HIV-1感染小鼠动物模型建立及体内整合前病毒定量分析

Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo

摘要:

近年来，多种新型抗人类免疫缺陷病毒(human immunodeficiency virus, HIV)疗法如基因治疗、广谱中和抗体以及衍生的嵌合抗原受体T细胞(chimeric antigen receptor modified T cell, CAR-T)疗法均迫切需要理想的动物模型及用于精准定量病毒基因组的方法。本研究通过双标HIV假病毒构建及感染获得HIV- ∆ENV-Jurkat-EGFP-mCherry稳转细胞，并以该细胞基因组作为整合前病毒的标准品，建立巢式荧光定量PCR (nested quantitative polymerase chain reaction, nested-qPCR)定量HIV整合前病毒。通过尾静脉注射健康人外周血单个核细胞(peripheral blood mononuclear cell, PBMC)到NOD/Prkdcscid/IL2rgnull (NPG)小鼠，通过监测小鼠外周血中hCD45+、hCD3+、hCD4+和hCD8+人源化细胞的比例来判断人源化小鼠模型构建是否成功。腹腔注射HIV NL4-3-NanoLuc病毒，随后通过小动物活体成像及分子病毒学评价HIV体内复制，结果表明，尾静脉注射正常人PBMC细胞至小鼠内3−5周后，实验组小鼠体内均检测到人源免疫细胞的浸润，建模5周后的人源化小鼠外周血中hCD45大于25%，即人源化小鼠模型构建成功。感染27 d后小动物活体成像能检测到病毒相关萤光素酶蛋白表达，分子病毒学结果表明在脾脏中病毒总DNA、RNA和整合前病毒DNA分别达到了18 000 copies/10⁶ cells、15 000 copies/μg RNA、15 000 copies/10⁶ cell。本研究证明了通过尾静脉注射正常人PBMC，可成功建立HuPBMC-NPG/严重联合免疫缺陷(severe combined immunodeficiency, SCID)人源化小鼠模型，并且HIV病毒可成功感染该模型。本研究建立了有效测定HIV整合前病毒DNA的方法，为艾滋病体内复制水平及病毒库大小和对多种新型抗HIV疗法的治疗效果的评价奠定了基础。

关键词:人源化小鼠;人类免疫缺陷病毒(HIV);NPG免疫缺陷小鼠;外周血单个核细胞(PBMC)

Abstract:

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3–5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/10⁶ cells, 15 000 copies/μg RNA, and 15 000 copies/10⁶ cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

Key words:humanized mice;human immunodeficiency virus (HIV);NOD/Prkdcscid/IL2rgnull (NPG) mice;peripheral blood mononuclear cell (PBMC)