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1、Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV,郑仲华 微生物学,猪CCL20趋化因子在抗猪瘟病毒的 DNA疫苗中的免疫调节作用,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 v
2、accine 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 rst injection.,这项工作的目的是探讨CCL20 趋化因子质粒表达是否可以提高DNA 疫苗表达E2 蛋白联合控制加强对猪瘟病毒的免疫应
3、答。免疫猪的 DNA 疫苗配方包含猪 CCL20 趋化因子，结果在疫苗第一次注射后的第36 天检测到同源诱导的猪瘟病毒的抗体水平。,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 pr
4、otected upon a lethal viral challenge (sterilizing protection).,引人注意的是，E2 DNA 疫苗和含猪 CCL20 质粒共同作用下的免疫动物，在致死量病毒的作用（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，并使之得到完全保护。这证实 CCL20 具有增加抗体免疫反应的作用。,2.Introduction,It is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and w
5、eakly 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 a
6、ctivating the chemokine receptor CCR6.,What is CCL20？,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 t
7、he Pestivirus Genus（瘟病毒属）of the Flaviviridae family（黄病毒科）.,Then,lets briefly know CSFV.,3.Materials and methods,4.Results,Fig.1.Induction of E2 specic antibodies after immunization and CSFV challenge. Open bars indicate pigs injected with pE2 and pCDNA3.1+ (pigs 13).Black bars indicate pigs injected
8、 with plasmids pE2 and pCCL20 (pigs 46).Grey bars indicate control pigs (pigs79). Values are expressed as meanstandard deviations.,Fig.2.Kinetics of specic CSFV IFN gamma-producing cells induced after immunization and CSFV challenge.Open bars indicate pigs injected with pE2 and pCDNA3.1+(pigs 13).Bl
9、ack bars indicate pigs injected with plasmids pE2 and pCCL20 (pigs46).Grey bars indicate control pigs(pigs79).Specic CSFV IFN gamma-producing cells are expressed as spots per 5105PBMC.Values are expressed as mean standard deviations,Fig.3.Lack of pyrexia upon CSFV challenge in pigs immunized with pE
10、2 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).,Fig.4.IFN alpha levels in serum at 3 and 7days post CSFV
11、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 deviat
12、ions.,Lack of detectable CSFV RNA in animals co-administered pE2 and pCCL20,The protection conferred by pE2 and pCCL20 mixtureassociates with a better induction of neutralizing antibodies upon challenge,5.Discussion,DNA immunization can be a useful tool for understanding the intrinsic immunological
13、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 adj
14、uvants to potentiate protective immune responses against different pathogens.,For CSFV, we have shown that immunization with E2 DNA vaccine induced specic T cell responses in the absence of neutralizing antibodies before viral challenge.Nevertheless, some vaccinated animals showed a delay in the ind
15、uction of neutralizing antibodies and they did not achieve enough clinical protection after CSFV challenge.This response was explained by an insufcient priming of the T and B lymphocytes after vaccination.,Taken into account the feasibility of DNA immunization and its protective capacity against CSF
16、V，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
17、, as indicated by the induction of antibodies against E2 glycoprotein in all immunized pigs.,On the contrary, the mean value of anti-E2 specic antibodies in pE2 group at 36 days p.i. was negative. After CSFV challenge, mean of E2-specic antibody response was positive at 3 days p.c. in animals inject
18、ed 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-e
19、xpression 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。,Recent studies have indicated
20、 that CCL20 chemokine could enhance the humoral response in mice in a DNA vaccine strategy. Our results conrm 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 wit
21、h 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。,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） Manua
22、l , 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OIE Manual（国际兽医手册）指示的相似，在经过pE2+pCCL20免疫的动物产生的中和抗体与经得起
23、的完全保护相关。这些动物既没有疾病症状，在血清中也检测不到RNA病毒。,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 ndings and virus det
24、ection in serum samples at 3 and 7 days post challenge.,相反，只有经过pE2免疫的猪在感染后第7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3和第7天的动物发热，轻微症状，病理发现和血清中病毒的测定相联系。,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(in
25、jected 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 (p
26、CDNA3.1+) in similar experiments.,On the other hand, animals injected only with pE2 showed induction of specic IFN-gamma-producing cells but an almost undetectable level of specic anti-E2 antibodies at 36 days p.i. These results suggest that the combined administration of pE2 and pCCL20 speeds up th
27、e induction of the specic 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pE2的动物产生特定-IFN产生细胞，但是在注射后
28、第36天几乎没有可测定的特定抗E2抗体的产生。这些结果表明联合控制的pE2和pCCL20加快诱导特殊体液应答。同样，CCL20在疫苗的效果与APC聚集诱导相关，进一步加强联合操纵和T、B细胞的活性，免疫后产生更强潜在的体液应答。,Previous reports have shown that pigs vaccinated with E2-DNA vaccine showed a specic induction of IFN-gamma expressing T cells after vaccination. Likewise, the frequency of CSFV-specic
29、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-specic IFN-gamma producing cells observed at 3 days p.c. in plasmid-i
30、njected groups.,早期报道表明经过E2-DNA疫苗免疫之后，-IFN表达T细胞的特定诱导。同样，CSFV特定-IFN产生细胞在病毒感染前与注射质粒组频数相似。感染后包括-IFN诱导控制病毒复制可以解释质粒注射组（pE2组）在感染后第3天观察到的CSFV特定-IFN产生细胞的应答的减少。,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 de
31、crease 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 im
32、munized with pE2 and pCCL20, compared to those of animals immunized with pE2 alone.,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 specic response associated with
33、 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 i
34、n serum measured by ELISA .,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 pig
35、s immunized with pE2 and non-vaccinated challenged animals .,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
36、 be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed.,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 C
37、SFV.To our knowledge, this is the rst 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CLL20趋化因子在由E2-DNA疫苗抗CSFV中增强细胞、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CLL20是潜在的能有效增强猪感染模型中DNA疫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。,Thank you！,