Mechanism of Potent Neutralization of HIV by IgA CD4i Antibody

描述（由申请人提供）：大量研究已明确证明，在自然HIV感染或接种疫苗期间，体液免疫系统一般无法产生功能有效的中和抗体。此外，大多数分离的功能性中和抗体通常具有天然产生的抗体中很少存在的结构。 抗体的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迫切使命。我们已经发现，CD 4 i抗体的IgA 1同种型转换变体F425 A1 g8显示出显著增加的中和活性，而在不存在sCD 4的情况下，任何IgG亚类几乎没有中和活性。此外，在一项小型的初步研究中，CD 4 i伊加在人源化小鼠模型中比抗体的IgG 1变体更有效地降低病毒载量。虽然已经清楚地表明，在HIV阴性的性工作者和HIV状态不一致的夫妇中存在伊加粘膜免疫应答，但这在HIV感染者中不易观察到。还已知的是，CD 4 i抗体的Fab片段或scFv分子在中和HIV方面比完整的IgG分子显著更有效，这可能是由于更小的片段接近HIV上的表位的增加的结果。然而，用于治疗的CD 4 i抗体片段的使用受到分子的短半衰期的限制，并且scFv通常不会响应于疫苗接种而产生。伊加的结构可以赋予片段类似的进入特性，具有更长的半衰期和作为免疫应答的一部分被引发的能力。的 CD 4 i表位代表病毒感染所必需的相对高度保守的表位。因此，我们假设CD 4 i抗体的伊加同种型通过其独特的分子结构可以增加针对HIV感染的中和活性。此外，我们提出，分泌型伊加的结构并不排除更广泛的IgA 1 Fab区的能力，从获得的CD 4 i表位的基础上公布的晶体结构。为了验证我们的假设并扩展我们对F425 A1 g8伊加的研究，我们建议再产生两种CD 4 i抗体的伊加变体，包括E51和17 b，以证实CD 4 i抗体伊加同种型的中和活性增加。我们不仅将比较IgG 1抗体与IgA 1和IgA 2变体的活性，还将包括分泌伊加。将根据体内活性的假设机制选择特定功能，包括直接病毒中和;上皮细胞传播（通过转胞吞作用评估）和参与效应子介导的HIV破坏（抗体依赖性细胞介导的病毒抑制，ADCVI）。除了功能测定外，还将在模拟体内条件的条件下评估构建体的稳定性。这些目标的成功完成将有助于理解病毒中和中的同种型结构-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.