应用ELISA和RT-PCR技术评价接种Capri Pox疫苗的犊牛外周血细胞介导的免疫应答
文献 Evaluation of Cell-mediated Immune Response in PBMCs of Calves Vaccinated by Capri Pox Vaccines Using ELISA and Real-time RT-PCR 发表在 Research in Molecular Medicine 原文链接
Abstract:
Background:
The analysis of antigen-specific cytokine expression has been considered to evaluate the immune responses and vaccines efficacy in recent years. The aim of this study was to compare the cell-mediated immune response characteristics of two Capri pox virus (CaPV) vaccines against lumpy skin disease in cattle.
Materials and Methods:
Two Capri pox virus vaccines were administered to dairy cows of two farms and followed up to 5 weeks post vaccination. These vaccines were live attenuated Goat pox virus (GTP) Gorgan strain (n=20) and Sheep pox virus (SPP) Romanian strain (n=20). Cell-mediated immune response of vaccinated calves was evaluated using in vitro lymphocyte proliferation and IFN-g and IL-4 release assay after stimulation with recall vaccine strains, and in vivo cytokine expression in PBMCs by real-time PCR.
Results:
Lymphocyte proliferation in GTP- and SPP-vaccinated groups began to increase till reached to its peak at third week post vaccination and then decreased in the weeks thereafter. Stimulation index in stimulated PBMCs in GTP-vaccinated calves was higher than SPP-vaccinated calves in all weeks, which indicated higher levels of immunogenicity produced by the GTP-vaccine in cattle. Also, in both vaccinated groups the peak release of IFN-g and IL-4 proteins in cultured PBMCs in response to recall antigen was detected at week 3 post vaccination. Although the mean of the cytokine release in GTP-vaccinated calves was higher than SPP-vaccinated calves in all weeks of experiment, a significant difference was only observed at week 3 post vaccination (P<0.05). In contrast, the IFN-g mRNA expression in PBMCs of vaccinated groups was induced early, peaked at week 1 post vaccination and decreased in the weeks thereafter, and this rate was higher in GTP-vaccinated calves compared with SPP-vaccinated calves in all weeks, but the significant difference was only found at week 3 post vaccination (P<0.05). However, the IL-4 mRNA expression showed delayed induction and peaked at week 3, and unlike the SPP group, it remained at this level in GTP group, until the end of experiment. Also this rate of expression in GTP-vaccinated calves was higher than SPP-vaccinated calves in all weeks and had a significant increase at week 5 post vaccination (P<0.05).
Conclusion:
The findings show that due to induction of high level cell-mediated immune response in live attenuated GTP vaccine compared to SPP vaccine, GTP vaccine has a good immunogenic response, and therefore can be a better choice for vaccination against lumpy skin disease.
摘要：
背景：
近年来，对抗原特异性细胞因子表达分析被认为是评价免疫应答和疫苗效果的重要手段。本研究目的是比较两种卡普里痘病毒（CaPV）疫苗对牛块状皮肤病的细胞介导免疫反应特性。
材料与方法：
两个农场的奶牛接种两种卡普里痘病毒疫苗，接种后5周随访。这些疫苗分别为山羊痘病毒(GTP)活弱毒株(20株)和绵羊痘病毒(SPP)罗马尼亚株(20株)。通过外淋巴细胞增殖法和疫苗株再刺激IFN-g和IL-4释放试验，评价接种疫苗后犊牛的细胞免疫应答，采用实时PCR技术检测细胞因子在PBMCs中的表达。
结果：
GTP和SPP疫苗接种组的淋巴细胞增殖率在接种后第三周开始增加直至达到高峰，然后在接下来的几周内下降。每周GTP免疫小牛PBMCs的刺激指数均高于SPP免疫小牛，表明GTP疫苗在牛体内具有较高的免疫原性。两个疫苗接种组在PBMCs中的IFN-g和IL-4蛋白表达均在免疫后的第3周达到最大值。每周GTP疫苗小牛的细胞因子释放的平均值都高于SPP免疫小牛，但是只有接种后的第3周，细胞因子的释放有显著差异(P<0.05)。免疫组PBMC中的IFN-g mRNA较早表达，在接种后第1周达到高峰，此后开始下降。每周GTP疫苗小牛的这一比率都高于SPP疫苗小牛，但是只有接种后的第3周，细胞因子的释放才有显著差异(P<0.05)。相比之下，IL-4 mRNA的表达受到延迟，在第3周达到高峰。与SPP组不同，GTP组的IL-4mRNA表达一直维持在最高水平，直到实验结束。同样，每周GTP疫苗小牛的细胞因子释放的平均值都高于SPP疫苗小牛，但是只有接种后的第5周，细胞因子的释放才有显著差异(P<0.05)。
结论：
结果表明，与SPP疫苗相比，GTP减毒活疫苗具有较高水平的细胞介导减毒活疫苗免疫应答能力和良好的免疫原性，可作为预防块状皮肤病疫苗的选择。
使用试剂原文信息：Cell-free supernatants were collected on the fourth day of culture (11, 13) and concentrations of IFN-γ and IL-4 cytokines were measured using commercially available ELISA-kits (USCN Life Science Inc. China).