Experimental Procedures

Preparation of OLP peptide pools:

• • • Spans 15 amino acids (ie. 15-mers)
    • Peptide offset: 5 amino acids apart

• Peptide pools:
  • PreCore/Core = 41 peptides
  • X = 29 peptides
  • Env - 1 =   peptides   1 – 181  (37 peptides)
  • Env – 2 =   peptides   186 - 376 (36 peptides - 3 peptides not synthesized: 241, 246, 251)
  • Pol-1 = peptides  1 – 206  (42 peptides)
  • Pol-2 = peptides  211 – 416  (42 peptides)
  • Pol-3 =  peptides 421 – 626  (42 peptides)
  • Pol-4 =  peptides  631 – 831 (41 peptides)

Preparation of 50X OLP peptide pool

• PreCore/Core 50x
  • 41 peptides x 10 µl /peptide = 410 µl of peptides + 590 ml Aim-V + Prim

• X 50x stimulation pool
  • 29 peptides x 10 µl /peptide =  290 µl of peptides + 710 ml Aim-V + Prim

• Envelope 50x stimulation pool
  • Env-1    37 peptides x 10 µl /peptide =  370 μl of peptides + 630 ml Aim-V + Prim
  • Env-2    36 peptides x 10 µl /peptide =  360 µl of peptides + 640 ml Aim-V + Prim

• Polymerase 50x stimulation pool
  • Pol-1 42 peptides x 10 µl /peptide =  420 µl of peptides + 580 ml  Aim-V + Prim
  • Pol-2 42 peptides x 10 µl /peptide =  420 µl of peptides + 580 ml  Aim-V + Prim
  • Pol-3 42 peptides x 10 µl /peptide =  420 µl of peptides + 580 ml  Aim-V + Prim
  • Pol-4 41 peptides x 10 μl /peptide =  410 μl of peptides + 590 ml  Aim-V + Prim

Ex Vivo IFNγ ELISpot – Cell prepping             

(General Protocol)

Materials:

• 10⁷ frozen PBMCs (per donor; maximum 6 donors per plate)
• 30ml polypropylene tube (one per donor sample)
• Eppendorf tubes
• HBSS medium (Gibco, Ref# 24020-117)
• AIM V medium (Gibco, Ref# 12055-091)
• Knockout serum Replacement (KSR) (Lifetech: 10828028)
• Primocin ( 1ml of 50mg/ml: InvivoGen)
• Human Blood type-AB serum (VWR, CA45001-062)
• 50X HBV overlapping peptides, genotype C (GenScript)
• CEF purified peptide library (GenScript): (USED to monitor treatment effect on unrelated virus-specific T cells)
• DMSO solution (Sigma, Ref# D8418)
• Dynabeads^TM Human T-activator CD3/28 (Gibco, Ref# 11131D) (USED as positive/Assay Control)

Day 1: Resting cells (pre-warm media)

1. Thaw 10⁷c with HBSS medium (as per protocol) in a 30ml polypropylene tube a. Minimum count required for experiment: 6 x 10⁶cells/donor
2. Centrifuge at 300xg for 5mins., aspirate
3. Resuspend at approximately 4x10⁶ cells/ml with AIM V (+2% human serum)
4. Take counting aliquot for pre-rest counts a. fix cell concentration if necessary (3.5-4.5x10⁶ cells/ml are suitable as well)
5. Incubate O/N in 37^oC incubator @ 5% CO₂
6. Coat IPFL plates, fridge O/N in 4^oC (as per manufacturer’s, see next pages)

Day 2a: Pulsing cells (pre-warm media)

1. 16-18h later, resuspend samples and take counting aliquot for post-rest counts
2. Aliquot two Eppendorf tubes with 4.5 x 10⁵cells each (per donor) a. 1 tube for HBV OLP stimulation, another for DMSO vehicle control
3. Centrifuge at 300xg for 5mins., aspirate
4. Resuspend both tubes with 84µl AIM V (+2% human serum) each
5. Pulsing scheme: final volume of 100µl a. HBV OLP sample: add 2µl of 250µg/ml/peptide per OLP pool (8 pools: 16µl) i. HBV OLP final concentration: 5µg/ml/peptide b. DMSO control sample: add 16µl of 19.38% DMSO (in AIM V) i. DMSO final concentration: 3.1% DMSO (equivalent to OLP sample)
6. Incubate for 1h in 37^oC incubator @ 5% CO₂
7. Block IPFL plate simultaneously (as per protocol, see next page)
8. Centrifuge both tubes of pulsed cells at 300xg for 5mins., aspirate a. Resuspend cells with 225µl AIM V (no serum) (ie. 2 x 10⁶cells/ml)
9. Aliquot two Eppendorf tubes with 8 x 10⁶cells each (per donor); centrifuge, aspirate a. Resuspend cells with 450µl AIM V (no serum) (ie. 4 x 10⁶cells/ml) b. Pool respectively with pulsed cells c. Wash tube with another 450µl AIM V (no serum) and pool respectively d. Final volume:25x10⁶ PBMCs in 1.125ml AIM V (ie. 2 x 10⁶cells/ml)
10. Aliquot 1.2 x 10⁶cells for CEF controls; centrifuge, aspirate a. Resuspend cells with 588µl AIM V and 12µl 50x CEF (ie. 2 x 10⁶ cells/ml)
11. Aliquot 1 x 10⁵cells for CD3/28 controls; centrifuge, aspirate a. Resuspend cells with 400µl AIM V and 0.4µl CD3/28 (ie. 2.5 x 10⁵ cells/ml)

Day 2b: Plating cells

1. Color scheme for plating: a. Blue wells: 100µl CD3/28 controls (2.5x10⁴cells per well) b. Purple wells: 200/200/100µl CEF controls (4x10⁵cells/2x10⁵cells per well) c. Red wells: 200µl DMSO controls (4x10⁵cells per well) d. Green wells: 200µl OLP pulsed cells (4x10⁵cells per well)
2. Plating totals: a. CD3/28: 5x10⁴cells in 3 wells b. CEF: 1x10⁶cells in 3 wells c. DMSO:2x10⁶cells in 5 wells d. OLP: 2x10⁶cells in 5 wells
3. Incubate for 20h in 37^oC incubator @ 5% CO₂

Day 3: Plate development (20 hours after plating cells)

1. Develop plate the next day (as per plate prep protocol)

Ex Vivo IFNγ ELISpot –Plate prepping

Materials:

• 96-well multiscreen PVDF filtration plate: Millipore; Cat. Num. MSIPS4W10
• α-Human IFN-γ capture ab: ImmunoSpot
• α-IFN-γ Biotin ab: ImmunoSpot
• Streptavidin-ALP: MABTEC AB; Code 3310-10
• KPL BCIP/NBT Phosphate substrate (KPL Substrate) (Sera Care: Cat No: 5420-0038)

Day 1: Plate coating

1. Prepare coating Ab: Stock 1mg/ml; Final 5µg/ml
   • Add 25µl of IFNγ capture Ab in 5ml of sterile PBS (for 45 wells)
2. Activate wells by adding 15µl of 35% ethanol NOTE: While activating the well with ethanol, don’t let it sit. No well, should be exposed to ethanol for more than 60 sec.
3. Wash the plate 6 times with sterile water
4. Place 100µl of coating Ab solution into each well
5. Parafilm and incubate plate at 4^oC O/N

Day 2: Setting up plate

1. Wash the plate 6 times with sterile water
2. Make blocking solution, AIM V – 10% KSR
   • Add 45ml of AIM V to 5ml of KSR
3. Add 100µl blocking solution into each well
4. Incubate the plate at room temperature for at least 30 minutes
5. Remove via flicking
6. Add PBMCs to the plate after pulsing (as per Cell prep protocol, Page 1)
7. Incubate the plate @ 37^oC O/N

Day 3: Plate development (24 hours after plating cells)

1. Prepare α-IFN-γ Biotin: Stock: 0.5mg/ml; Final: 0.5µg/ml
   • Add 5µl anti-human IFNγ mAb Biotin in 5ml sterile PBS (for 45 wells)
2. Wash the plate 6 times with PBS
3. Place 100µl of 0.5ug/ml α-IFN-γ solution into each well
4. Incubate plate at room temperature for 2 hours
5. Prepare Streptavidin-ALP: Stock: 0.5µg/ml; Final: 0.25ng/ml
   • Add 5µl Streptavidin-enzyme in 5ml sterile PBS (for 45 wells)
6. Wash the plate 6 times with non-sterile PBS
7. Add Streptavidin. Place 100µl solution into each well
8. Incubate the plate at room temperature for 30 minutes
9. Wash the plate 6 times with non-sterile PBS
10. Add 50µl of KPL Substrate solution into each well.
11. Incubate in the dark at room temperature for 20 minutes.
12. Remove plate underdrain and wash underside with running water, flick, and repeat ~10 time; flick dry as much as possible NOTE: Avoid flicking on paper towels to minimize dust/particulates in wells
13. Dry ~20-30mins in the BSC (with underside upwards) in the dark
14. Scan and count plates