最新獸醫(yī)專業(yè)英語

1、2020Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV豬CCL20趨化因子在抗豬瘟病毒的DNA疫苗中的免疫調(diào)節(jié)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 vaccine

2、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. 這項(xiàng)工作的目的是探討這項(xiàng)工作的目的是探討CCL20 CCL20 趨化因子質(zhì)粒表達(dá)是否可以提高趨化因子質(zhì)粒表達(dá)是否可以提高DNA

3、 DNA 疫苗表達(dá)疫苗表達(dá)E2 E2 蛋白聯(lián)合控制加強(qiáng)對豬瘟病毒的免疫應(yīng)答。蛋白聯(lián)合控制加強(qiáng)對豬瘟病毒的免疫應(yīng)答。免疫豬的免疫豬的 DNA DNA 疫苗配方包含豬疫苗配方包含豬 CCL20 CCL20 趨化因子，結(jié)果在疫苗趨化因子，結(jié)果在疫苗第一次注射后的第第一次注射后的第36 36 天檢測到同源誘導(dǎo)的豬瘟病毒的抗體水天檢測到同源誘導(dǎo)的豬瘟病毒的抗體水平。平。獸醫(yī)專業(yè)英語.ppt Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL

4、20 developed high titers of neutralizing antibodies against homologous and heterologous（同源和異源）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 疫苗和含豬疫苗和含豬 CCL20 CCL20 質(zhì)粒共同作質(zhì)粒共同作用下的免疫動物，在致死量病毒的作用（減毒保護(hù)）下用下的免疫動物，在致死量病毒的作用（減毒保護(hù)）下產(chǎn)

5、生對同源及異源豬瘟病毒株高效價(jià)的中和抗體，并使產(chǎn)生對同源及異源豬瘟病毒株高效價(jià)的中和抗體，并使之得到完全保護(hù)。這證實(shí)之得到完全保護(hù)。這證實(shí) CCL20 CCL20 具有增加抗體免疫反具有增加抗體免疫反應(yīng)的作用。應(yīng)的作用。獸醫(yī)專業(yè)英語.ppt2.Introduction It is a small cytokine belonging to the CC chemokine family. It is strongly chemotactic for lymphocytes and weakly attracts neutrophils（中性白細(xì)胞）. CCL20 is implicated i

6、n the formation and function of mucosal lymphoid tissues（粘膜淋巴組織） via chemoattraction of lymphocytes and dendritic cells towards the epithelial cells（上皮細(xì)胞） surrounding these tissues. CCL20 elicits its effects on its target cells by binding and activating the chemokine receptor CCR6.What is CCL20？獸醫(yī)專業(yè)

7、英語.ppt Classical swine fever virus is the causative agent（病原體） 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（正極） and a lipid envelope（脂質(zhì)包膜） that belongs to the Pestivirus Genus（瘟病瘟病毒屬毒屬）of the Flavivirida

8、e family（黃病毒科）.Then,lets briefly know CSFV.獸醫(yī)專業(yè)英語.ppt3.Materials and methodsCells and viruesConstruction of the plasmid expressing full-length CCL20 chemokineDNA immunization of pigsCSFV challendge獸醫(yī)專業(yè)英語.pptIsolation of porcine PBMC and Elispot assay fordetection CSFV-specific IFN-gamma producing ce

9、llsAntibody detectionRT-PCR real timeELISA for IFN-alpha and IFN-gamma detection from serum samples獸醫(yī)專業(yè)英語.ppt4.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 w

10、ith plasmids pE2 and pCCL20 (pigs 46).Grey bars indicate control pigs (pigs79). Values are expressed as meanstandard deviations.獸醫(yī)專業(yè)英語.pptFig.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+(pi

11、gs 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獸醫(yī)專業(yè)英語.pptFig.3.Lack of pyrexia upon CSFV challenge in pi

12、gs immunized with pE2 and pCCL20. Following CSFV challenge, rectal temperature was daily measured.Pyrexia was considered as rectal temperature 40 C. Pigs inoculated with pE2 and pCDNA3.1+ (13). Pigs inoculated with pE2 and pCCL20 (46).Pigs injected with PBS (79).獸醫(yī)專業(yè)英語.pptFig.4.IFN alpha levels in s

13、erum 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 exp

14、ressed as mean standard deviations.獸醫(yī)專業(yè)英語.pptLack of detectable CSFV RNA in animals co-administered pE2 and pCCL20獸醫(yī)專業(yè)英語.pptThe protection conferred by pE2 and pCCL20 mixtureassociates with a better induction of neutralizing antibodies upon challenge獸醫(yī)專業(yè)英語.ppt5.Discussion DNA immunization can be a u

15、seful 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（細(xì)胞因子）,chemokines（趨化因子）, co-stimulatory molecules and enhancers

16、of Dendritic Cells (DC) recruitment have been used as adjuvants to potentiate protective immune responses against different pathogens. 獸醫(yī)專業(yè)英語.ppt For CSFV, we have shown that immunization with E2 DNA vaccine induced specific T cell responses in the absence of neutralizing antibodies before viral cha

17、llenge.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.獸醫(yī)專業(yè)英語.ppt Taken into a

18、ccount 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 wi

19、th 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.獸醫(yī)專業(yè)英語.pptOn the contrary, the mean value of anti-E2 specific antibodies in pE2 group at 36 days p.i. was negative. After CSFV

20、 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, the

21、se antibodies neutralized a heterologous（異種的）CSFV strain with high titers, suggesting that co-expression of CCL20 expands the antigenic spectrum of the antibodies induced by E2.在pE2組的免疫后第36天，抗E2特定抗體的平均值顯示的是陰性。經(jīng)過CSFV的感染第3天，對比只免疫pE 2的動物，混合免疫pE2+pCCL20組，E2抗體的平均值顯示陽性。值得注意的是用pE2和pCCL20進(jìn)行誘導(dǎo)的免疫豬在攻毒后第7天、第13

22、天，有高水平中和抗體。當(dāng)然，這些抗體以一個(gè)高滴度中和異源CSFV株，意味著聯(lián)合表達(dá)的CCL20擴(kuò)大了由E2誘導(dǎo)的抗體的抗原圖譜。獸醫(yī)專業(yè)英語.ppt 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 th

23、e 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趨化因子可以增強(qiáng)經(jīng)過DNA疫苗免疫的小鼠體液應(yīng)答。我們的結(jié)果證實(shí)了這樣的一個(gè)小鼠模型實(shí)驗(yàn)。與此同時(shí)，揭示了CCL20趨化因子增強(qiáng)T細(xì)胞應(yīng)答和誘導(dǎo)豬中抗CSFV中和抗體的產(chǎn)生相關(guān)的能力。獸醫(yī)專業(yè)英語.ppt I

24、n the experiments here reported, after a lethal viral challenge similar to that indicated by the OIE（世界動物衛(wèi)生組織） 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,

25、 nor viral RNA could be detected in the sera. 這里的實(shí)驗(yàn)報(bào)告了致死性病毒感染后與這里的實(shí)驗(yàn)報(bào)告了致死性病毒感染后與OIE ManualOIE Manual（國際獸醫(yī)手冊）指示的相似，在經(jīng)過（國際獸醫(yī)手冊）指示的相似，在經(jīng)過pE2+pCCL20pE2+pCCL20免疫免疫的動物產(chǎn)生的中和抗體與經(jīng)得起的完全保護(hù)相關(guān)。這些的動物產(chǎn)生的中和抗體與經(jīng)得起的完全保護(hù)相關(guān)。這些動物既動物既沒有疾病癥狀沒有疾病癥狀，在血清中，在血清中也檢測不到也檢測不到RNARNA病毒病毒。獸醫(yī)專業(yè)英語.ppt Conversely, neutralizing antibody

26、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（短暫） peak of fever,mild clinical signs,pathological findings and virus detection in serum samples at 3 and 7 days post challenge. 相反，只有經(jīng)過相反，只有

27、經(jīng)過pE2pE2免疫的豬在感染后第免疫的豬在感染后第7 7天有較低水平天有較低水平的中和抗體。這個(gè)滴度的減少可以與感染后第的中和抗體。這個(gè)滴度的減少可以與感染后第3 3和第和第7 7天天的動物發(fā)熱，輕微癥狀，病理發(fā)現(xiàn)和血清中病毒的測定的動物發(fā)熱，輕微癥狀，病理發(fā)現(xiàn)和血清中病毒的測定相聯(lián)系。相聯(lián)系。獸醫(yī)專業(yè)英語.ppt This observation supports the relevance of the induction of a strong humoral response to confer a solid protection against CSFV. On the cont

28、rary, control animals(injected with PBS) showed severe clinical signs; pyrexia（發(fā)熱）,viremia（病毒血癥）and RNA of CSFV were detected by RT-PCR real time from serum samples analyzed.The clinical signs and the virus load（病毒載量）detected in these animals were similar to previously described for pigs inoculated（

29、接種）with empty plasmid (pCDNA3.1+) in similar experiments.獸醫(yī)專業(yè)英語.ppt 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 adminis

30、tration 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（抗原提呈細(xì)胞） that further enhance cooperation and activation of T and B cells, generating a more potent humoral response after v

31、accination.只接種只接種pE2pE2的動物產(chǎn)生特定的動物產(chǎn)生特定-IFN-IFN產(chǎn)生細(xì)胞，但是在注射產(chǎn)生細(xì)胞，但是在注射后第后第3636天幾乎沒有可測定的特定抗天幾乎沒有可測定的特定抗E2E2抗體的產(chǎn)生。這些結(jié)抗體的產(chǎn)生。這些結(jié)果表明聯(lián)合控制的果表明聯(lián)合控制的pE2pE2和和pCCL20pCCL20加快誘導(dǎo)特殊體液應(yīng)答。加快誘導(dǎo)特殊體液應(yīng)答。同樣，同樣，CCL20CCL20在疫苗的效果與在疫苗的效果與APCAPC聚集誘導(dǎo)相關(guān)，進(jìn)一步加聚集誘導(dǎo)相關(guān)，進(jìn)一步加強(qiáng)聯(lián)合操縱和強(qiáng)聯(lián)合操縱和T T、B B細(xì)胞的活性，免疫后產(chǎn)生更強(qiáng)潛在的體細(xì)胞的活性，免疫后產(chǎn)生更強(qiáng)潛在的體液應(yīng)答。液應(yīng)答。獸醫(yī)專業(yè)

32、英語.ppt 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 IF

33、N-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. 早期報(bào)道表明經(jīng)過早期報(bào)道表明經(jīng)過E2-DNAE2-DNA疫苗免疫之后，疫苗免疫之后，-IFN-IFN表達(dá)表達(dá)T T細(xì)胞的特定誘導(dǎo)。同樣，細(xì)胞的特定誘導(dǎo)。同樣，CSFVCSF

34、V特定特定-IFN-IFN產(chǎn)生細(xì)胞在病產(chǎn)生細(xì)胞在病毒感染前與注射質(zhì)粒組頻數(shù)相似。感染后包括毒感染前與注射質(zhì)粒組頻數(shù)相似。感染后包括-IFN-IFN誘誘導(dǎo)控制病毒復(fù)制可以解釋質(zhì)粒注射組（導(dǎo)控制病毒復(fù)制可以解釋質(zhì)粒注射組（pE2pE2組）在感染組）在感染后第后第3 3天觀察到的天觀察到的CSFVCSFV特定特定-IFN-IFN產(chǎn)生細(xì)胞的應(yīng)答的減產(chǎn)生細(xì)胞的應(yīng)答的減少。少。獸醫(yī)專業(yè)英語.ppt Principally in pigs immunized with pE2 alone those are positive to viral RNA detection at 3 and 7 days p.

35、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 a

36、ntibodies found at 7 days p.c. in animals immunized with pE2 and pCCL20, compared to those of animals immunized with pE2 alone.獸醫(yī)專業(yè)英語.ppt 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 da

37、ys p.c.) although CSFV specific response associated with IFN-gamma were found by Elispot（酶聯(lián)免疫斑點(diǎn)技術(shù)）.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 .獸醫(yī)專業(yè)英語.ppt Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN.Lower average values of IFN alpha were det