Name: Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection (Video) | JoVE Journal
Uploaded: 2020-01-31T15:00:00.000+00:00
Duration: 8 min 15 s
Description: Learn to establish a humanized NOG mouse model for preclinical HIV research using stem cell transplantation and intravaginal exposure. This protocol enables evaluation of viral strains and novel interventions.

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The authors would like to thank the Biomedicine Animal Facility staff at Aarhus University, particularly Ms. Jani K&#230;r for colony maintenance efforts and for tracking mouse weights. The authors would like to thank Professor Florian Klein for developing standard-of-care cART and for guidance. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pRHPA.c/2635 (cat# 11744) from Dr. John Kappes and Dr. Christina Ochsenbauer.

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</ol>

The authors declare no conflicts of interests.

The severely immunocompromised mouse strain NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) is extremely well suited for transplantation of human cells and tissues. Both innate and adaptive immune pathways in these mice are compromised. NOG and NSG mice harbor a Prkdcscid mutation that results in defective T and B cell function. Furthermore, these mice lack a functional interleukin-2 receptor &#947;-chain (common gamma chain, IL2rg) which is indispensable in ...

Human immunodeficiency virus (HIV) is a chronic infection with more than 37 million infected individuals worldwide1. Combination antiviral therapy (cART) is a life-saving therapy, but a cure is still warranted. Thus, there is a need for animal models that mirror the human immune system and its responses in order to facilitate continued research in HIV. Multiple types of humanized mice that are capable of supporting cell and tissue engraftment have been developed by transplanting human cells into severely immunodeficient mice2. Such humanized mice are susceptible to HIV infection and provide an important alternative to nonhuman primate simian immunodeficiency virus models, as they are cheaper and simpler to use than nonhuman primates. Humanized mice have facilitated research in HIV viral transmission, pathogenesis, prevention, and treatment3,4,5,6,7,8,9,10,11.
We present a flexible humanized model system for HIV research developed by transplanting cord blood derived human stem cells into mice of the NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) background. Besides being of non-fetal origin, the practical bioengineering of these mice is less technically demanding compared to the microsurgical procedures involved in the transplant of the blood-liver-thymus (BLT) construct.
We show how to establish HIV infection through intravaginal transmission and how to monitor the plasma viral load with a sensitive droplet digital PCR (ddPCR)-based setup. Subsequently, we describe the establishment of standard cART given as part of the daily mouse diet. The aim of these combined methods is to reduce stress to the animals and facilitate large-scale experiments where time spent handling each animal is limited12.
In humans, a CCR5&#916;32/wt or CCR5&#916;32/ &#916;32 genotype causes reduced susceptibility to HIV infection with transmitter/founder viruses13, and some precautions must be taken when bioengineering humanized mice with stem cells for the purpose of HIV studies. This is especially true in our region because naturally occurring variants in the CCR5 gene, particularly &#916;32 deletions, are more prevalent in Scandinavian and Baltic native populations compared to rest of the world14,15. Thus, our protocol includes an easy, high-throughput assay for screening donor hematopoietic stem cells for CCR5 variants prior to transplantation.
For the intravaginal exposure we chose the transmitter/founder R5 virus RHPA4259, isolated from a woman in an early stage of infection who was infected intravaginally16. We exposed the mice to a viral dose that was sufficient to yield successful transmission in the majority of mice, but below a 100% transmission rate. Choosing such a dose enables a sufficient dynamic range in transmission rate such that antiviral effects of a drug candidate can result in protected animals in HIV prevention experiments and decreased viral load for treatment studies.

<table><tbody><tr><td>Blue pad</td><td>VWR</td><td>56616-031</td><td>Should be sterilized prior to use</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A8022</td><td></td></tr><tr><td>CD19 (clone sj25c1) PE-Cy7</td><td>BD Bioscience</td><td>557835</td><td></td></tr><tr><td>CD3 (clone OKT3) FITC</td><td>Biolegend</td><td>317306</td><td></td></tr><tr><td>CD3 (clone SK7) BUV395</td><td>BD Bioscience</td><td>564001</td><td></td></tr><tr><td>CD34 (clone AC136) FITC</td><td>Miltenyi</td><td>130-113-740</td><td></td></tr><tr><td>CD4 (clone SK3) BUV 496</td><td>BD Bioscience</td><td>564652/51</td><td></td></tr><tr><td>CD45 (clone 2D1) APC</td><td>Biolegend</td><td>368511/12</td><td></td></tr><tr><td>CD8 (clone RPA-T8) BV421</td><td>BD Bioscience</td><td>562428</td><td></td></tr><tr><td>ddPCR Supermix for probes (no dUTP)</td><td>Bio-Rad</td><td>1863025</td><td></td></tr><tr><td>DMSO</td><td>Merck</td><td>10,02,95,21,000</td><td></td></tr><tr><td>DNAse</td><td>Sigma</td><td>D4263</td><td>For suspension buffer</td></tr><tr><td>dNTP mix</td><td>Life Technologies</td><td>R0192</td><td></td></tr><tr><td>Dulbeccos phosphate-buffered saline (PBS)</td><td>Biowest</td><td>L0615-500</td><td></td></tr><tr><td>EasySep Human Cord Blood CD34 Positive Selection Kit II</td><td>Stemcell</td><td>17896</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>FACS Lysing solution 10X</td><td>BD</td><td>349202</td><td>Dilute 1:10 in dH20 immediately before use</td></tr><tr><td>FACS tubes (Falcon 5 mL round-botton)</td><td>Falcon</td><td>352052</td><td></td></tr><tr><td>Fc Receptor blocking solution (Human Trustain FcX)</td><td>Biolegend</td><td>422302</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma</td><td>F8192-500</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare</td><td>17144002</td><td></td></tr><tr><td>Flowjo v.10</td><td></td><td></td><td></td></tr><tr><td>Gauze</td><td>Mesoft</td><td>157300</td><td>Should be sterilized prior to use</td></tr><tr><td>Heating lamp</td><td></td><td></td><td>Custom made</td></tr><tr><td>Hemacytometer (B&amp;uuml;rker-T&amp;uuml;rk)</td><td>VWR</td><td>DOWC1597418</td><td></td></tr><tr><td>Isoflurane gas</td><td>Orion Pharma</td><td>9658</td><td></td></tr><tr><td>LSR Fortessa X20 flow cytometer</td><td>BD</td><td></td><td></td></tr><tr><td>Microcentrifuge tubes, PCR-PT approved</td><td>Sarstedt</td><td>72692405</td><td></td></tr><tr><td>Mouse cART food</td><td>ssniff Spezialdi&amp;auml;ten GmbH</td><td>Custom made product</td><td></td></tr><tr><td>Mouse restrainer</td><td></td><td>Custom made product</td><td></td></tr><tr><td>Needle, Microlance 3, 30G &amp;frac12;&quot;</td><td>BD</td><td>304000</td><td></td></tr><tr><td>NOG mice NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac</td><td>Taconic</td><td>NOG-F</td><td></td></tr><tr><td>Nuclease-free water</td><td>VWR chemicals</td><td>436912C</td><td></td></tr><tr><td>Nucleospin 96 Virus DNA and RNA isolation kit</td><td>Macherey-Nagel</td><td>740691</td><td></td></tr><tr><td>PCR-approved microcentrifuge tubes</td><td>Sarstedt</td><td>72.692.405</td><td></td></tr><tr><td>Penicillin-Streptomycin solution 100X</td><td>Biowest</td><td>L0022-100</td><td></td></tr><tr><td>Phusion Hot Start II DNA polymerase</td><td>Life Technologies</td><td>F549S</td><td></td></tr><tr><td>Pipette tips, sterile, ART 20P Barrier</td><td>ThermoScientific</td><td>2149P</td><td></td></tr><tr><td>Proteinase K</td><td>NEB</td><td>100005398</td><td></td></tr><tr><td>QuantaSoft software</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>QX100 Droplet Generator</td><td>Bio-Rad</td><td>1886-3008</td><td></td></tr><tr><td>QX100 Droplet Reader</td><td>Bio-Rad</td><td>186-3003</td><td></td></tr><tr><td>RBC lysis solution</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>RNase-free DNAse size F + reaction buffer</td><td>Macherey-Nagel</td><td>740963</td><td></td></tr><tr><td>RNAseOUT Recombinant Ribonuclease inhibitor</td><td>ThermoScientific</td><td>10777-019</td><td></td></tr><tr><td>RPMI</td><td>Biowest</td><td>L0501-500</td><td>Dissolve in H20</td></tr><tr><td>Softject 1 mL syringe</td><td>Henke Sass Wolf</td><td>5010-200V0</td><td></td></tr><tr><td>Superscript III Reverse Transcriptase</td><td>ThermoFisher Scientific</td><td>18080044</td><td></td></tr><tr><td>Thermoshaker</td><td>VWR</td><td>89370-910</td><td></td></tr><tr><td>Trypane blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Ultrapure 0.5 EDTA, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575-020</td><td></td></tr><tr><td>Virkon S (virus disinfectant)</td><td>Dupont</td><td>7511</td><td></td></tr></tbody></table>

humanized nog mice for intravaginal hiv exposure and treatment of hiv infection

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

The gating strategy for the analysis of stem cell purity is depicted in Figure 1. Figure 1A–C shows the purified CD34+ population and Figure 1D–F the CD34- flow-through used to illustrate that the minimal amount of the CD34+ population is lost in the isolation process. The purity of isolated CD34+ stem cells was between 85%–95% with less than 1% T-cell contamination. Figure 1</s...

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

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JoVE Journal

Immunology and Infection

This video demonstrates an assay to evaluate the effectiveness of oral combined antiretroviral therapy (oral cART) in a human immunodeficiency virus-1 (HIV-1)-infected humanized mouse model. A combination of drugs targeting HIV-1, including nucleoside reverse transcriptase inhibitors and integrase strand transfer inhibitors, is administered along with a nutritional supplement and antibiotics. Following the consumption of this mixture by the humanized mice infected with HIV-1, the effective...

Oral Combinational Antiretroviral Therapy to Treat HIV-1 Infection in a Humanized Mouse Model

The video demonstrates a technique to generate a humanized mouse model with stem-cell-based engineered immunity against the human immunodeficiency virus (HIV). Human fetal thymus tissue and engineered hematopoietic stem cells (HSCs) are implanted into the kidney capsule of a severely immunocompromised mouse. The HSCs, which express chimeric antigen receptors (CARs) against HIV, are mixed in a gelatinous protein mixture that promotes their co-localization with thymus tissue. Post-procedure, the...

Constructing a Humanized Mouse Model with Engineered Immunity against HIV

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

This protocol describes how the combination of EcoHIV infection with Tmem119-EGFP mice offers a valuable biological system for investigating microglial alterations and viral reservoirs in rodent models of HIV-associated neurocognitive disorders.

Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Learn to establish a humanized NOG mouse model for preclinical HIV research using stem cell transplantation and intravaginal exposure. This protocol enables evaluation of viral strains and novel interventions.

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

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