口服联合抗逆转录病毒治疗HIV-1感染人源化小鼠
Summary
该协议描述了一种提供口服联合抗逆转录病毒药物的新方法，该方法成功抑制人源化小鼠中的HIV-1 RNA复制。
Abstract
人类免疫缺陷病毒（HIV-1）大流行继续在世界范围内有增无减地蔓延，目前还没有针对艾滋病毒的疫苗。虽然联合抗逆转录病毒疗法（cART）成功地抑制了病毒复制，但它不能完全根除艾滋病毒感染者的宿主。安全有效的HIV感染治愈策略需要多管齐下的方法，因此HIV-1感染动物模型的进步对于HIV治愈研究的发展至关重要。人源化小鼠概括了HIV-1感染的关键特征。人源化小鼠模型可以被HIV-1感染，并且可以通过cART方案控制病毒复制。此外，cART中断导致人源化小鼠的病毒迅速反弹。然而，对动物进行cART给药可能无效、困难或有毒，并且许多临床相关的cART方案无法得到最佳利用。除了对研究人员来说可能不安全之外，通过常用的强化日常注射程序施用cART还会通过动物的身体约束来引起压力。本文中描述的治疗HIV-1感染人源化小鼠的新型口服cART方法导致病毒血症抑制低于检测水平，增加CD4 +恢复率，并改善HIV-1感染人源化小鼠的整体健康状况。
Introduction
慢性人类免疫缺陷病毒（HIV）感染者的预期寿命通过联合抗逆转录病毒治疗（cART）显著提高1，2。cART成功地减少了大多数HIV-1慢性感染参与者的HIV-1复制并将CD4 + T细胞计数提高到正常水平3，从而改善了整体健康状况并显着减少了疾病进展4。然而，即使在急性感染期间开始 ART 时，潜伏的 HIV-1 储库也会建立5，6，7。在 ART 期间，储库持续多年，并且 ART 中断后病毒快速反弹是有据可查的8，9。接受抗逆转录病毒治疗的艾滋病毒感染者也容易患心血管疾病、癌症和神经系统疾病等合并症的风险更高10，11，12。因此，需要一种功能性的HIV治疗方法。HIV-1感染的动物模型在开发和验证新的HIV治疗策略方面具有明显的优势13，14，15。人源化小鼠作为一种小动物模型，可以在不同组织中提供多谱系人免疫细胞重建，从而可以密切研究HIV感染16，17，18，19。在人源化模型中，人源化骨髓-肝脏-胸腺（BLT）模型成功地概括了慢性HIV-1感染以及人类对HIV-1感染的功能性免疫反应20，21，22，23，24。因此，人源化BLT小鼠模型已被广泛用于HIV研究领域的各个方面的研究。人源化BLT小鼠不仅是重现持续HIV-1感染和发病机制的成熟模型，也是评估基于细胞疗法的干预策略的重要工具。目前的作者和其他人已经证明，人源化BLT小鼠模型概括了持续的HIV-1感染和发病机制25，26，27，并提供了评估基于细胞疗法的干预策略的工具28，29，30，31，32，33。
cART方案由每天服用的抗逆转录病毒药物组合组成，可抑制HIV-1复制，以至于成功治疗的个体的病毒载量在长期内仍然无法检测到34。用临床相关的cART方案治疗HIV感染的人源化小鼠的结果类似于在HIV-1感染的ART治疗个体中观察到的结果22：HIV-1水平被抑制在检测限以下，并且cART的中断导致HIV复制从潜伏库反弹35。皮下注射（SC）27，36，37或腹膜内（IP）37，38，39是人源化小鼠cART治疗的常用途径。然而，每天密集的注射会通过身体约束给动物带来压力40。它也是劳动密集型的，并且由于在使用锐器时暴露于艾滋病毒而增加，因此对研究人员来说可能是不安全的。口服给药是模仿HIV-1感染者服用的cART药物的吸收、分布和排泄的理想选择。口服给药通常涉及定制且通常费力的程序，将抗逆转录病毒药物放入灭菌（由于小鼠的免疫缺陷而必需）食物24，37，41或水42，43，44，45，46中，这可能与许多抗逆转录病毒药物在化学上相容，也可能不相容，或者导致小鼠不容易吃或喝的东西（这会影响体内的剂量和药物水平）。这里提出的新型经口cART给药方法超越了以前的给药尝试，因为它与不同类型的抗逆转录病毒药物相容，安全性和易于制备和给药，以及减少每日注射引起的动物压力和焦虑。
富马酸替诺福韦二吡呋酯 （TDF）、艾维特拉韦 （ELV） 和拉替拉韦 （RAL） 是水溶性较差的药物。有趣的是，在高脂肪食物中观察到TDF的生物利用度增加，这表明脂肪食物对脂肪酶的竞争性抑制可能为TDF47提供一定的保护。因此，选择DietGel Boost杯代替普通啮齿动物食物作为交付方法，基于其适度的脂肪含量（每100克20.3克），与普通啮齿动物食物（每100克10克10克）和典型的小鼠高脂肪饮食（每100克40-60克）相比48。一杯的总重量为75克;因此，每个杯子将包含足够五只小鼠在3天内食用的食物量，因此也包含足够的药物。
Protocol
匿名的人类胎儿组织是通过商业获得的。动物研究是根据加州大学洛杉矶分校和（加州大学洛杉矶分校）动物研究委员会（ARC）根据所有联邦，州和地方指南批准的协议进行的。具体而言，所有实验均按照美国国立卫生研究院（NIH）和国际实验动物护理评估和认证协会（AALAC）关于实验动物住房和护理的建议和指南进行，根据加州大学洛杉矶分校ARC协议编号2010-038-02B。所有手术均在氯胺酮（100mg / …
Representative Results
假设一只体重为25克的平均小鼠每天消耗4克食物，则通过口服摄入的每日药物剂量对应于2.88mg / kg TFV，83mg / kg FTC和768mg / kg RAL。为了测试与每日注射cART相比，优化的食物方案是否有毒并影响整体健康，在cART之前和期间通过口服或皮下注射每周监测小鼠体重。每组在cART给药前没有显着的体重差异（图1）。然而，在每日cART SC注射期间，小鼠重量不断下降。相比之下，DietGel中?…
Discussion
本文开发了一种口服cART给药方法，用于HIV-1感染的人源化小鼠，方法是在高营养食物中结合三种抗逆转录病毒药物。与每日注射给药相比，口服给药更易于使用，限制给药频率，减少动物处理，最大限度地减少压力并提高安全性55。到目前为止，只有少数针对人源化小鼠24，37，41的研究使用含有粉碎ART药物的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Romas Geleziunas博士和Jeff Murry博士以及吉利德的人们提供本研究中使用的抗逆转录病毒药物。这项工作由NCI 1R01CA239261-01（到厨房），NIH资助P30AI28697（加州大学洛杉矶分校CFAR病毒学核心，基因和细胞治疗核心，以及人源化小鼠核心），U19AI149504（PIs：Kitchen&Chen），CIRM DISC2-10748，NIDA R01DA-52841（到Zhen），NIAID R2120200174（PIs：Xie&Zhen），IRACDA K12 GM106996（Carrillo）。这项工作还得到了加州大学洛杉矶分校艾滋病研究所，詹姆斯·B·彭德尔顿慈善信托基金和麦卡锡家庭基金会的支持。
Materials
| 60 mm petri dish | Thermo Scientific Nunc | 150288 | For aliquoting ART food |
| APC anti-human CD8 Antibody | Biolegend | 344722 | For flow cytometry |
| BD LSRFortessa | BD biosciences | For flow data collection | |
| CD34 microbeads | Miltenyi Biotec | 130-046-702 | For NSG-BLT mice generation |
| Centrifuge tubes | Falcon | 14-432-22 | For dissolving ART |
| DietGel Boost | ClearH2O | 72-04-5022 | For making ART food |
| Elvitegravir | Gilead | Gifted from Gilead | |
| Emtricitabine | Gilead | Gifted from Gilead | |
| FITC anti-human CD3 Antibody | Biolegend | 317306 | For flow cytometry |
| Flowjo software | FlowJo | For flow cytometry data analysis | |
| HIV-1 forward primer: 5′-CAATGGCAGCAATTTCACCA-3′; | IDT | Customized | For viral load RT-PCR |
| HIV-1 probe: 5′-[6-FAM]CCCACCAACAGGCGGCCT TAACTG [Tamra-Q]-3′; |
IDT | Customized | For viral load RT-PCR |
| HIV-1 reverse primer: 5′-GAATGCCAAATTCCTGCTTGA-3′; | IDT | Customized | For viral load RT-PCR |
| Human fetal tissue | Advanced Bioscience Resources, Inc | ||
| Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ | The Jackson Laboratory | 5557 | For constructing the humanized mice |
| Pacific Blue anti-human CD45 | Biolegend | 304022 | For flow cytometry |
| PerCP anti-human CD4 Antibody | Biolegend | 300528 | For flow cytometry |
| QIAamp Viral RNA Kits | Qiagen | 52904 | For measuring viral load |
| Raltegravir | Merck | Gifted from Merck | |
| Sterile cell scrapers | Thermo Scientific | 179693 | For aliquoting ART food |
| TaqMan RNA-To-Ct 1-Step Kit | Applied Biosystems | 4392653 | For plasma viral load detection |
| Tenofovir disoproxil fumarate | Gilead | Gifted from Gilead | |
| Trimethoprim-Sulfamethoxazole | Pharmaceutical Associates | NDC 0121-0854-16 | For keeping ART food sterile. Each 5mL teaspoon contains 200 mg Sulfamethoxazole, USP 40 mg Trimethoprim, USP NMT 0.5% Alcohol |
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Cite This Article
Mu, W., Zhen, A., Carrillo, M. A., Rezek, V., Martin, H., Lizarraga, M., Kitchen, S. Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice. J. Vis. Exp. (188), e63696, doi:10.3791/63696 (2022).