公开(公告)号：US20100216120A1

公开(公告)日：2010-08-26

申请号：US12711070

申请日：2010-02-23

申请人： Candace Pert ,  Michael Ruff ,  Olivier Ducoudret ,  Lokesh Agrawal ,  Noel Baichoo

发明人： Candace Pert ,  Michael Ruff ,  Olivier Ducoudret ,  Lokesh Agrawal ,  Noel Baichoo

IPC分类号： C12Q1/70

CPC分类号： C12Q1/703

摘要： An assay to detect or quantify HIV infectious virus from clinically relevant cellular compartments, or reservoirs, in anti-retrovirally treated patients whose viral levels are low to undetectable is described. The method detects infectious virus in patients whose plasma viral loads are considered to be below the limit of current PCR based detection methods and thereby is more relevant for guiding treatment. A further advantage is that the method allows viral tropism to be directly determined in the presence of specific inhibitors of CCR5 or CXCR4. Drug sensitivity can also be directly determined without the need to laboriously recover patient virus by culture for extended time periods, a method that allows for viral selection or evolution, which is not desirable. Patient cells, like the blood mononuclear cells, or monocytes, are isolated and cultured in the presence of cytokines like CSF-1/M-CSF or GM-CSF. to promote their differentiation. Cells are activated with lectins, mitogenic antibodies, phorbol esters, Toll Receptor stimulation or inducers of NfKb or NFAT, followed by agents that induce viral release, like ATP or stimulation of autophagy with LiCl, spermidine, or rapamycin. A key aspect of the invention relates to the timing of the addition of these agents for optimal viral release. A further aspect of the invention relates to sensitive detection of released virus which can be accomplished by adding so-called reporter cells which are under control of the HIV TAT protein so that upon infection these cells synthesize proteins or enzymes that allow for the measurement of infectious particles.

摘要翻译： 描述了在病毒水平低到不可检测的抗逆转录病毒治疗的患者中检测或定量来自临床相关细胞区室或储库的HIV感染性病毒的测定法。 该方法检测血浆病毒载量低于目前基于PCR检测方法极限的患者中的感染性病毒，从而更有助于指导治疗。 另一个优点是该方法允许在CCR5或CXCR4的特异性抑制剂存在下直接测定病毒向性。 药物敏感性也可以直接确定，而不需要通过培养长时间地费力地恢复患者病毒，这是允许病毒选择或进化的方法，这是不希望的。 在细胞因子如CSF-1 / M-CSF或GM-CSF的存在下分离和培养患者细胞，如血液单核细胞或单核细胞。 促进他们的分化。 细胞被凝集素，有丝分裂抗体，佛波酯，Toll受体刺激或NfKb或NFAT的诱导剂激活，随后是诱导病毒释放的药剂，如ATP或用LiCl，亚精胺或雷帕霉素刺激自噬。 本发明的关键方面涉及添加这些试剂以获得最佳病毒释放的时间。 本发明的另一方面涉及释放的病毒的灵敏检测，其可以通过加入在HIV TAT蛋白质控制下的所谓的报道细胞来实现，使得在感染后，这些细胞合成允许测量感染性的蛋白质或酶 粒子。