靶定B 细胞受体的重组马立克氏病疫苗增强细胞免疫的机制研究

Marek's disease (MD) vaccines have been widely applied for several decades. However, the mechanism of immune protection induced by MD vaccine is still understood poorly. Our previous studies found that recombinant MD vaccine inducing specific antibodies could promote T cell response significantly, suppose that the status of B is associated with immune efficacy of MD vaccine. In order to understand how B cell mediates immune response induced by MD vaccine, in this study, the recombinant MD vaccine targeting B cell receptor (BCR) will be constructed by manipulating the MDV genome into bacterial artificial chromosomes (BAC). Then the avidity of recombinant MDV will be tested by monitoring the viral replication in B cell in vivo. Furthermore, the transcription and expression levels of immunoglobulin gene as well as membrane molecular gene of B cell isolated from chicken vaccinated with recombinant or parent vaccine will be investigated. To determine that activation and antigen-processing ability of B cell, the inflammatory cytokine secretion and expression of MHCII on B cell surface will be measured and the activation of BCR signaling transduction pathway will be analyzed as well. Subsequently, the release level of costimulatory molecule CD80 and expression of MHCI will be examined in order to evaluate effectors of cytotoxic T lymphocytes (CTL) response and explore the mechanism of delivering MDV antigen signals from B to T cell. The purpose of this study is to elucidate the molecular mechanism of better immune response induced by recombinant MD vaccine targeting B cell. The anticipated results will provide evidence for developing novel MD vaccine in the future.

马立克氏病（MD）疫苗被广泛应用数十年，但其诱导免疫保护的机制尚不十分清楚。我们研究发现，诱导特异性抗体的重组MD疫苗明显提高鸡体内的细胞免疫水平，推测B细胞的状态可能与MD疫苗免疫效力相关。为了解B细胞如何参与MD免疫应答，本项目拟利用细菌人工染色体(BAC)技术，构建靶定B细胞受体（BCR）的重组MDV CVI988；通过监测病毒的复制水平，测试重组MDV与B细胞的亲和力；进一步调查重组与疫苗病毒感染对B淋巴细胞免疫球蛋白及膜表面分子基因的转录和表达的影响；检测炎性细胞因子的分泌和MHCII的表达以及BCR信号通路的活化，分析B细胞活化程度和抗原呈递能力；测定MHCI和共刺激分子CD80的表达，评价病毒感染的B细胞刺激细胞毒性T细胞（CTL）反应的效果，探讨B、T细胞间进行MDV抗原信号的交流机制。预期阐明靶定B细胞的重组MD病毒诱导更强的细胞免疫应答的机制，为研发MD新疫苗提供依据

马立克氏病是鸡的一种由α疱疹病毒引起淋巴组织增生性肿瘤病，马立克氏病（MD）疫苗是世界上第一个肿瘤的疫苗，因此马立克氏病被认为是优秀的病毒性淋巴瘤研究模型，但人们对MD疫苗免疫的机理尚不清楚。前期研究表明，B细胞在MD病毒早期复制和诱导免疫应答时起关键作用，因此本项目围绕B细胞参与的MD疫苗免疫过程中的事件开展研究。我们首先利用细菌人工染色体(BAC)技术，构建靶定B细胞受体（BCR）的重组MDV CVI988-C3d；然后监测了重组病毒、亲本CVI988以及MDV强毒株在接种后3-7天的鸡脾脏和法氏囊淋巴细胞，增殖情况，发现重组病毒CVI988-C3d在脾脏淋巴细胞中的复制水平明显高于亲本MDV CVI988株，但均低于MDV强毒株RB1B的复制水平；同时发现，B细胞内与其活化和功能相关的基因转录与表达存在差异；尤其是不同马立克氏病毒诱导T、B淋巴细胞交流的细胞因子表达差异，包括IL-6、IL-1β、IL8L1、CCL4和CCL5等均在CVI988株免疫鸡的脾脏细胞中表达显著升高，说明B细胞中的疫苗病毒通过诱导产生趋化因子，更容易招募T细胞到感染MDV的B细胞周围，使病毒信号从B细胞传递到T细胞，促使T细胞活化成效应T细胞以发挥CTL作用；进一步的研究证实，B细胞表面分子CR2的表达受MDV疫苗毒（CVI988）和强毒(RB1B)抑制差异明显，即疫苗病毒可以通过较高CR2水平促进B细胞活化，从而增强T细胞免疫应答。总之，我们从不同方面阐明了B 细胞在 MD 疫苗诱导 T 细胞免疫应答的分子机制，为揭示 MD 弱毒疫苗激发宿主产生免疫保护的机理增加科学依据，为今后研究和开发新型高效 MD 疫苗奠定理论基础。此外，本项目还鉴定了鸡补体受体2并成功制备相应的单克隆抗体，为进一步研究B细胞表面特异性分子的生物学功能以及建立免疫检测新技术奠定基础。

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