Name: humanized nod/scid/il2r&#947;null (hu-nsg) mouse model for hiv replication and latency studies
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This work was supported by the National Institutes of Health [grant numbers R01AI29329, R01AI42552 and R01HL07470 to J.J.R.] and National Cancer Institute of the National Institutes of Health [grant number P30CA033572 to support City of Hope Integrative Genomics, Analytical Pharmacology, and Analytical Cytometry Cores]. The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV BaL virus.

All animal care and procedures have been performed according to protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC) held by the principal investigator of this study (Dr. John Rossi, IACUC #12034). Human fetal liver tissue was obtained from Advanced Bioscience Resources (Alameda, CA), a nonprofit organization, in accordance with federal and state regulations. The vendor has its own Institutional Review Board (IRB) and is compliant with human subject protection requiremen...

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Immunocompromised mice engrafted with human cells/tissue present human-like physiological characteristics and are a tremendous value for pathology, pathophysiology, and immunology studies concerning human-specific diseases. Among multiple strains of immunocompromised mice, the NOD.Cg-PrkdcscidIl2rgtm1Wji (NSG) model is the most immunodeficient due to its lack of both innate and adaptive immunity, as well as ablated mouse-specific cytokine signaling3,<sup c...

Because establishing suitable animal models for human diseases is key to finding a cure, appropriate animals models have long been pursued and improved over time. Multiple strains of immunocompromised murine models have been developed that permit the engraftment of human cells and/or tissues and the subsequent execution of humanized functions1,2. Such humanized mouse models are critical for investigations of human-specific diseases3,4,5.
Acquired immune deficiency syndrome (AIDS) resulting from infection with human immunodeficiency virus (HIV) is one example. Prior to the establishment of humanized mouse models, ethical and technical limitations confined HIV/AIDS preclinical animal studies to non-human primates3. However, the high expenses and requirements for specialized care for such animal hinder HIV/AIDS studies in typical academic settings. HIV primarily infects human CD4+ T-cells and impacts the development and immune responses of other human immune cells such as B-cells, macrophages, and dendritic cells6; therefore, small animal models transplanted with functional human immune systems are in high demand.
A breakthrough came in 1988, when CB17-scid mice with a Prkdcscid mutation were developed and showed successful engraftment of the human immune system1. The Prkdcscid mutation results in defective T- and B-cell functions and an ablated adaptive immune system in mice, thereby enabling the engraftment of human peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), and fetal hematopoietic tissues7,8. Nonetheless, low levels of engraftment are frequently observed in this model; possible causes are 1) residual innate immune activity modulated via natural killer (NK)-cells and 2) the late-stage development of mouse T- and B-cells (leakiness)5. The subsequent development of the non-obese diabetic (NOD)-scid mouse model achieved dramatic down-regulation of NK-cell activity; thus, it is able to support a higher level and more sustainable engraftment of human immune system components9. To further suppress or impede development of innate immunity, mouse models bearing truncation or total knockout of the interleukin-2 receptor &#947;-chain (Il2rg) in the (NOD)-scid background were established. Il2rg, also known as common cytokine-receptor &#947;-chain, is an indispensable component of various cytokine receptors10,11,12,13. Strains such as NOD.Cg-PrkdcscidIl2rgtm1Wji (NSG) and NODShi.Cg-PrkdcscidIl2rgtm1Sug (NOG) present robust disruption of mouse cytokine signaling and complete ablation of NK-cell development, in addition to severe impairment of adaptive immunity14,15,16.
Three humanized mouse models bearing a scid mutation and Il2rg knockout are frequently employed in HIV/AIDS research: the BLT (Bone marrow/Liver/Thymus) model, the PBL (Peripheral Blood Leukocyte) model, and the SRC (SCID Repopulating Cell) model3. The BLT model is created via surgical transplantation of human fetal liver and thymus under the mouse kidney capsule accompanied with intravenous injection of fetal liver HSCs3,17,18. The BLT mouse model offers high engraftment efficacy, development of human hematopoietic cells in all lineages, and establishment of a strong human immune system; additionally, T-cells are educated in a human autologous thymus and exhibit HLA-restricted immune responses4,5,17,19. However, the requirement for surgical procedures remains the major drawback of the BLT model. The PBL mouse model is established by intravenous injection with human peripheral lymphoid cells. The PBL model offers convenience and yields successful T-cell engraftment, but its application is limited due to insufficient B-cell and myeloid cell engraftment, low engraftment levels overall, and the onset of severe graft-versus-host disease (GVHD)3,20. The SRC mouse model is established through injection of human HSCs into newborn or young adult SCID mice. It exhibits average engraftment efficiency above 25% (assessed as peripheral blood CD45 percentage) and supports the multiple-lineage development of injected HSCs and the elaboration of an innate human immune system. However, the limitation of the SRC model is that the T-cell response is mouse H2-restricted instead of human HLA-restricted14,21.
The SRC mouse model is considered a facile and reliable model for preclinical HIV/AIDS small animal studies, exemplified by the consistent engraftment of a human immune system and successful hematopoietic development. We previously reported the establishment of a NSG Hu-SRC-SCID (hu-NSG) mouse model and described its application in HIV replication and latency studies22,23,24. This hu-NSG mouse model exhibits high levels of bone marrow homing, susceptibility to HIV infection, and recapitulation of HIV infection and pathogenesis. Additionally, the hu-NSG mouse model responds appropriately to combinatorial antiretroviral therapy (cART) and recapitulates plasma viral rebound upon cART withdrawal, confirming the establishment of an HIV latency reservoir25,26,27. This HIV latency reservoir is further substantiated by the production of replication-competent HIV viruses ex vivo induced by human resting CD4+ T-cells isolated from infected and cART-treated hu-NSG mice.
Herein, we describe the detailed protocol for establishment of the hu-NSG mouse model from neonatal NSG mice, including procedures related to HIV infection and cART treatment for latency development. We expect this protocol to offer a new set of approaches in HIV animal studies regarding HIV virology, latency, and treatment.

<table>
	<tbody>
		<tr>
			<td>CD34 MicroBead Kit, human</td>
			<td>MiltenyiBiotec</td>
			<td>130-046-703</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS2</td>
			<td>Stemcell Technologies</td>
			<td>07932</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-PrkdcscidIl2rgtm1Wji </td>
			<td>The Jackson Laboratory</td>
			<td>005557</td>
			<td>Order breeders instead of experimental mice</td>
		</tr>
		<tr>
			<td>IsoFlo</td>
			<td>Patterson Veterinary</td>
			<td>07-806-3204</td>
			<td>Order through animal facility, restricted item</td>
		</tr>
		<tr>
			<td>Clidox disinfectant</td>
			<td>Fisher Sicentific</td>
			<td>NC9189926</td>
			<td></td>
		</tr>
		<tr>
			<td>Wescodyne</td>
			<td>Fisher Sicentific</td>
			<td>19-818-419</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 80508 syringe/needle</td>
			<td>Hamilton</td>
			<td>80508</td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Blood collection tube (K2EDTA)</td>
			<td>BD Bioscience</td>
			<td>367843</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tube (Heparin)</td>
			<td>BD Bioscience</td>
			<td>365965</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tube (Heparinized)</td>
			<td>Fisher Sicentific</td>
			<td>22-362574</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Sigma Aldrich</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA mini kit</td>
			<td>Qiagen</td>
			<td>52906</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast VIrus 1-step Master Mix</td>
			<td>Thermofisher</td>
			<td>4444434</td>
			<td></td>
		</tr>
		<tr>
			<td>HIV-1 P24 ELISA (5 Plate kit)</td>
			<td>PerkinElmer</td>
			<td>NEK050B001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG from human serum</td>
			<td>Sigma Aldrich</td>
			<td>I4506-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>IgG from mouse serum</td>
			<td>Sigma Aldrich</td>
			<td>I5381-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>BB515 Mouse Anti-Human CD45 (clone HI30)</td>
			<td>BD Biosciences</td>
			<td>564586</td>
			<td>RRID: AB_2732068, LOT 6347696</td>
		</tr>
		<tr>
			<td>PE-Cy7 Mouse Anti-Human CD3 (Clone SK7)</td>
			<td>BD Biosciences</td>
			<td>557851</td>
			<td>RRID: AB_396896, LOT 6021877</td>
		</tr>
		<tr>
			<td>Pacific Blue Mouse Anti-Human CD4 (Clone RPA-T4)</td>
			<td>BD Biosciences</td>
			<td>558116</td>
			<td>RRID: AB_397037, LOT 6224744</td>
		</tr>
		<tr>
			<td>BUV395 Mouse Anti-Human CD8 (Clone RPA-T8)</td>
			<td>BD Biosciences</td>
			<td>563795</td>
			<td>RRID: AB_2722501, LOT 6210668</td>
		</tr>
		<tr>
			<td>APC-Alexa Fluor 750 Mouse Anti-Human CD14 (TuK4)</td>
			<td>ThermoFisher</td>
			<td>MHCD1427</td>
			<td>RRID: AB_10373536, LOT 1684947A</td>
		</tr>
		<tr>
			<td>PE Mouse Anti-Human CD19 (SJ25-C1)</td>
			<td>ThermoFisher</td>
			<td>MHCD1904</td>
			<td>RRID: AB_10373382, LOT 1725304B</td>
		</tr>
	</tbody>
</table>

humanized nod/scid/il2r&#947;null (hu-nsg) mouse model for hiv replication and latency studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

Flow cytometry analysis is frequently performed to validate the purity of isolated HSCs, evaluate engraftment levels, profile immune responses to viral infection, and survey cART efficacy. A typical antibody panel contains 4-6 individual fluorescently labeled antibodies; thus, a flow cytometer with multiple lasers and a wide selection of filters is crucial for achieving accurate results.
For initial engraftment validation, the h...

Watch this Scientific Journal Video about Humanized NOD/SCID/IL2rÎ³null hu-NSG Mouse Model for HIV Replication and Latency Studies at JoVE.com

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

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.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

These matters can help answer key questions in HIV/AIDS research, such as the molecular mechanism and the prognosis of HIV infection, and how and where the latency reservoir is established. The advantage of this technique is that it is non-invasive, and established a hu-NSG mice that must treat a multi-lineage development over transplanted human stem cells that are susceptible to HIV infection and are receptive to cART. Begin by filtering a digested, human, fetal, liver tissue, slurry sample through a sterile, 70 micrometer, nylon, mesh strainer to obtain a single cell suspension, and enrich for CD34+HSCs by magnetic activated cell sorting according to the manufacturer's instructions.After counting, re-suspend the HSC at a two-times-ten-to-the-seventh viable cells per milliliter concentration in fresh DPBS on ice. Next place an entire neonatal human NOD/SCID/gamma or NSG mouse litter into a sterile, pie-shaped, irradiation cage, and treat the animals with 200 to 250 centigrades of gamma ray from a cesium-137 radiation source. Then load the cells into a custom-made, Hamilton 80508 50-microliter syringe equipped with a 30-gauge needle, and inject each neonatal animal directly into the liver with five-times-ten-to-the-fifth viable human CD34+HSCs and 25 microliters of DPBS.Ten-to-twelve weeks post infection, spin down retro-orbital, peripheral, blood samples from each mouse by centrifugation, and transfer the plasma supernates to microcentrifuge tubes for negative 80 degrees Celsius storage as experimentally appropriate. For HSC engraftment validation, re-suspend the pellets with 200 microliters of red blood cell lysis buffer, and pull the cell suspensions in a 1.5 millimeter microcentrifuge tube for a ten-minute incubation at room temperature. Then collect the remaining white blood cells by centrifugation for two washes in 0.5 milliliters of fresh, 0.01 percent bovine serum albumin, or BSA, in DPBS per wash.After the second wash, block the cells with 100 microliters of blocking cocktail for twenty minutes at 4 degrees Celsius followed by labeling with the appropriate anti-body cocktail of interest at a 2 microliters of anti-body per one-times-one-to-the-sixth cells concentration. After 30 minutes at 4 degrees Celsius, wash the cells two times in fresh DPBS plus BSA, and evaluate the peripheral blood samples by flow cytometry according to standard protocols to quantify the percentage of human CD45+CD3+CD14+or CD19+cells per sample. Then calculate the ratio of CD4+to-CD8+T cells, and assess the viral load with in the plasma samples according to standard, quantitative, reverse transcriptase, PCR protocols.To assess the effects of HIV infection, deliver HIV bale virus to anesthetized, human, NSG mice with a greater than 20 percent human CD45+cell population in their peripheral blood, at a 200 nanograms of P24 per mouse concentration via intraperitoneal administration, collecting blood samples via retro-orbital bleeding every two weeks, and analyze both the CD4-to-CD8 ratio and the plasma viral load, as demonstrated. For viral suppression validation supply the infected, human NSG animals with fresh, combinatorial, anti-retroviral therapy, or cART, in sweetened water every week for 4 weeks, collecting blood samples via retro-orbital bleeding every two weeks and analyze both the CD4-to-CD8 ratio and the plasma viral load as demonstrated. To asses the viral rebound when the plasma viral loads are below the detection limit and the CD4-to-CD8 ratio is restored between 1.5 and 2.5 collect peripheral blood samples through retro-orbital bleeding every 2 weeks after cART withdrawal and evaluate the samples as demonstrated.During the initial engraftment validation, the human CD45+cell count should range from 20 to 80 percent, and the subsets of human leukocytes should appear as discrete populations by flow cytometric analysis. The ratio of CD4+to-CD8+cells remains between 1.5 and 2.5 in healthy individuals while significant CD4+T cell depletion is typically observed upon viral infection. Notably, the healthy ratio is restored in response to cART treatment.The detection of plasma viral loads by quantitative RT PCR throughout the course of the infection and the cART regimen can be plotted and used to evaluate the efficiency of the viral delivery and the anti-viral treatment, respectively. Though this method can provide insight into HIV/AIDS virology studies and therapeutic development, it can also be applied to other studies related to human disease, such as cancer, diabetes, and auto-immune diseases. The hu-NSG mouse model development paved the way for researchers in immunology and oncology to explore a new dysfunction and pathology in humans, particularly mechanistic and therapeutic studies targeting HIV chronic infection and the latency reservoir.

Research

JoVE Journal

Immunology and Infection

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

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...