2.1. Cells, antibodies and viruses
The yeast cell strain Saccharomyces cerevisiae
EBY100 was provided by Pacific Northwest National Laboratory (Richland, Wa). The mAbs D7, D8, F10, G17, H40, A66, D80, E90, H5-2A, 11A and 80R were isolated from a human phage display library [3
]. H5-2A was raised against monomeric HA0, and the other anti-H5 mAbs were obtained by panning with trimeric HA0 (highly pathogenic avian subtype H5N1 A/Vietnam/1203/04). NR2728, an HA-specific (A/Vietnam/1203/2004) mouse mAb, was obtained through the NIH Biodefense & Emerging Infections Research Resources Repository (Manassas, VA). The reassortant H5N1 avian influenza virus VNH5N1-PR8/CDC-RG, which contains HA and NA genes derived from A/Vietnam/1203/2004 was obtained from the Center for Disease Control (CDC, Atanta, GA) and propagated in Madin Darby Canine Kidney (MDCK) cells at The New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (NERCE) at Harvard Medical School (Boston, MA).
2.2. Construction of yeast surface display vectors
The full-length protein (HA0) and its subunits (HA1 and HA2) were cloned into a yeast display vector, pCTCON2 (from Dane Wittrup, Massachusetts Institute of Technology, Cambridge, MA). Briefly, HA0, HA1 or HA2 gene fragments of A/Vietnam/1203/2004 were PCR-amplified using the pAcGP67A-HA vector [3
] as template and specific primers and cloned into the pCTCON2 yeast vector in-frame with the endogenous yeast Aga2p signal peptide and aga2
gene at the N-terminal end and cMyc at the C-terminal end (). The resulting plasmids were transformed into competent yeast using the Frozen-EZ Yeast Transformation Kit (Zymo Research, Irvine, CA), which were then grown on synthetic dextrose plus casein amino acids (SD-CAA) agar plates under dual selection (Ura- and Trp-) at 30°C for 3 days. Single colonies were grown overnight in 5 ml SD-CAA medium (20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, 5.4 g/L Na2
, 8.6 g/L NaH2
O and 5 g/L casamino acids) at 30°C with shaking. Expressions of HA proteins were induced with galactose in SG-CAA medium (similar to SD-CAA medium except dextrose was replaced by galactose) at 20°C for 3 days.
Figure 1 Display of full-length HA0 and its subunits on yeast cell surface. A. Schematic of HA proteins displayed on the yeast surface (upper) and the gene construct expressing HA (full-length or each subunit) as a fusion protein with the Aga2 signal peptide (aga2SP), (more ...)
2.3. FACS analysis of HA expression on yeast surface
Surface expression of the HA0 and its subunits were confirmed by FACS (BD FacsCalibur, BD Biosciences, San Jose, CA; FlowJo software, Tree Star, Ashland, OR) using anti-cMyc directed towards the C-terminal tag. After induction (Section 2.2), cells were washed with PBS 0.5% BSA (PBS-B), probed with anti-cMyc (1:200 in PBS-B; 1 h, 25°C) and then stained with anti-chicken Alexa 488-conjugated antibody (Invitrogen, Carlsbad, CA; 30 min, 4°C). For binding specificity of surface-expressed HAs, the induced cells were incubated with anti-HA antibodies (1 h, 25°C), followed by a FITC-conjugated goat anti-human or anti-mouse IgG (30 min, 4°C).
2.4. Competitive binding of H5-2A and NR-2728
Yeast cells expressing the HA1 subunit were probed with unlabeled H5-2A or NR-2728 mAb for 1 h at 4°C. Unbound antibodies were removed after three washes with PBS-B. H5-2A and NR2728 were conjugated using an Alexa Fluor 647 mAb labeling kit (Invitrogen). Conjugated H5-2A or NR2728 was then added to the cells for 1 h at 4°C. Yeast cells were washed three times with PBS-B and analyzed by FACS.
2.5. Western blotting
Yeast surface-expressed HA proteins were resolved using SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% skim milk overnight, the blot was probed with anti-H5 polyclonal antibody (1 h, 25°C), followed by horseradish peroxidise (HRP)-conjugated anti-mouse IgG (1 h, 25°C). Proteins were detected using the West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and exposure to autoradiography film.
2.6. Construction of the epitope mapping library in yeast
A library of HA1 mutants was generated by an error-prone PCR method using the GeneMorph II random mutagenesis kit (Invitrogen). The HA1 subunit was amplified from pCTCON2-HA1 using the primers: pctcon2-MUT-F, 5′-CGACGATTGAAGGTAGATACCCATACGACGTTCCAGACTACGCTCTGCAG-3′; pctcon2-MUT-R, 5′-CAGATCTCGAGCTATTACAAGTCCTCTTCAGA AATAAGCTTTTGTTC-3′. Five micrograms of each gel-purified construct containing the mutant HA1 sequence were co-transformed with a NheI/BamHI-digested pCTCON2 vector into competent yeast cells and cultured on SD-CAA plates. The transformed cells were serially titrated on SD-CAA agar plates to determine the size of the library. The yeast library was grown in SD-CAA medium and induced to display proteins as described in Section 2.2.
2.7. Selection and fine epitope mapping by FACS
Surface expression of the HA1 mutant library was induced (Section 2.2) before each round of FACS selection for mutants binding only by mAb H5-2A or NR2728 mAbs after double staining with both antibodies (1 h, 25°C), followed by secondary PE-conjugated anti-human IgG and FITC-conjugated anti-mouse IgG (30 min, 4°C). Yeast cell populations binding exclusively to H5-2A or NR2728 were sorted on a DakoCytomation High Speed MoFloSorter (DaKoCytomation, Fort Collins, CO) and enriched in SD-CAA medium. After a second round of selection and sorting, the cells were plated to isolate individual clones and binding specificity confirmed by FACS. Plasmids from yeast clones binding exclusively to H5-2A or NR2728 mAbs were recovered (Zymoprep Yeast Plasmid Miniprep Kit, Zymo Research) and DNA sequenced using the primers pCTCON2-F:5′-GTTCCAGACTACGCTCTGCAGG and pCTCON2-R:5′-GATTTTGTTACATCTACACTGTTG.
2.8. Hemagglutination inhibition (HI) assay
The inhibition of hemagglutination by mAb H5-2A or NR2728 was performed to assess antibody response to the H5N1 virus. Two-fold dilutions of mAb samples were mixed with reassortant H5N1 virus at a concentration of 4 HA units per well and incubated for 30 min at RT. Fifty microliters of a 0.5% suspension of turkey RBCs were added to each well, and hemagglutination was assessed visually after 1 h.
2.9. Determination of linear versus conformational epitopes
For determination of linear versus conformational epitopes, the yeast surface-expressed HA1 subunit was denatured by heating the yeast cells at 80°C for 30 min. After chilling on ice for 20 min, cells were labeled with H5-2A or NR-2728 mAb, followed by a FITC-conjugated IgG for FACS analysis.