Raux H, Coulon P, Lafay F, Flamand A
Laboratoire de Génétique des Virus, CNRS, GIF, France.
Virology. 1995 Jul 10;210(2):400-8. doi: 10.1006/viro.1995.1356.
Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody.
在室温下孵育1小时期间，我们约15%的抗糖蛋白单克隆抗体（MAb）未能中和狂犬病病毒的感染性。在先前的研究中，我们已经证明，通过在酸性pH下孵育病毒，可以诱导糖蛋白群体发生大规模构象变化。这种构象变化是可逆的，因此病毒感染性不受酸性pH短暂暴露的影响。酸性或中性构型的糖蛋白比例取决于pH值，这意味着即使在中性pH下，一些糖蛋白也会短暂采用酸性构型，反之亦然。在此我们报告，我们的一些非中和性单克隆抗体识别病毒粒子表面糖蛋白的酸性形式。在pH 6.4下孵育病毒后，大多数糖蛋白处于酸性构型。在相同pH下与这些单克隆抗体进一步孵育1小时，导致更多免疫球蛋白附着在病毒上，从而诱导中和作用。通过在中性pH下孵育更长时间或在更高温度下孵育，也可以用相同的单克隆抗体诱导中和作用。可以选择对这些单克隆抗体中和具有抗性的突变体。赋予中和抗性的突变并不定位在先前描述的抗原位点，也不会在远处修饰这些位点。它们对病毒的致病力没有影响。要么它们位于表位中，要么它们修饰表位，使得在蛋白质的酸性构型下不再被抗体识别。或者，这些突变可能使蛋白质在其中性构型中稳定。此外，这些实验证实了我们先前的发现，即中和作用需要大量免疫球蛋白固定在病毒上，而与抗体识别的蛋白质区域无关。
