Induction of 2G12 neutralizing antibody by plant-derived HIVgp145

描述（由申请人提供）：令人失望的是，HIV感染和疫苗在激发中和初级分离株的抗体方面一直很差，因此鉴定高度免疫原性中和表位/s是最优先考虑的问题。最近已经产生了几种广泛交叉反应的人中和单克隆抗体，这些抗体组合在一起，已被证明可以被动地保护猕猴免受阴道SIV传播。其中2G12单克隆抗体的独特之处在于，它识别gp120外表面由多达三个寡甘露糖链（残基332,392和295）组成的表位。大多数受感染的个体不产生2g1样中和抗体，可能是因为糖蛋白通过分泌途径运输时，高甘露糖聚糖被修剪。为了验证这一概念，ProcCell利用植物表达系统生产了一种高甘露糖形式的HIV gp145，通过使用KDEL标签特异性地将HIV gp145合成到内质网，该标签可以阻断进入高尔基体和暴露于甘露糖酶。这些高甘露糖HIV Env分子比cho衍生的Env与2G12表现出更强的反应性，这表明高甘露糖和分泌形式的HIV Env之间存在重要的结构差异。当前提案的目的是产生分泌形式的gp145（包括复杂和高甘露糖聚糖的混合物），并比较高甘露糖形式的HIV gp145 Env与植物和细胞培养来源的分泌gp120/160分子的生化，功能和免疫学特性。植物源性蛋白将在转基因烟草植物和细胞系中产生，纯化后用于免疫豚鼠，以评估两种糖型的免疫原性差异。

DESCRIPTION (provided by applicant): HIV infections and vaccines have historically been disappointingly poor at eliciting antibodies that neutralize primary isolates, thus the identification of a highly immunogenic neutralizing epitope/s is of the highest priority. Recently several broadly crossreactive human neutralizing monoclonal antibodies have been generated, which in combination, have been shown to passively protect macaques against vaginal SIV transmission. One of these MAbs, 2G12 is unique in that it recognizes an epitope that is comprised of a cluster of up to three oligomannose chains (residues 332, 392 and 295) on the outer face of gp120. Most infected individuals do not make 2G1like neutralizing antibodies, perhaps because the high mannose glycans are trimmed as the glycoproteins traffic through the secretory pathway. To test this concept, ProcCell has used the plant expression system to produce a high-mannose form of HIV gp145 by specifically targeting synthesis of HIV gp145 to the endoplasmic reticulum using a KDEL tag which blocks exit into the Golgi and exposure to mannosidases. These high-mannose HIV Env molecules exhibited stronger reactivity with 2G12 than CHO-derived Env, suggesting important structural differences between high mannose and secreted forms of the HIV Env. The aim of the current proposal is to produce the secreted form of the gp145 (comprising a mix of complex and high mannose glycans) and to compare the biochemical, functional and immunological properties of the high-mannose form of HIV gp145 Env with both plant and cell culture-derived secreted gp120/160 molecule. Plant- derived proteins will be produced in transgenic tobacco plants and cell lines, purified and used to immunize guinea pigs to assess differences in immunogenicity of the two glycoforms. This approach represents an important new concept in optimizing vaccine efficiency by the induction of strong neutralizing 2G12-like antibodies and other potentially neutralizaing antibodies specific for yet unknown high mannose epitopes. It also highlights the usefulness of the plant expression systems in producing different glycoforms of proteins.