Rabbit Allotypes--Structure, Organization and Regulated

兔同种异型——结构、组织和调控

• 批准号: 7299903
• 负责人: rose G. mage
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7299903
• 关键词: Rabbit; Allotypes; Structure; Organization; Regulated

We study genes of the rabbit immune system using techniques of molecular biology and immunology. In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs)(1,2). Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. Rabbits with the rare b9 allotype are excellent sources for therapeutic monoclonal antibodies. We generated rabbit polyclonal antibodies from these rabbits. Broadly neutralizing antibodies against receptor-activated epitopes on the N-heptad repeat region (NHR) of HIV-1 gp41 are elicited during natural infection and by immunization. Our collaborator Dr. Michael Zwick rescued HIV-1-neutralizing anti-NHR antibodies from immune phage display libraries that were prepared from an HIV-1 infected individual, and from b9 rabbits that we immunized with a mimetic of the NHR inner trimeric coiled-coil previously described by Dr. C. Bewley, NIDDK. The human and rabbit monoclonal antibodies bind to overlapping epitopes on the NHR trimer, generate distinct binding profiles against a panel of gp41 mimetic proteins and neutralize primary HIV-1 from various clades with modest potency using a pseudotyped virus assay. An examination of the differing abilities of the antibodies to neutralize different viruses bearing identical NHR sequences has revealed a particular restriction to neutralization that appears to be steric in nature, rather than dependent on viral entry kinetics. Taken together, the results indicate that broadly neutralizing antibodies, albeit currently of limited potency, are elicited against the NHR trimer of HIV-1 gp41 during natural infection and by immunization. For vaccine design, the newly selected antibody panel can be used in optimizing the NHR trimeric coiled-coil to favorably display neutralizing epitopes (Nelson, J. D., Jensen, R., Bewley, C. A., Brunel, F. M., Louis, J. M., Clore, G. M., Mage, R. G., Dawson, P. E., Burton, D. R., and Zwick, M. B.: Broadly neutralizing antibodies against receptor-activated epitopes on the N-heptad repeat region of HIV-1 gp41 are elicited during natural infection and by immunization. AIDS Vaccine Meeting, Amsterdam, 2006). The laboratory of Dr. C. Rader (NCI) isolated monoclonal antibodies selective for all three members of the Nogo-66 receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. NgR1, NgR2, and NgR3 are primarily expressed by neurons in the central nervous system (CNS) and believed to limit axonal growth and sprouting following CNS injury. In an attempt to define the expression and decipher the function of individual members of the Nogo-66 receptor family, we generated selective rabbit polyclonal antibodies (3). We then used phage display technology to generate rabbit monoclonal antibodies (mAbs) from the same immune repertoires. We obtained mAbs with nanomolar affinity to epitopes that are specific for NgR1 and NgR2, respectively, but at the same time conserved between mouse, rat, and human orthologs. Employing phage display vector pC3C, a newly designed phagemid optimized for the generation and selection of Fab libraries with human constant domains, rabbit mAbs were selected from chimeric rabbit/human Fab libraries, characterized in terms of specificity, affinity, and amino acid sequence, and finally converted to chimeric rabbit/human IgG. Strong and specific recognition of cell surface bound Nogo-66 receptor family members by chimeric rabbit/human IgG was demonstrated using immunofluorescence microscopy and immunoprecipitation. The rabbit mAbs together with their amino acid sequences constitute a defined panel of species cross-reactive reagents in infinite supply for investigations toward a functional role of the Nogo-66 receptor family in and beyond the CNS (MS Under revision- Hofer, T., Tangkeangsirisin, W., Kennedy, M.G., Mage, Rose G., Raiker, S.J., Venkatesh, K., Lee, H. Giger, R and Rader, C.:Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity and produced with an improved phage display vector).

我们利用分子生物学和免疫学技术研究兔子免疫系统的基因。在小鼠和人类等物种中，通过在免疫球蛋白 VHDJH 和 VLJL 重排中使用不同的 VH 和 VL 基因产生组合多样性可能是一抗库的主要贡献者。在兔子中，组合机制对重链多样性的贡献很小，因为只有少数 VH 基因被重排和表达。这类似于鸡抗体的形成。兔阑尾和鸡法氏囊是主要淋巴器官，其中 B 细胞抗体库主要通过类似基因转换的过程在生发中心发育。与鸡一样，大多数兔 B 淋巴细胞中的 3 素数 VH1 基因发生重排。体细胞超突变和基因转换有助于幼兔阑尾或胚胎和幼鸡法氏囊的初级多样化，也有助于生发中心 (GC) 免疫反应过程中的次级多样化(1,2)。 用于产生治疗性单克隆抗体和人源化的兔免疫库 兔免疫库长期以来一直是诊断性多克隆抗体的丰富来源。现在，它作为治疗性单克隆抗体的来源也具有广阔的前景。具有罕见 b9 同种异型的兔子是治疗性单克隆抗体的极好来源。我们从这些兔子中产生了兔多克隆抗体。在自然感染和免疫过程中，会引发针对 HIV-1 gp41 N 七肽重复区 (NHR) 上受体激活表位的广泛中和抗体。我们的合作者 Michael Zwick 博士从免疫噬菌体展示文库中拯救了 HIV-1 中和性抗 NHR 抗体，这些抗体是从 HIV-1 感染个体制备的，以及我们用 NHR 内部三聚体卷曲螺旋模拟物免疫的 b9 兔中，之前由 NIDDK 的 C. Bewley 博士描述过。人和兔单克隆抗体与 NHR 三聚体上的重叠表位结合，针对一组 gp41 模拟蛋白产生不同的结合谱，并使用假型病毒测定以适度的效力中和来自不同分支的初级 HIV-1。对抗体中和带有相同 NHR 序列的不同病毒的不同能力的检查揭示了对中和的特殊限制，这种限制本质上是空间位阻的，而不是依赖于病毒进入动力学。总而言之，结果表明，在自然感染和免疫过程中，会引发针对 HIV-1 gp41 的 NHR 三聚体的广泛中和抗体，尽管目前效力有限。对于疫苗设计，新选择的抗体组可用于优化 NHR 三聚体卷曲螺旋，以有利地展示中和表位（Nelson, J. D.、Jensen, R.、Bewley, C. A.、Brunel, F. M.、Louis, J. M.、Clore, G. M.、Mage, R. G.、Dawson, P. E.、Burton, D. R. 和 Zwick, M. B.：广泛 HIV-1 gp41 N-七肽重复区域上受体激活表位的中和抗体是在自然感染和免疫过程中引发的。艾滋病疫苗会议，阿姆斯特丹，2006 年）。 C. Rader 博士 (NCI) 实验室分离出对 Nogo-66 受体 (NgR) 家族所有三个成员具有选择性的单克隆抗体。 NgR 家族成员是参与中枢神经系统发育、可塑性和再生的细胞表面蛋白。 NgR1、NgR2 和 NgR3 主要由中枢神经系统 (CNS) 中的神经元表达，并被认为在 CNS 损伤后限制轴突生长和发芽。为了定义 Nogo-66 受体家族各个成员的表达并破译其功能，我们生成了选择性兔多克隆抗体 (3)。然后，我们使用噬菌体展示技术从相同的免疫库中生成兔单克隆抗体 (mAb)。我们获得了对 NgR1 和 NgR2 特异性表位具有纳摩尔亲和力的 mAb，但同时在小鼠、大鼠和人类直向同源物之间保守。采用噬菌体展示载体pC3C，一种新设计的噬菌粒，针对具有人恒定结构域的Fab文库的生成和选择进行了优化，从嵌合的兔/人Fab文库中选择兔单克隆抗体，并根据特异性、亲和力和氨基酸序列进行表征，最后转化为嵌合的兔/人IgG。使用免疫荧光显微镜和免疫沉淀证明了嵌合兔/人 IgG 对细胞表面结合的 Nogo-66 受体家族成员的强烈和特异性识别。兔单克隆抗体及其氨基酸序列构成了一组无限供应的物种交叉反应试剂，用于研究中枢神经系统内外 Nogo-66 受体家族的功能作用（MS 正在修订中 - Hofer, T.、Tangkeangsirisin, W.、Kennedy, M.G.、Mage、Rose G.、Raiker, S.J.、Venkatesh, K., Lee, H. Giger, R 和 Rader, C.：针对 Nogo-66 受体家族成员的嵌合兔/人 Fab 和 IgG，针对物种交叉反应性进行选择，并使用改进的噬菌体展示载体生产。