Immunoglobulin CDR-H3 and neutralizing antibodies to HIV

免疫球蛋白 CDR-H3 和 HIV 中和抗体

• 批准号: 7458507
• 负责人: Harry William Schroeder
• 金额: $ 23.15万
• 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 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458507
• 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; Range; Reading Frames; Regulation; Reporting; Research; Resources; Role; 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（由适用提供）：在针对HIV-1包膜GP120/GP41糖蛋白的中和抗体（NAB）中，2F5和4E10在其效力和广泛中和活性方面脱颖而出。两种抗体均针对GP41雄鹿的膜代理外部区域（MPER）。不幸的是，在患者中很少引起2F5和4E10样的抗MPer NAB [3; 70]。这两个NAB都与规范不同，因为它们的每个抗原结合位点都包含长长的疏水性H链互补性决定区域3（CDR-H3）。在先前的研究中，我们已经表明，具有疏水性CDR-H3S的免疫球蛋白通常从常规的B细胞库中剔除。因此，我们建议很难引起2f5-和4e10样的NAB，因为由于这种正常过程，健康个体中这种类型的抗原结合位点的频率往往非常低。我们将检验以下假设：使用长长的疏水性CDR-H3间隔强迫富集将促进针对HIV-1 MPER的NAB的产生。我们还建议检验完整的假设，即在抗MPer NAB中使用这些长，疏水的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的响应有响应，因为它的重点是HIV-1疫苗开发中对B细胞和抗体调节的基本免疫学研究。我们提出的研究针对HIV-1包膜糖蛋白GP120/GP41的关键构成表位。他们专注于阐明基本的免疫力学，通过这种免疫机制可以在未感染的个体中引起有效且可靠的中和抗体反应。他们解决了引起NAB的耐受性和自身免疫的问题。而且，我们的研究促进了两位既定基本B细胞免疫学家Drs之间的合作。 Schroeder和Kearney；和两名艾滋病毒专家，博士。海恩斯和肖。我们创建了一个小鼠的菌株，即？ DFL16.1也比其他鼠标DH序列更长两到四个密码子。这是？d-d fs小鼠会产生多克隆曲目，它不仅富含疏水性CDR-H3s，而且还用于更长的CDR-H3S。为了测试长，疏水CDR-H3s在允许生成广泛中和的抗HIV抗体中的作用，Schroeder博士将挑战突变体？d-d-d-d- d- d- d- d- d- d-d-d-d-d-d-d-dfl和野生型balb/c小鼠，并获得了从haynes博士获得的MPER免疫。为了开始测试耐受性的作用，Schroeder博士还将在BALB/C，C57BL/6和C57BL/6 SLE1/SLE1/SLE1/SLE2/SLE3先生背景上挑战突变体和野生型DH基因座小鼠，每种背景逐渐增加了自身反应性抗体的易感性。乔治·肖（George Shaw）博士将评估这些小鼠的血清中和中和抗体的存在。博士。 Schroeder和Kearney将产生那些表达中和MPER抗体并获得单克隆抗体的小鼠的杂交瘤，以进行进一步研究。这些抗MPer单克隆抗体将进一步筛选神经化和自动反应性。将检查它们的结合特性。最后，将其V结构域克隆并测序，以进一步将神经化与CDR-H3长度或疏水性之间的关系相关联。事实证明，能够引起对艾滋病毒中和抗体的抗体的疫苗的产生比最初预期的更加困难[7; 44]。 HIV包膜尖峰在病毒感染中起关键作用，针对尖峰底部的MPER区域的抗体广泛且可能中和。不幸的是，这些抗体不仅极为罕见。它们也倾向于自动反应。这些抗体结合HIV的位点含有一种结合结构，该结构往往比其他抗体中的大多数此类结合结构更大且富含脂肪氨基酸。我们已经使用遗传技术来迫使小鼠表达富含结合结构以及更长，更脂溶性结合结构的抗体。为了测试这些特征将在多大程度上产生中和MPER抗体，我们建议用HIV MPER免疫剂挑战突变小鼠，分离出抗MPER抗体，测试这些抗体是否中和HIV，并进一步将结构与功能相关。