Origin of Antibodies with Long CDR-H3 Loops in HIV Infection & Autoimmune Disease

HIV 感染中具有长 CDR-H3 环的抗体的起源

• 批准号: 7496335
• 负责人: Felix J Breden
• 金额: $ 13.69万
• 依托单位: SIMON FRASER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496335
• 关键词: AIDS Vaccines; Accounting; Acute; Address; Affinity; Amino Acids; Antibodies; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Autologous; B cell repertoire; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Binding; Blood; Bone Marrow; Cardiolipins; Cells; Charge; Chronic; Complementarity Determining Regions; DNA Sequence; DNA Sequence Databases; Development; Differentiation Antigens; Drug Formulations; Employee Strikes; Epitopes; Family; Frequencies; Gene Rearrangement; Genes; HIV; HIV Antigens; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; HIV-1 vaccine; Health; Human; Immunodominant Epitopes; Immunoglobulin D; Immunoglobulin G; Immunoglobulin M; Infection; Laboratories; Length; Masks; Memory; Memory B-Lymphocyte; Nature; Outcome; Patients; Peptides; Peripheral; Phenotype; Plasma Cells; Population; Process; Proteins; Protocols documentation; Public Health; Range; Reporting; Research Personnel; Sequence Analysis; Serum; Site; Somatic Mutation; Sorting - Cell Movement; Specificity; Stretching; Structure; Structure of germinal center of lymph node; Systemic Lupus Erythematosus; Testing; V3 Loop; Vaccine Design; Vaccines; Variant; Viral; Virus; Work; autoreactivity; base; chronic autoimmune disease; design; env Gene Products; immunogenic; interest; neutralizing antibody; research study; response

DESCRIPTION (provided by applicant): HIV-1 infection induces neutralizing (Nt) antibodies (Abs) against one's infecting virus, yet rarely do such Abs neutralize a broad viral spectrum. A few broadly(b)-Nt Abs have been discovered: 4E10, 2F5, b12, 447-52D and 2G12; all but 2G12 have longer-than-usual CDR-H3 hypervariable loops (H3s). Typically, H3 is crucial to antigen contact; yet the crystal structures of 4E10, 2F5 and 447-52D show that, while prominent, their H3s either contact marginal residues of their peptide-epitopes or do not contact these epitopes at all. Haynes et al. (2005) reported that most of the bNt MAbs bind to self-antigens (e.g., 2F5 and 4E10 bind cardiolipin; CL), and speculated they are autoAbs that arose through a process of positive selection on autoantigen followed by recruitment by Env into the adaptive Ab response. Under this hypothesis, bNt Abs arise when tolerance is broken, allowing self-reactive B cells to survive, and Abs with long H3s to emerge. Thus, it was proposed that, to produce bNt Abs, HIV-1 vaccines must break tolerance and produce self-reactive Abs. Our preliminary studies indicate that if 4E10 and 2F5 are autoAbs they are unusual, as their affinity for CL is 100-1000-fold weaker than for their HIV-1 peptide epitopes. We also did not detect increased CL reactivity in 12 bNt sera from HIV+ donors nor in 20 other HIV+ sera, compared to HIV- controls. As it is claimed that long H3s reflect a loss of tolerance, we produced a DNA sequence database of 450 human MAbs of known specificity, obtained mainly from people with autoimmune disease, chronic and acute infection, and found that long H3s exist mainly among anti-protein subsets of the autoimmune and chronic Abs; among the latter, long-H3 Abs are not restricted to HIV infection. From this we propose 3 hypotheses: (1) Long-H3 Abs are produced during HIV-1 infection through loss of tolerance during B-cell development. This predicts that long-H3 Abs accumulate in the IgM+/IgD+, mature, naive B cell compartment, and among IgG+ memory B cells; bNt sera should have significant autoreactivity. (2) Long-H3 Abs arise during antigen-driven processes. These Abs should be present only in the IgG+ memory B cell and plasma cell compartments, and bNt sera should not to be autoreactive. (3) HIV antigens select long-H3 Abs from normal Ab repertoires; this has the same predictions as (2). To test these alternatives, we plan to analyze sera and PMBCs collected from 12 untreated HIV+ donors whose sera are bNt, 12 HIV+ donors whose sera are not broadly neutralizing, 12 patients with SLE, and 14 healthy donors. B cells will be sorted by phenotype into naive, memory, and plasma cell pools and if possible into single, HIV-specific B and plasma cells; expressed mu or gamma VH genes from each B cell pool and from single cells will be sequenced and analyzed for H3 length, somatic mutations, and gene usage. Sera and Abs produced by antigen-specific cells will be analyzed on microarrays bearing autoantigens, HIV-1 antigens, and markers of polyreactivity. We expect to determine if long-H3 Abs are present in B-cell subsets in HIV-1 infection vs. a truly autoimmune state, and whether distinct autoreactivities or generalized polyreactivity is present in bNt sera. The results should help to clarify if HIV vaccines should be designed to break tolerance. PUBLIC HEALTH RELEVANCE: One approach to AIDS vaccine design uses HIV-1-neutralizing antibodies. These antibodies are rare and their structures are considered unusual; moreover, questions have arisen concerning their ability to react with self . The proposed work will address whether HIV-neutralizing antibodies are unusual and/or self-reactive , and should clarify whether induction of unusual, self-reactive antibodies is a necessary consideration in AIDS vaccine design.

描述（由申请人提供）：HIV-1感染诱导针对感染病毒的中和（Nt）抗体（Ab），但此类Ab很少中和广泛的病毒谱。已经发现了几种广泛的（B）-Nt Ab：4 E10、2F 5、b12、447- 52 D和2G 12;除了2G 12之外，所有的都具有比通常更长的CDR-H3高变环（H3）。通常，H3对于抗原接触是至关重要的;然而4 E10、2F 5和447- 52 D的晶体结构显示，虽然突出，但它们的H3要么接触它们的肽-表位的边缘残基，要么根本不接触这些表位。Haynes等（2005）报道了大多数bNt MAb结合自身抗原（例如，2F 5和4 E10结合心磷脂; CL），并推测它们是通过对自身抗原进行阳性选择，然后被Env募集到适应性Ab应答中的过程产生的自身Ab。在这一假设下，当耐受性被破坏时，bNt抗体产生，允许自身反应性B细胞存活，并出现具有长H3的抗体。因此，有人提出，为了生产bNt抗体，HIV-1疫苗必须打破耐受性并产生自反应抗体。我们的初步研究表明，如果4 E10和2F 5是autoAb，它们是不寻常的，因为它们对CL的亲和力比它们对HIV-1肽表位的亲和力弱100-1000倍。与HIV-对照相比，我们在来自HIV+供体的12份bNt血清和20份其他HIV+血清中也未检测到CL反应性增加。由于声称长H3反映了耐受性的丧失，我们产生了450个已知特异性的人单克隆抗体的DNA序列数据库，主要从患有自身免疫性疾病、慢性和急性感染的人中获得，并且发现长H3主要存在于自身免疫性抗体和慢性抗体的抗蛋白亚群中;在后者中，长H3抗体不限于HIV感染。由此，我们提出了3个假设：（1）长H3抗体在HIV-1感染期间通过B细胞发育期间的耐受性丧失而产生。这预测了长H3抗体在IgM+/IgD+、成熟、幼稚B细胞区室和IgG+记忆B细胞中积累; bNt血清应具有显著的自身反应性。(2)长H3抗体在抗原驱动过程中产生。这些抗体应仅存在于IgG+记忆B细胞和浆细胞区室中，并且bNt血清不应是自身反应性的。(3)HIV抗原从正常Ab库中选择长H3 Ab;这与（2）具有相同的预测。为了测试这些替代方案，我们计划分析从12名未经治疗的HIV+供体（其血清是bNt）、12名HIV+供体（其血清不是广泛中和的）、12名SLE患者和14名健康供体收集的血清和PMBC。将B细胞按表型分选至初始、记忆和浆细胞池中，如可能分选至单个HIV特异性B和浆细胞中;将对每个B细胞池和单个细胞中表达的mu或γ VH基因进行测序，并分析H3长度、体细胞突变和基因使用情况。将在携带自身抗原、HIV-1抗原和多反应性标志物的微阵列上分析抗原特异性细胞产生的血清和Ab。我们希望确定是否长H3抗体存在于B细胞亚群中的HIV-1感染与真正的自身免疫状态，以及是否存在不同的自身反应性或广义的多反应性bNt血清。这些结果应该有助于澄清艾滋病毒疫苗是否应该被设计成打破耐受性。 公共卫生相关性：艾滋病疫苗设计的一种方法是使用HIV-1中和抗体。这些抗体是罕见的，它们的结构被认为是不寻常的;此外，关于它们与自身反应的能力也出现了问题。拟议的工作将解决艾滋病毒中和抗体是否不寻常和/或自身反应，并应澄清是否诱导不寻常的，自身反应性抗体是艾滋病疫苗设计的必要考虑。