Immunoglobulin CDR-H3 and neutralizing antibodies to HIV

免疫球蛋白 CDR-H3 和 HIV 中和抗体

• 批准号: 7623056
• 负责人: Harry William Schroeder
• 金额: $ 27.83万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623056
• 关键词: Acquired Immunodeficiency Syndrome; Address; Alleles; Amino Acids; Antibodies; Antibody Formation; Antigens; Arginine; Autoantibodies; Autoimmunity; B cell repertoire; B-Lymphocytes; Binding; Binding Sites; Biological Assay; Bone Marrow; CCR5 gene; CXCR4 gene; Cardiolipins; Charge; Codon Nucleotides; Collaborations; Commit; Complementarity Determining Region III; Complex; Data; Development; Epitopes; Fatty acid glycerol esters; Fostering; Frameshift Mutation; Frequencies; Gene Targeting; Generations; Genetic Predisposition to Disease; Genetic Techniques; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV vaccine; HIV-1; Human; Hybridomas; Hydrophobicity; Immune; Immune system; Immunization; Immunoglobulins; Immunologic Deficiency Syndromes; Immunologist; Immunology; Inbred BALB C Mice; Individual; Infection; Laboratories; Length; Life; Light; Lipid Binding; Liposomes; Measures; Medical; Membrane; Membrane Lipids; Modeling; Monoclonal Antibodies; Mouse Strains; Mus; Mutant Strains Mice; Nucleotides; Patients; Peptides; Persons; Phospholipids; Play; Predisposition; Process; Production; Property; Reading Frames; Regulation; Reporting; Research; Resources; Role; SLEB1 gene; SLEB2 gene; SLEB3 gene; Serum; Site; Specificity; Staging; Structure; Surface; Systemic Lupus Erythematosus; Testing; Treatment Cost; Tyrosine; Vaccines; Valine; Viral; Virion; Virus Diseases; Work; anti-dsDNA antibodies; antigen binding; antiretroviral therapy; autoreactivity; base; cohort; complementarity-determining region 3; congenic; cross reactivity; env Gene Products; expectation; experience; falls; mutant; neutralizing antibody; novel; pandemic disease; pressure; prevent; receptor; receptor binding; sugar; vaccine development; virus envelope

DESCRIPTION (provided by applicant): Among neutralizing antibodies (Nabs) directed against the HIV-1 envelope gp120/gp41 glycoprotein, 2F5 and 4E10 stand out for their potency and broadly neutralizing activity. Both antibodies are directed against the membrane proximal external region (MPER) of the gp41 stalk. Unfortunately, 2F5 and 4E10-like anti-MPER Nabs are rarely elicited in patients [3;70]. Both of these Nabs differ from the norm in that each of their antigen binding sites contain a long, hydrophobic H chain complementarity determining region 3 (CDR-H3). In previous studies we have shown that immunoglobulins with hydrophobic CDR-H3s are normally culled from the conventional B cell repertoire. We thus propose that 2F5- and 4E10-like Nabs are difficult to elicit because as a result of this normal process the frequency of this type of antigen binding site in healthy individuals tends to be very low. We will test the hypothesis that forced enrichment for use of long, hydrophobic CDR-H3 intervals will promote the production of Nabs directed against the HIV-1 MPER. We also propose to test the complementary hypothesis that the use of these long, hydrophobic CDR-H3 intervals in anti-MPER Nabs may require release from normal tolerance mechanisms. The application is responsive to the RFA in that its focus is basic immunology research on B cell and antibody regulation in the context of HIV-1 vaccine development. Our proposed studies target a critical conserved epitope on the HIV-1 envelope glycoprotein gp120/gp41. They focus on elucidating basic immune mechanisms by which an effective and robust neutralizing antibody response can be elicited in uninfected individuals. They address the issue of tolerance and autoimmunity in eliciting Nabs. And, our studies foster collaboration between two established basic B cell immunologists, Drs. Schroeder and Kearney; and two experts in HIV, Drs. Haynes and Shaw. We have created a strain of mouse, ?D-D¿FS, where we force use of hydrophobic CDR-H3s by committing B cells to the use of a hydrophobic DH reading frame, valine-enriched DFL16.1 RF2. DFL16.1 is also two to four codons longer than the other mouse DH sequences. Thus, the ?D-D¿FS mouse creates a polyclonal repertoire that is not only enriched for hydrophobic CDR-H3s, but for longer CDR-H3s as well. To test the role of long, hydrophobic CDR-H3s in permitting the generation of broadly neutralizing anti-HIV antibodies, Dr. Schroeder will challenge mutant ?D-D¿FS BALB/c mice, as well as control ?D-DFL and wild-type BALB/c mice, with MPER immunogens obtained from Dr. Haynes. To begin to test the role of tolerance, Dr. Schroeder will also challenge mutant and wild-type DH locus mice on BALB/c, C57BL/6, and C57BL/6 sle1/sle2/sle3 congenic backgrounds, each of which progressively increases susceptibility to the generation of autoreactive antibodies. Dr. George Shaw will evaluate the sera from these mice for the presence of neutralizing antibodies. Drs. Schroeder and Kearney will generate hybridomas from those mice that express neutralizing MPER antibodies and obtain monoclonal antibodies for further study. These anti-MPER monoclonal antibodies will be further screened for neutralization and for autoreactivity. Their binding properties will be examined. Finally, their V domains will be cloned and sequenced to further correlate the relationship between neutralization and CDR-H3 length or hydrophobicity. The creation of vaccines capable of eliciting neutralizing antibodies to HIV has proven more difficult than first expected [7;44]. The HIV envelope spike plays a key role in viral infection, and antibodies directed against the MPER region at the base of the spike are broadly and potently neutralizing. Unfortunately, these antibodies are not only extremely uncommon; they also tend to be autoreactive. The site at which these antibodies bind HIV contains a binding structure that tends to be both larger and enriched for fat soluble amino acids than most such binding structures in other antibodies. We have used genetic techniques to force mice to express antibodies enriched for longer binding structures as well as for longer, more fat soluble binding structures. To test the extent to which these features will enable generation of neutralizing MPER antibodies, we propose to challenge our mutant mice with HIV MPER immunogens, isolate anti-MPER antibodies, test whether these antibodies neutralize HIV, and further correlate structure with function.

描述（由申请人提供）：在针对 HIV-1 包膜 gp120/gp41 糖蛋白的中和抗体 (Nab) 中，2F5 和 4E10 因其效力和广泛的中和活性而脱颖而出，这两种抗体都针对膜近端外部区域 (MPER)。不幸的是，2F5 和 4E10 样抗 MPER Nab 很少在患者体内引发。 [3;70]. 这两种 Nab 与标准抗体的不同之处在于，它们的每个抗原结合位点都包含一个长的疏水性 H 链互补决定区 3 (CDR-H3)。 -H3 通常是从传统 B 细胞库中挑选出来的，因此我们认为很难引出 2F5 和 4E10 样的 Nab，因为这种正常过程的结果是这种类型的 Nab 的频率较高。我们倾向于检验以下假设：使用长疏水性 CDR-H3 间隔进行强制富集将促进针对 HIV-1 MPER 的 Nab 的产生。互补的假设是，在抗 MPER Nab 中使用这些长的疏水性 CDR-H3 间隔可能需要从正常的耐受机制中释放出来。该应用对 RFA 做出了响应，因为其重点是 B 细胞和抗体调节的基础免疫学研究。 HIV-1 的背景我们提出的研究针对 HIV-1 包膜糖蛋白 gp120/gp41 上的一个关键保守表位，重点是阐明基本免疫机制，通过该机制可以在未感染个体中引发有效且强大的中和抗体反应。而且，我们的研究促进了两位基础 B 细胞免疫学家 Schroeder 博士和 Kearney 博士以及两位 HIV 专家之间的合作。海恩斯和肖我们创造了一种老鼠，“D-D”。 FS，我们通过使 B 细胞使用疏水性 DH 阅读框来强制使用疏水性 CDR-H3，富含缬氨酸的 DFL16.1 RF2 也比其他小鼠 DH 序列长两到四个密码子。因此，“D-D” FS 小鼠创建了一个多克隆库，不仅富含疏水性 CDR-H3，而且还富含更长的 CDR-H3。为了测试长疏水性 CDR-H3 在产生广泛中和性抗 HIV 抗体中的作用，Dr.施罗德将挑战变种人“D-D” FS BALB/c 小鼠以及对照 ?D-DFL 和野生型 BALB/c 小鼠，使用从 Haynes 博士获得的 MPER 免疫原。为了开始测试耐受性的作用，Schroeder 博士还将挑战突变型和野生型。 BALB/c、C57BL/6 和 C57BL/6 sle1/sle2/sle3 同源背景上的 DH 型基因座小鼠，每种基因的易感性逐渐增加George Shaw 博士将评估这些小鼠的血清中是否存在中和抗体，Kearney 博士将从这些小鼠中产生表达中和 MPER 抗体的杂交瘤，并获得单克隆抗体以供进一步研究。 -MPER 单克隆抗体将被进一步筛选中和和自身反应性，最后，它们的 V 结构域将被克隆和测序，以进一步关联中和和自身反应之间的关系。 CDR-H3 长度或疏水性。事实证明，能够引发 HIV 中和抗体的疫苗比最初预期的要困难 [7;44]。不幸的是，这些抗体不仅极其罕见，而且往往具有自身反应性。这些抗体与 HIV 结合的位点往往更大且富集。脂溶性氨基酸我们使用遗传技术来迫使小鼠表达富含更长结合结构以及更长、更脂溶性结合结构的抗体，以测试这些特征能够在多大程度上产生中和作用。 MPER 抗体，我们建议用 HIV MPER 免疫原挑战我们的突变小鼠，分离抗 MPER 抗体，测试这些抗体是否中和 HIV，并进一步将结构与功能关联起来。