Babas T, Le Grand R, Dormont D, Bahraoui E
Laboratoire d'Immunovirologie des Lentivirus des Primates, UFR SVT, Université Paul Sabatier, Toulouse, France.
AIDS Res Hum Retroviruses. 1997 Sep 1;13(13):1109-19. doi: 10.1089/aid.1997.13.1109.
Twelve monoclonal antibodies (MAbs), TB1 to TB12, were produced against a soluble vaccinia recombinant envelope glycoprotein (gp140) from simian immunodeficiency virus SIVmac251. These MAbs recognized SIV gp140 with a relatively high affinity (K0.5 from 6.7 x 10(-8) to 4 x 10(-9) M). All the MAbs except TB9, TB11, and TB12 cross-reacted with HIV-2 envelope glycoproteins, but none of the 12 MAbs recognized those from HIV-1. Using a panel of 87 overlapping synthetic peptides containing 20 amino acid residues, with an overlap of 10 amino acids and spanning the entire primary sequence of gp140, 3 linear epitopes were identified. The first mapped with a neutralizing MAb, TB12, which recognized a linear sequence around amino acids 28-31 within the N-terminal end of the external envelope glycoprotein. The two other new nonneutralizing MAbs recognized linear epitopes around amino acid sequence 380-381 by MAbs TB1, TB2, and TB3, and at the transmembrane glycoprotein amino acids 581-600 by MAb TB6. Seven of the 12 MAbs, TB4, TB5, TB7-9, TB10, and TB11, failed to bind the linear synthetic peptides in ELISA. Moreover, among these seven MAbs only MAbs TB4, TB5, TB9, and TB10 failed to recognize SIV envelope glycoproteins in Western blot (WB) or ELISA after reduction of disulfide bridges by dithiothreitol (DTT), suggesting that they are directed against conformational or discontinuous epitopes. It is of interest to note that MAb TB10 can block the binding of gp140 to the CD4 receptor when the MAb is previously incubated with gp140. Consistent with this result, MAb TB10 cannot bind to gp140 that has been previously complexed with the CD4 receptor. All these results suggest that MAb TB10 recognizes a conformational or discontinuous epitope overlapping or close to the CD4-binding site. These properties are probably implicated in the neutralizing activity observed with this MAb.
针对来自猴免疫缺陷病毒SIVmac251的可溶性痘苗重组包膜糖蛋白（gp140），制备了12种单克隆抗体（MAb），即TB1至TB12。这些单克隆抗体以相对较高的亲和力（K0.5为6.7×10^(-8)至4×10^(-9)M）识别SIV gp140。除TB9、TB11和TB12外，所有单克隆抗体均与HIV-2包膜糖蛋白发生交叉反应，但这12种单克隆抗体均未识别HIV-1的包膜糖蛋白。使用一组由87个重叠的含20个氨基酸残基的合成肽组成的肽库，肽段间重叠10个氨基酸，覆盖gp140的整个一级序列，确定了3个线性表位。第一个表位由中和性单克隆抗体TB12定位，它识别外膜糖蛋白N端氨基酸28 - 31周围的线性序列。另外两个新的非中和性单克隆抗体，MAb TB1、TB2和TB3识别氨基酸序列380 - 381周围的线性表位，MAb TB6识别跨膜糖蛋白氨基酸581 - 600处的线性表位。12种单克隆抗体中的7种，即TB4、TB5、TB7 - 9、TB10和TB11，在ELISA中未能与线性合成肽结合。此外，在这7种单克隆抗体中，只有MAb TB4、TB5、TB9和TB10在通过二硫苏糖醇（DTT）还原二硫键后，在蛋白质印迹法（WB）或ELISA中未能识别SIV包膜糖蛋白，这表明它们针对的是构象或不连续表位。值得注意的是，当MAb TB10预先与gp140孵育时，它可以阻断gp140与CD4受体的结合。与此结果一致，MAb TB10不能与先前已与CD4受体复合的gp140结合。所有这些结果表明，MAb TB10识别一个与CD4结合位点重叠或接近的构象或不连续表位。这些特性可能与该单克隆抗体所观察到的中和活性有关。
