In vivo therapeutic efficacies and neural toxicity of F9170 in SHIV-infected rhesus macaques

1. Determination of does for F9170 administration

1.1 Intravenously inject two female ICR mice (6- to 8-week-old) with 100 μl of peptide F9170 (20 mg/kg). 

1.2 Collect 50 μl of blood of mice via saphenous bleeding at 0, 15, 30 min, and 1.5, 3, 6, 9, 12, 24 hours after injection (the detailed bleeding operation was explicitly presented in JOVE Blood Withdrawal II: https://www.jove.com/science-education/10247/blood-withdrawal-ii). 

1.3 Place the blood samples at room temperature for coagulation for 1 h. Collect the serum after centrifugation at 3,000 g for 10 min. 

1.4 Assess the serum concentration of F9170 in these samples by LC-MS/MS. 

1.5 Under conversion based on body surface area, a concentration that is supposed to result in a Cmax of ~10 μM, a safe and effective concentration for peptide F9170 in entire in vitro studies, was chosen for rhesus macaque model.

2. Efficacies of F9170 in SHIV-infected rhesus macaques

2.1 Eight experimental female rhesus macaques (8 to 10 years old) were confirmed to be negative for SIV, SRV, herpes B virus, and simian T-lymphotropic virus.

2.2 Expand virus (SHIVSF162P3) on macaque PBMCs. Add one milliliter of stock virus to 20 ml of macaque PBMCs (10⁵/ml). Culture the cells in DMEM containing 10% fetal bovine serum for 3-4 days. Collect the viruses in the supernatant by a centrifugation at 3,000 rpm for 10 min and an filtration by 45 μm filter. Aliquot the harvested viruses and store them at -80 ^oC freezer.

2.3 Determine the TCID₅₀. Sequentially add a series of dilutions at 1:10 of the original virus sample (100 μl) to 3 x 10⁴cells in 100 μl culture medium in 96-well plate. After 3-day culture, calculate the TCID₅₀ by the cytopathic effect.

2.4 Inoculate intravenously with 1000 x TCID₅₀ of viruses to every rhesus macaque.

2.5 Collect blood samples periodically. Perform the qRT-PCR assay for measurement of plasma viral loads (the sequences for primers and probes are shown in Table 1, the cycling protocol: 48°C for 30 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, and 60°C for 1 min).

2.6 Establish stable chronic SHIV infection for 6 months.

2.7 Randomly assign eight macaques to two treatment groups (four monkeys per group). Intramuscularly inject one milliliter of 0.9% saline solution or F9170 in saline solution (3 mg/kg) twice daily for the first week and once daily for the second week.

2.9 Perform the qRT-PCR assay for measurement of plasma viral loads (the sequences for primers and probes are shown in Table 1, the following cycling protocol: 48°C for 30 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, and 60°C for 1 min).

Table 1 Primers and probes for detection of viral loads by qRT-PCR

3. Neural toxicity assessment of F9170 in SHIV-infected rhesus macaques

3.1 Execute euthanasia of the eight macaques in the above test and another uninfected macaque with ketamine hydrochloride.

3.2 Dissect out frontal cortex of the macaque brains for the following assessment.

3.3 Fix the brain sample in formalin for 24 hours at 4 ^oC. Embed the fixed samples in paraffin.

3.4 Sequentially place the paraffin slices into xylene I for 15 min, xylene II for 15min, xylene III for 15 min, ethanol I for 5 min, ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and distilled water.

3.5 Antigen recovery: place tissue slices in a box filled with citric acid buffer (pH 6.0). Heat the box in the microwave oven (medium heat for 10 min, cooling at room temperature for 10 min, low heat for 7 min). Naturally cool the slices and put the slices in PBS buffer (pH 7.4). Shake and wash the slices for 3 times.

3.6 Block endogenous peroxidase: place the slices in 3% hydrogen peroxide solution at room temperature for 25 min. Wash the slices in PBS (pH 7.4) for 3 times.

3.7 Cover the samples with 3% BSA and culture them at room temperature for 30 min. 

3.8 Gently shake off the BSA and add 1:500 dilution of anti-NeuN antibody (ab177487, Abcam) or 1:1000 dilution of anti-GFAP antibody (GB11096, Servicebio) on the slice. Culture the slices in a wet box at 4 ^oC overnight.

3.9 Wash the slices in PBS (pH 7.4) for 3 times and add 1:200 solution of HRP-labeled anti-rabbit antibody (G1213, Servicebio). Culture the slices at room temperature for 50 min.

3.10 Wash the slices in PBS (pH 7.4) for 3 times and add formulated DAB substrate. Observe the slices under microscope. Rinse the slices with tap water to terminate the process.

3.11 Culture the slices with Harris hematoxylin at  room temperature for 3 min. Sequentially rinse the slices with tap water, 1% ethanol in HCl, tap water, ammonium hydroxide, and tap water.

3.12 Sequentially place the slices into 75% ethanol for 5 min, 85% ethanol for 5 min, ethanol I for 5 min, ethanol II for 5 min, xylene I for 5 min. Dry the slices in room temperature and seal them with mounting medium.

3.13 Scan these slices under microscope. Two random fields of view were analyzed per section. The NeuN+ and GFAP+ areas were measured with ImageJ (https:// imagej.nih.gov/ij/).

NeuN-staining macaque brain

GFAP-staining macaque brain 

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