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

描述(由申请人提供)：HIV-1感染可诱导针对感染病毒的中和抗体(NT)，但此类抗体很少能中和广泛的病毒谱。已经发现了几个广泛的(B)-NT抗体：4E10、2F5、B12、447-52d和2G12；除2G12外，所有的抗体都有比通常更长的CDR-H3高变环(H3)。通常情况下，H3对抗原接触是至关重要的；然而，4E10、2F5和447-52d的晶体结构表明，虽然突出，但它们的H3要么接触其多肽表位的边缘残基，要么根本不接触这些表位。Haynes等人。(2005)报道了大多数BNT单抗与自身抗原结合(如2F5和4E10结合心磷脂；CL)，推测它们是通过对自身抗原进行阳性选择然后被Env重新招募到适应性抗体反应中而产生的自体抗体。在这一假设下，当耐受性被打破时，BNT抗体出现，允许自我反应的B细胞存活，并出现具有长H3s的抗体。因此，有人提出，要生产BNT抗体，HIV-1疫苗必须打破耐受性，产生自我反应抗体。我们的初步研究表明，如果4E10和2F5是自体抗体，它们是不寻常的，因为它们对CL的亲和力比它们的HIV-1多肽表位弱100-1000倍。与HIV对照组相比，我们也没有检测到来自HIV+捐赠者的12份BNT血清和其他20份HIV+血清中CL活性的增加。由于长H3s被认为反映了一种耐受性的丧失，我们建立了一个已知特异性的450个人类单抗的DNA序列数据库，这些单抗主要来自自身免疫性疾病、慢性和急性感染的人，发现长H3s主要存在于自身免疫和慢性单抗的抗蛋白亚群中；在后者中，Long-H3单抗并不局限于HIV感染。在此基础上，我们提出了3个假说：(1)长H3抗体是在HIV-1感染过程中通过B细胞发育过程中的耐受性丧失而产生的。这预示着Long-H3Abs聚集在成熟的、未成熟的IgM+/IGD+的B细胞和免疫球蛋白G+的记忆性B细胞之间；BNT血清应该具有显著的自身反应性。(2)Long-H3抗体产生于抗原驱动的过程中。这些抗体应该只存在于免疫球蛋白G+记忆B细胞和浆细胞隔室中，并且BNT血清不应该发生自身反应。(3)HIV抗原从正常抗体谱系中选择Long-H3抗体；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12名未经治疗的HIV+捐赠者的血清和PMBC，这些捐赠者的血清是BNT，12名HIV+捐赠者的血清没有广泛中和，12名SLE患者和14名健康捐赠者。B细胞将按表型分为幼稚细胞库、记忆性细胞库和浆细胞库，如果可能，还将分为单个HIV特异性B细胞库和浆细胞；每个B细胞库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、体细胞突变和基因用途。由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抗原和多反应标记的微阵列上进行分析。我们希望确定在HIV-1感染和真正的自身免疫状态下，B细胞亚群中是否存在Long-H3抗体，以及BNT血清中是否存在明显的自身反应或普遍的多反应。这一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是不寻常的和/或自我反应的，并应澄清诱导不寻常的自我反应抗体是否是艾滋病疫苗设计中的必要考虑因素。

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.