The Bordetella strain used in this study was the B. pertussis Tahoma I strain (ATCC # BAA-587) grown on Bordet-Gengou agar supplemented with 15% sheep blood (BD Biosciences). Plasmid vectors for transient expression of antibodies in CHO-K1 cells were purified from the E. coli strain DH5α. Transformation of the antibody plasmid vectors into DH5α was by electroporation. Cells successfully transformed were selected on 2XYT plates containing ampicillin.
3.2.3 Antigens and antibodies 3.2.3.1 PTx
Native PTx (in glycerol or lyophilized) or genetically modified PTx (PTg) was purchased from List Biological Laboratories, Inc. (Campbell, CA). Native PTx was also obtained through BEI Resources, NIAID, NIH: B. pertussis Toxin, Salt-free, NR-31826. A soluble, truncated version of the PTx S1 subunit, PTx-S1-220 expressed using the pAK400 plasmid was purified from E. coli as described (Sutherland and Maynard 2009). 3.2.3.2 Antibodies
The anti-PTx antibodies were transiently expressed on a laboratory scale by protein A/G affinity chromatography as described for purification of the anti-FHA antibodies (Chapter 2). Human intravenous pertussis immunoglobulin (P-IGIV) (Bruss, Malley et al. 1999) was obtained from the Massachusetts Public Health Biologic Laboratory. The previously characterized murine anti-PTx neutralizing antibodies, 1B7 and 11E6 (Sato, Sato et al. 1987, Sato and Sato 1990) were purified as described (Sutherland and Maynard 2009). Humanized versions of 1B7 and 11E6 with similar affinity and epitope specificity as the parent murine antibodies were purified from the
culture media of stably transfected CHO cells by Catalent (Somerset, NJ) (Nguyen et al., 2014. In review). HRP and fluorescent conjugated secondary antibodies were obtained from Southern Biotech and Thermoscientific respectively.
3.2.4 Phylogenetic analyses
The DNA sequences of the isolated anti-PTx antibodies were analyzed similar to the analyses done for the anti-FHA antibodies (Section 2.2.5). Briefly, NCBI/IgBLAST was used to map the VDJ or VJ sequences of each VH or Vĸ respectively to the closest human germline sequence from the IMGT database by selecting the hit with the highest score. The score is determined after adding points for each match and subtracting points for each mismatch (Ye, Ma et al. 2013). The gene segment pairings (VDJ or VJ) for each genetic locus (H or K) were compared. The frequencies of pairing for the putative VDJ or VJ are presented as a heat map. The individual gene families and alleles were also analyzed by indicating the frequencies in a pie chart. To compare the full VH or Vĸ, the
sequences of the isolated antibodies were aligned using Phylogeny.fr (Dereeper, Guignon et al. 2008) to generate phylogenetic trees.
3.2.5 Thermal stability analysis
Thermal stability of the purified antibodies was analyzed as described for the anti- FHA antibodies (Chapter 2). Briefly, three replicates of 100 µg/ml solutions of the antibodies were prepared in PBS using the protein thermal shift dye (Life Technologies) according to the manufacturer’s protocol. The antibodies were heated from 25oC to 90oC with a scanning rate of 1oC/min increments. The melting temperature (Tm) was
fluorescence. The data was fit to a sigmoidal four parameter (4PL) non-linear regression model using Graph pad prism 5. For antibodies which exhibited complex melting paths, the temperatures at 10% of the maximum fluorescence, defined as the Tonset temperature,
or the average of the minimum and maximum fluorescence, Taverage was determined.
3.2.6 Antigen specific responses 3.2.6.1 Indirect ELISA assays
Indirect capture ELISAs were performed as described for FHA (Chapter 2) with the following modifications. PTx holotoxin final concentration wase 1 µg/ml. PTx A- subunit and PTx B-subunit final concentrations were 0.25 µg/ml and 0.85 µg/ml respectively. Briefly, serially diluted antibodies were added to the plates after blocking with 5% milk in wash buffer. Subsequently, HRP-conjugated anti-human Fc antibodies (Southern Biotech) diluted 1:1000 were used to detect bound antibodies. The plates were developed with TMB substrate (ThermoScientific) and quenched with 1N HCl. Absorbances were read at 450 nm in a SpectraMax M5 instrument (Molecular Devices) using the SoftMax Pro v5 software. The relative binding affinities of antibodies to respective coated antigens were determined by estimation of the concentration of each antibody required to achieve 50% maximal binding at saturation (EC50). The EC50 values
were determined from fitting the binding curves to the 4 parameter logistic (4PL) non- linear curve model using the GraphPad Prism 5 software.
3.2.6.2 Competition ELISA assays
The ability of the antibodies to compete with competitor antibody for binding to PTx was evaluated by a competition ELISA as described for FHA (Chapter 2) with the following modifications. The competitor antibody was either a biotinylated or a murine antibody. Due to low concentrations, the anti-PTx antibodies were biotinylated with 1000 or 5000 molar excess EZ-link Sulfo-NHS-LC-Biotin following the manufacturer’s instructions. Functional binding of the biotinylated antibodies to PTx was confirmed. The biotinylated antibody was detected with streptavidin-HRP, while the murine antibodies were detected with a polyclonal goat-anti-mouse antibody preparation.
3.2.6.3 Simultaneous binding of antibody to PTx bound to fetuin
Fetuin was used as model soluble receptor to detect antibodies bound to a PTx- fetuin complex using a method adapted from (Kenimer, Kim et al. 1989). High binding ELISA plates (Costar) were coated with 50 µL/well of 10 µg/mL fetuin (Sigma) at in PBS. After overnight incubation at 4˚C, the plates were blocked with 50 µL/well blocking buffer (PBS + 5% BSA) at room temperature for 1 hour. Plates were washed three times in wash buffer (PBS, + 0.05% Tween-20) after each incubation step. Serially diluted PTx was then added to the fetuin coat and incubated further overnight. Antibodies at a concentration of 1 µg/ml antibody were then added and incubated for 1 hour at room temperature. Anti-human Fc HRP (Jackson Immunoresearch) was added at a 1:1000 dilution in wash buffer and incubated at room temperature for another hour. Signal was developed using the TMB Substrate Kit (Thermo-Scientific), the reaction stopped with 1N HCl, and the plate read using a SoftMax Pro v5 (Molecular Devices) at 450 nm. All plated volumes were 50 µL/well.
3.2.6.4 Western blot
To detect antibody epitopes on PTx subunits, 0.5 µg PTx was first boiled for 2 minutes in reducing SDS buffer to dissociate PTx into its constituent subunits. PTx was then electrophoresed in a 12% gel, and then transferred to a PVDF membrane at 20V, 4oC overnight. The membranes were blocked with PBS + 5 % milk, washed with PBS- tween, incubated with 25 µg of antibody, and subsequently with HRP-conjugated secondary antibodies. Binding to PTx subunits was detected using a colorimetric TMB substrate kit (Fisher scientific).
3.2.6.5 Expression of antibody epitopes on whole B. pertussis
The expression of anti-PTx epitopes on live B. pertussis was determined as for FHA (Chapter 2). Briefly, B. pertussis harvested from 3 day cultures grown on Bordet Gengou agar, were resuspended in PBS with 5% glycerol (PBSG), to an OD600/mL of 1 after washing with PBS. The cells were then incubated with 1 µg of each antibody in 100 µL at room temperature. Bound antibodies were detected after washing with a 1:500 dilution of goat anti human Fc Alexa Fluor 647 conjugate (Jackson Immunoresearch). 10 000 events per treatment were collected on a BD SLRII Fortessa flow cytometer. The cell associated fluorescence for a gated population of the cells was determined using the FlowJo_V10 software.