Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

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 requirements. Human PBMCs are isolated from discarded peripheral blood specimens from anonymous healthy donors from City of Hope Blood Donor Center (Duarte, CA), with no identification regarding age, race, gender, or ethnicity. IRB#/REF#: 97071/075546

1. General Aseptic Practice

1. Perform tissue culture experiments in designated laminar flow cabinets.
2. Keep tissue culture medium and supplements under sterile conditions and filter using a 0.22-µm filtration unit prior to use.
3. Preserve prepared human HSCs in sterile tubes and keep on ice until injection.
4. As a result of severe immune deficiency, handle NSG mice aseptically. Wear a disposable surgery gown, hair cap, face mask, shoe covers, and gloves to prevent contamination. Sanitize the bench surface, irradiation holder, and induction chamber for anesthesia with chlorine dioxide based sterilant (e.g., Clidox) before and after experiments.
5. Apply petroleum-based eye ointment after retro-orbital bleeding to prevent dry eyes.
6. Change to antibiotic-containing diet to prevent infection due to retro-orbital bleeding.

2. Handling HIV Virus, Infected Rodents, and Virus-containing Blood/Tissue Samples

CAUTION: HIV is a class 3 human pathogen; the handling rules must be followed exactly.

1. Handle HIV virus-containing samples in a designated BSL2+/3 laboratory space. Lock virus-containing reagents securely in a secondary container during transportation. Disinfect the outer surface of all containers by thorough wiping with 10% bleach and isopropanol prior to transportation.
2. Keep all virus-containing wastes in designated waste containers with 2-3 layers of biohazardous waste bags. Prior to waste disposal, disinfect by generously spraying the waste with iodine/ethoxylated nonylphenol solution (e.g., Wescodyne) and autoclave.
3. Wear personal protective equipment: hair cap, face cover, anti-splash face shield, shoe covers, disposable surgery gown, and double-layer gloves.
4. Handle infected rodents with extreme caution. Conduct injections and blood collections while mice are under anesthesia (Steps 5.1-5.2, 6.3-6.5, 7.3, and 8.1-8.2).

3. Isolation of Hematopoietic Stem Cells

1. Prepare collagenase/dispase solution for tissue digestion.
   1. Dissolve collagenase/dispase powder to make stock solution at a concentration of 100 mg/mL, and store collagenase/dispase stock solution in 150 µL aliquots at -20 °C.
   2. For working collagenase solution, dilute 150 µL of collagenase/dispase stock with 15 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin.
      NOTE: The final concentration of collagenase/dispase for tissue digestion is 1 mg/mL.
2. With a sterile razor blade, cut fetal liver tissue (16-24 weeks gestation) into small pieces (approximately 2-3 mm³ in size) followed by digestion with collagenase/dispase solution (1 mg/mL) for 30 minutes at room temperature. Adjust the volume of digestion solution so that all tissue pieces are fully submerged. Use the back end of the syringe plunger to gently grind the tissue sample to facilitate digestion.
3. Pass the digestion mixture through a 70-µm sterile nylon mesh filter to acquire a single cell suspension.
4. Enrich for CD34+ HSCs using a MACS system per manufacturer's instruction.
5. Count the viable percentage of enriched CD34+ HSCs using a hemacytometer²⁸ and proceed to intrahepatic injection into neonatal NSG mice.
6. Freeze the remaining enriched CD34+ HSCs in freezing media (e.g., CryoStor CS2) and preserve cells in a liquid nitrogen tank. Upon future use, recount the viable percentage of thawed CD34+ HSCs prior to injection. With the use of freezing media, post-thaw viability is between 80 to 90%.

4. Intrahepatic Injection of Human CD34+ HSCs in Neonatal NSG Mice

NOTE: Neonatal NSG mice 2-3 days from birth are most suitable for this procedure, as they are strong enough to endure irradiation at half-lethal dose, while young enough to have totally impaired immune systems. No anesthesia is required for the HSC injection.

1. Place the whole litter of neonatal NSG mice (typically 5-10 mice, both genders) in a sterile pie-shaped irradiation cage and expose to a total dose of 200-250 cGy gamma-ray radiation from a Caesium-137 radiation source. Ensure that radiation dose is within the range, as significant weight loss or even death can be observed when NSG mice are exposed to high doses of irradiation.¹²
   NOTE: Radiation is a health hazard, personal protection against radiation should be taken.
2. Following irradiation, inject each neonatal NSG mouse with 5 x 10⁵ viable human CD34+ HSCs using a syringe/needle setup (Figure 1). Directly inject cells into the liver.

5. Engraftment Validation Through Retro-Orbital Bleeding and Flow Cytometry Analysis

1. At 10-12 weeks post-HSC injection, anesthetize mice using an isoflurane/oxygen inhalation apparatus. Adjust the oxygen flow to maintain isoflurane percentage at 4-5%. Anesthesia takes 3-5 minutes, depending on individual mouse. Properly anesthetized mice show a slow and deep breathing pattern, and no reaction to toe-pinching stimulation.
2. Collect 50-100 µL of peripheral blood from each mouse through retro-orbital sampling²⁸. Store blood samples in K2EDTA or heparinized blood collection tubes to prevent coagulation.
   NOTE: Blood collection tubes are required to have anticoagulant coating so that mouse PBMCs can be isolated from whole blood for flow cytometry analysis. EDTA is known to inhibit enzymatic reactions such as PCR and qRT-PCR; thus, if downstream enzymatic reactions are required, use a heparinized blood collection apparatus.
3. Centrifuge blood samples at 2,000 x g for 20 minutes at 4 °C to pellet blood cells.
   1. Transfer the plasma supernatants to new microcentrifuge tubes after centrifugation and store at -80 °C if cytokine analysis is required.
   2. Lyse the red blood cells (RBCs) within the blood cell pellet at room temperature for 10 minutes using Red Blood Cell Lysis Buffer (Table of Materials).
      NOTE: If plasma samples are not required, directly lyse the whole blood with lysis solution without centrifugation.
4. Pellet remaining blood cells after RBC lysis at 300 x g for 5 minutes at 4 °C; aspirate the supernatant. Wash the cell pellets with 0.01% BSA containing DPBS, centrifuge at 300 x g for 5 minutes at 4 °C and aspirate supernatant. Repeat the washing step one more time.
5. Block cells with 100 µL of 1x blocking cocktail (Table 3 for recipe) for 20 minutes at 4 °C.
6. After blocking, directly add each antibody solution into the cell suspension at a concentration of 2 µL/10⁶ cells and incubate at 4 °C for 30 minutes. Antibodies for engraftment validation are: anti-human CD45 (leukocytes, RRID: AB_2732068), anti-human CD3 (T-cells, RRID: AB_396896), anti-human CD4 (helper T-cells, RRID: AB_397037), anti-human CD8 (cytotoxic T-cells, RRID: AB_2722501), anti-human CD14 (monocytes, RRID: AB_10373536), and anti-human CD19 (B-cells, RRID: AB_10373382). For suggested flow panel, please see Table 4 and Table of Materials for catalog numbers, RRIDs and lot numbers.
   NOTE: Antibody staining in blocking solution eliminates non-specific binding of the anti-human antibodies toward mouse surface markers, as several surface markers share homology between human and mouse.
7. Isolate human PBMCs from healthy donors and prepare single-stained flow cytometry compensation controls using purified human PBMCs. Alternatively, use fluorescence-labeled microspheres as compensation controls²⁹.
8. Analyze the peripheral blood samples using a flow cytometer and quantify the percentage of human CD45+ cells, human CD3+ cells, human CD14+ cells, and human CD19+ cells from total peripheral blood cells.
   1. For downstream HIV infection and latency studies, calculate the percentage of CD4+ T-cells and CD8+ T-cells within the CD3+ cells.
      NOTE: Typically, a CD4:CD8 ratio between 1.5 and 2.5 is observed prior to HIV infection³⁰.

6. Analysis of HIV Infection of hu-NSG Mice and Plasma Viral Load Using qRT-PCR

1. Propagate HIV BaL viral stock in human PBMCs from healthy donors, harvest at day 10 post-infection, and titrate using p24 ELISA kit³¹.
2. Select hu-NSG mice with more than 20% human CD45+ cells in the peripheral blood for HIV infection.
3. Anesthetize hu-NSG mice using an isoflurane/oxygen inhalation apparatus (Step 5.1).
4. After confirming animal is anesthetized, inject HIV BaL virus at a dose of 200 ng of p24 per mouse through intraperitoneal route.
   NOTE: Be extremely cautious with the needle, do not reuse needles, and discard immediately after use into designated sharp container. Disinfect the area of injection after virus administration.
5. Three weeks post-infection, anesthetize hu-NSG mice and collect peripheral blood through retro-orbital bleeding using heparinized capillary tubes and collection tubes.
6. Separate plasma and blood cells by centrifugation at 2,000 x g for 20 minutes.
7. Lyse RBCs. Block and stain remaining blood cells with antibodies for flow cytometry analysis (Section 5.3-5.8).
   NOTE: Flow cytometry analysis should focus on comparing CD4:CD8 ratio before and after infection. Significant decrease in CD4+ cell count is expected, as the CD4 antigen serves as a co-receptor for viral entry.
8. Use plasma samples to analyze the HIV viral load using qRT-PCR. Isolate viral RNA from plasma samples using a viral RNA mini kit. Perform qRT-PCR with HIV-1 LTR-specific primers and probe sets (sequence details in Table 5), using manufacturer's protocol.

7. Oral Administration of cART and Validation of Viral Suppression (optional)

NOTE: This step is optional for investigations regarding HIV prognosis, virology, or infection-related pathophysiology during the acute infection phase. cART treatment of HIV-infected hu-NSG is used to recapitulate HIV latency among human patients receiving cART. Successfully HIV-infected hu-NSG mice are given cART for 4 weeks. The cART regimen consists of tenofovir disoproxil fumarate (TDF; 300 mg/capsule), emtricitabine (FTC; 200 mg/capsule), and raltegravir (RAL; 400 mg/capsule). The dose of cART for treating HIV-infected hu-NSG mice is adjusted according to body surface area (Table 1, Equation 1, Table 2)³².

1. Calculate the amount of each medication required for each cage during one week of treatment, taking into consideration the volume of the water bottle, the number of mice in each cage, and an average daily water intake of 4 mL per mouse.
   NOTE: The key point in this step is to ensure that each mouse acquires its daily dose within 4 mL of drinking water.
2. Grind all three medications with adjusted doses into fine powder and dissolve the powder mixture in sweetened water (e.g., Medidrop Suralose). Change the water bottle every week with freshly dissolved cART cocktail supplied in sweetened water.
   NOTE: The drug powder may not dissolve immediately, shake vigorously to achieve a homogeneous suspension before returning the bottle to the holding cage.
3. Collect peripheral blood samples via retro-orbital bleeding every two weeks and analyze both the CD4:CD8 ratio using flow cytometry (Section 5.3-5.8) and the plasma viral load using qRT-PCR (Section 6.6).
   NOTE: Typically after 4 weeks of treatment, the plasma viral load decreases to below the detection limit and a typical CD4:CD8 ratio is restored.

8. Validation of Viral Rebound upon cART Withdrawal (optional)

NOTE: This step is critical in validating the latency model, as viral rebound upon cART withdrawal provides direct evidence of a latency reservoir. It is also recommended to serve as a control experiment for therapeutic investigations on HIV rebound suppressants.

1. Plan for cART withdrawal after plasma viral loads from peripheral blood samples are below the detection limit and the CD4:CD8 ratio is restored to a range between 1.5 and 2.5.
2. Collect peripheral blood samples through retro-orbital bleeding every 2 weeks after cART withdrawal. Closely monitor the change in plasma viral loads (Section 6.6) as well as the CD4:CD8 ratio (Section 5.3-5.8).