Purification of Cap-PCV2
The DNA coding sequence of Cap-PCV2 (GenBank: ABD42929) was cloned into the pET-16b vector (Novagen), according to Salgado et al. . The N-terminus 6x Histidine tagged-viral protein was expressed in Escherichia coli strain BL21-CodonPlus (DE3) - RIL (Agilent Technologies). Protein purification was performed by affinity chromatography using a HisTrap crude FF (GE), according to the manufacturer’s recommendations in a FPLC system (AKTA TM purifier). The purity of the recombinant protein was checked in a 12% SDS-PAGE gel.
Selection of scFv clones
A human scFv phage library containing 2x106 antibody fragment sequences  was used to select antibodies against the recombinant and purified Cap-PCV2. Two selection rounds were carried out to amplify the scFv library in E. coli strain XL1 Blue (Stratagene) with the concomitant infection of the helper VCSM13 phage . In total, each well in a 96-well microplate (Nunc) was sensitized with 4 μg of Cap-PCV2 in 0.1 M bicarbonate buffer (pH 9.6), and incubated for 14 hours at 4°C. The blocking step was performed in phosphate buffer saline (PBS) containing 3% bovine serum albumin (BSA) for 1 hour at 37°C. Subsequently, 50 μL of each phage solution was added to an individual well and the plate was incubated for 2 hours at 37°C. The plate was then washed 10 times with PBS containing 0.005% (v/v) Tween 20 (PBS-T). Finally, phages were eluted in 50 μL of 0.1 M HCl solution (pH 2.2), and subsequently neutralized with 2 M of Tris-HCl buffer (pH 9.0).
Induction of scFv peptides expression
Plasmids were isolated from previously selected E. coli strain XL1 Blue and used to transform E. coli strain Top 10 (Invitrogen) competent cells . Bacterial cells were then plated in a solid Luria-Bertani (LB) medium containing 100 μg.ml−1 carbenicillin. After 12 hours of incubation at 37°C, transformed colonies were transferred to a deep 96-well plate containing Super Broth (SB) medium supplemented with 2M glucose (v/v) and 100 ug.ml−1 carbenicillin. The plate was then incubated for 12 hours at 37°C and 250 rpm. After centrifugation at 2250 g for 10 minutes, supernatants were discarded and pellets were resuspended in SB medium containing 2.5 mM isopropyl β-D-thiogalactopyranoside inducer (IPTG) (Sigma), 100 μg.ml−1 carbenicillin and 2 M glucose. The plate was later incubated for another 12 hours at 30°C and 250 rpm. Finally, the plate was centrifuged at 4000 g for 10 minutes at 4°C and supernatants were collected for analysis of expression of scFv clones.
Analyses of protein expression and specificity of scFv clones
To analyze protein expression in the scFv clones, 10 μL of the supernatants derived from scFv clones diluted in 40 μL bicarbonate buffer (pH 9.6) were individually immobilized on microtiter wells of a MaxiSorp™ plate (Nunc). To determine the specificity of clones, each well of a MaxiSorp™ plate (Nunc, Denmark) was sensitized with 0.8 μg of Cap-PCV2 or BSA (negative control), both diluted in bicarbonate buffer, followed by addition of scFv clones induced supernatants into each well. In both analyses, we used a peroxidase conjugated anti-HA antibody (1:1000) (Roche, Switzerland). Color development was achieved by adding a solution containing H2O2, orthophenylenodiamine (OPD), and 0.1 M citrate buffer (pH 5.0). Readout was performed at a wavelength of 492 nm by an Enzyme-Linked Immunosorbent Assay (ELISA) plate reader.
Purified 6x Histidine tagged-Cap-PCV2 was subjected to a 12% SDS-PAGE gel, and after electrophoresis proteins were transferred to a nitrocellulose membrane (GE) using a Mini Trans-Blot® electrophoretic transfer (Bio-Rad) according to the manufacturer’s instructions. The membrane was first incubated with blocking buffer for 1 hour at room temperature. Afterwards, the membrane was washed with PBS-T and then cut in strips which were incubated for 1 hour in 3 mL of F1 and F5 scFv clones supernatants, commercial anti-PCV2 polyclonal antibody (VMRD) (positive control) or PBS (negative control). After washing three times with PBS-T, the respective antibodies were added: anti-HA peroxidase conjugated antibody (Roche, Switzerland) (1:2000) to the negative control, F1 and F5 scFv clones; and anti-swine IgG peroxidase conjugated secondary antibody (Sigma) (1:10,000) to the positive control. Immunoreactive protein bands were revealed with 3.3 tetrahydrochloride (DAB) and 0.015% (v/v) hydrogen peroxide in PBS.
Selection of Cap-PCV2 mimetic peptides
We performed the phage display screening using anti-PCV2 polyclonal antibodies (VMRD), an in-house rabbit anti-PCV2 polyclonal antibody purified by the caprylic acid precipitation method , and supernatants derived from F1 and F5 scFv clones.
Phage display libraries, obtained from New England Biolabs, Inc., Beverly, MA,
contained peptide sequences (7-mer or 12-mer) inserted in the pIII minor coat protein N-terminus of the M13 bacteriophage. The Ph.D.-C7C and the Ph.D.-12 libraries consisted of approximate 1.2x109 and 1.9x109 independent clones, respectively. Ph.D.-C7C and Ph.D.-12 libraries were screened through biopanning in a solution containing either protein G magnetic beads (Invitrogem) to retrieve the antibody-phage complex or nickel charged magnetic agarose beads (Qiagen) to retrieve 6x Histidine tagged-scFv fragments.
Biopannings were performed with three rounds of selection when phages were screened using swine serum, and two rounds of selection when phages were screened with scFv fragments. A combination of 1x1011 viral particles of Ph.D.-12 with either 4x108 beads/mL-antibody (swine serum) or beads-scFv were incubated for 1 hour at 37°C. After incubation, beads were washed 10 times with PBS-T and the unbound phage particles were discarded, followed by competitive elution of bound phages with 50 µg of purified Cap-PCV2 for 10 minutes at room temperature. Eluted phages were amplified in E. coli strain ER2738 (New England Biolabs) and purified using PEG-NaCl precipitation. After each round of biopanning, isolated bacterial colonies containing amplified phage clones were grown in a microtiter plate and titrated essentially as described by Barbas III et al. .
The biopanning using rabbit polyclonal antibodies was performed in two steps. The first step consisted of a subtractive step to remove phages that bound non-specifically on the magnetic beads. The second step consisted of retrieving those phages that expressed peptides bound to antibodies against Cap-PCV2. The removal of non-specific phages, a combination of 1x1011 viral particles of Ph.D.-C7C with 4x108 beads/mL-antibody derived from the negative serum for PCV2 was performed. After 1 hour of incubation at 37°C, the supernatant was used for positive selection, which was performed with microspheres bound to positive rabbit serum IgG for PCV2 antigen, as previously done for Ph.D.-12.
Sequencing and in silico analysis
The DyEnamic ET Dye Terminator Cycle Sequencing Kit (GE Healthcare) and a MegaBaceTM sequencer 1000 (GE Healthcare) were used to sequence the heavy and light chains of the scFv clones previously selected by ELISA using Cap-PCV2 as the coating protein. Phage DNA sequences encoding the recombinant peptides selected from the Ph.D. were sequenced. The primers used were the following: MMB4 (5’-TCC TCC GCT GGC TAT GTG GTT T-3’) for the light chain, MMB5 (5’-CGT TTG CCA TTT CAT AAT TCT C-3’) for the heavy chain of scFv clones, and -96 M13 (5’-CCCTCATTAGTTAGCGCGTAACG-3’) for the phage display peptides. The scFv nucleotide sequences were analyzed using IgBlast (http://www.ncbi.nlm.nih.gov/igblast). The peptides sequences were aligned with Cap-PCV2 amino acid sequence using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). The three dimensional structures of the Cap-PCV2 found in the Protein Data Bank (PDB), e.g. 3R0R, and the PCV2 capsid sequence were used for structural inferences using the program PyMOL (DeLano Scientific).
PoliSorp microplates (Nunc) were coated with each clone at a concentration of 5.0x1010 phages/mL, previously diluted in bicarbonate buffer, pH 9.6, and incubated at 4°C for 14 hours. Plates were blocked for 1 hour at 37°C with 10% (w/v) non-fat dry milk in PBS, followed by three washes with PBS. Positive or negative rabbit antibodies for PCV2 were previously incubated with M13 phage (wild-type) (1011 phages/mL) and then added to microplates at 1:400 dilution in blocking solution and incubated for 1 hour at 37°C. After washing three times with PBS, a commercial anti-rabbit IgG conjugated to peroxidase (Sigma) at 1:2000 dilution in blocking solution was added to each well. Color development was achieved by adding a solution containing H2O2, OPD, and 0.1 M citrate buffer (pH 5.0). Readout was performed at a wavelength of 492 nm. Wild-type M13 phage was used as a negative control. Each clone was tested in duplicate.
Four synthetic peptides (PS14, PS34, PF1, and PC12) (Table. 1) were designed from the selected phages and they were chemically synthesized and conjugated to Keyhole Limpet Hemocyanin (KLH) using PEPTIDE 2.0 (Peptide 2.0 Inc., USA).
All experimental procedures were conducted in compliance with the ethical principles of the Brazilian Academy of Animal Experimentation and approved by the Animal Research Ethics Committee of the Universidade Federal de Viçosa under the protocol number 39/2012.
The two following immunization experiments were conducted in mice: (1) using selected phages as antigens, and (2) using the conjugated synthetic peptides as antigens.
Experiments were carried out in 4–6 weeks old male BALB/c mice. In the first trial, mice were divided into 8 groups of 5 mice each. Immunizations were performed with three subcutaneous injections with intervals of 15 days for each and using the following phages: S14, S34, C12, FV7, and F1. Each inoculum contained 1010 phages and 25% aluminum hydroxide as an adjuvant. The negative control inoculums contained PBS and wild-type M13 phage. The positive control contained Cap-PCV2 recombinant protein. In the second trial, mice were divided into 6 groups of 6 mice each. Immunizations were performed with three subcutaneous injections with intervals of 15 days for each and using the following peptides, 100 µg per dose: PS14, PS34, PC12, and PF1 conjugated to KLH. The negative and positive controls contained PBS and purified cap-PCV2 protein, respectively. Saponin (Sigma) at 100 μg per dose was used as an adjuvant in combination with the peptides and the controls. Blood samples were collected 45 days after immunization with phages, and at 0, 15, 30, and 45 days after immunization with peptides. Sera were later examined for the presence of specific antibodies.
Detection of IgG, IgG1, and IgG2a antibodies for Cap-PCV2
A 96-well Maxisorp microtiter plate (Nunc) was coated with 0.36 ng/well of Cap-PCV2 diluted in bicarbonate buffer. After incubation for 14 hours at 4°C, wells were blocked with 5% BSA in PBS. Sera from animals inoculated with either phage or peptides were diluted in blocking buffer at 1:20 and 1:50, respectively, before being added to the plate, followed by incubation at 37°C for 1 h. After washes, peroxidase-conjugated anti-mouse IgG, IgG1, or IgG2a antibodies (Sigma), diluted at 1:5000, were added to the plate and incubated for 1 hour at 37°C. Color development was achieved by adding a solution containing H2O2, OPD, and 0.1M citrate buffer (pH 5.0). Readout was performed at a wavelength of 492 nm. Each sample was tested in triplicate.
Evaluation of swine serum response by PC12 peptide based ELISA
To investigate whether the PC12 peptide can be used as a serological tool for detecting PCV2 infection, twenty-seven positive swine serum and 14 negative swine serum samples which had been validated by the peroxidase monolayer assay (IPMA) were used for the evaluation . In addition, 20 swine which had not been vaccinated in the field were naturally infected with PCV2, and swine sera were collected for evaluation at 52, 102, and 166 days old. To conduct in house ELISA, 5 ug/well of PC12 or PCV2-Cap in bicarbonate buffer was immobilized on a 96-well Maxisorp microtiter plate (Nunc). After incubation for 14 hours at 4°C, wells were blocked with 5% BSA in PBS. Sera from swine were diluted in blocking buffer at 1:50 and then added to the plate for 1 h at 37°C. After washes, peroxidase-conjugated anti-pig IgG antibodies (Sigma), diluted at 1:5000, were added to the plate and incubated for 1 hour at 37°C. Color development was achieved by adding a substrate solution containing H2O2, OPD, and 0.1 M citrate buffer (pH 5.0). Readout was performed at a wavelength of 492 nm by an ELISA plate reader (company name). Each sample was tested in triplicate.
Differences between groups were analyzed using ANOVA test, followed by the Dunnett’s test. Statistical analyses were done using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). P values that were less than 0.05 were considered statistically significant.