Arthur L O, Bess J W, Chertova E N, Rossio J L, Esser M T, Benveniste R E, Henderson L E, Lifson J D
AIDS Vaccine Program, SAIC/Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
AIDS Res Hum Retroviruses. 1998 Oct;14 Suppl 3:S311-9.
Although most viral vaccines used in humans have been composed of live attenuated viruses or whole killed viral particles, the latter approach has received little attention in research on experimental primate immunodeficiency virus vaccines. Inactivation procedures involving heat or formalin appear to adversely affect the viral envelope proteins. Recently we have inactivated human immunodeficiency virus type 1 (HIV-1) with the compound 2,2'-dithiodipyridine (Aldrithiol-2, Aldrich, Milwaukee, WI), which inactivates infectivity of retroviruses by covalently modifying the nucleocapsid zinc finger motifs. HIV-1 inactivated with Aldrithiol-2 retained the conformational and functional integrity of the viral and virion-associated cellular proteins on the viral membrane. We have extended our studies of zinc finger targeted inactivation to simian immunodeficiency virus (SIV) and evaluated the feasibility of applying the procedures to large scale (>30 l) production and purification of the primate immunodeficiency viruses. There was no detectable residual infectivity of SIV after treatment with 1 mM Aldrithiol-2 (>5 logs inactivation). Treatment with Aldrithiol-2 resulted in extensive reaction with the nucleocapsid protein of treated virus, as shown by immunoblot and high-performance liquid chromatography (HPLC) analysis. As expected, the virion gp120SU appeared to be completely unreactive with Aldrithiol-2. Sucrose gradient purification and concentration procedures resulted in little loss of viral infectivity or virion-associated gp120SU. When tested in a gp120-CD4 dependent cell binding assay, the inactivated virus bound to cells comparably to the untreated virus. Analysis of gp120-CD4 mediated postbinding fusion events showed that the inactivated virus could induce CD4-dependent fusion with efficiencies similar to the untreated virus. Inactivation and processing of primate immunodeficiency viruses by methods described here results in highly concentrated virus preparations that retain their envelope proteins in a native configuration. These inactivated virus preparations should be useful in whole killed-particle vaccine experiments as well as laboratory reagents to prepare antisera, including monoclonal antibodies, and to study noninfective virion-cell interactions.
尽管用于人类的大多数病毒疫苗是由减毒活病毒或全灭活病毒颗粒组成，但后一种方法在实验性灵长类免疫缺陷病毒疫苗研究中很少受到关注。涉及加热或福尔马林的灭活程序似乎会对病毒包膜蛋白产生不利影响。最近，我们用化合物2,2'-二硫代二吡啶（Aldrithiol-2，Aldrich，密尔沃基，威斯康星州）灭活了1型人类免疫缺陷病毒（HIV-1），该化合物通过共价修饰核衣壳锌指基序来灭活逆转录病毒的感染性。用Aldrithiol-2灭活的HIV-1保留了病毒膜上病毒及与病毒粒子相关的细胞蛋白的构象和功能完整性。我们将锌指靶向灭活的研究扩展到了猴免疫缺陷病毒（SIV），并评估了将该程序应用于大规模（>30升）生产和纯化灵长类免疫缺陷病毒的可行性。用1 mM Aldrithiol-2处理后，未检测到SIV有残留感染性（>5个对数级的灭活）。免疫印迹和高效液相色谱（HPLC）分析表明，用Aldrithiol-2处理导致处理后的病毒核衣壳蛋白发生广泛反应。正如预期的那样，病毒粒子gp120SU似乎与Aldrithiol-2完全无反应。蔗糖梯度纯化和浓缩程序导致病毒感染性或与病毒粒子相关的gp120SU损失很小。在gp120-CD4依赖性细胞结合试验中进行测试时，灭活病毒与未处理病毒一样能与细胞结合。对gp120-CD4介导的结合后融合事件的分析表明，灭活病毒能以与未处理病毒相似的效率诱导CD4依赖性融合。通过本文所述方法对灵长类免疫缺陷病毒进行灭活和处理，可得到高度浓缩的病毒制剂，其包膜蛋白保持天然构象。这些灭活病毒制剂可用于全灭活颗粒疫苗实验以及制备抗血清（包括单克隆抗体）的实验室试剂，并用于研究非感染性病毒粒子与细胞的相互作用。
