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

In this work, all animal care and procedures were performed according to protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine (protocol numbers 1018017, 1018018, and 0318009).

1. Human CD34⁺ HSC engraftment of newborn mice

1. Always use disposable personal protection equipment (PPE), including sterile scrubs, gloves, dedicated shoes, shoe covers, mask, goggles, hair/beard bonnet, and sterile lab coats.
2. Re-suspend 1 x 10⁶ of frozen CD34⁺ HSCs (see Table of Materials) in 10 mL of RPMI 1640 media 10% FBS under a certified biosafety cabinet and maintain sterile conditions.
3. Use 10 µL of the suspension to count (to assure the presence of the expected number of cells) and check cell viability by trypan blue exclusion staining in a hemocytometer.
4. Centrifuge at 400 x g for 15 min at room temperature (RT). Discard the supernatant and resuspend in cold 1x PBS to the required concentration (1.4 x 10⁵ of CD34⁺ HSCs in 50 µL). Keep the cells on ice until injection.
5. Place pups (NS γ-chain^null pups of both genders from 1–4 days old) into a sterile 100 mm² Petri dish along with a small amount of bedding material from the breeder cage. Additionally, rub clean bedding between hands to further mask any foreign odors on the recipient pups.
   NOTE: This minimizes foreign odors being impregnated onto the pups, thereby improving the chances of mothers accepting their pups back into cages after the procedure.
6. Put the Petri dish into a clean transport cage and place the cage in a clean mouse microisolator cage to protect pups from the environment. Cover the transport cage with a cover pad to avoid exposure of animals to external light while in transit to the irradiation room.
7. Irradiate pups with 100 cGy whole body irradiation (WBI) by exposure to a 137 Cs source. Clean the interior of the irradiator with disinfectant solution, place mice in the irradiator, and turn on the turntable so that all pups are irradiated homogeneously. Close the irradiator and press the power switch to initiate the irradiation process. Wait until the irradiation time is completed and immediately remove the mice.
   NOTE: Since irradiation does not generate stress in the mice, previous anesthesia is not required.
8. 2–4 h after the irradiation procedure, place pups in a biosafety cabinet inside a chilled sterile Petri dish covered with sterile gauze on ice, until anesthetized (~5–10 min). Sufficient anesthesia is achieved when gross movements cease.
9. Load the syringe with an attached needle (29 G, 0.5") with 50 µL of the cell suspension and 1.4 x 10⁵ of CD34⁺ HSCs under the certified biosafety cabinet.
10. For the engraftment via hepatic injection, restrain pups with the thumb and index fingers. To minimize the mouse restraint (~30–45 s), let one investigator hold the pup and administer the injection, and have the second investigator load the syringe with the HSC suspension. Clean the injection site with 70% alcohol and deliver 50 µL of cells directly into the liver. Use a shallow needle angle when injecting to avoid completely piercing the liver. As a control, inject mice with 50 µL of 1x PBS into the liver.
11. Place the pups on a pre-warmed sterile Petri dish covered with sterile gauze for 1–5 min to allow recovery. Pre-warm the dish using an infrared warming pad for rodents at 20 °C to ensure that pups will not be over-warmed.
12. Immediately before returning the pups to their parents, apply a small amount of menthol- and eucalyptus-based ointment, using the thumb and index fingers, to the snout of both parents to avoid cannibalism or rejection of the pups.
13. Check cages every day, looking for any signs of graft-versus-host disease (GVHD) in the pups such as dry skin, no feeding, rash, and alopecia. Euthanize the animals if any of these signs are observed.
14. Wean mice at 3 weeks of age, grouping them by gender. Do not put more than 5 animals per cage. Verify engraftment in the peripheral blood by flow cytometry at 14 weeks of age.
    NOTE: The success rate of engraftment is between 80%–100%.

2. Human PBMC engraftment of juvenile mice

1. For the acute and reactivation models, inject 6–8 week-old NS γ-chain^null mice intraperitoneally with human PBMCs derived from a healthy donor or HIV-infected patient who was under ART, respectively. In both models, include mice injected with PBMCs from a healthy donor without HIV infection (sham) as controls.
2. Layer 15 mL of whole blood in 5 mL of sterile density gradient medium into a 50 mL conical tube.
3. Centrifuge at 400 x g for 30 min at RT, without brakes, to avoid the buffy coat from becoming mixed with the density gradient medium.
4. Carefully collect the fraction of mononuclear cells (between the density gradient medium and supernatant) and transfer the buffy coat to a 15 mL centrifuge tube containing 10 mL of 1x PBS. Centrifuge at 300 x g for 10 min at RT.
5. Discard the supernatant and remove the remaining red blood cells by lysing with 5 mL of ACK buffer added to the pelleted cells. Incubate for 4 min at RT.
6. Centrifuge at 300 x g for 10 min at RT. Discard the supernatant and resuspend in 10 mL of 1x PBS or RPMI 1640 medium.
7. Use 10 µL of the cell suspension to count the cells and check viability by trypan blue exclusion staining in a hemocytometer. Typically, 1–2 x 10⁶ cells are obtained for each 1 mL of blood, with more than 95% viability.
8. Centrifuge at 300 x g for 10 min at RT. Discard the supernatant and adjust the cell number to the required concentration (in this case, 3.5 x 10⁶ cells in 200 µL of 1x PBS).
9. Load the syringe with an attached needle (28 G, 0.5") with 3.5 x 10⁶ of PBMCs in 200 µL of 1x PBS under a certified biosafety cabinet.
10. Remove the mouse from the cage and hold it by the tail so that it can grip the mesh, applying gentle traction backward. Then, place the index and thumb fingers on the shoulders of the animal, grabbing the loose skin of the neck and using the middle finger to stabilize its back.
11. Slide the mouse head back so that its back is above the head. This allows the viscera in the abdominal cavity to be displaced backward and reduces the risk of puncturing the internal organs during the injection.
12. Clean the injection site with 70% alcohol.
13. Penetrate the syringe used in step 2.9 through the abdominal wall and aspirate before injecting the cells, if any material is aspirated, remove the syringe and discard it. Otherwise, inject the cells slowly in the intraperitoneal cavity, remove the syringe and discard it. Inject 1x PBS to control mice.
14. Return the animal to its cage.
15. Verify engraftment in peripheral blood by flow cytometry at 3 weeks post-injection.

3. Post-engraftment care

1. Identify mice by ear tagging.
2. Observe the mice used in these experiments closely 2x per day after each procedure for clinical signs of distress.
3. After human cells transplantation, monitor mice for GVHD. For monitoring GVHD symptoms in newborn, juvenile, and adult mice, evaluate animals for the appearance of skin disease (i.e., color, dryness, rash, alopecia), in addition to body weight measure. Evaluate animals that show these signs by a veterinarian to consider early euthanasia.

4. HIV infection procedure and sham infection procedure

NOTE: For the chronic and acute models, mice are infected with the HIV BaL reference strain at week 14 and week 3 post-engraftment, respectively. Injections with HIV is administered intraperitoneally into the lower abdominal quadrants.

1. Perform the loading process of the virus/PBS into syringes, using a 28 G 0.5" needle, in BSL2 cabinets following ABSL2 procedures. The total amount of virus injected is 15,000 median tissue culture infectious dose (TCID₅₀) in 200 µL of sterile RPMI 1640.
2. Remove the mouse from the cage and hold it by its tail so that it can grip the mesh, applying gentle traction backward. Then, place the index finger and thumb on the shoulders of the animal, grabbing the loose skin of the neck and using the middle finger to stabilize the back.
3. Slide the mouse head back so that its back is above its head. This allows the viscera in the abdominal cavity to be displaced backward and reduces the risk of puncturing internal organs during injection.
4. Clean the mice with a pre-wetted alcohol pad in the lower left/right quadrant of the abdomen. Inject 15,000 TCID₅₀ of the HIV BaL virus contained in 200 µL of sterile RPMI 1640.
5. After injection, return the mouse to its home cage.

5. Blood collection by retroorbital puncture

NOTE: Retroorbital bleeding allows for the fast collection of blood, thereby reducing the overall collection time and increasing the stability of human lymphocyte markers. Use EDTA tubes to collect mice blood.

1. In the chronic model, at 14 weeks post-HSC injection, collect the blood via retroorbital vein. In the acute and reactivation models, perform this procedure at 3 weeks post-PBMC injection.
2. Anesthetize the animals using 250 µL of isoflurane inhalation prior to blood collection in a biosafety hood class B2 that is ducted externally.
3. Dispense the Isoflurane into cotton pads under a wire mesh in a clear 1 L jar in a biosafety cabinet vented outside of the building. The use of the mesh ensures that the animals do not contact the isoflurane-soaked pad, which can cause skin irritation and potential overdosing since isoflurane is also absorbed through the skin. Also, put a soft paper towel between the mesh and animal to avoid limbs injuries.
4. Once the jar is saturated with isoflurane (approximately 1 min after adding it), introduce the animal and observe the respiratory rate, which will increase then decrease. Check for the clinical indication of a deep plane of anesthesia, which includes the lack of a righting reflex (upon tipping jar gently) and lack of gross movements. Start the bleeding procedure as soon as the animal is completely relaxed and lacking the toe pinch reflex.
   NOTE: Since Isoflurane evaporates, dispense more drug if no signs of anesthesia are observed.
5. For the retroorbital bleeding, press the mouse external jugular vein caudal to the mandible with the thumb, and with the same hand, gently elevate the upper eyelid with the index finger.
6. Insert a hematocrit tube into the medial canthus of the eye and direct it in a ventrolateral direction until the blood starts fluxing.
7. Collect at least 100 µL of blood. Once the desired volume of blood is obtained (a volume no more than the 1% of the body weight of the animal), discontinue the external jugular pressure and remove the hematocrit tube.
8. Assure that the hemostasis is complete by applying the direct pressure on the eye using a sterile gauze for a minimum of 30 s.
9. Apply tetracaine drops in the eye. Monitor the animal until it has completely recovered from anesthesia and place it back in the cage.
   NOTE: The 100 µL of collected blood is used for the evaluation of the level of engraftment of human CD45⁺ and other blood cell populations, as well as for the evaluation of plasma viral load.

6. Screening of engraftment level and flow cytometry analysis

1. Follow a conventional flow cytometry staining protocol for whole blood, which includes the incubation of fluorochrome-labeled anti-human antibodies (for suggested flow panel, see Table of Materials), followed by the lysis of red blood cells and washing steps^13,15.
   NOTE: For the screening of the level of engraftment, include an anti-human CD45 antibody. For the comparison, an anti-mouse CD45 antibody may also be used. Include compensation controls as well as a human blood sample stained with the same antibody mix, unstained mouse and human blood samples, and non-humanized control to test cross-reactivity of the reagents. After staining, there is always some background signal; however, all positive signals are clearly distinguished from negative and cross-reactive controls.
2. In an appropriate flow cytometer, acquire at least 10,000 events on the lymphocyte gate (FSC-A vs. SSC-A). For flow cytometry analysis, after duplicate exclusion, determine the percentage of human CD45⁺ cells as well as other cell populations of interest.

7. Evaluation of plasma viral load

1. Evaluate the viral load in HIV infected animals 1x per week after infection.
2. After the retroorbital bleeding (approximately 100 µL), obtain plasma by collecting the supernatant after centrifugation of the anticoagulated blood at 3,500 x g for 3 min in a microcentrifuge. The pellet is used for blood cell phenotyping.
3. Use a commercial viral RNA extraction kit (see Table of Materials) to obtain RNA from 40 µL of plasma.
4. Convert RNA into cDNA using the first strain synthesis mix (see Table of Materials) and HIV gag primer SK431.
5. Perform quantitative real-time PCR using HIV Gag primers SK38/SK39 and fluorescent green dyes (e.g., SYBR green) as described in previous studies^13,25.

8. Administration of antiretroviral therapy

1. Administer oral ART at least 3 weeks after infection, when high viral load is observed, in the chronic, acute, and reactivation models.
2. Calculate the doses of tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and raltegravir (RAL), according to Km values of 37 and 3 for humans and mice, respectively²⁶. Typically, the human-equivalent doses of TDF, FTC, and RAL are 61.7 mg/kg/day, 40.7 mg/kg/day, and 164 mg/kg/day, respectively.
3. For administration in drinking water, crush drug tablets and add the respective amount in the water bottle, ensuring that each mouse in the cage acquires its daily dose. Since the drug powder may form sediment in the bottle, periodically shake the water bottle to achieve homogeneous suspension.
4. Change the water bottle every week with freshly dissolved drugs.
5. Collect the blood via retroorbital vein every week or every 2 weeks after ART initiation to evaluate the changes in viral load and CD4:CD8 ratio.

9. Mouse euthanasia, collection of secondary lymphoid organs, and isolation of mononuclear cells

1. Euthanasia is performed in the three humanized mouse models, periodically along infection time, or at the end of the experiment.
2. Perform the euthanasia of adult mice by CO₂ asphyxiation, followed by the cervical dislocation. For asphyxiation, use a non-precharged chamber, dispense CO₂ from a commercial cylinder with fixed pressure regulator and in line restrictor controlling the gas flow within 20%-30% of the chamber volume/minute to comply with 2013 AVMA guidelines.
3. Maintain the CO₂ flow for >60 s monitoring respiratory arrest (which may take up to 5 min), followed by the cervical dislocation to assure euthanasia.
4. Euthanize neonates <7 days old by a physical method (i.e., using sharp scissors).
5. Visualize axillary, mediastinal, and mesenteric lymph nodes (which are typically observed) and extract them with tweezers and sharp scissors. Also extract the spleen, located in the upper left side of the peritoneal cavity.
6. Deposit the lymphoid tissues in 1.5 mL centrifuge tubes containing sterile RPMI 1640 medium.
7. Immediately process the lymphoid tissues in a 70 μm pore-size nylon cell strainer, collecting the cells in a 50 mL tube. Do not aspirate the tissues.
8. Wash the cells with 5 mL of RPMI 1640 medium supplemented with 1% FBS to facilitate cells filtering.
9. After the tissue disaggregation, centrifuge the cells suspension at 3,500 x g for 10 min in a microcentrifuge.
10. Discard the supernatant and resuspend the cells with 500 µL of 1x PBS.
11. Transfer the cells suspension into a 1.5 mL centrifuge tube containing 500 µL of sterile density gradient medium.
12. Centrifuge at 3,500 x g for 3 min in a microcentrifuge, without brake, to prevent the buffy coat from mixing with the density gradient medium
13. Carefully collect the fraction of mononuclear cells (between the density gradient medium and supernatant) and transfer the buffy coat to a 1.5 mL centrifuge tube containing 500 µL of 1x PBS. Centrifuge at 3,000 x g for 3 min.
14. Remove the remaining red blood cells by lysing with 500 µL of ACK buffer, incubating for 4 min at RT.
15. Centrifuge at 3,000 x g for 3 min. Discard the supernatant and resuspend in 1 mL of 1x PBS or medium.