INCREASE/gp120 IMMUNOGENICITY/LINKED ALPHA-GAL EPITOPES

描述(申请人提供)：该项目的总体目标是开发一种新型的gp120疫苗，该疫苗利用天然的抗Gal抗体(Ab)作为天然佐剂，有效地针对抗原提呈细胞(APC)的gp120。该疫苗由gp120组成，设计用于在这种糖蛋白的许多N(天冬酰胺)连接的碳水化合物链上表达多个α-半乳糖表位(Galphal/Alpha1-3 Galbeta1-4GlcNAc-R)。HIV疫苗必须有效地针对APC，才能引起保护性免疫反应。将gp120上的碳水化合物改变为α-半乳糖表位将通过消除gp120的负电荷和靶向APC FcGamma受体来显著增加APC对抗原的摄取。我们建议利用我们生产的重组α-1，3-半乳糖基转移酶(α-1，3-GT)将CHO细胞产生的重组gp120上的多个唾液酸(SA)单元转化为α-半乳糖表位(简称gpl20(GAL))。用gpl20/Alpha/Gal免疫很可能导致与天然的抗Gal抗体在原位形成免疫复合体，Gal抗体是人类体内含量最丰富的抗体，占循环免疫球蛋白的1%。我们推测gpl20/α/Gal与抗Gal之间形成的免疫复合物是针对APC的免疫分子，因为复合的抗Gal抗体的Fc部分与APC上的FcGamma受体结合。这导致了gp120多肽的加工和呈现(包括交叉呈现)的增加，从而有效地激活了CD4+和CD8+T细胞。我们的目的是在模拟人类相关免疫参数的3GT基因敲除小鼠的实验动物模型系统中，获得gp120(Gal)比gp120增强免疫原性的原理证明。具体地说，我们建议：1.对gp120/α/Gal进行大量的加工和纯化，以用于疫苗。2.比较gp120/α/Gal疫苗与gp120疫苗在增强小鼠体液和细胞免疫应答方面的优势。如果我们在这个初步项目中发现gp120/α/ga疫苗比gp120更具免疫原性，我们将在未来的研究中通过将gp120与HIV的p18或p24融合来扩大抗原靶点的范围。然后，我们还将与一个研究猴子免疫反应的小组合作，以确定该疫苗在灵长类动物模型中的效力。

DESCRIPTION (provided by applicant): The overall objective of this project is to develop a novel type of gp120 vaccine that exploits the natural anti-Gal antibody (Ab) as a natural adjuvant effectively targeting gp120 to antigen presenting cells (APC). This vaccine is comprised of gp120 engineered to express multiple alpha-gal epitopes (Galphal/alpha1-3 Galbeta1-4GlcNAc-R) on the many N(asparagine)-linked carbohydrate chains of this glycoprotein. HIV vaccines have to be effectively targeted to APC in order to elicit a protective immune response. Changing carbohydrates on gp120 to alpha-gal epitopes will significantly increase uptake of antigen by APC by both eliminating the negative charge of gp120 and by targeting gp120 to APC Fcgamma receptors. We propose to convert the multiple sialic acid (SA) units on recombinant gp120 produced in CHO cells, into alpha-gal epitopes (referred to as gpl20(gal) by the use of recombinant alpha1,3 galactosyltransferase (alpha 1,3 GT) that we produce. Vaccination with gpl20/alpha/gal is likely to result in in situ formation of immune complexes with the natural anti-Gal Ab, the most abundant Ab in humans constituting 1% of circulating IgG. We hypothesize that the immune complexes formed between gpl20/alpha/gal and anti-Gal target the vaccinating molecules to APC, since the Fc portion of the complexed anti-Gal IgG binds to Fcgamma receptors on APC. This results in increased processing and presentation (including cross presentation) of gp120 peptides for effective activation of CD4+ and CD8+ T cells. Our aim is to obtain a proof of principle for the increased immunogenicity of gp120(gal in comparison with that of gp120 in an experimental animal model system of (l,3GT knockout mice which simulates pertinent immune parameters in humans. Specifically, we propose: 1. To process and purify large amounts of gp120/alpha/gal for use as vaccine. 2. To demonstrate the superiority of the gp120/alpha/gal vaccine for increasing humoral and cellular immune response in mice in comparison with gp120 vaccine. If we find, in this preliminary project, that gp120/alpha/gal vaccine is significantly more immunogenic than gp120, we will, in future studies, increase the range of antigenic targets by fusing gp120 with p18 or p24 of HIV. We will then also collaborate with a group studying immune responses in monkeys to determine the efficacy of this vaccine in a primate model.