Cell lines and plasmid clones
Huh-7.5 cells were obtained from the laboratory of Dr. Charles M Rice (Center for the Study of Hepatitis C, The Rockefeller University, New York) and cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, San Diego, CA) with high glucose supplemented with non-essential amino acids, sodium pyruvate and 5% fetal bovine serum. A full-length JFH-1 clone and replicon plasmid (pSGR-JFH1) were obtained from Dr. Takaji Wakita [37
] (National Institute of Infectious Diseases, Tokyo, Japan). Full-length and sub-genomic JFH1-GFP was constructed by inserting the coding sequence of green fluorescence protein in the NS5A gene described before [38
Development of stable Huh-7.5 cell lines expressing IgG1 antibody
The construction of plasmid vector pFab-CMV-NS3 (L+H) containing light chain, heavy chain, CH2-CH3, and part of the hinge region for a germline immunoglobulin 1 was described previously [30
]. This vector allows conversion of recombinant Fab antibody into a complete IgG1 antibody in the transfected cells. Huh-7.5 cells were electroporated with 10 μg of pFab-CMV (H+L) plasmid DNA. After 24 hours, cells were selected with a growth medium containing G-418 (500 μg/ml). A control stable Huh-7.5 cell line was prepared that expressed IgG1 antibody targeted to influenza A viruses (clone Fab-9) [39
]. These two Huh7.5 lines stably expressing intracellular antibody were cultured in a growth medium containing G-418 (500 μg/ml). Intracellular expression of IgG1 antibody in these two stable cell lines was confirmed by immunofluorescence microscopy. Briefly, cells cultured in chamber slides were washed with phosphate-buffered saline (PBS) pH 7.4 twice, air-dried and fixed with chilled acetone for 5 min. The cells were permeabilized by the treatment with 0.05% saponin for 10 min at room temperature. Blocking was performed with 5% fetal bovine serum (FBS) diluted in a minimum essential medium for 5 min at room temperature. The slides were washed with PBS thrice for 5 min each. The cells were incubated with goat anti-human phycoerythrin conjugated antibody (anti-human-IgG-γ chain specific-R-Phycoerythrin, Sigma-Aldrich, Saint Louis, MO) at 1:50 dilution (in DMEM+5% FBS) for 1 hour. When staining was completed, the slides were washed three times with PBS and mounted with hoechst dye (H33342, Calbiochem, Darmstadt, Germany) at a concentration of 10 μg/ml prepared in water containing 50% glycerol. Finally, the slides were examined under a fluorescence microscope at 563 nm for the red fluorescence and 340 nm for blue fluorescence. For each area, two sets of pictures were generated. Superimposing blue with red fluorescence using the Abode Photoshop computer software (V 7.0) generated the final image.
The effect of an intracellular antibody expression in Huh-7.5 cells on the replication of full-length JFH1-GFP RNA genome was examined by using a transfection based replication assay. Full-length in vitro HCV-GFP RNA transcripts were prepared from XbaI digested linearized pJFH1-GFP plasmid by using a commercially available MEGA script kit (Ambion Inc, Austin, TX). The HCV RNA pellet was re-suspended in nuclease free water and 20 μg aliquots of this RNA was stored at -80°C. Approximately, 2 × 107 cells were re-suspended in 400 μl of serum free DMEM, mixed with 20 μg of in vitro transcribed RNA and was electroporated using a Gene Pulser Xcell apparatus (Bio-Rad Laboratories Inc. Hercules, CA) with the condition 260 V, 960 μF. Following this step, cells were cultured in DMEM with 10% fetal bovine serum. The expression of GFP due to HCV replication was monitored under a fluorescence microscope (Olympus 1 × 70) with every 24 hours interval and the images were captured using an Olympus DP-71 digital camera. Positive- and negative-strand HCV-RNA in the transfected cells was detected by ribonuclease protection assay (RPA). Briefly, total RNA was isolated from the HCV transfected cells every 24 hours by the GITC method and subjected to RPA using a probe targeted to the 5'UTR of HCV. The same amounts of the RNA extracts were subjected to RPA for GAPDH mRNA. To detect HCV RNA, we prepared a plasmid construct called pCR-II-NT-218, which have the sequence of 79-297 nucleotides of 5'UTR sequence of the JFH-1 clone. This plasmid was linearized with HindIII enzyme and positive strand RNA probe was prepared using T7 RNA polymerase to detect the HCV negative strand RNA. Likewise, this plasmid was linearized with the Xba I restriction enzyme and Sp6 RNA polymerase was used to prepare a negative-strand RNA probe for the detection of a positive-strand HCV RNA. We used a linearized plasmid pTRI-GAPDH-human antisense control template was used to prepare probe to detect GAPDH mRNA using Sp6 RNA polymerase (Ambion Inc., Austin, TX).
The effect of intracellular antibody expression on production of infectious HCV was examined by multicycle infectivity assay [38
]. Huh-7.5 cells were transfected with 20 μg of in vitro transcribed full-length JFH1-GFP RNA by electroporation method. After 96 hours, cells were collected by scraping and lysed by four rounds of freeze-thaw cycles. The cell lysates were clarified by centrifugation at 3400 rpm for five minutes. The clear supernatant was collected and titer of HCV in the supernatant was determined by real-time RT-PCR using a primer set targeted to the 5'UTR. Tissue culture infective dose (TCID50 and MOI) of the virus stock was determined using 10-fold serial dilution of the virus containing supernatant using 2-well Lab-Tek chamber slides (Nalge-Nunc International, Rochester, New York). Huh-7.5 cells stably expressing intracellular antibody were seeded at a density of 1 × 106
cells/100 mm plate. The next day the culture medium was removed and cells were infected with 3 ml of culture supernatant containing infectious virus (5 × 105
virus particles/ml, MOI 1.5). After overnight incubation, the cells washed three times using 10 ml of PBS and incubated with 10 ml of complete growth medium. Cell free culture supernatants were collected after 96 hours, clarified by centrifugation. Three ml of culture supernatants was used to infect new batch of antibody expressing Huh-7.5 cells. The infectivity assay was performed up to four cycles each using the identical procedure. At the end of infectivity assay, RNA was isolated from 1 ml of culture supernatants and HCV RNA titer was measured by real-time RT-PCR.
Real time reverse transcription polymerase chain reaction (RT-PCR)
Real time RT-PCR was performed to quantify HCV RNA levels in the infected cell culture using a published protocol [40
]. The 243 bp HCV DNA was amplified from the RNA extract by reverse transcription polymerase chain reaction using the outer sense (OS) primer 5'-GCAGAAAGCGCCTAGCCATGGCGT-3' (67-90) and outer anti-sense (OAS) primer 5'-CTCGCAAGCGCCCTATCAGGCAGT-3' (287-310). First the complementary DNA synthesis was performed from positive-strand HCV-RNA using an outer anti-sense primer (OAS) targeted to the highly conserved 5'UTR region of HCV in 20 μl volume. Briefly, 2 μg of total cellular RNA were mixed with 1 μl OAS primer (200 ng/μl), denaturized at 65°C for 10 minutes and annealed at room temperature. Avian myeloblastosis virus (AMV) reverse transcriptase (10 U) (Promega, Madison, WI) was added and incubated at 42°C for 60 minutes in the presence of 50 mmol/L Tris, pH 8.3, 50 mmol/L ethylenediaminetetraacetic acid (EDTA), 500 nmol/L dNTP, 250 nmol/L spermidine, and 40 U RNasin (Promega, Madison, WI). The cDNA was stored at -20°C until use. SYBR Green real time PCR amplification was performed in 20 μl of volume containing 10 μl of SYBR Green ER qPCR SuperMix, 1 μl (250 ng/ul) of sense and antisense primer with 4 μl of cDNA and 4 μl of distilled water. All samples were run in triplicate. The amplification was carried out using the standard program recommended by Bio-Rad Laboratory that includes: 50°C for 2 minutes, 95°C for 8 minutes, then additional 50 cycles wherein each cycle consists of a denaturation step at 95°C for 10 seconds, and annealing and extension step at 60°C for 30 seconds. At the end of the amplification cycles, melting temperature analysis was performed by a slow increase in temperature (0.1°C/s) up to 95°C. Amplification, data acquisition, and analysis were performed on CFX96 Real-Time instrument using CFX manager software (Bio-Rad, Hercules, CA).
Construction of adenovirus vector containing IgG1 antibody
A replication defective adenovirus construct carrying the gene for the recombinant human antibody (Ad-IgG1) was prepared using standard PCR and cloning methods. We used the pFab-CMV-NS3 (L+H) plasmid construct to prepare a recombinant adenovirus vector (30). The cloning process was carried out in multiple steps. First, the heavy chain antibody expression cassette containing the CMV promoter-antibody ORF, Fd termination sequence and heavy chain polyadenylation sequences were PCR amplified and assembled in pGEM-7Z(f+) vector (Promega, Madison, WI) using XhoI and BamHI sites. An unique HindIII site was introduced before the XhoI and BamHI site such that the entire antibody expression cassette can be excised from the pGEM-7Z(f+) plasmid and inserted into the adenovirus pShuttle Vector (QBIOgene, AES1020, without CMV promoter) using a unique HindIII site. After cloning, the exact orientation of the heavy chain antibody expression cassette was confirmed by DNA sequence analysis. At the second step, the antibody light chain gene was inserted into the same plasmid shuttle (pShuttle vector without CMV promoter) using two PCR fragments. The first light chain CMV promoter-leader sequence and light chain ORF was cloned into a pShuttle plasmid using Kpn1 and XbaI sites. Then the remaining light chain termination sequence and polyadenylation sequences were introduced into the same pShuttle vector using XbaI sites. The orientation of the CMV promoter of the light chain and heavy chain reading frame in the resulting plasmid was confirmed by restriction analysis. The recombinant plasmid is called pShuttle CMV NS3 (H+L). The antiviral effect of recombinant antibody clone was confirmed again by transfection into a replicon cell line. The pShuttle CMV NS3 (H+L) antibody gene was then transferred into the adenoviral genome (pAdEasy, QBiogene, Carlsbad, CA) by homologous recombination. The recombinant adenovirus plasmid was examined by restriction digestion analysis. The recombinant adenovirus plasmid was linearized with a PacI restriction enzyme and transfected to QBI-293 cells (QBiogene, Carlsbad, CA). After several weeks, the recombinant adenovirus was plaque purified and amplified on 293 cells. Large-scale purification of the recombinant adenovirus was performed by CsCL gradient centrifugation. The titer of recombinant adenovirus (virus particles/ml) was determined by using absorbance at optical density at 260 nm, a standard protocol supplied in the kit. A recombinant adenovirus carrying firefly luciferase (Ad-Luc) was used as a control to exclude the non-specific effect of recombinant adenovirus infection on HCV replication.
Huh-7.5 cells were cultured in chamber slides and after 24 hours they were infected with a different concentration of recombinant adenovirus (Ad-IgG1). The intracellular expression of a recombinant antibody was examined by an immunostaining method using goat anti-human antibody (Sigma-Aldrich, Saint Louis, MO). Briefly, Huh-7.5 cells cultured in chamber slides were washed with phosphate-buffered saline (PBS) pH 7.4 twice, air-dried and fixed with chilled acetone for five minutes. The cells were permeabilized by the treatment with 0.05% saponin for 10 minutes at room temperature. Blocking was performed with 5% normal goat serum diluted in a minimum essential medium for 30 minutes at room temperature. Blocking for endogenous biotin-avidin was performed using an avidin/biotin blocking kit (Vector Laboratories Inc., Burlingame, CA). Blocking for endogenous peroxidase was carried out with 0.9% H2O2 for 30 minutes at room temperature. The cells were incubated with a biotin conjugated goat anti-human IgG antibody (1:500 dilution, Sigma-Aldrich, Saint Louis, MO). The slide was then washed three times and incubated with an anti-mouse biotin conjugated antibody (1:1000) for one hour at room temperature. The slides were then washed and incubated for 30 minutes with Elite avidin-biotin peroxidase complex (Vector Laboratories Inc., Burlingame, CA). The slides were reacted with diaminobenzidine for 10 minutes. Counterstaining was performed with hematoxylin for one minute. After dehydration, the slides were mounted with per mount and observed by light microscopy.
To make sure that the intracellular expressed antibody molecule processed accurately, western blot analysis was performed using lysates prepared from the adenovirus infected Huh-7.5 cells. Immunoblotting was performed with a peroxidase conjugated rabbit anti-human antibody at a dilution of 1:500. (Sigma-Aldrich, Saint Louis, MO). The membrane was developed using the enhanced-chemiluminescence detection kit (ECL kit) (Amersham Pharmacia, Piscataway, NJ) and was exposed to chemiluminescent-sensitive film (Kodak Rochestor, NY).