Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

Summary

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

Abstract

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.

Introduction

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 infection^5,6....

Protocol

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....

Results

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, .......

Discussion

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 mice^24,37,41 have used fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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