EPITOPE MAPPING OF HIV PROTEINS USING PROTEOLYTIC FOOT PRINTING AND MS

工作总结：人类免疫缺陷病毒表位图谱 (HIV)对感染的诊断和发展都很重要 获得性免疫缺陷综合症的疫苗和疗法 （艾滋病）。 我们一直在探测HIV蛋白gp 120和p24上的表位。 HIV进入宿主细胞的第一步是结合 包膜糖蛋白gp 120与细胞受体CD 4的结合。 GP120 消除保护性免疫反应的主要成分， 人类和黑猩猩体内的艾滋病病毒。 gp 120及其合成肽 作为潜在的候选疫苗。 同样，HIV p24 在感染艾滋病毒时产生第一批抗体。 由于艾滋病毒感染 艾滋病的进展，同时抗p24抗体减少， 抗体滴度 有人提出， 引发针对gp 120和p24的抗体可能有助于对抗HIV 感染 因此，了解p24和p25上的抗原决定簇， gp 120，特别是引发保护性抗体形成的那些， 在疫苗的研发中是极其重要的。我们结合了 蛋白水解足迹和MALDI/MS来定位天然表位 抗体识别的蛋白质。 在该方法中，蛋白质亲和- 与固定化抗体结合的抗体被蛋白水解裂解， 通过洗涤除去未结合的片段。 绑定的片段包含 表位的特征在于直接分析固定的 通过MALDI/MS，我们已经确定了重组抗体的核心表位 HIV p26经单克隆抗体13-102-100鉴定为 残基102-112。 亲环素A结合区也含有 在这些残留物中。我们还确定了抗原决定簇 被多克隆抗体识别的包膜糖蛋白gp 120 针对蛋白质的C末端产生。 抗C- 末端在体外经常显示保护作用。我们目前正在绘制 gp 120上的表位，其被从HIV血清中获得的MAb识别 特征为缓慢进展者的感染个体，即，一个 可能产生保护性抗体的人。我们也 定位HIV p24上的不连续表位。 后一个示例利用 蛋白水解足迹结果结合已知的分子 结构以鉴定表位中涉及的氨基酸。

Summary of Work: Mapping epitopes of the Human Immunodeficiency Virus (HIV) is important for the diagnosis of infection and for the development of vaccines and therapeutics for Acquired Immune Deficiency Syndrome (AIDS). We have been probing epitopes on the HIV proteins gp120 and p24. The initial step in the entry of HIV into the host cell is binding of the envelope glyco-protein gp120 to the cellular receptor CD4. Also gp120 elicits the major components of the protective immune response against HIV in humans and chimpanzees. gp120 and its synthetic peptides have been investigated as potential vaccine candidates. Similarly, HIV p24 elicits the first antibodies upon HIV infection. As the HIV infection progresses to AIDS, there is a simultaneous reduction in anti-p24 antibody titer. It has been proposed that a combination vaccine eliciting antibodies to both gp120 and p24 may be useful in combating HIV infection. Thus, knowledge of the antigenic determinants on p24 and gp120, especially those eliciting the formation of protective antibodies, is extremely important in the development of a vaccine.We have combined proteolytic footprinting and MALDI/MS to map epitopes on the native proteins recognized by antibodies. In this method, proteins affinity- bound to an immobilized antibody are proteolytically cleaved and the unbound fragments are removed by washing. The bound fragments containing the epitope are characterized by directly analyzing the immobilized antibody by MALDI/MS. We have identified the core epitope on recombinant HIV p26 identified by the monoclonal antibody 13-102-100 as being residues 102-112. The cyclophilin A binding region is also contained within these residues. We have also determined the antigenic determinant of the envelope glycoprotein gp120 recognized by a polyclonal antibody raised against the C-terminus of the protein. Antibodies against the C- terminus often show protective effects in vitro. We are currently mapping an epitope on gp120 recognized by a MAb obtained from sera of an HIV infected individual characterized as a slow progressor, i.e., an individual who may be producing protective antibodies. We are also mapping a discontinuous epitope on HIV p24. This latter example utilizes the proteolytic footprinting results combined with the known molecular structure to identify amino acids involved in the epitope.