Induction of 2G12 neutralizing antibody by plant-derived HIVgp145

描述（由申请人提供）：历史上，艾滋病毒感染和疫苗在激发中和原代分离株的抗体方面一直令人失望，因此鉴定高免疫原性中和表位是最优先考虑的事情。最近已经产生了几种广泛交叉反应的人中和单克隆抗体，其组合已经显示出被动保护猕猴免受阴道SIV传播。这些MAb之一，2G 12是独特的，因为它识别由gp 120外表面上的一簇至多三个寡甘露糖链（残基332、392和295）组成的表位。大多数感染者不产生2G 1样中和抗体，可能是因为高甘露糖聚糖在糖蛋白通过分泌途径运输时被修剪。为了测试这一概念，ProcCell已经使用植物表达系统来产生高甘露糖形式的HIV gp 145，其通过使用KDEL标签特异性地将HIV gp 145的合成靶向到内质网，该KDEL标签阻断进入高尔基体和暴露于甘露聚糖酶。这些高甘露糖HIV Env分子与2G 12的反应性比CHO衍生的Env更强，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。本提案的目的是生产分泌形式的gp 145（包含复合物和高甘露糖聚糖的混合物），并比较高甘露糖形式的HIV gp 145 Env与植物和细胞培养物来源的分泌型gp 120/160分子的生物化学、功能和免疫学特性。植物来源的蛋白质将在转基因烟草植物和细胞系中产生，纯化并用于免疫豚鼠以评估两种糖型的免疫原性差异。 这种方法代表了通过诱导强中和2G 12样抗体和对未知高甘露糖表位特异性的其他潜在中和抗体来优化疫苗效率的重要新概念。它还强调了植物表达系统在生产不同糖型蛋白质中的有用性。

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.