C179 monoclonal antibody (A/H2N2 subtype) was purchased from Takara Bio Inc (Otsu, Shiga, Japan); Mouse monoclonal antibody (IV.C102) against influenza virus A strain H1N1 was from Santa Cruz (Santa Cruz, CA, USA); Purified H3N2 goat polyclonal IgG specific to influenza A/Texas 1/77 was from Virostat (Portland, ME, USA); SIV sera were prepared from patients hospitalized by swine-origin influenza virus A/2009 and their binding activities were tested by ELISA. Endotoxin was removed by purification with polymyxin B chromatography. Endotoxin levels were < 0.1 unit/μg of protein by the Limulus Amebocyte Lysate QCL-1000 pyrogen test (Cambrex).
Phage display libraries
Ph.D.-7, Ph.D.-12 and Ph.D.-C7C were produced by New England Biolabs, Inc (Ipswich, MA, USA), with random linear 7-mer, 12-mer or cyclic 7-mer peptides fused to minor coat proteins (pIII) of M13 filamentous phages.
Screening of phage libraries for H2N2 antibody-reactive phages
C179 (0.2 ml, 10 μg/ml) was coated on three wells of 24-wells microplate at 4°C overnight. The coated wells were blocked with 2% bovine serum albumin (BSA) at 37°C for 1 h, then washed with Tris•HCl buffer solution (TBS) containing 0.1% Tween-20 (TBST) for 6 times. Ten microliter Ph.D.-7 (2 × 1011 pfu), Ph.D.-12 (1.5 × 1011 pfu) and Ph.D.-C7C (2 × 1011 pfu) libraries diluted with 0.2 ml TBST were dropped into the coated wells respectively. The incubation wells were rocked gently for 30 min, followed by discarding nonbinding phages. The binding phages were eluted with 0.2 M Glycine-HCl (pH 2.2), 1 mg/ml BSA and neutralized with 1 M Tris•HCl (pH 9.1). The eluted phages were used to infect log-phase bacteria ER2738, concentrated by PEG precipitation and submitted to the second round of selection. The following selections were performed as above except that the Tween-20 concentration was raised from 0.1% to 0.5% in the wash steps. After 3 rounds of selection, the eluted phages were used for plaque isolation. 32 plaque clones were amplified for ELISA test and single-strand DNA preparation.
Screening of phage libraries for H1N1 antibody-reactive phages
IV.C102 (0.2 ml, 10 μg/ml) was coated on three wells of 24-wells microplate at 4°C overnight. The blocking and bio-spanning procedures were carried as did in C179 antibody, except that the binding phages were eluted with IV.C102 (0.2 ml, 10 μg/ml). After 3 rounds of selection, the eluted phages were used for plaque isolation.
Screening of phage libraries for H3N2 antibody-reactive phages
H3N2 polyclonal antibody (30 μg/ml) and goat IgG (100 μg/ml) were coated respectively. After blocking with BSA and washing with TBST for 6 times, Ph.D.-7, Ph.D.-12 and Ph.D.-C7C libraries were added into the goat IgG-coated wells for 30 min incubation. Then, the nonbinding phages were transferred to H3N2 polyclonal antibody-coated wells for additional 30 min incubation. After that, the non-binding phages were discarded, and the binding phages were eluted with H3N2 polyclonal antibody. The eluted phages were amplified in bacteria ER2738 and used in the following selections as above.
Screening of phage libraries for swine-origin influenza antibody-reactive phages
Goat anti-human IgG (100 μg/ml) was coated on six wells. After blocking, three wells were added with diluted SIV sera (1:20), the others were added with human IgG (100 μg/ml). Then they were placed at 4°C overnight and washed with TBST for 6 times. Peptide phage display libraries were added into the IgG wells for 30 min incubation. Subsequently, the nonbinding phages solutions were transferred to the sera wells for 30 min incubation. Then, the binding phages at the sera wells were eluted with goat anti-human IgG. The eluted phages were used for plaque isolation at the end of the 3rd selection.
Binding specificity of the selected phage by ELISA and DNA sequencing
Ninety-six-well plates were coated with mAb and BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight, and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and allowed to bind at 37°C for 1 h. After the unbounded phages were removed with 5% TBST, the bound phages were detected by incubation with peroxidase-labelled murine anti-M13 antibodies (Pharmacia). The bound peroxidase was determined by incubation with Opheny lenediamine dihydrochloride (Pierce Chemicals) in buffer (30 mM citrate, 70 mM Na2HPO4, and 0.02% H2O2, pH 5.5). When the reaction was stopped by the addition of 3 N HCl, A450 nm was determined with an ELISA reader (BioRad). All the assays were carried out in triplicate.
The phage from the 3rd biopanning eluate was cloned for immune-analysis. The nucleotide sequence of the gene III insert was determined as the instruction manual. The amino acid sequence of the insert was deduced from the nucleotide sequence and was compared with native influenza A. The sequences that appeared > 3 times among the selected phage clones were classified as the consensus sequence. The aligned amino acid sequences shared by three or more identical amino acids within the dodecapeptides (heptapeptides) were determined as the mimotopes of the matched protein sequences.
Multi-mimotope gene synthesis
The 7- and 12-mer mimotopes of H1N1, H2N2, H3N2 and SIV were linked by GSGGS with the mimotope sequences of SIV7-SIV12-H1N17-H1N112-H3N27- H3N212-H2N27-H2N212. Each mimotope represents the peptide with the highest frequency on phage surface. The codon usage was optimized by species preference and GC content. The gene was synthesized with EcoR I/BamH I enzyme site by Sangon (Shanghai, China).
The multi-mimotope gene was cut with EcoR I and BamH I endonucleases, and ligated separately into precut pGEX-2 T-1, pGEX-4 T-1 and pET43a (+) plasmids. The ligation products were used to transform competent Trans 109 E. coli cells, which were selected on LB-AMP agar plates at 37°C for 12 h. Three AMP-resisitant clones were picked randomly for plasmids extraction, EcoR I/BamH I digestion and gene sequencing. The confirmed plasmids with correct insert were transformed into competent BL21 (DE3) E. coli cells for protein expression. Four transformed bacteria BL21 (DE3) clones were picked from LB-AMP agar plates for culturing overnight in LB-AMP medium. The resultant bacteria were inoculated into 5 ml fresh medium, cultured to mid-log growth phase for protein expression induction with 1 mM IPTG. After 12 h induction, the bacteria sample was aliquot for SDS-PAGE analysis.
One hundred milliliters of BL21 (DE3) E. coli cells containing pET43a (+)-multi-mimotope plasmids were induced with IPTG for 12 h. The resultant medium was centrifuged with 8000× g for 10 min and the pellet was resuspended into 10 ml of 20 mM TBS (pH 7.9). The cells were broken by ultrasounding with 100 W for 100 s, followed by centrifuging to remove the cell debris. The supernatant was filtered through 0.22 μm membrane and then loaded onto pre-equilibrated Ni2+-NTA-resin. Then, the resin was rinsed by TBS containing 5 mM imidazole; the binding protein was eluted by TBS containing variable imidazole.
In vitro binding
The recombinant multi-mimotope was coated on 96-well microplate with concentration of 10 μg/ml at 4°C overnight, followed by blocking with 2% BSA at 37°C for 2 h. The bait antibodies including C179, H1N1 monoclonal antibody, H3N2 polyclonal antibody and SIV sera were added into wells separately to incubate with coated protein at 37°C for 2 h. The wells were washed with PBST for 6 times. Then, HRP-conjugated secondary antibodies were added for binding at 37°C for 2 h. The TMB solution was then added into the wells for color development, which was stopped with 3 N HCl. The unrelated protein BSA was coated as control protein to determine the binding specificity of multi-mimotope to the antibodies.
To evaluate the potential of the selected mimotopes as experimental vaccine candidates, purified phage mimotopes were used to immunise female inbred specific-pathogen-free BALB/c mice through intraperitoneal administration.
The multi-mimotope protein was concentrated to 1 mg/ml and injected intraperitoneally (50 μg, 100 μg, 200 μg per mouse) or subcutaneously (100 μg per mouse) into BALB/c mice (9 per group) as emulsion (1:1) with complete Freund's adjuvant (CFA) for the first immunization and with incomplete Freund's adjuvant (IFA) for the booster injection at 14 days later. The control group were injected with PBS. Ten days after the booster injection, except for control group, other 5 groups were challenged with 2 × 103 f.f.u A/Puerto Rico/8/1934 (H1N1) by intranasal inoculation of 50 μl per mouse. Mice were weighed on the day of virus challenge and then every three days for two weeks. Two weeks after the challenge, lungs were removed for pathological examination. Blood samples were taken to measure serum Ab titers by ELISA. Animals were conducted and approved by the Institutional Animal Care and Use Committee of the Academy of Military Medical Science, under protocol number 0054921. All experiments were performed according to institutional guidelines.
Serum Ab assay by ELISA
The concentrations of IgG Abs against H1N1 influenza virus were measured by ELISA. Purified antigen was coated on the microtitre plates (100 μl/well, 5 μg/ml in coating solution, 0.1 M sodium bicarbonate, pH 9.6) (Corning, Corning, NY, USA) and incubated at 4°C overnight. Serial 2-fold dilutions of sera (100 μl/well) from each group of unimmunized or immunized and immunized infected were incubated for 1 h at 37°C. Goat anti-mouse IgG HRP (1:10,000 dilution with washing buffer) was used to detect IgG Abs and O-phenylenediamine dihydrochloride (Pierce Chemicals) was used as substrate for HRP and the reaction was monitored at an absorption of 492 nm using an ELISA reader (Labsystems Multiskan, Finland).
The lung tissue pathological examination
Lung tissue samples were fixed in 10% formalin and embedded with paraffin,sections were cut at 5 μm thickness and were stained with hematoxylin eosin (HE).
The data from test groups were evaluated by Student'st-test. The survival rates of mice in test and control groups were compared by using Fisher's exact test. All differences were considered significant at P values0.05.