Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR

Materials and Reagents

1. Pipette tips (Neptune, 1,000 μl, 200 μl, 20 μl, 10 μl, DNase-/RNase-free & Biozym, premium tips 1000 μl, 200 μl, 10 μl)
2. 1.5 ml microcentrifuge tube (Sarstedt AG & Co.KG, SafeSeal tube, catalog number: 72.706)
3. 0.2 ml microcentrifuge tube (Greiner Bio-one, Sapphire PCR tube, catalog number: 683271)
4. Hard-Shell PCR plates 96-well, thin-wall (Bio-rad, catalog number: HSP9601)
5. Primary human hepatocytes (PHH) (obtained from Hannover medical school or prepared in University Hospital Heidelberg) (Possible commercial vendors of PHH are Lonza, Biopredic, BioIVT, ThermoFisher, however susceptibility to HBV may vary), HepaRG^NTCP and HepG2^NTCP cell lines (Qu et al., 2018)
6. Plasmid pSHH2.1 (Cattaneo et al., 1983), available upon request from the corresponding author
7. Primers:
   Primer p1040: 5’-GTGGTTATCCTGCGTTGAT-3’
   Primer p1996: 5’-GAGCTGAGGCGGTATCT-3’
   p1578: 5’-CCGTGTGCACTTCGCTTCA-3’
   p1867: 5’-GCACAGCTTGGAGGCTTGA-3’
   p1583: 5’-TGCACTTCGCTTCACCT-3’
   p2301: 5’-AGGGGCATTTGGTGGTC-3’
8. Probe
   Probe p1085: 5’-FAM-AGTTGGCGAGAAAGTGAAAGCCTGC-TAMRA-3’
9. Trypsin [0.25% Trypsin-EDTA (1x)] (Invitrogen, Gibco, catalog number: 25200-056), 4 °C
10. NucleoSpin Tissue kit (Macherey-Nagel, catalog number: 740952.250)
11. Buffer T1 (Macherey-Nagel, catalog number: 740940.25)
12. Proteinase K (Macherey-Nagel, catalog number: 740506), -20 °C
13. Buffer B3 (Macherey-Nagel, catalog number: 740920)
14. Ethanol absolute (VWR chemicals, catalog number: 20821.330)
15. Buffer BW (Macherey-Nagel, catalog number: 740922)
16. Buffer B5 Concentrate (Macherey-Nagel, catalog number: 740921)
17. T5 exonuclease (New England Biolabs, catalog number: M0363), -20 °C
18. NEBuffer 4 (New England Biolabs, catalog number: B7004), -20 °C in aliquots
19. PerfeCTa qPCR Toughmix (Quanta Biosciences, catalog number: 95112-012), -20 °C in aliquots
20. SYBR Green Supermix (Bio-Rad, catalog number: 172-5121), -20 °C in aliquots
21. Myrcludex B (Bachem), available upon request from the corresponding author, -80 °C in aliquots
22. Recombinant human Interferon-α-2a (PeproTech, catalog number: 300-02AA), -80 °C in aliquots
23. Human Interferon-α-2a (PBL Assay Science, catalog number: 11100-1), -80 °C in aliquots
24. Nuclease-free water (B. Braun Melsungen, Aqua ad iniectabilia Braun)
25. NaCl (Carl Roth GmbH, catalog number: 9265.2)
26. KCl (Sigma-Aldrich, catalog number: 31248)
27. Na₂HPO₄·2H₂O (Sigma-Aldrich, catalog number: 04272)
28. KH₂PO₄ (Sigma-Aldrich, catalog number: P9791)
29. Tris-base (Carl Roth GmbH, catalog number: 4855.2)
30. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: 1.02950)
31. Fetal bovine serum gold (PAA Laboratories GmbH, catalog number: A15-151), -20 °C
32. Penicillin/Streptomycin (Thermo Fisher Scientific, catalog number: 15140-122), 4 °C
33. L-glutamine (Thermo Fisher Scientific, catalog number: 25030-024), 4 °C
34. MEM non-essential amino acids (Thermo Fisher Scientific, catalog number: 11140-035), 4 °C
35. Recombinant insulin (Sigma-Aldrich, catalog number: 91077C-1G), -20 °C in aliquots
36. Hydrocortisone 21-hemisuccinate sodium salt (Sigma-Aldrich, catalog number: H4881), -20 °C in aliquots
37. Culture medium (see Recipes)
38. 10x PBS buffer (see Recipes)
39. 5 mM Tris-HCl (see Recipes)
    

Equipment

1. Pipettes (Gilson P1000, P200, P20; Eppendorf P2.5)
2. Cell counter (Bio-Rad, TC20^TM Automated Cell Counter, catalog number: 1450102)
3. Thermomixer (Eppendorf, ThermoMixer C, catalog number: 5382000015)
4. Microcentrifuge (Thermo Fisher Scientific, Heraeus^TM Pico^TM 21 Centrifuge, catalog number: 75002553)
5. Thermocycler (Labrepco, Biometra T3000 Thermocycler 48, catalog number: 050-723)
6. qPCR thermocycler (Bio-Rad, C1000 Touch^TM Thermal cycler with 96-well Fast Reaction Module, catalog number: 1851196)
   

Software

1. CFX96 Real-time System
   (Bio-Rad, http://www.bio-rad.com/en-us/product/cfx96-touch-real-time-pcr-detection-system)
2. CFX Manager^TM Software
   (Bio-Rad, http://www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000)
   

Procedure

Figure 1. Working flowchart of this protocol. Susceptible cells (PHH, HepaRG^NTCP, HepG2^NTCP) infected with high mges (> 300) of HBV are lysed. A. Lysates are incubated at 70 °C in the presence of proteinase K and later loaded on silica column and eluted. B. T5 exonuclease (5 units/1 h) removes host genomic DNA (black) and HBV replicative intermediates (rcDNA, dslDNA, etc.) (green) and preserves cccDNA (red) intact. C. cccDNA is amplified by p1040/p1996 and detected by probe p1085.

1. HBV DNA extraction from in vitro infected hepatocytes (Figure 1A)
   
   1. For infection performed in a 24-well plate (culture area: 2 cm²), trypsinize and resuspend infected PHH, differentiated HepaRG^NTCP or HepG2^NTCP cells (infection procedure see Ni and Urban, 2017) in 1 ml of culture medium. Take 10 μl of the resuspension for cell count by a cell counter (see Equipment). Calculate total cell numbers.
      Notes:
      
      1. At confluency, cells in one well of a 24-well plate approximately correspond to 180,000 PHH, 200,000 HepaRG^NTCP and 500,000 HepG2^NTCP cells.
      2. When smaller or larger plates are used, volumes of the respective buffers given in the protocol should be adjusted according to the respective culture area (cm²) of a well.
   2. Spin down the cells (900 x g, 5 min) at room temperature. Wash cells with 1 ml of PBS twice. Spin down the cells and carefully aspirate PBS (Optional: freeze cell pellet at -20 °C for long-term storage). For DNA extraction, manufacturer’s manual of the NucleoSpin Tissue kit (740952.250) is adapted with a minor modification regarding the incubation time in Step A2 (1 h instead of 10~15 min) and the elution volume in Step A6 (50 μl instead of 100 μl). Add 200 μl of lysis buffer T1, pipet up and down and incubate at room temperature for 10 min. Add 25 μl of proteinase K and 200 μl of lysis buffer B3, vortex and incubate the lysate at 70 °C for 1 h with shaking (500 rpm).
      Note: After proteinase K digestion, it is not necessary to re-centrifuge the lysates. Immediately perform the next step.
   3. Add 210 μl of ethanol (96%-100%) to the sample and vortex vigorously for a few seconds. Remove buffer from the lid by a short centrifugation and apply the samples to the column. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through.
   4. Add 500 μl of wash buffer BW. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through.
   5. Add 600 μl of wash buffer B5 (pre-add indicated volume of ethanol to the Buffer B5 Concentrate) to the column. Centrifuge (11,000 x g, 1 min) at room temperature. Discard flow-through. Repeat this step once.
   6. Remove residual ethanol from the column (13,000 x g, 1 min). Place the column into a 1.5 ml microcentrifuge tube. Add 50 μl of pre-warmed (70 °C) elution buffer. Incubate at room temperature for 1 min. Centrifuge (11,000 x g, 1 min). Freeze eluted DNA samples at -20 °C until use or analyze them immediately.
      Notes:
      
      1. Elution buffers are nuclease-free water or 5 mM Tris-HCl (pH = 8.0). Do not use buffers with EDTA that may inhibit enzymatic activity in the next step.
      2. DNA concentration in eluates is 200-1,000 ng/μl.
      3. Preserve at least 10 μl of the sample without T5 exonuclease digestion for quantification of β-globin (internal standard) for normalization use.
   
   
2. T5 exonuclease hydrolysis of total DNA elutes (Figure 1B)
   
   1. Assemble the reaction components in a 0.2 ml microcentrifuge tube following the table as shown:
      
      
      

   Note: Do not exceed unit and incubation time of T5 exonuclease since overdigestion leads to partial loss of cccDNA.

   2. Incubate the reaction at 37 °C for 1 h and inactivate the enzyme at 70 °C for 20 min in a thermocycler with heat-lid supply.
   3. Proceed quantification by real-time qPCR or freeze the products at -20 °C until use.
      
3. Quantification of cccDNA relative to human β-globin (single-copy gene) by real-time qPCR (Figure 1C)
   
   1. Quantification of cccDNA
      
      1. Dilute primers and probe to 10 μM:
         
         FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine
         
         
      2. Prepare the following qPCR reactions in a 96-well Hard-Shell PCR plate:
         
         ^#for absolute quantification, load 2 µl of each 1:10 serial-diluted plasmid (pSHH2.1: 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10² copies/μl) as templates on the same plate.
         
         
      3. Run qPCR program below:
         
         
         
      4. Read absolute cccDNA copy numbers in the CFX Manager^TM Software. Calculate cccDNA copies per well.
   2. Quantification of human β-globin prior to T5 exonuclease digestion
      
      1. Dilute primers at 10 μM:
         
         
         
      2. Prepare qPCR reactions in a 96-well plate:
         
         
         
      3. Start qPCR program below:
         
         
         
      4. Read levels of human β-globin. Normalize cccDNA copies per well using values of human β-globin. Calculate cccDNA copies per cell (copies per well divided by cell numbers per well). For instance, if cell numbers in a well of 24-well plate are 4.2 x 10⁵ (as determined in Procedure A1) and cccDNA copies per well are 8.52 x 10⁵ (as determined in Procedure C), cccDNA copies per cell are 2.03.

Data analysis

cccDNA copy number per cell or infected cell is calculated according to Figure 2. The cccDNA level in in vitro infected cells can be determined similarly as two examples shown in Figure 3 and Qu et al., 2018. Note that to discriminate signals from the inoculum, we highly recommend using an entry inhibitor (e.g., Myrcludex B) as a control. Routinely Myrcludex B control gives a value below 0.05 copy per infected cell.


Figure 2. Schematic diagram of the calculation of absolute cccDNA copy numbers. A. On the day of experiment, cell number in the well is determined. B. Total DNA samples are extracted as suggested in Procedure. C. Here shows a proper T5 exonuclease digestion as shown in Procedure. D. Absolute cccDNA copies and E level of human β-globin are quantified, respectively as shown in Procedure. “cccDNA copies/cell” is “cccDNA copies/well” divided by “cell numbers”. Optional: F. If calculation of copies per infected cell is required, additional wells have to be arranged in parallel on the same plate during the infection. On the day of DNA extraction, cells in the wells are fixed and subjected to an immunofluorescence assay (HBcAg visualization using DAKO B0586 antibody, etc.) to determine infectivity (%: number of HBcAg positive cells divided by number of total cells) (Qu et al., 2018). “cccDNA copies/infected cell” is the “cccDNA copies/cell” divided by “infectivity (%)”.


Figure 3. cccDNA levels in three in vitro. models and upon IFN-α-2a and Myrcludex B treatment. A. PHH, differentiated HepaRG^NTCP and HepG2^NTCP cells were infected with HBV at mges/cell of 500. Myrcludex B (1 μM), an entry inhibitor blocking cccDNA formation, was co-administered with HBV inoculum during infection. On Day 7 post infection, cccDNA copies per infected cell were determined. Data shown in triangles were collected from three independent experiments. B. HepG2^NTCP cells were infected with HBV at mges/cell of 500 with mock treatment (untreated), co-treated with Myrcludex B (1 μM) during infection, or co- and post-treated with IFN-α-2A at 100 ng/ml purchased from PeproTech and PBL Assay Science. On Day 7 post infection, cccDNA copies per infected cell were analyzed. Statistics: P < 0.01 untreated versus IFN-treated; P = 0.828 PeproTech versus PBL.

Notes

The primer pair (pp1040-1996) is specific for the formed cccDNA in genotype D. Genotype D is the HBV genome in HepAD38 cell line. Users should consider this issue if they try patient-derived serum to perform in vitro infection using this protocol since HBV in patients can be any genotype and pp1040-1996 does not bind the formed cccDNA of other genotypes.
   Genbank accession numbers of other genotypes: A (HE974370.1); B (AB540582.1); C (AB540584.1); E (HE974384.1); F (HE974369.1); G (AP007264.1); H (AB846650.1). pp1578-1867 and pp1583-2301 (Qu et al., 2018) allowing quantification of eight HBV genotypes (A-H) are suggested below. (Table 1)

Table 1. List of primer mismatches to HBV genotypes. The numbers of base-pair mismatch of each forward and reverse primers to eight HBV genotypes are summarized below. Green squares: perfect binding; yellow squares: one base-pair mismatch still allowing template binding; red squares: not binding.

F: forward primer; R: reverse primer. #: The probe of this protocol is not pan-genotypic and has mismatch number as shown: A(1), B(2), C(0), D(0), E(0), F(6), G(1), H(4).

Recipes

1. Culture medium
   For HepG2^NTCP cells: DMEM supplemented with 10% fetal calf serum (heat-inactivated), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids. For infection, add 2% dimethyl sulfoxide
   For PHH and HepaRG^NTCP cells: William’s medium E supplemented with 10% fetal calf serum (heat-inactivated), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids, 5 μg/ml recombinant insulin, 50 μM hydrocortisone. For differentiation and infection, add 1.5% dimethyl sulfoxide
2. 10x PBS
   Dissolve 80 g of NaCl, 2 g of KCl, 11.5 g of Na₂HPO₄·2H₂O and 2 g of KH₂PO₄ in 800 ml of H₂O, adjust pH to 7.4, refill H₂O to 1 L
3. Tris-HCl (1 M)
   Dissolve 121.14 g of Tris-base in 800 ml of H₂O, adjust pH to 8.0 and refill H₂O to 1 L
   

Acknowledgments

This protocol was adapted from Qu et al., 2018. We herein acknowledge financial supports by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–Project number 272983813–SFB/TRR179 (B.Q. and S.U.) and German Center for Infection Research (DZIF) TTU Hepatitis projects 05.704 (S.U.). We thank Pascal Mutz (DKFZ, Germany) and Florian WR Vondran (MHH, Germany) for providing PHH. We are grateful to Xue Li for critical reading of this manuscript.

Competing interests

Prof. Dr. Stephan Urban, the corresponding author, holds patents and intellectual property on Myrcludex B.

Ethics

Following written informed consent of the patients, PHH were isolated from liver specimens obtained after partial hepatectomy.

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