We would like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study. This work was funded by NCI 1R01CA239261-01 (to Kitchen), NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504 (PIs: Kitchen &#38; Chen), CIRM DISC2-10748, NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie &#38; Zhen), IRACDA K12 GM106996 (Carrillo). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation.

Anonymized human fetal tissue was acquired commercially. Animal research was carried out according to protocols approved by the University of California, Los Angeles, and (UCLA) Animal Research Committee (ARC) in accordance with all federal, state, and local guidelines. Specifically, all the experiments were carried out in accordance with the recommendations and guidelines for housing and care of laboratory animals of the National Institutes of Health (NIH) and the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International under UCLA ARC Protocol Number 2010-038-02B. All surgeries were performed under ketamine (100 mg/kg)/xylazine (5 mg/kg) and isoflurane anesthesia (2-3 vol%) and all efforts were made to minimize animal pain and discomfort.
1. Humanized mice infected with HIV-1
NOTE: Humanized mice were constructed as previously described in30,31,49. The protocol is briefly described below.
<ol>
	<li>Purify CD34+ hematopoietic progenitor cells from the human fetal liver via anti-CD34 microbeads according to the manufacturer&#39;s protocol.</li>
	<li>Anesthetize 6-8-week-old NOD/SCID/IL2R&#947;&#8722;/&#8722; (NSG) male and female mice and sub-lethally irradiate (2.7 Gy) before the surgery.</li>
	<li>Implant thymus, derived from the same donor as the fetal liver, under the kidney capsule along with the liver.</li>
	<li>Following implantation, inject mice with 0.5 million to one million CD34+ cells, intravenously.</li>
	<li>After 8-10 weeks, collect 100 &#181;L of mouse blood via retro-orbital bleed50 into microcentrifuge tubes containing 5 &#181;L of EDTA and centrifuge at 350 x g for 3 min.</li>
	<li>Store the plasma at -80 &#176;C to monitor viral load after the mouse has been infected with HIV-1. Add 2 mL of 83% NH4C solution and incubate for 5 min at room temperature to lyse red blood cells.</li>
	<li>Add 10 mL of RPMI with 10% Fetal Bovine Serum (FBS) to stop lysis. Spin at 300 x g for 5 min.</li>
	<li>Aspirate supernatant. Stain cells with antibody panel (see Table of Materials) and analyze by flow cytometry to check human immune cell engraftment.</li>
	<li>Infect mice exhibiting more than 50% of circulating CD45+ cells by retro-orbital vein injection51,52 with at least 200 ng of p24 of an HIV-1 strain (i.e., NFNSXSL930,53,54) using an insulin syringe. Collect blood biweekly for flow cytometry analysis and to measure the viral load.</li>
</ol>
2. Preparation of ART drugs
<ol>
	<li>Weigh individual drugs; for example, to make 10 food cups with cART, use sterile cell scrapers to weigh out 250 mg of FTC (Emtricitabine), 375 mg of TDF, and 500 mg of RAL or ELV into individual sterile 15 mL centrifuge tubes in a biosafety cabinet.</li>
	<li>Add 1 mL of DMSO into 250 mg FTC tube (final concentration of 250 mg/mL), add 1.5 mL of DMSO into 375 mg TDF tube (final concentration of 250 mg/mL), and add 1mL of DMSO into 500 mg RAL or ELV tube (final concentration of 500 mg/mL). Stir or pipet the drug mixture until fully dissolved and a clear solution is obtained.</li>
	<li>Use a 0.22 &#181;M pore size hydrophilic PVDF membrane filter to sterilize solutions with a sterile syringe. Individual drug solutions can be stored at -20 &#176;C for 12 weeks.</li>
	<li>When ready to use, freshly thaw one aliquot of each drug solution at 37 &#176;C until the solution becomes clear. Mix well using a pipet.</li>
	<li>Combine drugs and mix well to make up master mix: 1 mL of FTC in DMSO, 1.5 mL of TDF in DMSO, and 1 mL of ELV or RAL in DMSO. 
	NOTE: This amount will make 10 food cups.</li>
	<li>Add 350 &#181;L of cART master mix solution into one cup to make one DietGel Boost cART cup.</li>
	<li>Add 0.75 mL of Trimethoprim-Sulfamethoxazole (0.48 mg/mL final concentration) into the cup.</li>
	<li>Stir thoroughly using 1 mL sterile pipette tips.</li>
	<li>Aliquot the food cup containing cART from the original cup with a micro spatula onto a 60 mm Petri dish as needed. Weigh the food on a scale to calculate the amount of food cup containing cART for each cage according to the number of mice.</li>
</ol>
3. Administration of ART drugs to HIV-1 infected mice
<ol>
	<li>Remove regular chow from the cage and replace it with a food cup containing cART. 
	NOTE: On average, a mouse will eat up to 5 g of food per day. Approximately one food cup can be administrated to five mice for 2 days.</li>
	<li>Refresh cART food three times per week.</li>
	<li>Weigh used cups to monitor intake. Weigh mice weekly to confirm consumption.</li>
</ol>
4. Monitor viral load by real-time PCR
<ol>
	<li>Assess human immune cells (CD4 and CD8 T cell levels) and HIV-1 replication in BLT mice every 2 weeks by retro-orbital bleeding. Harvest plasma by following instructions in steps 1.5-1.8.</li>
	<li>Monitor plasma viral loads of mice infected with HIV-1 before and during oral cART administration for 8 weeks. Extract plasma viral RNA from plasma using a viral RNA extraction kit and quantify it by real-time PCR using the primers and probes (see Table of Materials) as previously described27,30,31. Use the following cycling protocol: 48 &#176;C (15 min), 95 &#176;C (10 min), then cycling 95 &#176;C (15 s), 60 &#176;C (1 min) for 45 cycles.</li>
</ol>
5. Assess CD4/CD8 ratios by flow cytometry
<ol>
	<li>Prepare single-cell suspensions from peripheral blood of biweekly bleeds following steps 1.5-1.8.</li>
	<li>Stain cells with surface markers and analyze by flow cytometry. Use the following surface marker antibodies27,30,43,49 in flow cytometry: CD45 (clone HI30), CD8 (clone SK1), CD3 (clone OKT3), CD4 (clone RPA-T4)27,30,42,49.</li>
</ol>

SK is a founder of CDR3 Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

An oral cART administration method is developed here for HIV-1 infected humanized mice by combining three antiretroviral drugs within high nutrient food. Compared to administration by daily injections, oral delivery is easier to use, limits administration frequency, reduces animal handling, minimizes stress, and improves safety55. Up to this point, only a few studies in humanized mice24,37,41 have used fo...

The life expectancy of chronic human immunodeficiency virus (HIV)-infected individuals has significantly improved with combinational antiretroviral treatment (cART)1,2. cART successfully reduces the HIV-1 replication and increases CD4+ T cell counts to normalcy in the majority of HIV-1 chronically infected participants3, resulting in improved overall health and dramatically reduced disease progression4. However, the latent HIV-1 reservoir is established even when ART is initiated during acute infection5,6,7. Reservoirs persist over years during ART and rapid viral rebound after ART interruption is well-documented8,9. People living with HIV on ART are also predisposed to a higher risk of comorbidities such as cardiovascular disease, cancer, and neuro disorders10,11,12. Therefore, a functional cure for HIV is needed. Animal models for HIV-1 infection offer obvious advantages in developing and validating novel HIV cure strategies13,14,15. Humanized mice, as a small animal model, can provide multilineage human immune cell reconstitution in different tissues, which allows for the close study of HIV infection16,17,18,19. Among humanized models, the humanized bone marrow-liver-thymus (BLT) model successfully recapitulates chronic HIV-1 infection as well as functional human immune responses to HIV-1 infection20,21,22,23,24. Therefore, the humanized BLT mouse model has been widely used to investigate various aspects in the HIV research field. Humanized BLT mice are not only well-established models for the recapitulation of persistent HIV-1 infection and pathogenesis, but also consequential tools for the evaluation of cell therapy-based intervention strategies. The current authors and others have demonstrated that the humanized BLT mice model recapitulates persistent HIV-1 infection and pathogenesis25,26,27 and provides tools to evaluate cell therapy-based intervention strategies 28,29,30,31,32,33.
cART regimens consisting of combinations of antiretroviral drugs that are taken daily suppress HIV-1 replication to the point that the viral load in successfully treated individuals remains undetectable over long term34. The outcomes of treating HIV-infected humanized mice with clinically relevant cART regimens resemble those observed in HIV-1 infected ART-treated individuals22: HIV-1 levels are suppressed below the limits of detection and interruption of cART results in a rebound of HIV replication from the latent reservoir35. Subcutaneous (SC)27,36,37 or intraperitoneal (IP)37,38,39 injection is the route commonly used for cART treatment in humanized mice. However, intensive daily injection induces stress to animals by physical restraint40. It is also labor-intensive and potentially unsafe for researchers due to increased exposure to HIV while using sharps. Oral administration is ideal to mimic the absorption, distribution, and excretion of cART drugs that are taken by HIV-1-infected individuals. Oral administration typically involves customized and often laborious procedures to put the antiretroviral drugs in sterilized (necessary due to the immunodeficiency of the mice) food24,37,41 or water42,43,44,45,46, which may or may not be chemically compatible with many antiretroviral drugs, or result in something the mice would not readily eat or drink (which would affect dose and drug levels in the body). The novel peroral cART administration method proposed here surpasses previous delivery attempts due to its compatibility with different types of antiretroviral drugs, safety and ease of preparation and administration, and reduction of animal stress and anxiety resulting from the daily injection.
Tenofovir disoproxil fumarate (TDF), Elvitegravir (ELV), and Raltegravir (RAL) are poorly water-soluble drugs. Interestingly, increased bioavailability of TDF is observed with fatty foods, suggesting that competitive inhibition of lipases by fatty food may provide certain protection for TDF47. Therefore, DietGel Boost cups were selected to replace regular rodent chow as the method of delivery based on their modest fat content (20.3 g per 100 g) as compared to regular rodent chow (10 g per 100 g) and a typical mouse high-fat diet (40-60 g per 100 g)48. The total weight of one cup is 75 g; thus, each cup will contain the amount of food, and therefore drug, sufficient for five mice over 3 days.

<table><tbody><tr><td>60 mm petri dish</td><td>Thermo Scientific Nunc</td><td>150288</td><td>For aliquoting ART food</td></tr><tr><td>APC anti-human CD8 Antibody</td><td>Biolegend</td><td>344722</td><td>For flow cytometry</td></tr><tr><td>BD LSRFortessa</td><td>BD biosciences</td><td></td><td>For flow data collection</td></tr><tr><td>CD34 microbeads</td><td>Miltenyi Biotec</td><td>130-046-702</td><td>For NSG-BLT mice generation</td></tr><tr><td>Centrifuge tubes</td><td>Falcon</td><td>14-432-22</td><td>For dissolving ART</td></tr><tr><td>DietGel Boost</td><td>ClearH2O</td><td>72-04-5022</td><td>For making ART food</td></tr><tr><td>Elvitegravir</td><td>Gilead</td><td></td><td>Gifted from Gilead</td></tr><tr><td>Emtricitabine</td><td>Gilead</td><td></td><td>Gifted from Gilead</td></tr><tr><td>FITC anti-human CD3 Antibody</td><td>Biolegend</td><td>317306</td><td>For flow cytometry</td></tr><tr><td>Flowjo software</td><td>FlowJo</td><td></td><td>For flow cytometry data analysis</td></tr><tr><td>HIV-1 forward primer: 5&amp;prime;-CAATGGCAGCAATTTCACCA-3&amp;prime;;</td><td>IDT</td><td>Customized</td><td>For viral load RT-PCR</td></tr><tr><td>HIV-1 probe: 5&amp;prime;-[6-FAM]CCCACCAACAGGCGGCCT&lt;br/&gt; TAACTG [Tamra-Q]-3&amp;prime;;</td><td>IDT</td><td>Customized</td><td>For viral load RT-PCR</td></tr><tr><td>HIV-1 reverse primer: 5&amp;prime;-GAATGCCAAATTCCTGCTTGA-3&amp;prime;;</td><td>IDT</td><td>Customized</td><td>For viral load RT-PCR</td></tr><tr><td>Human fetal tissue</td><td>Advanced Bioscience Resources, Inc</td><td></td><td></td></tr><tr><td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td><td>The Jackson Laboratory</td><td>5557</td><td>For constructing the humanized mice</td></tr><tr><td>Pacific Blue anti-human CD45</td><td>Biolegend</td><td>304022</td><td>For flow cytometry</td></tr><tr><td>PerCP anti-human CD4 Antibody</td><td>Biolegend</td><td>300528</td><td>For flow cytometry</td></tr><tr><td>QIAamp Viral RNA Kits</td><td>Qiagen</td><td>&amp;nbsp;52904</td><td>For measuring viral load</td></tr><tr><td>Raltegravir</td><td>Merck</td><td></td><td>Gifted from Merck</td></tr><tr><td>Sterile cell scrapers</td><td>Thermo Scientific</td><td>179693</td><td>For aliquoting ART food</td></tr><tr><td>TaqMan RNA-To-Ct 1-Step Kit</td><td>Applied Biosystems</td><td>4392653</td><td>For plasma viral load detection</td></tr><tr><td>Tenofovir disoproxil fumarate</td><td>Gilead</td><td></td><td>Gifted from Gilead</td></tr><tr><td>Trimethoprim-Sulfamethoxazole</td><td>Pharmaceutical Associates</td><td>NDC 0121-0854-16</td><td>For keeping ART food sterile. Each 5mL teaspoon contains&lt;br/&gt; 200 mg Sulfamethoxazole, USP&lt;br/&gt; 40 mg Trimethoprim, USP&lt;br/&gt; NMT 0.5% Alcohol</td></tr></tbody></table>

oral combinational antiretroviral treatment in hiv-1 infected humanized mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

Assuming an average mouse weighing 25 g consumes 4 g of food per day, the daily drug dose through oral intake corresponds to 2.88 mg/kg TFV, 83 mg/kg FTC, and 768 mg/kg RAL. To test whether the optimized food regimen is toxic and influences overall health compared to daily injection of cART, mice weight was monitored weekly before and during cART through oral or subcutaneous injection. There were no significant weight differences before cART administration in each group (Figure 1). However, ...

Watch this Scientific Journal Video about Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice at JoVE.com

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

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

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

oral-combinational-antiretroviral-treatment-hiv-1-infected-humanized

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Immunology and Infection

2.3K Views.  University of California, Los Angeles. Dit protocol beschrijft een nieuwe methode om orale combinatie antiretrovirale geneesmiddelen, of ART, te leveren die met succes hiv-RNA-replicatie in gehumaniseerde muizen onderdrukt. De nieuwe orale ART-methode overtreft eerdere leveringspogingen vanwege de veiligheid, het gemak van voorbereiding en toediening en verminderde dierlijke stress als gevolg van een dagelijkse injectie. De voorgestelde orale toedieningsmethode om HIV-RNA-replicatie bij gehumaniseerde muizen te onderdrukken, zal z...

Video: Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

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 viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

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.

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 ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

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

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

Medicine

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).

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 ...

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 their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

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.

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

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

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

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.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

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.

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 ...

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

Source: Mu, W., et al. <a href="https://app.jove.com/v/63696">Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice.</a> J. Vis. Exp. (2022).
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 suppression of viral replication is assessed through periodic examinations of the mice.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Source: Dagur, R. S. et al., <a href="https://app.jove.com/v/58645">Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Constructing a Humanized Mouse Model with Engineered Immunity against HIV

Pour the thymus pieces and medium from the flask into a 60-millimeter dish. Chill some positive displacement pipette tips by placing them in open 1.5-milliliter sterile screw-cap tubes on ice. Also, place the cell pellets and a volume of gelatinous protein mixture, such as Matrigel, on ice.
Anesthetize the recipient mice with an injectable anesthetic according to an approved animal protocol. Check the anesthesia level of the mouse by squeezing a paw. The absence of a reaction to the pinch indicates a surgical plane of anesthesia.
Shave the left side of each mouse from hip to shoulder, between the center of the back and the stomach. Subcutaneously, inject 6 milligrams per kilogram of diluted carprofen into the animal&#39;s shoulder or inguinal triangle. Flush the cannula of the trocar with PBS in a 60-millimeter dish. Using a pair of blunt, curved forceps, place a piece of thymus from the 60-millimeter dish just inside the opening of the cannula, then pull back on the trocar to aspirate the tissue into the cannula.
Next, use a positive displacement pipette and a chilled tip to add 5 microliters of cold gelatinous protein mixture into the tube with the cell pellet, and gently stir to generate the cell suspension. Pipette the cell suspension into the opening of the cannula and slowly pull back on the trocar to load the needle.
Swab the shaved area of the mouse with povidone-iodine followed by isopropanol three times. Determine the darkest spot under the skin, which indicates the location of the spleen. The kidney is approximately 5 millimeters dorsal to the spleen.
After ensuring a suitable level of anesthesia by the absence of a reaction to the paw pinch, use curved forceps to lift the skin, and use surgical scissors to make a 15-millimeter incision in the skin parallel to the spleen. Then, make a similar cut in the peritoneum layer below.
In males, the kidney is easily visible, thus extruded simply by pressing on the abdomen. Support the kidney with a hemostat or a pair of curved blunt forceps. In females, the ovaries tend to block the kidney from easy extraction. Using a hemostat, pick up the ovary and carefully expose the kidney.
Use needle-tipped forceps to pluck a tiny hole at the posterior end of the kidney capsule. Slide the trocar into this hole and along the kidney until the opening of the cannula is completely covered by the kidney capsule. Gently extrude the tissue under the kidney capsule and pull the trocar back out. Use a curved forceps to make sure the thymus piece does not come out with the needle.
Next, lift up the peritoneum with the forceps and gently use the hemostat to push the kidney back into place. Tie one double-knotted stitch with surgical suture in the peritoneum. Also, place two auto clips wound clips to close the skin.
Now, mix the transduced CD34-positive cells and aspirate 0.5 x 106 cells in a 100-microliter volume into an insulin syringe. Inject these cells into the mouse through retro-orbital vein injection.
After injection, swab a small drop of eye lubricant onto each eye and lay the mouse on its side back in a cage. After implanting all the mice, confirm that the animals have regained consciousness and are ambulatory before returning them to the housing room.

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