EPITOPE MAPPING OF HIV PROTEINS USING PROTEOLYTIC FOOT PRINTING AND MS

工作总结：绘制人类免疫缺陷病毒(HIV)的表位图对于诊断感染以及开发获得性免疫缺陷综合征(AIDS)的疫苗和治疗方法非常重要。我们一直在探索HIV蛋白gp120和p24的表位。HIV进入宿主细胞的第一步是将包膜糖蛋白gp120与细胞受体CD4结合。此外，gp120还在人类和黑猩猩身上引发了针对艾滋病毒的保护性免疫反应的主要组成部分。Gp120及其合成肽已被研究为潜在的候选疫苗。同样，HIV p24在感染HIV时会引发第一批抗体。随着HIV感染进展为AIDS，抗p24抗体效价同时降低。有人提出，一种既能产生gp120抗体又能产生p24抗体的联合疫苗在对抗HIV感染方面可能是有用的。因此，了解p24和gp120上的抗原决定因素，特别是那些引起保护性抗体形成的决定因素，对于疫苗的开发是极其重要的。我们结合了蛋白质分解足迹和MALDI/MS来绘制抗体识别的天然蛋白质的表位图。在这种方法中，与固定抗体亲和结合的蛋白质被蛋白水解性切割，并通过洗涤去除未结合的片段。含有表位的结合片段通过MALDI/MS直接分析固定化抗体来表征。我们目前正在将从HIV感染者血清中分离的人单抗识别的gp120表位进行定位。这些HuMAb已被证明在体外中和实验室的艾滋病毒毒株，但往往不表现出对初级分离株的很强的中和性能。了解gp120上识别的表位及其中和特性对于疫苗开发是必要的。在识别gp120的人单抗1331D的情况下，用ELISA法鉴定的表位是一个简单的线性表位。然而，我们确定，事实上，单抗识别了天然蛋白质上的一个不连续的表位。这些结果也解释了为什么识别gp120 C末端的两个不同的人单抗通过免疫荧光映射到相同的核心肽上表现出不同的分支特异性。我们还开发了一种用于提高对不纯抗体的敏感性的方法，该方法基于最初使用的固定捕获抗体，该抗体将捕获感兴趣的抗体。然后，将捕获的抗体与目标抗体进行交联。显著降低了MALDI光谱中的化学背景，从而提高了灵敏度。

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 glycoprotein 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 are currently mapping epitopes on gp120 recognized by human MAbs isolated from sera of HIV infected individuals. These huMAbs have been shown to neutralize laboratory strains of HIV in vitro, but often do not demonstrate strong neutralizing properties against primary isolates. Knowledge of the epitopes recognized on gp120 in combination with their neutralizing properties is necessary for vaccine development. In the case of one specific human MAB, 1331D, recognizing gp120, the epitope identified by ELISA was a simple linear epitope. We determined, however, that the MAb, in fact, recognized a discontinuous epitope on the native protein. these results also explain why two different human MAbs recognizing the C-terminus of gp120 map to the same core peptides by immunoflorescence show different clade specificities. We have also developed a methodology for improved sensitivity for use with impure antibodies based on initial use of an immobilized capture Ab that will capture the Ab of interest. The capture Ab is then crosslinked to the Ab of interest. Significantly reduced chemical background in the MALDI spectra leads to increased sensitivity.