Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/VHH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/VHH from a humanized-camel VH/VHH display library. VH/VHH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed VHH hallmark and were designated VHH6 and VHH24; other clones were conventional VH, designated VH9 and VH13. All VH/VHH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, VHH6 and VHH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/VHH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.


Introduction
The NS5B protein has RNA-dependent RNA polymerase (RdRp) activity which is pivotal for de novo RNA synthesis of hepatitis C virus (HCV). The protein is an attractive target of developing anti-HCV agents [1]. Similar to other polymerases, the NS5B resembles human right hand structure consisting of finger, thumb, and palm domains [1]. The polymerase active site is located in the palm [1]. NS5B acquires two different crystal forms: active closed-form-I and inactive open-form-II [1]. The closed conformation mediated by anchoring of ?1 and ?2 subdomain loops of fingers to the thumb is believed to regulate entering of RNA template and ribonucleotide (rNTP) substrate into the catalytic cavity during RNA replication [2]. NS5B lacking a hydrophobic C-terminal 55 amino acid residues (NS5BD55) has higher polymerase activity than the full-length NS5B [3].
There is no vaccine against HCV infection. Combined pegylated-interferon (PEG-IFN) and ribavirin is used for intervening of the chronic hepatitis C progression to the end stage liver diseases including liver cirrhosis and hepatocellular carcinoma [4]. Rationales are to enhance the host immunity and inhibit the viral RNA synthesis. Weekly IFN injection and daily oral ribavirin are necessary throughout the 24-48 week treatment course in order to expect effectiveness [4]. Even with such intensive treatment, the success rate is only about 50% due to tolerance of some HCV genotypes (1 and 4) [5]. Many patients do not comply with this regimen, partly because of the severe adverse side effects. Moreover, the treatment cost is beyond affordability of many infected individuals of the developing part of the world where HCV infection is a real problem. As such, novel anti-HCV agent with improved treatment efficacy and safety and less expensive warrants development. Recently, telaprevir and bocepprevir which are HCV protease inhibitors have been approved by US FDA [6] but these drugs are not yet widely available.
Recently, sera of camelids were found to contain not only the conventional four chain-immunoglobulin G (IgG) but also heavy chain antibody (HCAb) which each molecule consists of heavy (H) chain homodimers. The HCAb is soluble in serum in spite of the fact that the H chains do not have the linked light (L) chain partners. This is because the HCAb has mutated some hydrophobic amino acids at the former interface between the variable heavy chain domain (VH) and the variable light chain domain (VL) to be more hydrophilic; thus reducing aggregation [7][8][9]. This area is located on immunoglobulin framework-2 (FR2) of the antigen binding domain of HCAb, designated V H H in order to differentiate from the VH of the conventional four chain antibody. The V H H FR2 area contains a tetrad amino acid hallmark, i.e., F/ Y42, E49, R/C50 and G/L52 which substitute for V42, G49, L50, and W52 of the conventional VH [7][8][9]. Besides, the third complementarity determining region (CDR3) of the V H H is exceptionally long and can extend to cover the FR2 hydrophobic region; thus increasing solubility of the V H H [7][8][9]. Recently humanized-V H H phage display library was constructed from naïve camel immunoglobulin genes. The camel VH/V H H coding genes were humanized by using human primer directed PCR amplification [10]. Phage clones secreted V H H specific to botulinum neurotoxin were selected from the library. One V H H (V H H17) neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A by inserting its CDR3 into the emzymatic groove of the target enzyme and blocked specifically the catalytic enzymatic activity [10]. This functional mechanism of antibody has never been possible by large molecular sized conventional antibody. More recently, humanized-camel VH/V H H that bound specifically to Cobra (Naja kaouthia) venom phospholipase A2 and inhibited the enzyme activity was reported [11]. It is now generally accepted that the V H H is a potent enzyme inhibitor [12]. In this study, cell penetrable humanized VH/V H H, synonym single domain antibodies (SdAb), that bound specifically to HCV NS5B and interfered with the native RdRp catalytic activity inside the HCV infected cells leading to inhibition of the HCV replication were produced. To our knowledge this is the first report on HCV polymerase neutralization by cell penetrable humanized-VH/ V H H.

Recombinant NS5BD55 Protein
Complementary DNA (cDNA) was synthesized from RNA extracted from the serum of patient infected with genotype 3a HCV (kindly provided by Professor Yong Poovorawan, Chulalongkorn Hospital, Bangkok) which is the predominant serotype. The cDNA was used as a template for amplification of NS5BD55 cDNA by polymerase chain reaction (PCR). Oligonucleotide primers specific to nucleotide sequence coding for HCV NS5BD55 protein were designed from the genotype 3a HCV nucleotide sequence of the database (GenBank NC_009824). The PCR amplicon was cloned into pET23b + vector between EcoRI and XhoI sites and the recombinant plasmid was introduced into BL21 (DE3) E. coli. Transformed E. coli was grown and induced to overexpress the recombinant protein by 0.2 mM isopropyl-b-D-1thiogalactopyranoside (IPTG). The recombinant NS5BD55 was purified from the bacterial lysate by using Ni-NTA beads (Invitrogen) and verified by gel-based liquid chromatographytandem mass spectrometry [13].

RdRp Activity of the Recombinant NS5BD55
Enzyme linked immunosorbent assay (ELISA) was used for determining RdRp activity of the NS5BD55 by detecting an incorporation of biotinylated-cytosine triphosphate (CTP) (Invitrogen) into an RNA template in the presence of the NS5BD55. The SLD3 RNA (59 GGGCUUGCAUAGCAAGUCUGAGACC 39) [14] was used as RNA template and the procedure described previously [15] was followed with modification. The 59 end of the SLD3 RNA (800 pM) was attached covalently to the surface of the Nucleolink module (Thermo Scientific Nunc, UK) via carbodiimide condensation [16]. Polymerase reaction mixture (80 mL) (300 nM of NS5BD55; 20 mM sodium glutamate, pH 8.2; 4 mM MgCl 2 ; 12.5 mM DTT; 0.5% (v/v) Triton X-100; 2 mM MnCl 2 ; 40 units RNase inhibitor; 200 mM each ATP, UTP, GTP, and biotinylated-CTP) was added to the SLD3 RNA coated well and incubated at 37uC for 2 hours. Polymerase reaction mixture containing heparin (2 mM) which is polymerase quencher [17] was included in the assay as the RdRp inhibition control. Nonincorporated rNTPs were washed away before adding with streptavidin-horseradish peroxidase (HRP) conjugate (Southern Biotech, USA), followed by 2,29-azino-di (3-ethylbenzthiazoline-6sulfonate (ABTS) substrate (KPL, USA). Wells containing buffer and normal BL21 (DE3) E. coli lysate were included as blank and negative control, respectively. Optical density at absorbance 405 nm (OD 405 nm ) of the content of each well was determined.

Humanized-camel VH/V H H Phage Display Library
The library was constructed previously [10] using total RNA extracted from peripheral blood mononuclear cells of a naive male camel (Camelus dromedarius) and messenger RNA (mRNA) was reverse transcribed to cDNA. The gene fragments encoding variable domains of the camel VH/V H H were PCR amplified using the cDNA as template and 14 forward and 3 reverse degenerate primers designed from all families of human immunoglobulin genes [18]. The human primer directed-camel vh/v h h cDNA amplicons were ligated into a pCANTAB5E phagemid vector and introduced into competent TG1 E. coli cells. The complete phage particles displaying humanized-camel VH/V H H with integrated vh/v h h in the phage genomes were rescued from by co-infecting the vh/v h h-phagemid transformed E. coli with a helper phage.

Phage Biopanning and Preparation of the Humanized-VH/V H H
A single round phage biopanning for selecting phage clones that displayed NS5BD55 bound-VH/V H H was performed as described previously [10] using 10 mg of purified NS5BD55 as the panning antigen. Antigen bound phages were supplemented with log phase grown HB2151 E. coli. The phagemid transformed E. coli clones were selected on LB agar plate containing 100 mg/mL ampicillin and 2% glucose. E. coli clones carrying recombinant vh/v h hphagemids were screened by PCR using phagemid specific R1 and R2 primers [18]. Selected clones were grown individually under 0.5 mM IPTG induction and VH/V H H proteins in bacterial lysates were partially purified by ion exchange (DEAE) column chromatography. Amount of VH/V H H in each preparation was standardized.

Specific Binding of VH/V H H to NS5BD55
Specific binding of VH/V H H to NS5BD55 was determined using indirect ELISA and Western blot analysis (WB) [10,18]. For ELISA, one mg of NS5BD55 and antigen control, i.e., bovine serum albumin (BSA) was immobilized separately in wells of an ELISA plate. After blocking the empty sites on well surface with 3% BSA in PBS, standardized VH/V H H contained in lysates of transformed E. coli were added to appropriate wells and kept at 37uC for 1 hour. Unbound VH/V H H were removed; the bound VH/V H H in each well was detected by adding rabbit anti-E tag antibody (Abcam, UK), goat anti-rabbit immunoglobulin-HRP conjugate (Southern Biotech), and ABTS substrate (KPL), respectively, with washing with phosphate buffered saline, pH 7.4 containing 0.5% Tween-20 (PBST) between steps. E. coli clones which their lysates gave OD 405 nm to NS5BD55 two times higher than to the BSA were selected.
For WB, NC strip blotted with SDS-PAGE separated-NS5BD55 was blocked with 5% skim milk in Tris buffered saline (TBS) and kept at 25uC for 1 hour. After washing with TBS containing 0.05% Tween-20 (TBST), the NC strip was incubated with E. coli lysate containing standardized VH/V H H at 25uC for 1 hour. The NS5BD55-VH/V H H reactive band was revealed by using rabbit anti-E tag antibody (Abcam), goat anti-rabbit immunoglobulinalkaline phosphatase (AP) conjugate (Southern Biotech) and BCIP/NBT substrate (KPL).

ELISA Inhibition for Screening VH/V H H that Inhibited NS5BD55 RdRp Activity
Test mixtures, i.e., partially purified VH/V H H mixed individually with NS5BD55, and control mixtures, i.e., NS5BD55 mixed with irrelevant V H H that specific to botulinum neurotoxin type A, V H H17 [10] (background inhibition control) and NS5BD55 mixed with antibody diluent (negative inhibition control or blank) were prepared. The mixtures were added separately to ELISA wells containing immobilized SLD3 RNA template. The polymerase reaction mixture prepared as above was added to each well and the ELISA procedure was similarly completed. OD 405 nm of the content of the test and control wells were measured against blank. Less OD 405 nm of the tests compared to the background and negative inhibition controls indicated that the VH/V H H could neutralize specifically the NS5BD55 RdRp activity.

Production of Cell-penetrable VH/V H H
Gene sequence coding for the VH/V H H that inhibited the NS5BD55 RdRp in vitro was subcloned to PEN-pET23b + plasmid backbone [19] in order to produce cell-penetrable VH/V H H. The vh/v h h sequences were cloned into the recombinant plasmid vector at downstream of the DNA sequence coding for a 16 amino acid cell-penetrating peptide, i.e., penetratin (PEN) via SfiI and NotI sites and introduced into BL21 (DE3) E. coli. PEN-VH/V H H fusion proteins were produced from transformed bacteria and purified by using Ni-NTA beads [19].

Inhibition of HCV Replication in Huh7 Cells by Cellpenetrable VH/V H H
The plasmid pJFH-1 containing full-length cDNA of the JFH-1 HCV of genotype 2a (GenBank AB047639) was kindly provided by Dr. Takaji Wakita, Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan and Professor Ralf Bartenschlager, Department of Molecular Virology, University of Heidelberg, Germany. To generate genomic HCV RNA, the pJFH-1 was linearized with XbaI endonuclease (Fermentas) and transcribed in vitro using Megascript T7 kit (Ambion, USA). The transcribed RNA (10 mg) was put into Huh-7 cells (4.0610 6 cells) by electroporation (single pulse at 0.27 kV, 100 milliseconds) using eukaryotic electroporation mode by ElectroporatorH (Eppendorf). The JFH1 RNA transfected Huh7 cells were immediately transferred to 40 mL of complete (serum supplemented) DMEM and seeded into wells of 12-well culture plate (Corning), 2.0610 5 cells/well. After 24 hours, the cell monolayer was washed with PBS and added with complete DMEM containing 10 and 20 mg of purified PEN-VH/V H H of individual E. coli clones and irrelevant PEN-V H H17 (specific to botulinum neurotoxin type A) [10]. Cells added with 50 nM Ribavirin +100 units of PEG-IFN served as positive inhibition control. Infected Huh7 cell monolayer added with the medium alone served as negative inhibition control.
After 5 days, total RNA was extracted from the spent culture medium and the cell monolayer using Trizol TM reagent (Invitrogen). Copy number of HCV 59 UTR (230 bp) in 900 ng of total RNA in each extract was determined by quantitative real-time PCR [20] using 1-step Brilliant II SYBR green qRT-PCR master mix kit (Agilent Technologies, USA). A standard curve was constructed from Ct of ten-fold dilutions of the pJFH-1 carrying full-length cDNA of the JFH-1 HCV genotype 2a (ranged from 2.79610 7 to 0.02 copies). Ct of each sample was expressed as log 10 of RNA copies/mL calculated from the standard curve. Similar experiments were performed by culturing the pJFH-1 RNA transfected Huh7 cells in medium containing 20 mg of PEN-VH/V H H and PEN-V H H17 and ribavirin + PEG-IFN controls. The numbers of HCV foci in the pJFH-1 RNA transfected cells were enumerated by staining the cells (after washing) with mouse anti-HCV core antibody (Abcam); rabbit anti-mouse immunoglobulin-alkaline phosphatase conjugate and BCIP/NBT substrate were used as foci revelation reagents. Means 6 SD of the numbers of HCV foci from 100 microscopic fields (magnification 10620) in the PEN-VH/V H H treated cells were compared with the infected cells (negative inhibition control), infected cells treated with ribavirin + PEG-IFN (positive inhibition control) and the cells treated with irrelevant V H H17 (background inhibition control). The amounts of HCV core antigen in the culture supernatants of all treatments on day 5 were determined by using QuickTiter TM HCV Core Antigen ELISA kit (Cell Biolabs, Sandiego, USA).

LDH Assay
Cytotoxicity of individual PEN-VH/V H H on naïve Huh7 cells was determined by using CytoTox 96H non-radioactive cytotoxicity (LDH) assay (Promega, USA).

Restriction Fragment Length Polymorphism (RFLP) of vh/ v h h Sequences
RFLP of MvaI digested DNA sequences coding for the individual VH/V H H were determined by 14% polyacrylamide gel electrophoresis followed by ethidium bromide staining [13].

Amino Acid Sequences, Immunoglobulin Frameworks (FRs) and Complementarity Determining Regions (CDRs) of the VH/V H H
The vh/v h h were sequenced and amino acids were deduced. All protein sequences were multiply aligned by ClustalW. Immunoglobulin frameworks (FRs) and complementarity determining regions (CDRs) of each VH/V H H were predicted by using the International Immunogenetics information system (IMGT) [21].

Mimotope Searching
Ph.D.-12 TM phage display peptide library (New England Biolabs, USA) was used to determine VH/V H H bound phage mimotopes as described previously [10]. The mimotope peptide sequences were deduced from the phage DNA sequences by DNAMAN software version 4.15. The mimotopes were classified into groups by using Phylogeny ClustalW [22]. The sequences of the same mimotope group were multiply aligned with HCV NS5B sequence (Accession no. NP_751928) by Kalign [23].

ELISA Inhibition Assay for Validation of the Phage Mimotopes
Phage clones displaying the representative mimotopes of mimotope groups were propagated in ER2738 E. coli and the titers of the amplified phages were determined according to manufacturer's instruction (New England Biolabs, USA). Phage mimotope preparations (50 mL) at various amounts (10 6 , 10 7 and 10 8 plaque forming unit; pfu) were mixed individually with fixed amount of 50 mL VH/V H H (5 mg) and incubated at 37uC for 1 hour. The VH/V H H mixed with M13KO7 phage served as background binding control. NS5BD55 coated wells added with the VH/V H H served as 100% binding (maximum binding). After washing, rabbit anti-E tag antibody, goat anti-rabbit immunoglobulin-HRP conjugate and ABTS substrate were added respectively. OD 405 nm of the content of each wells were determined. The % ELISA inhibition was calculated: % ELISA inhibition = [(OD 405 nm of maximum binding 2 OD 405 nm of test) 4 (OD 405 nm maximum control)] 6 100.

Homology Modeling and Molecular Docking
Deduced amino acid sequences of NS5B and VH/V H H were subjected to basic local alignment search (BLAST). The sequences with maximum identities were used as templates for homology modeling. The constructed models were validated by using PROCHECK [24]. Three dimensional structure of the protein complex was predicted by protein docking technique. ZDOCK and RDOCK modules embedded on Discovery Studio program were used for docking. NS5B and VH/V H H were set as input receptor and input ligand, respectively. Each ZDOCK docking result was subjected to structure refinement by using RDOCK module. After RDOCK calculation, the dock pose with lowest RDOCK energy was analyzed for the binding interaction.

Statistical Analysis
Means and standard deviations of three independent experiments were used for comparison between tests and controls. P values ,0.05 of unpaired t-test was considered significant difference.

Recombinant NS5BD55
NS5BD55 (,60 kDa) of HCV genotype 3a was successfully produced and purified from the lysate of a selected transformed BL21 (DE3) E. coli carrying the recombinant NS5BD55-pET23b + plasmids. Deduced amino acid sequence of the NS5BD55 showed 98% identity to the sequence of HCV genotype 3 NS5B protein (Accession no. YP_001491557.1) and approximately 80% identity to the NS5B protein sequences of various other HCV genotypes including 1a, 1b, 6b, 6c and 6m ( Figure S1). The protein was verified by LC-MS/MS as the HCV NS5B (data not shown).
SLD3 RNA and biotinylated-CTP based-ELISA showed that the recombinant NS5BD55 acquired RdRp activity (Figure 1). The colorimetric values of the newly synthesized biotinylated RNA increased steadily when the amounts of NS5BD55 were increased from 50 to 600 nM. The RdRp activity of the NS5BD55 was quenched by the presence of heparin (2 mM) which was known to be the polymerase trapping reagent.   (Figure 2A) as well as by WB ( Figure 2B). The vh/ v h h sequences of the 10 clones revealed 10 different DNA banding patterns (RFLP) ( Figure 3A). Multiple alignments showed that all clones had different amino acid sequences especially at the CDR domains ( Figure 3B). The deduced amino acid sequences of two clones had the characteristic amino acid tetrad of V H H; they were designated clones V H H6 and V H H24, while the other clones had conventional VH feature; thus, designated clones VH1, VH3, VH8, VH9, VH13, VH18, VH20, and VH25 [7,10]. Sequences of these 10 humanized-camel VH/V H H showed high homology with human VH sequences (Table 1).

Inhibition of HCV Replication in Huh7 Cells by Cellpenetrable VH/V H H
The pJFH-1 RNA transfected-Huh7 cells cultured in the medium containing 10 and 20 mg of VH13 had significantly less HCV RNA inside the cells ( Figure 5A, bar 3) and in culture supernatants ( Figure 5B, bar 3) than the transfected cells cultured in the medium alone (negative inhibition control) and the cells treated with irrelevant PEN-V H H17 (background inhibition control) (p#0.01). Both extracellular and intracellular HCV RNA of cells exposed to 20 mg of PEN-V H H6 and PEN-V H H24 were also less than both controls. Nevertheless, the amounts of the HCV RNA in the culture fluids of the cells exposed to 10 mg of the two antibodies were not different from both controls. PEN-VH9 had much less inhibitory activity on the HCV RNA replication than the other three antibodies. The means 6 SD of the viral RNA detected inside the cells exposed to VH13, V H H6 and V H H24 were as low as the amounts in the cells treated with ribavirin + PEG-IFN (positive inhibition control).
Numbers of HCV foci in HCV RNA transfected Huh7 cells of all treatments were expressed as means 6 SD in 100 microscopic fields (magnification 2006) ( Figure 5C). The numbers of the foci in the infected cells exposed to 20 mg of PEN-VH13, PEN-V H H6, PEN-V H H24 and ribavirin + PEG-IFN were not different and were less than the negative and the background inhibition controls. Figure 5D shows the amounts of HCV core antigen (ng/mL) in cell culture supernatants of pJFH-1 RNA transfected Huh7 cells. Correlation of the intracellular amounts of the SdAbs with their inhibitory activity on the HCV replication was not clearly demonstrated ( Figure 4) although the PEN-VH9 which had the least cellular entering capacity had the lowest HCV inhibitory activity.
The PEN-VH/V H H at the amounts up to 10 mM did not cause any detectable LDH leakage from the Huh7 cells after 24 hour incubation indicating their innocuousness ( Figure S3).

VH/V H H Bound Phage Mimotopes and Tentative Epitopes of the VH/V H H on NS5B
The 12 mer peptides deduced from genomes of the phages that bound to VH/V H H (phage mimotopes) could be classified into several homology groups; individual mimotope peptides are shown in Table S1. Sequences of each mimotope group were aligned with NS5B sequence of the database to locate the tentative VH/V H H peptide epitopes on the HCV NS5B ( Figure 5A

Inhibition of the VH/V H H Binding to the NS5BD55 by Phage Mimotopes
Representative results of the ELISA inhibition for determining the ability of phage mimotopes in inhibiting the VH/V H H binding to the NS5BD55 are shown in Figure S2

Interface Binding of VH/V H H with the NS5B
The amino acid sequence of NS5B genotype 3a has 73% identity with hepatitis C virus NS5B RNA polymerase (PDB code 2HAI). The Ramachandran plot of structure of NS5B was validated by using PROCHECK, and it has no residue in disallowed region (complete match). The Ramachandran plots of  Figure S4. The docked pose of NS5B and VH9, VH13, V H H6 and V H H24 are shown in Figure 6B. All antibodies interacted with all three domains of the NS5B and covered the RdRp catalytic groove.

Discussion
There is a clinical need for safer, less expensive and more effective anti-HCV agents. The new agents should be highly tolerable to viral antigenic variation and requires shorter treatment duration than the current protocol. NS5B with inherent RdRp activity is one of the novel anti-HCV targets. Incapacitating the protein activity interrupts the HCV infectious cycle by halting replication. Many small molecular pharmacologic agents that target the NS5B RdRp have been developed and some have reached clinical trials. Nevertheless, most of them caused undesirable side effects and/or rapid selection of drug resistant HCV mutants [25]. Besides, their RdRp inhibition tends to be HCV genotype specific [26]. Alternative approach was used in this study to develop specific inhibitor of the intracellular NS5B RdRp activity.
An antibody molecule has multiple CDRs which contact many residues of either linear or conformational epitope of the target antigen. Thus, specific antibody can tolerate not only single point mutation but also multiple nucleotide mutations or even several amino acid alteration of the antigen [27]. Antibody basedimmunotherapy has been practiced for treatment of viruses, intoxications, envenomation and inflammations including autoimmune disorder and allergy [28]. Intact antibody molecules or their engineered fragments with reduced immunogenicity, i.e., Fab, ScFv and SdAb have been developed in the past few decades and many have been approved for clinical use [28]. SdAb are highly potent enzyme inhibitors which directly insert CDRs, particularly CRD3, into the catalytic groove [10]; the mechanism which conventional antibody is lacking. Due to small sizes (15)(16)(17)(18)(19)(20), the sdAb has higher penetrating ability and better in vivo tissue distribution than their larger size counterparts (150 kDa IgG or 25-35 kDa ScFv). Recently, a humanized-camel VH/V H H phage display library was constructed [10]. VH/V H H coding sequences of this library had high homology to human VH coding sequences [10]. Thus, VH/V H H derived from this library should have negligible immunogenicity in human recipient.
In this study, bacterially expressed recombinant NS5BD55 of genotype 3a HCV which retained the RdRp activity [3] was successfully produced and conveniently purified. Thus, the recombinant protein was suitable for use as an antigen in a single round phage bio-panning for selecting phage clones that bound to the protein from the established humanized-camel VH/V H H phage display library. The advantages of single over multiple round phage-panning had been discussed previously [10,13]. The NS5BD55-bound phages recovered from the panning might be either antigen specific phages (bound to the antigen via the displayed VH/V H H) or non-specific phages (adhered to the antigen by means of phage coat proteins). Therefore, specific binding of the soluble VH/V H H prepared from lysates of the recombinant phagemid transformed bacteria to the NS5BD55 had to be verified using the indirect ELISA and WB. According to the set positive criteria of the assays, 10 vh/v h h-phagemid transformed E. coli clones were selected. The vh/v h h sequences of these clones had 10 different DNA banding patterns (RFLP) after digesting with MvaI endonuclease indicating high diversity among them. This was confirmed by DNA sequencing. Each vh/v h h was a complete coding sequence of an antigen binding variable (V) domain containing four immunoglobulin framework segments (FRs 1-4) and three CDR segments (CDRs 1-3) ( Figure 3B). FR2 sequences of two clones showed characteristic amino acid tetrad of V H H; thus they were V H H. The FR2 sequences of the remaining Figure 3. Different RFLP patterns of DNA sequences coding for the VH/V H H of clones no. 1,3,6,8,9,13,18,20,24, and 25, respectively. (A). Multiple alignment of amino acid sequences for determining immunoglobulin frameworks (FRs) and complementarity determining regions (CDRs) of the 10 VH/V H H by using the International Immunogenetics Information System sever (B). Clones no. 6 and no. 24 have the tetrad amino acid hallmark of V H H in FR2 (bold letters) and were designated V H H6 and V H H24; the rests were conventional VH, designated VH1, VH3, VH8, VH9, VH13, VH18, VH20, and VH25 Asterisk indicates identical amino acids; colon indicates conserved amino acid substitution; and dot indicates a semiconserved amino acid substitution. doi:10.1371/journal.pone.0049254.g003  [29][30][31].
Because the antibody must reach the intracellular NS5B in order to inhibit the de novo RdRp activity, the DNA sequences coding for all four VH/V H H clones were linked molecularly to a DNA sequence coding for a 16 amino acid cell penetrating peptide (penetratin, PEN) in a plasmid backbone constructed previously in our laboratory [19]. The cell penetrable VH/V H H (transbodies) specific to NS5B, i.e., PEN-VH9, PEN-VH13, PEN-V H H6 and PEN-V H H24, were successfully produced as bacterial inclusions by the respective transformed E. coli. Fortunately, after refolding all of the transbodies still retained the NS5B RdRp inhibitory activity similar to their original molecules.When added to the cell culture medium of Huh7 cells transfected with the JFH1 RNA the cell penetrable VH/V H H suppressed replication of the HCV RNA replicon, albeit in different degrees, as shown by reduction of both intracellular and released viral RNA copies. Quantification of the HCV phenotypes inside the respective VH/V H H exposed transfected cells and in the culture fluids conformed to the results on the viral RNA detection. Overall data indicate the crossgenotypic inhibitory activity of the transbodies. Unfortunately genomic replicons of other HCV genotypes are not available for testing the cross-genotype inhibition. Nevertheless, the high amino acid sequence homology was observed among NS5B proteins of different HCV genotypes deposited in the Genbank database. Moreover, all HCV genotypes share identical RdRp Dx 4-5 D and GDD motifs of the catalytic groove [32]. Thus, it is optimistically envisaged that the cell penetrable VH/V H H produced in this study should also cross-neutralize the RdRp activity of other heterologous HCV genotypes.
Phage peptides bound to the NS5B specific-VH/V H H (mimotopes) were searched for predicting the locations of the NS5B protein interacted by the antibodies which would enlighten the molecular RdRp inhibitory mechanism of the antibodies. Multiple alignments of the mimotopes indicated that the VH/V H H bound to discontinuous (conformational) epitopes on the NS5B molecule which the antibody contact residues scattered on the polymerase, either on thumb and palm (VH9) or on palm, thumb and finger domains) [33]. The findings by indirect ELISA inhibition that representative phage clones displaying the mimotope groups could inhibit the VH/V H H binding to the NS5BD55 indicate the amino acids on the HCV polymerase analogous to the mimotope peptides are the presumptive VH/V H H epitopes. The multiple contact points of the VH/V H H to the target NS5B would render them high tolerability to the HCV mutations [27]. It is noteworthy that the mimotopes of the clone VH9 which conferred the least HCV replication inhibition among the four antibody clones are located on the thumb and palm only, while mimotopes of the other clones interacted also with the interconnecting loop between fingers and thumb (VH13), a-helix that links fingers and palm (V H H6) or fingers (V H H24). It is known that the RdRp HCV is active under the closed configuration (forming a tunnel for template and ribonucleotides accommodation) of the polymerase protein formed by anchoring of the ?1 and ?2 loops of the fingers and the thumb [2]. Thus it is plausible that the observed higher inhibitory activity of the VH13, V H H6 and V H H24 than the VH9 on the HCV replication was due to their interference with the RdRp tunnel formation which was likely to be more readily than the VH9.
The results of homology modeling and molecular docking confirmed that the VH/V H H mediated interface binding to the NS5B molecule by occupying multiple areas around the RdRp template channel [34] and also covered the RdRp active site. The findings presumptively indicate that the NS5B specific VH/V H H suppressed the polymerase activity by preventing accessibility of the template/substrate to the enzyme catalytic cavity which consequently incapacitated the HCV RNA replication.
A unique feature that distinguishes the active HCV RdRp from the other polymerases is the closed hand conformation of the former as opposed to the openhand structure of the latter. Alteration of the closed configuration of HCV RdRp impaired the RNA synthetic activity [35]. Legacy from evolutional studies have documented the un-relatedness of eukaryotic RdRp, viral RdRp and DNA-dependent RNA polymerase (DdRp) [36]. The b' subunit of the DdRp and eukaryotic RdRp contain a signature motif DbDGD (b is a bulk residue) which contributes to the polymerase catalytic activity via divalent cation coordination, whereas the core catalytic groove of HCV RdRp contains Dx 4-5 D and GDD motif in the palm domain [37]. No other similarity was detected between DdRp and RdRp [38]. Moreover, the biochemical activity of the HCV RdRp is also different enough from the host DNA polymerases. The HCV NS5B does not express in normal human cells. Thus, the HCV NS5B specific cell penetrable VH/V H H that interfere HCV RdRp function produced in this study should not inhibit the host cellular enzymes and should be harmless. Preliminary result of LDH assay performed on Huh7 cells indicated that the cell penetrable VH/ V H H at the amount as high as 10 mM were not toxic to the cells implying the antibody innocuousness. Supporting Information Figure S1 Multiple alignments of the cloned NS5B amino acid sequence with the NS5B sequences of various HCV genotypes/ subtypes of the database. The homology of the cloned sequence with the heterologous genotype/subtype sequences was approximately 80%. (TIF)  (Table S1) at 10 6 , 10 7 and 10 8 pfu were separately mixed with V H H6 (5 mg) before adding to the ELSA well containing immobilized NS5BD55 (10 mg). The % ELISA inhibition was calculated. The results indicate that the mimotopes could inhibit the V H H6 binding to NS5BD55 implying that the mimotopes carried the amino acid residues analogous to the native HCV NS5B polymerase which validated the mimotope search results. (TIF) Figure S3 Results of LDH assay for determining cellular toxicity of 10 mM of VH9, VH13, V H H6 and V H H24 (1-4, respectively) on Huh7 cells after 24 hour incubation. Maximum LDH release control is shown in (5). All antibody preparations did not cause significant release of the LDH from the cells indicating the innocuousness of the preparations. (TIF) Figure S4 Ramachandran plots of the VH/V H H models. VH9, VH13, V H H6 and V H H24 had 2.8%, 0%, 4% and 0% of residue in disallowed region, respectively. The highest identity sequences used for protein homology modeling of VH9, VH13, V H H6 and V H H24 are sequences of PDB codes 2GCY, 2H32, 1VHP and 1F2X, respectively. (TIF) Table S1 Phage mimotope groups, respective 12-mer peptides displayed on the phages that bound to VH9, VH13, V H H6 and V H H24 and mimotope-matched peptides (tentative epitopes of the antibodies) on NS5B primary protein sequence. (DOC) Figure 6. Tentative locations of amino acid residues on NS5B primary sequence matched with the respective VH9, VH13, V H H6 and V H H24 phage mimotopes, i.e., tentative epitopes of the antibodies. Fingers, palm, thumb, loop interconnecting fingers and thumb, a-helix linking palm and fingers, and b-loop insertion are colored in blue, red, green, cyan, black, and dark green, respectively. B, Hypothetical models showing binding sites of the ribbons of VH9 (orange), VH13 (magentas), V H H6 (yellow), and V H H24 (grayish blue) on molecular surface of NS5B RNA duplexes. Finger, palm and thumb of the NS5B are colored in blue, red, and green, respectively. doi:10.1371/journal.pone.0049254.g006