetic peptide can be considered as TB vaccine candidates or effective components in TB vaccines.
评估结核分枝杆菌（MTB）的HSP16.3及其合成肽诱导的小鼠对MTB感染的免疫反应和抵抗力。
BALB/c小鼠在尾根部皮下免疫3次，间隔2周。HSP16.3蛋白和合成肽的剂量均为每次50微克。用单剂量卡介苗（BCG，5×10⁶CFU/小鼠）免疫小鼠。通过酶联免疫吸附测定（ELISA）分析首次免疫后0、2、4、6、8周获得的血清中特异性抗体的浓度以及第8周获得的血清滴度。末次免疫后4周，处死每组8只小鼠，制备脾细胞单细胞悬液，其中一些用于经HSP16.3刺激的MTT比色法检测淋巴细胞增殖，其余细胞用于夹心ELISA检测IFN-γ水平。每组剩余小鼠经静脉注射10⁵个结核分枝杆菌H(37)Rv菌落形成单位（CFU）进行攻击，感染4周后处死，通过将匀浆组织的系列稀释液接种于Middlebrook 7H10琼脂平板来测定脾脏和肺中的细菌数量。采用LSD-t检验评估均值差异的统计学意义。
HSP16.3蛋白和肽的特异性抗体水平在前4周迅速升高，后几周适度升高。3个实验组（HSP16.3蛋白+DDA+MPL、合成肽+DDA+MPL和合成肽+IFA）的平均抗体特异性滴度均高于卡介苗组。3个实验组的脾淋巴细胞增殖指数（SI）（分别为3.13±0.18、3.21±0.21和2.40±0.15）分别显著高于卡介苗组（1.67±0.12）和生理盐水组（1.04±0.9）。HSP16.3蛋白+DDA+MPL组（3.13±0.18）和合成肽+DDA+MPL组（3.21±0.21）的SI高于合成肽+IFA组（2.40±0.15）。3个实验组诱导的IFN-γ水平[（182±6）、（194±9）和（179±8）mg/L]低于卡介苗组[（275±10）mg/L]，但高于生理盐水组[（71±3）mg/L]。3个实验组诱导的IFN-γ水平未显示出明显差异。虽然HSP16.3蛋白+DDA+MPL、合成肽+DDA+MPL和合成肽+IFA诱导的保护作用均显示出对MTB H(37)Rv感染的脾脏或肺部抵抗力（脾脏细菌对数载量：6.74±0.14、6.60±0.13和6.81±0.28；肺部细菌对数载量：5.81±0.21、5.74±0.27和6.65±0.32），但它们均不比传统卡介苗好（脾脏和肺部细菌对数载量：5.95±0.17和5.62±0.23）。
HSP16.3及其合成肽均可被视为结核病疫苗候选物或结核病疫苗中的有效成分。
