Protective immunity against HIV: the role of IgG subclass and glycosylation

描述(由申请人提供)：自然杀伤细胞、单核细胞、巨噬细胞或树突状细胞上的免疫球蛋白Fc受体(Fc？Rs)可能在预防慢病毒感染中发挥关键作用。例如，当Fc？R结合和抗体依赖的细胞介导的病毒抑制(ADCVI)发生时，广中和单抗(IgG1B12)为猕猴提供了更全面的保护，使其免受SIV的阴道攻击。此外，人的重组gp120(Rgp120)免疫可引起ADCVI抗体反应，其大小与性传播艾滋病毒感染率的降低有关。因此，一种有效的HIV疫苗很可能不仅取决于抗体和抗原之间的相互作用，而且还取决于Fc？Rs和它们的Fc配体之间的相互作用。我们将研究抗体的两个生物学特性，即Ig G亚类和Fc糖基化，它们影响其与Fc？RS结合的能力，从而抑制HIV-1。由于IgG2与大多数Fc？Rs结合较差，且唾液酸或岩藻糖含量减少增加了Fc-Fc？R亲和力，我们将检验下列假设：1)rgp120疫苗对IgG2的应答因个体而异，rgp120疫苗激发的ADCVI活性与gp120特异性IgG2水平成反比；2)rgp120免疫激发的抗gp120抗体与FC糖基化模式有关，Fc糖基化是ADCVI活性的决定因素；3)修改IgG1B12的唾液酸和岩藻糖含量将增加中和和ADCVI活性的效力和广度。我们的长期目标是构建能够激发抗体的疫苗，从而最大限度地利用Fc？RS。我们将完成以下具体目标：1)检测rgp120免疫后产生的gp120特异性免疫球蛋白亚类，确定亚类与ADCVI活性之间的关系。利用免疫受试者的血清，gp120特异性免疫球蛋白亚类将通过酶联免疫吸附试验进行定量。亚类分布将与先前测量的ADCVI反应相关；2)定量rgp120诱导的Ig G Fc中唾液酸和岩藻糖的含量，并确定Fc糖基化与抗HIV-1活性之间的关系。将应用凝集素ELISA法和其他方法来确定gp120亲和纯化的Ig G的Fc糖基化模式，特别是岩藻糖化和唾液酸化的频率；以及3)分离广谱反应单抗IgG1 B12的低唾液酸和低岩藻糖变异体，并确定Fc糖基化变化对抗HIV-1活性的影响。岩藻糖和唾液酸含量将被修改，中和和ADCVI函数将被测量。很可能疫苗反应故意偏向于亚类或Fc糖形式的特定分布。考虑到开发广泛反应的疫苗诱导抗体的障碍，通过改变Fc来改善抗体功能的努力是艾滋病毒疫苗开发的一种有希望的、或许是必要的补充方法。我们建议研究抗体的某些特性，这可能对预防艾滋病毒感染至关重要。这类研究可能导致改进抗体抑制艾滋病毒能力的方法，并最终开发出有效的艾滋病毒/艾滋病疫苗。

DESCRIPTION (provided by applicant): Engagement of IgG Fc receptors (Fc?Rs) on natural killer cells, monocytes, macrophages, or dendritic cells may play a critical role in preventing lentivirus infection. For example, a broadly neutralizing monoclonal antibody (IgG1 b12) provides far more complete protection of macaques against vaginal challenge with SHIV when Fc?R engagement and antibody-dependent cell-mediated virus inhibition (ADCVI) are allowed to occur. Moreover, recombinant gp120 (rgp120) immunization of humans elicits an ADCVI antibody response whose magnitude correlates with reduced rates of sexually transmitted HIV infection. Thus, it is likely that an effective HIV vaccine will depend not only on interactions between antibody and antigen but also on interactions between Fc?Rs and their Fc ligands. We will investigate two biological properties of antibody, IgG subclass and Fc glycosylation, that impact its ability to bind to Fc?Rs and consequently to inhibit HIV-1. Since IgG2 binds poorly to most Fc?Rs and decreased sialic acid or fucose content increases Fc-Fc?R affinity, we will test the following hypotheses: 1) the IgG2 response to rgp120 vaccine varies between individuals, and the ADCVI activity elicited by rgp120 vaccine is inversely proportional to gp120-specific IgG2 levels; 2) anti-gp120 antibodies elicited by rgp120 immunization vary with respect to Fc glycosylation pattern, and Fc glycosylation is a determinant of ADCVI activity; and 3) modifications in the sialic acid and fucose content of IgG1 b12 will increase the potency and breadth of neutralizing and ADCVI activity. Our long-term goal is to construct vaccines eliciting antibodies that optimally engage Fc?Rs. We will accomplish the following specific aims: 1) Measure gp120-specific IgG subclasses elicited by vaccination with rgp120, and determine the association between subclass and ADCVI activity. Using sera from vaccinated subjects, gp120- specific IgG subclasses will be quantified by ELISA. Subclass distribution will be correlated with ADCVI responses measured previously; 2) Quantify the sialic acid and fucose content in IgG Fc elicited by rgp120, and determine the association between Fc glycosylation and anti-HIV-1 activity. Lectin ELISA and other methods will be applied to determine Fc glycosylation patterns-particularly the frequency of fucosylated and sialylated glycans - of gp120 affinity-purified IgG; and 3) Isolate low sialic acid and low fucose variants of the broadly reactive mAb IgG1 b12, and determine the affect of Fc glycosylation changes on anti-HIV-1 activity. Fucose and sialic acid content will be modified and neutralizing and ADCVI functions measured. It is likely that vaccine responses could be purposefully biased toward a specific distribution of subclasses or Fc glycoforms. Given the obstacles to developing broadly reactive, vaccine-induced antibodies, efforts at improving antibody function by altering Fc represents a promising, and perhaps essential, complementary approach to HIV vaccine development. We propose studying certain properties of antibody that might be crucial for preventing HIV infection. Such studies may lead to methods of improving the ability of antibody to inhibit HIV and eventually to the development of effective HIV/AIDS vaccines.