【作者】 孙英军；

【导师】 张志东；

【作者基本信息】 中国农业科学院 ， 兽医， 2012， 硕士

【摘要】 目前商品化的口蹄疫疫苗大多是化学灭活苗，该疫苗在预防和控制乃至根除口蹄疫过程中发挥重要作用。口蹄疫灭活疫苗主要依靠体液免疫发挥作用，细胞免疫和非结构蛋白免疫的作用往往被忽视，同时灭活疫苗也存在免疫持续期短的缺点。众多科研人员认为CD4+T细胞应答在口蹄疫保护性应答中具有重要意义，并且认为免疫持续期的长短与病毒特异性的CD4+T细胞数量有一定的关联。融合口蹄疫病毒非结构蛋白2B基因后可以显著提高腺病毒载体疫苗诱导CD4+和CD8+T淋巴细胞应答的水平。在FMDV非结构蛋白上鉴定的大量的CD4+、CD8+T细胞表位，也进一步证明非结构蛋白在诱导细胞免疫方面具有不可忽视的作用。本研究利用结构蛋白相同，而非结构蛋白不同的毒株，在豚鼠和猪体内进行免疫差异分析，具体如下：(1)在豚鼠体内的免疫应答差异和保护率差异本研究选用反向遗产操作技术构建的与野生型毒株(Mya98/BY/2010)相同P1基因而非结构蛋白和前导蛋白不同的重组病毒(分别命为Re-Mya98/BY/2010和Mya98/BY/2010)作为抗原，选用相同剂量病毒液，经BEI灭活后与4种佐剂乳化制备不同的疫苗作为免疫原免疫豚鼠。利用流式细胞仪检测外周血液中CD4+T细胞和CD8+T细胞亚群的含量，ELISA方法检测豚鼠血清中抗体应答，在免疫28天时进行攻毒实验。抗原剂量相同的条件下，Re-Mya98/BY/2010和Mya98/BY/2010诱导的抗体应答之间没有显著性差异(P>0.05)。两种抗原均能诱导CD4+和CD8+T细胞应答，CD4+T细胞应答没有显著性差异(P>0.05)。Re-Mya98/BY/2010比Mya98/BY/2010更有效的诱导CD8+T细胞亚群分化。免疫Re-Mya98/BY/2010疫苗的保护率分别达到100%，83.33%，100%和50%，而免疫Mya98/BY/2010疫苗保护率仅为20%，0，17%和25%，结果表明融合了不同的非结构蛋白的重组口蹄疫病毒抗原能够诱导与保护率相关的CD8+T细胞应答。(2)在猪体内的免疫应答差异和保护率差异挑选免疫效果较好的，ISA206佐剂乳化的口蹄疫灭活疫苗进行猪体实验，采用同样的方法监测免疫猪的细胞免疫和体液免疫水平。两种疫苗的攻毒保护率都达到100%。液相阻断ELISA结果表明两种抗原在诱导抗体应答上并无显著差异(P>0.05)。Re-Mya98/BY/2010诱导更多数量CD3+T淋巴细胞分化。第7天，免疫Re-Mya98/BY/2010的实验组CD4+和CD8+T淋巴细胞含量升高，并且显著高于免疫Mya98/BY/2010抗原的实验组(P<0.05)。上述结果表明，完整的非结构蛋白可以增强猪体早期的CD4+和CD8+T细胞应答。(3)差异肽段对猪和牛淋巴细胞刺激差异分析我们合成了非结构蛋白上的28条差异肽段，利用基于CFSE的淋巴细胞增殖实验探讨造成免疫差异的原因。实验发现：Re-Mya98/BY/2010与Mya98/BY/2010前导蛋白上2-11氨基酸和3D上2001-2014位氨基酸具有刺激猪源淋巴细胞增殖的作用，前导蛋白上2-11位氨基酸和3A上1493-1510位氨基酸具有刺激牛源淋巴细胞增殖的作用。位于前导蛋白、3A、3D上的多肽刺激猪源或牛源淋巴细胞增殖的比率与其他多肽具有极显著差异(P<0.05)。用前导蛋白上的2-11的肽段免疫FMDV致敏的猪，发现该多肽都能刺激猪外周血中CD8+T细胞亚群分化。结果表明在两种口蹄疫病毒抗原前导蛋白和3A上可能存在具有差异性的T细胞表位。本实验结果表明，重组的口蹄疫病毒能够诱导较高的体液免疫和细胞免疫，进一步说明非结构蛋白在诱导免疫应答方面具有显著的作用。包含流行毒株衣壳的重组口蹄疫疫苗能诱导保护性免疫应答，具有应用于口蹄疫免疫防控的前景。更多还原

【Abstract】 The commercial foot-and-mouth (FMD) vaccines comprise chemically inactivated virus particlesincorporated in adjuvant.and play an important role in prevention, control and eradication of FMD inendemic regions of the world. It is known that the vaccine-induced protection is generally correlatedwith high levels of neutralizing antibodies. However, knowledge of cell-mediated immune responsesand immune efficacy of NSP in protective immunity to FMDV is limited. CD4+T cell responses aresuggested to play an important role in protection against FMDV, but link between the duration ofabsolute immunity and amount of antivirus specific CD4+T cell remins unclear. Recent studiesdemonstrate the presence of FMDV-specific CD4+and CD8+T cell response in both infected andvaccinated animals. Furthermore, FMD virus (FMDV) NSP2B was found to be able to improve theefficacy of adenovirus-vectored FMDV capsid subunit vaccine against FMDV serotype O and inducesignificant increase in the percentage of specific-CD8+and CD4+. Idenification of CD4+and CD8+T cellepitopes in FMDV NSP indicates that NSP may play an immprotant role in cell mediated immunityaginst FMDV. In present study, recombinant FMDV Re-Mya98/BY/2010and wild-type strainMya98/BY/2010with the identical structure protein but different NSP were used to immunized guineapigs and pigs, and immune response in these animals were then evaluated.(1) Differences of immune response and protection rate in immunized guinea pigsThe recombinant virus Re-Mya98/BY/2010was constructed by replacing the structuralprotein-coding gene of the O vaccine strain, with that of the wild type strain Mya98/BY/2010. Both therecombinant and wild-type viruses was inactivated with BEI and formulated with four differentadjuvants (EMULSIGEN-BCL, EMULSIGEN-D, ISA206, POLYGEN). The guinea pigs werevaccinated muscularly and challenged with Mya98/BY/2010at28day post vaccination (28dpv).Cellular and humoral immune responses at different days post vaccination were analyzed. Bothrecombinant virus Re-Mya98/BY/2010and wild-type viruse Mya98/BY/2010were able to inducestrong anti-FMDV antibody response, but there was no difference in level of antibody in both groups ofvaccinated guinea pigs (P>0.05) regardless of adjuvants. Interestingly, a significant antigen specificCD4+and CD8+T cell response was detected at21dpv with the stronger CD8+T cell response in thegroup of guinea pigs vaccinated with the recombinant virus. Of four groups of guinea pigs vaccinatedwith Re-Mya98/BY/2010, three groups had a statistically significant higher antigen specific-CD8+T cellresponse at21dpv when compared to those in guinea pigs vaccinated the wild-type strainMya98/BY/2010(P<0.05). However, there was no significant difference in the CD4+Tcell response(P>0.05). The protection rate in guinea pigs vaccinated with the recombinant virus Re-Mya98/BY/2010with four difference adjuvants (EMULSIGEN-BCL, EMULSIGEN-D, ISA206, POLYGEN) was100%,100%,83.33%and50%, respectively while the guinea pigs vaccinated with FMDVMya98/BY/2010was20%,0%,17%and25%, respectively, and the difference of protection rateinduced by the two strains was statistically significant (P<0.01). These results indicate that inclusion ofthe complete NSP may be able to improve the efficacy of vaccine-induced specific-CD8+T cell responses which enhance vaccine-induced protection.(2) Differences of immune response and protection rate in immunized pigsTwo chemically inactivated whole-virus preparations which were emulsified with ISA206wereselected and used to vaccine pigs (n=5/group) based on the results in immunized guinea pigs. Cellularand humoral immune responses at different days post vaccination were investigated in immunized pigs.There was no difference of the protection rate observed between pigs vaccinated with recombinant virusRe-Mya98/BY/2010and pigs with wild-type viruse Mya98/BY/2010(P>0.05), and the two vaccinesoffered clinical protection. Both recombinant virus Re-Mya98/BY/2010and wild-type viruseMya98/BY/2010induced strong anti-FMDV antibody response, but there was no difference in level ofantibody response (P>0.05). However, a significant antigen specific-CD3+T cell response was detectedin the group of pigs vaccineated with Re-Mya98/BY/2010. Furthermore, in this group, a statisticallysignificant stronger antigen specific-CD4+and CD8+T cell response was detedted at as early as7dpv.These results indicate that the complete NSP has a positive effect on induction of specific-CD4+T andCD8+T cell responses of pigs in the early stages of the adaptive immune response.(3) Proliferative response of PBMC isolated from FMDV infected pigs and bovines to stimulationwith different NSP peptidesTo address the different immunogenicity of NSP from Re-Mya98/BY/2010and Mya98/BY/2010,14overlapping NSP peptides based on the sequence of FMDV strain Re-Mya98/BY/2010andMya98/BY/2010were synthesed and tested in proliferation assays using lymphocytes from pigs andbovine experimentally infected with different strains of FMDV. Peptides located on NSP3D and L wererecognized by PBMC from infected pigs while, peptides located on NSP3A(1493-1510) and L(2-11)induced a significant proliferative response in PBMCs isolated from FMDV-infected bovines,. The Tlymphocyte proliferative response stimulated by3A(1493-1510),3D(2001-2014) and L(2-11) wasdifferent with those stimulated by other peptides analyzed by statistical analusis (P<0.05). Thesignificant diffrernce was also observed on the amount of T lymphocytes stimulated by peptides whichare located in the same position of Re-Mya98/BY/2010and Mya98/BY/2010(P<0.05). To investigatethe immunogenicity of peptides located in Leader Proteinase, L(2-11) was immunized in infected pigswith FMDV, and the higher CD8+T cell response was observed compared to negative control (P<0.05).In conclusion, the results demonstrate that the recombinant FMDV Re-Mya98/BY/2010was ableto induce both strong humoral and cellular immune responses, supporting the concept that a role ofFMDV NSP in induction of the immune response. Furthermore, the results demonstrate thatrecombinant vaccines containing the capsid of field isolates can be successfully produced and is able toinduce protective immune responses.更多还原