Mechanism of Potent Neutralization of HIV by IgA CD4i Antibody

描述(由申请人提供)：许多研究清楚地表明，在自然感染艾滋病毒或接种疫苗期间，体液免疫系统一般无法产生有效的功能中和抗体。此外，分离出的大多数功能性中和抗体通常具有自然产生的很少存在的结构。 抗体。了解抗体结构对中和活性的影响是被动免疫治疗实际应用中的一项紧迫任务。我们发现，CD4i抗体的IgA1同型转换变异体F425A1g8显示出显著增强的中和活性，而在缺乏sCD4的情况下，任何Ig G亚类都几乎没有中和活性。此外，在一项小型先导性研究中，在人源化小鼠模型中，CD4I IgA在降低病毒载量方面比抗体的IgG1变体更有效。虽然已经清楚地表明，在HIV阴性的性工作者和HIV状态不和谐的夫妇中存在IgA粘膜免疫反应，但在HIV感染者中并不容易观察到这一点。因此，很难辨别粘膜IgA在预防或抑制HIV感染中的作用。也已知，CD4i抗体的Fab片段或ScFv分子在中和HIV方面明显比完整的Ig G分子更有效，这可能是由于较小的片段更容易接触到HIV上的表位。然而，CD4i抗体片段用于治疗的使用受到分子半衰期短的限制，而且通常不会产生单链抗体对疫苗的反应。IgA的结构可能赋予片段类似的访问特性，具有更长的半衰期和作为免疫反应的一部分被激发的能力。这个 CD4i表位是病毒感染过程中相对保守的表位。因此，我们推测，IgA亚型的CD4i抗体通过其独特的分子结构可以增强抗HIV感染的中和活性。此外，我们认为，分泌型IgA的结构并不排除更广泛的IgA1 Fab区基于已公布的晶体结构获得CD4i表位的能力。为了验证我们的假设并扩大我们对F425A1g8 IgA的研究，我们建议再产生两个CD4i抗体的IgA变体，包括E51和17b，以证实IgA亚型CD4i抗体的中和活性增加。我们不仅将比较IgG1抗体与IgA1和igA2变异体的活性，还将包括分泌型IgA。具体功能将根据体内活跃的假设机制进行选择，包括直接中和病毒；通过上皮细胞传播(通过跨细胞作用评估)，以及参与效应器介导的艾滋病毒破坏(抗体依赖细胞介导的病毒抑制，ADCVI)。除了功能分析外，还将在模拟体内条件的条件下评估构建体的稳定性。这些目标的成功实现将有助于理解病毒中和中和的同型结构-功能关系，并直接有助于HIV疫苗的开发和用于预防和/或治疗HIV的被动免疫疗法的开发。

DESCRIPTION (provided by applicant): Numerous studies have clearly demonstrated the general inability of the humoral immune system in developing functionally effective neutralizing antibodies during natural HIV infections or vaccinations. Further, most of the functional neutralizing antibodies isolated usually possess structures that rarely exist in naturally produced antibodies. Understanding the structural effect of antibody on neutralizing activity is an urgent mission in the practical application of passive immunotherapy. We have found that the IgA1 isotype-switch variant of the CD4i antibody, F425A1g8, displays a significantly increased neutralizing activity, whereas there is little neutralizing activity by any of the IgG subclasses i the absence of sCD4. Moreover, in a small pilot study, CD4i IgA was more effective at reducing viral load in a humanized mouse model than the IgG1 variant of the antibody. While it has clearly been shown that there is an IgA mucosal immune response in HIV-negative sex workers and HIV-status discordant couples, this is not readily observed in HIV infected individuals. Thus, it has been difficult to discern the role of mucosal IgA in preventing or inhibiting HIV infection.It is also known that Fab fragments or scFv molecules of CD4i antibodies are significantly more effective at neutralizing HIV than the intact IgG molecule, likely the result of increased access o the smaller fragments to the epitope on HIV. However, the use of CD4i antibody fragments for therapy is limited by the short half-life of the molecules and scFvs are not normally produced in response to vaccination. The structure of IgA may confer similar access properties of the fragment with a longer half-life and the ability to be elicited as part of the immune response. The CD4i epitope represents a relatively highly conserved epitope integral to viral infection. Therefore, we hypothesize that IgA isotypes of CD4i antibodies through their unique molecular structure can increase the neutralizing activity against HIV infection. Further, we propose that the structure of secretory IgA does not preclude the ability of the more extended IgA1 Fab region from gaining access to the CD4i epitope based on published crystal structures. To test our hypotheses and extend our research on the F425A1g8 IgA, we propose to generate two more IgA variants of CD4i antibodies including E51 and 17b, to confirm the increased neutralizing activity of IgA isotypes of CD4i antibodies. We will not only compare the activity of IgG1 antibodies with IgA1 and igA2 variants, but will also include secretory IgA. Specific functions will be selected based on hypothesized mechanisms active in vivo and include direct viral neutralization; transmission by epithelial cells (as assessed by transcytosis), and participation in effector mediated destruction of HIV (antibody dependent cell-mediated viral inhibition, ADCVI). In addition to functional assays, the stability of the constructs will be assessed under conditions that mimic in vivo conditions. Successful completion of these aims will help to understand the isotype structure-function relation in viral neutralization and directl contributes to HIV vaccine development and the development of passive immunotherapy for prevention and/or treatment of HIV.