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: 7629770
• 负责人: Felix J Breden
• 金额: $ 19.72万
• 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 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7629770
• 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; 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; public health relevance; 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（Abs），但这种抗体很少能中和广泛的病毒谱。一些广泛(b)-Nt抗体已被发现：4E10、2F5、b12、447-52D和2G12；除了2G12外，所有的CDR-H3高变量循环（H3s）都比通常的长。通常，H3对抗原接触至关重要；然而，4E10、2F5和447-52D的晶体结构表明，它们的H3s要么与肽表位的边缘残基接触，要么根本不与这些表位接触。Haynes等人（2005）报道，大多数bNt单克隆抗体与自身抗原结合（例如，2F5和4E10与心磷脂结合；CL），并推测它们是自身抗体，通过对自身抗原的积极选择，然后被Env募集到适应性抗体反应中产生。根据这一假设，当耐受性被打破时，bNt抗体就会产生，从而允许自反应性B细胞存活，并产生具有长H3s的抗体。因此，我们提出，为了产生bNt抗体，HIV-1疫苗必须打破耐受性，产生自身反应性抗体。我们的初步研究表明，如果4E10和2F5是自身抗体，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对照组相比，我们也未发现来自HIV阳性供者的12份bNt血清和其他20份HIV阳性血清中CL反应性增加。由于有人声称长H3s反映了耐受性的丧失，我们建立了450个已知特异性的人单克隆抗体的DNA序列数据库，主要来自自身免疫性疾病、慢性和急性感染的人群，发现长H3s主要存在于自身免疫性和慢性抗体的抗蛋白亚群中；在后者中，长h3抗体并不局限于HIV感染。由此，我们提出了3种假设：(1)Long-H3抗体是在HIV-1感染过程中通过b细胞发育过程中耐受性的丧失而产生的。这表明长h3抗体在IgM+/IgD+、成熟、幼稚B细胞室和IgG+记忆B细胞中积累；nt血清应具有显著的自身反应性。(2) Long-H3抗体在抗原驱动过程中产生。这些抗体应该只存在于IgG+记忆B细胞和浆细胞区室中，并且bNt血清不应该是自反应性的。(3) HIV抗原从正常抗体库中选择长h3抗体；这与(2)有相同的预测。为了测试这些替代方案，我们计划分析12名未经治疗的HIV+供者的血清和pmbc，这些供者的血清是nt， 12名HIV+供者的血清不是广泛中和，12名SLE患者和14名健康供者。B细胞将按表型分为初始细胞池、记忆细胞池和浆细胞池，如果可能的话，将分为单个hiv特异性B细胞和浆细胞；从每个B细胞池和单个细胞中表达的mu或gamma VH基因将被测序并分析H3长度，体细胞突变和基因使用。抗原特异性细胞产生的血清和抗体将在带有自身抗原、HIV-1抗原和多反应性标记物的微阵列上进行分析。我们希望确定HIV-1感染的b细胞亚群中是否存在长h3抗体与真正的自身免疫状态，以及nt血清中是否存在明显的自身反应性或普遍的多反应性。这一结果应该有助于澄清HIV疫苗是否应该设计成打破耐受性。

项目成果

Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

• DOI: 10.4161/mabs.28421
• 发表时间: 2014-05
• 期刊: mAbs
• 影响因子: 5.3
• 作者: Almagro JC;Gilliland GL;Breden F;Scott JK;Sok D;Pauthner M;Reichert JM;Helguera G;Andrabi R;Mabry R;Bléry M;Voss JE;Laurén J;Abuqayyas L;Barghorn S;Ben-Jacob E;Crowe JE Jr;Huston JS;Johnston SA;Krauland E;Lund-Johansen F;Marasco WA;Parren PW;Xu KY
• 通讯作者: Xu KY