Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

All cord blood samples were obtained in strict accordance with locally approved protocols, including informed consent of anonymous donation by the parents. All animal experiments were approved and performed in strict accordance with Danish national regulations under the license 2017-15-0201-01312.

CAUTION: Handle HIV exposed mice and blood with extreme caution. Decontaminate all surfaces and liquids that have been in contact with HIV with a confirmed HIV disinfectant (Table of Materials).

1. Isolation of human CD34+ stem cells

1. Collect cord blood samples in EDTA-coated blood collection tubes after planned Caesarean sections or vaginal births and according to local ethical approvals.
2. Isolate PMBCs from cord blood by density-gradient separation according to the manufacturer's protocol.
3. Isolate CD34+ cells from the PBMC population by first pre-enriching with antibodies against common markers for mature cells, which induces crosslinking of cells of undesired lineages with red blood cells. This is followed by CD34+ cell enrichment using magnetic beads, according to the manufacturer's protocol.
   1. Determine live cell count by standard trypan blue exclusion by resuspending 10 µL of cell suspension in 90 µL of trypan blue. Add 10 µL of this solution to a hemacytometer and count non-blue cells, according to the manufacturer's protocol.
   2. Viably cryopreserve CD34+ cells in 1 mL of 10% DMSO in fetal bovine serum (FBS) until the day of mouse transplantation.
   3. Viably cryopreserve a small fraction of both isolated (CD34+) and flow-through cells (CD34-) separately for assessing CD34+ stem cell purity (~30,000 cells of each sample). Alternatively, test the purity on freshly enriched cells (see step 2 below).
   4. Freeze a fraction of non-pelleted flow-through (CD34-) for determination of CCR5Δ32 status. Cells can be frozen directly without conditioned freezing solution, but the presence of red blood cells in the pellet can inhibit the subsequent PCR if the flow-through is pelleted.

2. Assessing CD34+ stem cell purity via flow cytometry

1. Thaw the isolated cells (CD34+) and flow-through cells (CD34-). Wash the cells by resuspending the cells from each vial in 9 mL of room temperature (RT) FACS buffer, consisting of 2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS).
2. Centrifuge for 5 min at 300 x g at RT to pellet cells.
3. Pour off the supernatant, resuspend the cells in the remaining liquid and transfer to FACS tubes. Repeat the washing step with 3 mL of FACS buffer. After the second centrifugation, pour off the supernatant and resuspend the cells in the remaining liquid.
4. Add 5 µL of Fc Receptor blocking solution (Table of Materials) and leave for 10 min at RT. Do not wash off the Fc Receptor blocking solution.
5. Add the mix containing predetermined volumes of antibodies against human CD3 (clone SK7) BUV395, CD34 (clone AC136), FITC, and CD45 (clone 2D1) APC (Table 1). Leave the cells for 30 min at RT in the dark. The fluorophores must be chosen based on parameters that can be assessed with available flow cytometers without requiring a compensation matrix.
6. Wash the cells with 3 mL of FACS buffer.
7. Centrifuge for 5 min at 300 x g at RT to pellet the cells.
8. Pour off the supernatant and resuspend the cells in the remaining liquid.
   1. Repeat this washing step 2x to ensure all unbound antibodies have been removed.
9. Record the samples on the flow cytometer (Table of Materials) and perform data analysis with appropriate software. The gating strategy is presented in Figure 1A–F.

3. Genetic screening for CCR5Δ32 variants in cord blood

1. Incubate 1.25 µL of non-pelleted flow-through with 11.25 µL of PCR mix containing 200 µM of dNTP mix, 0.01 U/µL high fidelity DNA polymerase, and the forward and reverse primers detailed in Table 2.
   1. Adjust the volume with nuclease-free water to approximately 12.5 µL for each PCR reaction.
2. Amplify the genomic fragments with the PCR cycling program detailed in Table 3.
3. Separate the PCR products on a 2% agarose gel¹³.
   1. PCR products from the wild type alleles and the Δ32 alleles yield PCR fragments of 196 base pairs and 164 base pairs bands respectively, making them easily distinguishable by gel electrophoresis¹³ (Figure 1G).

4. Intravenous stem cell transplant

NOTE: Having one person prepare the cells in the laboratory and another person prepare the mice and workspace for transplants is an efficient approach.

1. In an animal facility, 4–6 h before the planned transplantation of the stem cells, irradiate 6–7 week-old female NOD.Cg-Prkdc^scid Il2rg^tm1Sug/JicTac (NOG) mice (Table of Materials) with 0.75 Gy with a Cs¹³⁷ source. The best preconditioning dose may vary based on mouse age, source of radiation, and other factors. This process conditions the animals for successful engraftment with human stem cells.
2. In an animal facility, prepare the flow bench workspace and all reagents before bringing the mice or cells into the workspace.
   1. Place a sterile blue pad to cover the working surface of the flow bench. Prepare sterile gauze and a sharps container.
   2. Place a heating lamp disinfected with 70% ethanol in the flow bench with an empty sterile mouse cage underneath the heat.
3. In the laboratory, thaw isolated CD34+ cells and dilute them in 9 mL of 37 °C plain RPMI.
4. Centrifuge the cells at 350 x g for 5 min at RT, discard the supernatant by aspiration, and resuspend the pellet in 1 mL of plain RPMI at 37 °C.
5. Determine the cell count by trypan blue exclusion, and adjust the volume to 200 µL per mouse. Make extra to take into account possible loss due to the subsequent handling steps.
   1. Prepare to transplant 75,000 CD34+ cells per 200 µL into each mouse.
   2. The cells can be kept at 4 °C during transport to the animal facility before the transplant. Avoid keeping the cells on ice to reduce aggregation or clumping.
6. In the animal facility, bring the cage with the mice into the flow bench and transfer the mice to the cage under the heating lamp to dilate the blood vessels. Leave one end of the cage away from the heat source so that the mice can move away from the heat upon becoming warm. Mice that have moved to the end of the cage, away from the heat source, are sufficiently warm for a successful tail vein injection.
7. Load a 1 mL lubricated syringe to above the 800 µL mark with suspended CD34+ cells. Using a lubricated 1 mL syringe will dramatically ease the intravenous injection and increase the precision of this technique.
8. Attach a 30 G 13 mm needle and prepare the needle and syringe for injection. This order of operation allows for the syringe to be loaded quicker while protecting the integrity of the cells to be transplanted given the possible damage that can occur during the rapid aspiration of cells through such a small gauge needle. Fill the needle hub with liquid by pressing the plunger and remove liquid down to the 200 µL mark of the needle.
9. Place a heated mouse (step 4.6) in a restrainer used for giving IV injections. Carefully inject 200 µL of the cell suspension into the tail vein of the mouse. Spend 2 s performing the plunge and keep the needle inserted for approximately 2 s after completion of the injection. This ensures that the cells have migrated adequately far from the injection site prior to removal of the needle.
10. As necessary, wipe the mouse tail with sterile gauze to remove any visible blood. Put the mouse back into its non-heated home cage. Repeat the injection procedure with the remaining mice.

5. Blood collection and processing for analysis

NOTE: Human cell engraftment in the peripheral blood can be evaluated via flow cytometry 3–5 months after human stem cell transplantation.

1. Draw blood samples from the mice using local IACUC-approved techniques.
2. Collect a maximum of 70–100 µL of total blood into sterile PCR microcentrifuge tubes containing 10 µL of 0.5 M EDTA (pH = 8.0) to avoid coagulation of blood.

6. Evaluation of human engraftment via flow cytometry

1. Transfer 40–50 µL of blood to FACS tubes.
2. Add 5 µL of Fc Receptor blocking solution to prevent nonspecific binding of antibodies and leave for 10 min at RT.
3. Add the mouse anti-human antibody mix containing CD4 (clone SK3) BUV 496, CD8 (clone RPA-T8) BV421, CD3 (clone OKT3) FITC, CD19 (clone sj25c1) PE-Cy7, CD45 (clone 2D1) APC (Table 4) and leave to stain in the dark at RT for 30 min. Fluorophores must be chosen based on parameters that can be assessed with the available flow cytometers without requiring a compensation matrix.
4. Add 2 mL of an appropriate red blood cell lysing buffer to each tube to lyse the red blood cells. Use a lysis buffer specifically formulated for antibody staining prior to red blood cell lysis (a suitable example is given in the Table of Materials). Vortex briefly to ensure equal distribution of the cells in the lysing solution and leave for 10 min at RT. Equal distribution of the cells is important.
5. Add 2 mL of FACS buffer to stop the lysis reaction.
6. Centrifuge for 5 min at 300 x g at RT to pellet the cells.
7. Pour off the supernatant and vortex gently until the cells are resuspended.
8. Add 3 mL of FACS buffer and centrifuge for 5 min at 300 x g at RT.
9. Pour off the supernatant and resuspend the cells.
10. Record the samples on an appropriate flow cytometer and analyze using appropriate software (Table of Materials). Representative analysis and results are depicted in Figure 2 and Figure 3.

7. Intravaginal HIV exposure

NOTE: The virus used for intravaginal exposure of the mice can be produced using previously published protocols¹⁷. The virus is kept at -80 °C and transported between locations while stored on dry ice following locally approved protocols. The virus is stored on dry ice until immediately before the exposure of the mice. The virus can be diluted into plain RPMI (avoid RPMI that has antibiotics or serum additives) to achieve the appropriate concentration immediately prior to exposure (21,400 IUs were used for this IVAG exposure). Once generated, keep the diluted stock on wet ice throughout the procedure to avoiding freeze-thaw cycles that would occur if the diluted virus was placed back on dry ice once thawed.

1. Prepare all the equipment and flow bench workspace as presented in Figure 4 before bringing the mice or virus into the flow bench (similar to step 4.2).
   1. Place the heating lamp focus in the center of the workspace where the mouse will be located during the HIV exposure procedure. The heating lamp will ensure no decrease in body temperature of the mice. Other equipment that controls temperature can also be used, (e.g. a heated gel pad or a circulating warm water blanket, according to local IACUC regulations¹⁸).
   2. Bring sterile 20 µL pipette tips and an appropriate pipette into the bench. Place a container with liquid disinfectant (Table of Materials) in the bench for immediate inactivation of materials and liquids that have been in contact with the virus.
2. Place a mouse into a chamber with 3% isoflurane gas and paper towels. This percentage of gas will anesthetize the animals within 2–4 min. As with all other materials used with immunodeficient mice, the anesthesia apparatus must be properly disinfected prior to use.
3. Once anesthetized, transfer the mouse to a sterile blue pad under the heating lamp. Insert the mouse snout into a mask supplying continuous 3% isoflurane gas to maintain anesthesia. Hold the mouse at the base of the tail, stomach facing up, with your hand supporting the mouse back as depicted in Figure 4.
4. With a sterile pipette tip, stimulate the genital area by gently stroking upwards towards the anus to induce emptying of the rectum, relieving pressure on the vagina.
5. Carefully bare the vaginal opening by wrapping the mouse tail across your fingers such that the vulva naturally opens, perhaps with slight nudging using a sterile pipette tip.
6. Change the pipette tip and pipette 20 µL of virus atraumatically into the mouse vagina without creating bubbles. Do not insert the tip deep into the vagina. Rather, with the vulva opened, place the pipette tip at the level of the vaginal opening, avoid going deeper to eliminate the potential for abrasions during the inoculation process, release the virus, and allow gravity to pull the virus into the vagina. Alternatively, use a 22 G 1.25 mm blunt-end, straight needle, as described in Veselinovic et al.⁶.
7. Retain the mouse in this position with the vagina facing up for 5 min after exposure to avoid gravity-induced leakage of the virus suspension.
   1. Carefully place the mouse into the home cage, taking care to place the mouse on its back.

8. Processing of blood samples prior to viral load analysis

1. Collect blood as described in section 5 above.
2. Centrifuge the blood samples for 5 min at 500 x g at RT to separate the plasma and cells.
3. Collect 40 µL of plasma for viral load measurement into a new sterile PCR-approved microcentrifuge tube and store at -80 °C for at least 1 h until further processing. It is important to freeze all samples before RNA extraction to avoid the risk of bias from comparing RNA levels in samples that have not been frozen prior to RNA isolation to samples frozen prior to RNA isolation.
4. Adjust the volume of blood back to the original volume by adding 40 µL of suspension media (PBS with 2.5% bovine serum albumin, 50 U/mL penicillin G and streptomycin, and 10 U/mL DNase, sterile-filtered at 0.22 µm).
5. Transfer 15 µL of the adjusted blood volume to a new PCR-approved microcentrifuge tube.
6. Add 1 mL of 1x RBC lysis solution (Table of Materials), vortex, and incubate for 10 min at RT.
7. Centrifuge for 1 min at 9,600 x g at RT to pellet cells.
8. Aspirate supernatant and leave only the tiny white cell pellet, because red blood cell contamination can inhibit PCR.
9. Store pellet at -80 °C for at least 1 h until further processing.
   NOTE: Optionally, any remaining blood from step 8.4 can be used for flow cytometry analysis, as described above in step 6.

9. DNA extraction using a proteinase K extraction method

1. Extract DNA from peripheral cell pellets (generated in step 8.8) using a proteinase K extraction method as described below. This method has been demonstrated to extract the highest DNA yield from a small volume of blood such as that required for the serial blood collections utilized in this study¹⁹.
2. Add 25 μL of proteinase K (20 μg/mL) to 1 mL of 0.1M Tris buffer.
3. Vortex the proteinase K solution briefly.
4. Add 50 μL of the proteinase K solution to each cell pellet to be digested.
5. Mix by pipetting up and down and confirm the resuspension of the cell pellet.
6. Shake on a thermoshaker (Table of Materials) at 400 rpm (depending on the instrument) at 56 °C for 1 h. Tape tubes down to hold them in place, if necessary.
7. Immediately and in the same thermoshaker, inactivate proteinase K with a temperature shift to 95 °C while the shaking continues for an additional 20 min.
8. Vortex each sample.
9. Place each sample at -80 °C for a minimum of 30 min.
10. Thaw, then centrifuge the samples at 17,000 x g for 1 min at RT to pellet unwanted cellular fragments.
11. Place the DNA-containing supernatant into a new microcentrifuge tube.
12. The DNA template is ready for PCR. The DNA templates can be stored at -80 °C.

10. RNA extraction, cDNA synthesis, and ddPCR quantification of viral RNA

1. Isolate RNA from thawed mouse plasma with a virus RNA isolation kit following the manufacturer's protocol (Table of Materials).
2. After addition of the sample to the column, add an on-column DNase treatment step to ensure removal of all DNA in the plasma sample.
   1. For each sample, add 95 µL of RNase-free DNase solution (mix 2 µL RNAse-free DNase and 98 µL reaction buffer) to the column and incubate for 15 min at RT.
3. Store the RNA samples at -80 °C for at least 1 h before further processing. It is important to freeze all samples after RNA extraction do avoid the risk of bias when comparing samples that have not been frozen to samples that were frozen.
4. Synthesize cDNA using a reverse transcriptase step using reagents as described previously²⁰. Make sure to add 0.5 µL of an RNase inhibitor to the cDNA reaction to avoid degradation of the RNA.
   1. Perform cDNA synthesis with the program detailed in Table 5.
5. Store the cDNA samples at -80 °C for at least 1 h. It is important to freeze all samples after cDNA synthesis do avoid the risk of bias when comparing samples that have not been frozen to samples that were frozen.
6. Prepare samples for ddPCR as follows²⁰:
   1. Mix 3 µL of the cDNA sample with 11 µL of the ddPCR probe mixture (no dUTP)²⁰, 250 nM minor groove-binding probe, and 900 nM of each of the forward and reverse primers as detailed in Table 6.
   2. Adjust the total PCR volume to 22 µL with nuclease-free water.
7. Emulsify the PCR mixes with Droplet Generation Oil for Probes on a droplet generator according to the manufacturer's protocol and previous descriptions²⁰.
8. Run the PCR program as detailed in Table 7.
   NOTE: The primer/probe sequences and PCR programs displayed here have been specifically designed and optimized for sensitive detection of the HIV strain RHPA4259. Primer and probe sequences can easily be adjusted to detect any other HIV strain of choice.
9. Detect droplet fluorescence from the samples on a droplet reader, and analyze the results with appropriate software according to the manufacturer's protocol.

11. Treatment with cART-containing chow

1. Feed mice with pellets containing a standard cART regimen containing 4,800 mg/kg raltegravir (RAL), 720 mg/kg tenofovir disoproxil fumarate (TDF), and 520 mg/kg emtricitabine (FTC)²¹ (Table 8). These doses were determined assuming that a mouse weighs 25 g and eats 4 g of chow per day. This corresponds to a daily dose of 768 mg/kg RAL, 2.88 mg/kg TDF, and 83 mg/kg FTC²¹.
2. Use a cART diet that has been prepared by an external vendor (See Table of Materials) of prescription drugs. Other companies could potentially also produce this regimen. Use a cART diet colored red to easily distinguish it from ordinary mouse chow.
   1. Produce a control chow diet without cART in a standard brown color for easy distinction.
3. For initiation of cART, prepare sterile mouse cages with the addition of cART-containing chow diet, and then transfer the mice from the old cage to the new cage.
   1. Monitor the weights of the mice and consumption of cART-containing chow by visual inspection to ensure that the mice are adjusting to the change.