Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV猪CCL20趋化因子在抗猪瘟病毒的DNA疫苗中的免疫调节作用兽医专业英语 1.Abstract The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation,that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. 这项工作的目的是探讨这项工作的目的是探讨CCL20 CCL20 趋化因子质粒表达是否可以提高趋化因子质粒表达是否可以提高DNA DNA 疫苗表达疫苗表达E2 E2 蛋白联合控制加强对猪瘟病毒的免疫应答。免蛋白联合控制加强对猪瘟病毒的免疫应答。免疫猪的疫猪的 DNA DNA 疫苗配方包含猪疫苗配方包含猪 CCL20 CCL20 趋化因子，结果在疫苗第趋化因子，结果在疫苗第一次注射后的第一次注射后的第36 36 天检测到同源诱导的猪瘟病毒的抗体水平。天检测到同源诱导的猪瘟病毒的抗体水平。兽医专业英语 Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous（同源和异源）CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). 引人注意的是，引人注意的是，E2 DNA E2 DNA 疫苗和含猪疫苗和含猪 CCL20 CCL20 质粒共同作质粒共同作用下的免疫动物，在致用下的免疫动物，在致死量死量病毒的病毒的作用作用（减毒保护）下（减毒保护）下产生对同源及异源猪瘟病毒株高效价的中和抗体产生对同源及异源猪瘟病毒株高效价的中和抗体，并并使使之之得到完全保护。这证实得到完全保护。这证实 CCL20 CCL20 具有增加抗体免疫反具有增加抗体免疫反应的作用。应的作用。兽医专业英语2.Introduction It is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and weakly attracts neutrophils（中性白细胞）. CCL20 is implicated in the formation and function of mucosal lymphoid tissues（粘膜淋巴组织） via chemoattraction of lymphocytes and dendritic cells towards the epithelial cells（上皮细胞） surrounding these tissues. CCL20 elicits its effects on its target cells by binding and activating the chemokine receptor CCR6.What is CCL20？兽医专业英语 Classical swine fever virus is the causative agent（病原体） of classical swine fever (CSF), one of the most important swine infectious diseases. CSFV is an icosahedral(二十面的) RNA virus with (+) polarity（正极） and a lipid envelope（脂质包膜） that belongs to the Pestivirus Genus（瘟病毒属瘟病毒属）of the Flaviviridae family（黄病毒科）.Then,lets briefly know CSFV.兽医专业英语3.Materials and methodsCells and viruesConstruction of the plasmid expressing full-length CCL20 chemokineDNA immunization of pigsCSFV challendge兽医专业英语Isolation of porcine PBMC and Elispot assay fordetection CSFV-specific IFN-gamma producing cellsAntibody detectionRT-PCR real timeELISA for IFN-alpha and IFN-gamma detection from serum samples兽医专业英语4.ResultsFig.1.Induction of E2 specific antibodies after immunization and CSFV challenge. Open bars indicate pigs injected with pE2 and pCDNA3.1+ (pigs 13).Black bars indicate pigs injected with plasmids pE2 and pCCL20 (pigs 46).Grey bars indicate control pigs (pigs79). Values are expressed as meanstandard deviations.兽医专业英语Fig.2.Kinetics of specific CSFV IFN gamma-producing cells induced after immunization and CSFV challenge.Open bars indicate pigs injected with pE2 and pCDNA3.1+(pigs 13).Black bars indicate pigs injected with plasmids pE2 and pCCL20 (pigs46).Grey bars indicate control pigs(pigs79).Specific CSFV IFN gamma-producing cells are expressed as spots per 5105PBMC.Values are expressed as mean standard deviations兽医专业英语Fig.3.Lack of pyrexia upon CSFV challenge in pigs immunized with pE2 and pCCL20. Following CSFV challenge, rectal temperature was daily measured.Pyrexia was considered as rectal temperature 40C. Pigs inoculated with pE2 and pCDNA3.1+ (13). Pigs inoculated with pE2 and pCCL20 (46).Pigs injected with PBS (79).兽医专业英语Fig.4.IFN alpha levels in serum at 3 and 7days post CSFV challenge.Open bars indicate pigs injected with pE2 and pCDNA3.1+(pigs13).Black bars indicate pigs injected with plasmids pE2 and pCCL20 (pigs46).Grey bars indicate control pigs (pigs 79).Levels of IFN-alpha are expressed as Units of IFN-alpha/mL of serum.Values are expressed as mean standard deviations.兽医专业英语Lack of detectable CSFV RNA in animals co-administered pE2 and pCCL20兽医专业英语The protection conferred by pE2 and pCCL20 mixtureassociates with a better induction of neutralizing antibodies upon challenge兽医专业英语5.Discussion DNA immunization can be a useful tool for understanding the intrinsic immunological mechanisms involved in protection against a given pathogen or to map immunogenic protective determinants. Illustrating the versatility of DNA immunization, plasmid-based cytokines（细胞因子）,chemokines（趋化因子）, co-stimulatory molecules and enhancers of Dendritic Cells (DC) recruitment have been used as adjuvants to potentiate protective immune responses against different pathogens. 兽医专业英语 For CSFV, we have shown that immunization with E2 DNA vaccine induced specific T cell responses in the absence of neutralizing antibodies before viral challenge.Nevertheless, some vaccinated animals showed a delay in the induction of neutralizing antibodies and they did not achieve enough clinical protection after CSFV challenge.This response was explained by an insufficient priming of the T and B lymphocytes after vaccination.兽医专业英语 Taken into account the feasibility of DNA immunization and its protective capacity against CSFV，we determined to use this methodology as a tool to analyze the potential of swine CCL20 chemokine to enhance the immune response elicited against E2 of CSFV in pigs. Here, we have shown that co-injection of pCCL20 with pE2 in domestic pigs results in an enhancement of the humoral response elicited, as indicated by the induction of antibodies against E2 glycoprotein in all immunized pigs.兽医专业英语On the contrary, the mean value of anti-E2 specific antibodies in pE2 group at 36 days p.i. was negative. After CSFV challenge, mean of E2-specific antibody response was positive at 3 days p.c. in animals injected with the mixture of pE2 + pCCL20 in comparison with the pE2 injected pigs.Remarkably, pigs immunized with pE2 and pCCL20 elicited,at days 7 and 13 p.c., high levels of neutralizing antibodies. Also, these antibodies neutralized a heterologous（异种的）CSFV strain with high titers, suggesting that co-expression of CCL20 expands the antigenic spectrum of the antibodies induced by E2.在pE2组的免疫后第36天，抗E2特定抗体的平均值显示的是阴性。经过CSFV的感染第3天，对比只免疫pE 2的动物，混合免疫pE2+pCCL20组，E2抗体的平均值显示阳性。值得注意的是用pE2和pCCL20进行诱导的免疫猪在攻毒后第7天、第13天，有高水平中和抗体。当然，这些抗体以一个高滴度中和异源CSFV株，意味着联合表达的CCL20扩大了由E2诱导的抗体的抗原图谱。兽医专业英语 Recent studies have indicated that CCL20 chemokine could enhance the humoral response in mice in a DNA vaccine strategy. Our results confirm the role of CCL20 to increase antibody-mediated responses previously described in the mouse model . At the same time the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs is suggested. 最近的研究表明CCL20趋化因子可以增强经过DNA疫苗免疫的小鼠体液应答。我们的结果证实了这样的一个小鼠模型实验。与此同时，揭示了CCL20趋化因子增强T细胞应答和诱导猪中抗CSFV中和抗体的产生相关的能力。兽医专业英语 In the experiments here reported, after a lethal viral challenge similar to that indicated by the OIE（世界动物卫生组织） Manual , the levels of neutralizing antibodies found in pigs immunized with pE2 and pCCL20 are related to the complete protection afforded. These animals neither developed signs of disease, nor viral RNA could be detected in the sera. 这里的实验报告了致死性病毒感染后与这里的实验报告了致死性病毒感染后与OIE OIE ManualManual（国际兽医手册）指示的相似，在经过（国际兽医手册）指示的相似，在经过pE2+pE2+pCCLpCCL2020免疫的动物产生的中和抗体与经得起的完全免疫的动物产生的中和抗体与经得起的完全保护相关。这些动物既保护相关。这些动物既没有疾病症状没有疾病症状，在血清中，在血清中也检测也检测不到不到RNARNA病毒病毒。兽医专业英语 Conversely, neutralizing antibody titers after challenge were lower in pigs immunized with pE2 alone at 7 days p.c. This titer reduction could be associated with the development of a transient（短暂） peak of fever,mild clinical signs,pathological findings and virus detection in serum samples at 3 and 7 days post challenge. 相反，只有经过相反，只有经过pE2pE2免疫的猪在感染后第免疫的猪在感染后第7 7天有较低水平天有较低水平的中和抗体。这个滴度的减少可以与感染后第的中和抗体。这个滴度的减少可以与感染后第3 3和第和第7 7天天的动物发热，轻微症状，病理发现和血清中病毒的测定的动物发热，轻微症状，病理发现和血清中病毒的测定相联系。相联系。兽医专业英语 This observation supports the relevance of the induction of a strong humoral response to confer a solid protection against CSFV. On the contrary, control animals(injected with PBS) showed severe clinical signs; pyrexia（发热）,viremia（病毒血症）and RNA of CSFV were detected by RT-PCR real time from serum samples analyzed.The clinical signs and the virus load（病毒载量）detected in these animals were similar to previously described for pigs inoculated（接种）with empty plasmid (pCDNA3.1+) in similar experiments.兽医专业英语 On the other hand, animals injected only with pE2 showed induction of specific IFN-gamma-producing cells but an almost undetectable level of specific anti-E2 antibodies at 36 days p.i. These results suggest that the combined administration of pE2 and pCCL20 speeds up the induction of the specific humoral response. Likely,the effect of CCL20 on vaccination was related to the induction of recruitment of APC（抗原提呈细胞） that further enhance cooperation and activation of T and B cells, generating a more potent humoral response after vaccination.只接种只接种pE2pE2的动物产生特定的动物产生特定-IFN-IFN产生细胞，但是在注射产生细胞，但是在注射后第后第3636天几乎没有可测定的特定抗天几乎没有可测定的特定抗E2E2抗体的产生。这些结抗体的产生。这些结果表明联合控制的果表明联合控制的pE2pE2和和pCCL20pCCL20加快诱导特殊体液应答。加快诱导特殊体液应答。同样，同样，CCL20CCL20在疫苗的效果与在疫苗的效果与APCAPC聚集诱导相关，进一步加聚集诱导相关，进一步加强联合操纵和强联合操纵和T T、B B细胞的活性，免疫后产生更强潜在的体细胞的活性，免疫后产生更强潜在的体液应答液应答。兽医专业英语 Previous reports have shown that pigs vaccinated with E2-DNA vaccine showed a specific induction of IFN-gamma expressing T cells after vaccination. Likewise, the frequency of CSFV-specific IFN-gamma-producing cells at Pre-challenge was similar in plasmid-injected groups. The involvement of IFN-gamma induced in controlling viral replication after challenge may explain the decrease in the response associated with CSFV-specific IFN-gamma producing cells observed at 3 days p.c. in plasmid-injected groups. 早期报道表明经过早期报道表明经过E2-DNAE2-DNA疫苗免疫之后，疫苗免疫之后，-IFN-IFN表达表达T T细胞的特定诱导。同样，细胞的特定诱导。同样，CSFVCSFV特特定定-IFN-IFN产生细胞在产生细胞在病病毒毒感染前与注射质粒组感染前与注射质粒组频数频数相似。感染后包括相似。感染后包括-IFN-IFN诱诱导导控制控制病毒复制可以解释质粒注射组病毒复制可以解释质粒注射组（pE2pE2组）组）在感染在感染后第后第3 3天观察到的天观察到的CSFVCSFV特定特定-IFN-IFN产生细胞产生细胞的应答的减的应答的减少少。兽医专业英语 Principally in pigs immunized with pE2 alone those are positive to viral RNA detection at 3 and 7 days p.c. A reasonable hypothesis to explain the decrease in the number of IFN-gamma-producing cells in the group of pE2 may be due to a redistribution of circulating cells in blood to tissues to halt viral replication there. On the other hand, these results could be related to the levels of neutralizing antibodies found at 7 days p.c. in animals immunized with pE2 and pCCL20, compared to those of animals immunized with pE2 alone.兽医专业英语 In contrast, none of the animals showed detectable levels of IFN-gamma in serum samples tested by ELISA in the analyzed times (36 days p.i., and at 3, 7 and 13 days p.c.) although CSFV specific response associated with IFN-gamma were found by Elispot（酶联免疫斑点技术）.Probably the number of IFN-gamma producing cells may not be high enough to produce detectable levels in blood.These results corroborate previous reports where no correlation was found between the number of IFN-gamma-producing cells and the cytokine levels in serum measured by ELISA .兽医专业英语 Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN.Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge compared to the average values detected in pigs immunized with pE2 and non-vaccinated challenged animals .兽医专业英语 These results agree with previous reports in the literature where elevated levels of serum IFN alpha in infected pigs and virus replication or persistence have been described. The levels of IFN-alpha detected in pigs immunized with pE2 can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed.兽医专业英语 Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV.To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the induction of immune response and protection against virus challenge in swine infection model.我们的结果强调了猪我们的结果强调了猪CLL20CLL20趋化因趋化因子在由子在由E2-DNAE2-DNA疫苗抗疫苗抗CSFVCSFV中增强细胞、中增强细胞、体液和抗病毒应答的辅助作用。就我体液和抗病毒应答的辅助作用。就我们所知，这是报道关于猪们所知，这是报道关于猪CLL20CLL20是潜是潜在的能有效增强猪感染模型中在的能有效增强猪感染模型中DNADNA疫疫苗诱导产生免疫应答和保护受病毒感苗诱导产生免疫应答和保护受病毒感染的猪的第一份报告。染的猪的第一份报告。兽医专业英语Thank you！兽医专业英语此课件下载可自行编辑修改，供参考！此课件下载可自行编辑修改，供参考！感谢你的支持，我们会努力做得更好！感谢你的支持，我们会努力做得更好！