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

Protokół

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

1. Humanized mice infected with HIV-1

NOTE: Humanized mice were constructed as previously described. The protocol is briefly described below.

1. Purify CD34+ hematopoietic progenitor cells from the human fetal liver via anti-CD34 microbeads according to the manufacturer's protocol.
2. Anesthetize 6-8-week-old NOD/SCID/IL2Rγ−/− (NSG) male and female mice, and sub-lethally irradiate (2.7 Gy) before the surgery.
3. Implant thymus, derived from the same donor as the fetal liver, under the kidney capsule along with the liver.
4. Following implantation, inject mice with 0.5 million to one million CD34+ cells, intravenously.
5. After 8-10 weeks, collect 100 µL of mouse blood via retro-orbital bleed into microcentrifuge tubes containing 5 µL of EDTA and centrifuge at 350 x g for 3 min.
6. Store the plasma at -80 °C to monitor viral load after the mouse has been infected with human immunodeficiency virus-1 (HIV-1). Add 2 mL of 83% NH4C solution and incubate for 5 min at room temperature to lyse red blood cells.
7. Add 10 mL of RPMI with 10% Fetal Bovine Serum (FBS) to stop lysis. Spin at 300 x g for 5 min.
8. Aspirate supernatant. Stain cells with an antibody panel (see Table of Materials), and analyze by flow cytometry to check human immune cell engraftment.
9. Infect mice exhibiting more than 50% of circulating CD45+ cells by retro-orbital vein injection with at least 200 ng of p24 of an HIV-1 strain (i.e., NFNSXSL9) using an insulin syringe. Collect blood biweekly for flow cytometry analysis and to measure the viral load.

2. Preparation of ART drugs

1. Weigh individual drugs; for example, to make 10 food cups with cART, use sterile cell scrapers to weigh out 250 mg of Emtricitabine (FTC), 375 mg of Tenofovir disoproxil fumarate (TDF), and 500 mg of Raltegravir (RAL) or Elvitegravir (ELV) into individual sterile 15 mL centrifuge tubes in a biosafety cabinet.
2. Add 1 mL of dimethyl sulfoxide (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.
3. Use a 0.22 µM pore size hydrophilic polyvinylidene fluoride (PVDF) membrane filter to sterilize solutions with a sterile syringe. Individual drug solutions can be stored at -20 °C for 12 weeks.
4. When ready to use, freshly thaw one aliquot of each drug solution at 37 °C until the solution becomes clear. Mix well using a pipet.
5. 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.
6. Add 350 µL of cART master mix solution into one cup to make one DietGel Boost cART cup.
7. Add 0.75 mL of Trimethoprim-Sulfamethoxazole (0.48 mg/mL final concentration) into the cup.
8. Stir thoroughly using 1 mL sterile pipette tips.
9. 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.

3. Administration of ART drugs to HIV-1 infected mice

1. 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.
2. Refresh cART food three times per week.
3. Weigh used cups to monitor intake. Weigh mice weekly to confirm consumption.

Materiały

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