用于阴道内艾滋病毒暴露和艾滋病毒感染的人性化 NOG 小鼠
Summary
我们开发了一种基于干细胞移植、阴道内人体免疫缺陷病毒暴露和滴数字PCR RNA的人性化和人体免疫缺陷病毒感染NOG小鼠模型的生成和评估方案量化。
Abstract
人化小鼠为研究人类免疫缺陷病毒（HIV）病毒学和测试抗病毒药物提供了一个复杂的平台。该协议描述了在成年NOG小鼠中建立人体免疫系统。在这里，我们解释从分离脐带血衍生人类CD34+细胞及其随后静脉移植到小鼠，到通过艾滋病毒感染操作模型，联合抗逆转录病毒疗法的所有实际步骤（cART）和血液取样。约75，000 hCD34+细胞静脉注射到小鼠和人类血化水平，也称为人化，在外周血液纵向估计数月的流动细胞测定。共有75，000个hCD34+细胞在外周血中产生20%~50%的人类CD45+细胞。小鼠易感染阴道内艾滋病毒，血液可每周取样一次进行分析，并每月两次，持续一次。该协议描述了使用滴数字PCR（ddPCR）定量血浆病毒载量的测定。我们展示了如何在饮食中使用标准的护理CART方案来有效地治疗小鼠。以常规小鼠小周的形式交付cART是实验模型的一个重大改进。该模型可用于全身和局部暴露前预防化合物的临床前分析，以及检测新疗法和艾滋病毒治疗策略。
Introduction
人体免疫机能丧失病毒（HIV）是一种慢性感染，全世界有3700多万感染者。联合抗病毒疗法（cART）是一种拯救生命的疗法，但治愈仍有必要。因此，需要动物模型来反映人类免疫系统及其反应，以便促进艾滋病毒的持续研究。通过将人类细胞移植到严重免疫缺陷的小鼠2中，已经开发出多种能够支持细胞和组织移植的人性化小鼠。这种人性化的小鼠容易感染艾滋病毒，为非人类灵长类动物模拟免疫缺陷病毒模型提供了重要的替代品，因为它们比非人类灵长类动物更便宜、更易于使用。人化小鼠促进了HIV病毒传播、发病机制、预防和治疗3、4、5、6、7、8、9、10、11的研究。
我们提出了一个灵活的人性化模型系统，通过移植脐带血衍生的人类干细胞到NOD小鼠中，为HIV研究开发。Cg-PrkdcscidIl2rgtm1Sug/JicTac （NOG） 背景。除了非胎儿来源外，这些小鼠的实际生物工程在技术上比移植血-肝-胸腺（BLT）结构的显微手术要求低。
我们展示了如何通过阴道内传播建立HIV感染，以及如何监测血浆病毒载量与敏感滴数字PCR（ddPCR）为基础的设置。随后，我们描述了作为日常小鼠饮食的一部分而给出的标准cART的建立。这些组合方法的目的是减轻动物的压力，促进大规模实验，其中处理每只动物的时间是有限的12。
在人类中，CCR5[32/wt]或CCR5+32/ +32基因型导致 HIV 感染传播者/创始人病毒13的易感性降低，在生物工程将具有干细胞的小鼠用于HIV研究时，必须采取一些预防措施。这在我们地区尤其如此，因为CCR5基因中自然发生的变异，特别是#32缺失，在斯堪的纳维亚和波罗的海本地种群中比世界其他地区更普遍14，15。因此，我们的方案包括一种简单、高通量的测定，用于在移植前筛选供体造血干细胞的CCR5变异。
对于阴道内暴露，我们选择了发射器/创始人R5病毒RHPA4259，从感染早期被感染的妇女分离出来，她被感染在阴道内16。我们让小鼠接触的病毒剂量足以使大多数小鼠成功传播，但传播率低于100%。选择这种剂量可以使传播速率有足够的动态范围，使候选药物的抗病毒效应可以在艾滋病毒预防实验中导致受保护动物，并减少治疗研究的病毒载量。
Protocol
所有脐带血样本均严格按照当地批准的协议进行，包括父母匿名献血的知情同意。根据许可证 2017-15-0201-01312，所有动物实验均严格按照丹麦国家法规进行。 注意：小心处理暴露的艾滋病毒小鼠和血液。用经证实的HIV消毒剂（材料表）清除所有与艾滋病毒接触的表面和液体。 1. 人类CD34+干细胞的分离 在计划剖腹产或阴道分娩后，在EDTA?…
Representative Results
图1描述了干细胞纯度分析的浇注策略。图 1A_C显示了纯化 CD34+ 总体，图 1D_F CD34- 流通用于说明在隔离过程中丢失的 CD34+ 总体的最小数量。分离的CD34+干细胞纯度在85%~95%之间，T细胞污染率低于1%。图1G描绘了一个成人控制捐赠者的CCR5带，其基因型为CCR5+32/wt，其次是两个<…
Discussion
严重免疫功能低下的小鼠菌株NOD。Cg-PrkdcscidIl2rgtm1Sug/JicTac （NOG） 非常适合人体细胞和组织移植。这些小鼠的先天和适应性免疫途径都受到损害。NOG和NSG小鼠有Prkdcscid突变，导致有缺陷的T和B细胞功能。此外，这些小鼠缺乏功能白细胞介素-2受体β链（常见的伽马链，IL2rg），这是许多关键细胞因子（如IL-2、IL-4、IL-7、IL-9、IL-15和IL-21）结合复合物?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢奥胡斯大学生物医药动物设施的工作人员，特别是Jani Kür女士的殖民地维护工作和跟踪老鼠重量。作者要感谢弗洛里安·克莱因教授制定护理标准CART和指导。以下试剂是通过NIH艾滋病试剂项目，艾滋病司，NIAID，NIH：pRHPA.c/2635（cat# 11744）从约翰·卡佩斯博士和克里斯蒂娜·奥奇森鲍尔博士那里获得。
Materials
| Blue pad | VWR | 56616-031 | Should be sterilized prior to use |
| Bovine serum albumin (BSA) | Sigma | A8022 | |
| CD19 (clone sj25c1) PE-Cy7 | BD Bioscience | 557835 | |
| CD3 (clone OKT3) FITC | Biolegend | 317306 | |
| CD3 (clone SK7) BUV395 | BD Bioscience | 564001 | |
| CD34 (clone AC136) FITC | Miltenyi | 130-113-740 | |
| CD4 (clone SK3) BUV 496 | BD Bioscience | 564652/51 | |
| CD45 (clone 2D1) APC | Biolegend | 368511/12 | |
| CD8 (clone RPA-T8) BV421 | BD Bioscience | 562428 | |
| ddPCR Supermix for probes (no dUTP) | Bio-Rad | 1863025 | |
| DMSO | Merck | 10,02,95,21,000 | |
| DNAse | Sigma | D4263 | For suspension buffer |
| dNTP mix | Life Technologies | R0192 | |
| Dulbeccos phosphate-buffered saline (PBS) | Biowest | L0615-500 | |
| EasySep Human Cord Blood CD34 Positive Selection Kit II | Stemcell | 17896 | |
| EDTA | Invitrogen | 15575-038 | |
| FACS Lysing solution 10X | BD | 349202 | Dilute 1:10 in dH20 immediately before use |
| FACS tubes (Falcon 5 mL round-botton) | Falcon | 352052 | |
| Fc Receptor blocking solution (Human Trustain FcX) | Biolegend | 422302 | |
| Fetal bovine serum | Sigma | F8192-500 | |
| Ficoll-Paque PLUS | GE Healthcare | 17144002 | |
| Flowjo v.10 | |||
| Gauze | Mesoft | 157300 | Should be sterilized prior to use |
| Heating lamp | Custom made | ||
| Hemacytometer (Bürker-Türk) | VWR | DOWC1597418 | |
| Isoflurane gas | Orion Pharma | 9658 | |
| LSR Fortessa X20 flow cytometer | BD | ||
| Microcentrifuge tubes, PCR-PT approved | Sarstedt | 72692405 | |
| Mouse cART food | ssniff Spezialdiäten GmbH | Custom made product | |
| Mouse restrainer | Custom made product | ||
| Needle, Microlance 3, 30G ½" | BD | 304000 | |
| NOG mice NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac | Taconic | NOG-F | |
| Nuclease-free water | VWR chemicals | 436912C | |
| Nucleospin 96 Virus DNA and RNA isolation kit | Macherey-Nagel | 740691 | |
| PCR-approved microcentrifuge tubes | Sarstedt | 72.692.405 | |
| Penicillin-Streptomycin solution 100X | Biowest | L0022-100 | |
| Phusion Hot Start II DNA polymerase | Life Technologies | F549S | |
| Pipette tips, sterile, ART 20P Barrier | ThermoScientific | 2149P | |
| Proteinase K | NEB | 100005398 | |
| QuantaSoft software | Bio-Rad | ||
| QX100 Droplet Generator | Bio-Rad | 1886-3008 | |
| QX100 Droplet Reader | Bio-Rad | 186-3003 | |
| RBC lysis solution | Biolegend | 420301 | |
| RNase-free DNAse size F + reaction buffer | Macherey-Nagel | 740963 | |
| RNAseOUT Recombinant Ribonuclease inhibitor | ThermoScientific | 10777-019 | |
| RPMI | Biowest | L0501-500 | Dissolve in H20 |
| Softject 1 mL syringe | Henke Sass Wolf | 5010-200V0 | |
| Superscript III Reverse Transcriptase | ThermoFisher Scientific | 18080044 | |
| Thermoshaker | VWR | 89370-910 | |
| Trypane blue | Sigma | T8154 | |
| Ultrapure 0.5 EDTA, pH 8.0 | ThermoFisher Scientific | 15575-020 | |
| Virkon S (virus disinfectant) | Dupont | 7511 |
Referências
- Skelton, J. K., Ortega-Prieto, A. M., Dorner, M. A Hitchhiker’s guide to humanized mice: new pathways to studying viral infections. Immunology. 154 (1), 50-61 (2018).
- Denton, P. W., Krisko, J. F., Powell, D. A., Mathias, M., Kwak, Y. T. Systemic Administration of Antiretrovirals Prior to Exposure Prevents Rectal and Intravenous HIV-1 Transmission in Humanized BLT Mice. PLoS ONE. 5 (1), 8829 (2010).
- Zou, W., et al. Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4 + CD8 + thymocytes. Retrovirology. 9 (1), 44 (2012).
- Berges, B. K., Akkina, S. R., Folkvord, J. M., Connick, E., Akkina, R. Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2 -/- γc -/- (RAG-hu) mice. Virology. 373 (2), 342-351 (2008).
- Veselinovic, M., Charlins, P., Akkina, R. Modeling HIV-1 Mucosal Transmission and Prevention in Humanized Mice. Methods Mol Biol. , 203-220 (2016).
- Neff, C. P., Kurisu, T., Ndolo, T., Fox, K., Akkina, R. A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice. PLoS ONE. 6 (6), 20209 (2011).
- Neff, P. C., Ndolo, T., Tandon, A., Habu, Y., Akkina, R. Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model. PLoS ONE. 5 (12), 15257 (2010).
- Veselinovic, M., et al. HIV pre-exposure prophylaxis: Mucosal tissue drug distribution of RT inhibitor Tenofovir and entry inhibitor Maraviroc in a humanized mouse model. Virology. 464-465, 253-263 (2014).
- Akkina, R., et al. Humanized Rag1-/-γc-/- mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes. PLoS ONE. 6 (6), 20169 (2011).
- Zhou, J., et al. Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice. Molecular Therapy. 19 (12), 2228-2238 (2011).
- Balcombe, J. P., Barnard, N. D., Sandusky, C. Laboratory routines cause animal stress. Contemporary Topics in Laboratory Animal Science. 43 (6), 42-51 (2004).
- Trecarichi, E. M., et al. Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals. AIDS Research and Therapy. 3 (1), (2006).
- Novembre, J., Galvani, A. P., Slatkin, M. The geographic spread of the CCR5 Δ32 HIV-resistance allele. PLoS Biology. 3 (11), 1954-1962 (2005).
- Solloch, U. V., et al. Frequencies of gene variant CCR5-Δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national DKMS donor centers. Human Immunology. 78 (11-12), 710-717 (2017).
- Ochsenbauer, C., et al. Generation of Transmitted/Founder HIV-1 Infectious Molecular Clones and Characterization of Their Replication Capacity in CD4 T Lymphocytes and Monocyte-Derived Macrophages. Journal of Virology. 86 (5), 2715-2728 (2012).
- Andersen, A. H. F., et al. Long-Acting, Potent Delivery of Combination Antiretroviral Therapy. ACS Macro Letters. 7 (5), 587-591 (2018).
- Caro, A. C., Hankenson, F. C., Marx, J. O. Comparison of thermoregulatory devices used during anesthesia of C57BL/6 mice and correlations between body temperature and physiologic parameters. Journal of the American Association for Laboratory Animal Science JAALAS. 52 (5), 577-583 (2013).
- Gatlin, J., Padgett, A., Melkus, M. W., Kelly, P. F., Garcia, J. V. Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector. Human Gene Therapy. 12 (9), 1079-1089 (2001).
- Leth, S., et al. HIV-1 transcriptional activity during frequent longitudinal sampling in aviremic patients on antiretroviral therapy. AIDS. 30 (5), 713-721 (2016).
- Halper-Stromberg, A., et al. Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice. Cell. 158 (5), 989-999 (2014).
- Rothenberger, M. K., et al. Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption. Proceedings of the National Academy of Sciences. 112 (10), 1126-1134 (2015).
- Rongvaux, A., et al. Human Hemato-Lymphoid System Mice: Current Use and Future Potential for Medicine. Annual Review of Immunology. 31 (1), 635-674 (2013).
- Walsh, N. C., et al. Humanized Mouse Models of Clinical Disease. Annual Review of Pathology: Mechanisms of Disease. 12 (1), 187-215 (2017).
- Denton, P. W., García, J. V. Humanized mouse models of HIV infection. AIDS Reviews. 13 (3), 135-148 (2011).
- Denton, P. W., Søgaard, O. S., Tolstrup, M. Using animal models to overcome temporal, spatial and combinatorial challenges in HIV persistence research. Journal of Translational Medicine. 14 (1), (2016).
- Andersen, A. H. F., et al. cAIMP administration in humanized mice induces a chimerization-level-dependent STING response. Immunology. 157 (2), 163-172 (2019).
- Tanaka, S., et al. Development of Mature and Functional Human Myeloid Subsets in Hematopoietic Stem Cell-Engrafted NOD/SCID/IL2r KO Mice. The Journal of Immunology. 188 (12), 6145-6155 (2012).
- Quan, P. L., Sauzade, M., Brouzes, E. DPCR: A technology review. Sensors (Switzerland). 18 (4), (2018).
- Denton, P. W., et al. Generation of HIV Latency in Humanized BLT Mice. Journal of Virology. 86 (1), 630-634 (2012).
- Li, Y., et al. A human immune system mouse model with robust lymph node development. Nature Methods. 15 (8), 623-630 (2018).
- Satheesan, S., et al. HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy. Journal of Virology. 92 (7), 02118 (2018).
- Bachmanov, A. A., Reed, D. R., Beauchamp, G. K., Tordoff, M. G. Food intake, water intake, and drinking spout side preference of 28 mouse strains. Behavior Genetics. 32 (6), 435-443 (2002).
- Shultz, L. D., et al. Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r null humanized mice. Proceedings of the National Academy of Sciences. 107 (29), 13022-13027 (2010).
- Willinger, T., et al. Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung. Proceedings of the National Academy of Sciences. 108 (6), 2390-2395 (2011).
- Hanazawa, A., et al. Generation of human immunosuppressive myeloid cell populations in human interleukin-6 transgenic NOG mice. Frontiers in Immunology. 9, (2018).
- Huntington, N. D., et al. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. The Journal of Experimental Medicine. 206 (1), 25-34 (2009).
- Rongvaux, A., et al. Development and function of human innate immune cells in a humanized mouse model. Nature Biotechnology. 32 (4), 364-372 (2014).
Citar este artigo
Andersen, A. H. F., Nielsen, S. S. F., Olesen, R., Mack, K., Dagnæs-Hansen, F., Uldbjerg, N., Østergaard, L., Søgaard, O. S., Denton, P. W., Tolstrup, M. Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection. J. Vis. Exp. (155), e60723, doi:10.3791/60723 (2020).