The influence of recombinant production on the immunologic behavior of birch pollen isoallergens

Abstract

Background: Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.

Methodology: Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.

Conclusion: The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.

Figure 1. Circular dichroism analysis of Bet v 1.0401.

CD spectra of Bet v 1.0401 batch A were recorded in 10 mM sodium phosphate after addition of sodium hydroxide pH 13 (dashed line) and after refolding in 10 mM sodium phosphate pH 7.4, Spectra of batch B were recorded in 10 mM sodium phosphate pH 7.4, 0.5 M Urea (dashed dot dotted line) and after removal of the Urea in 10 mM were recorded in 10 mM Data are presented as mean residue molar ellipticity [Θ]_MRW at a given wavelength and baseline corrected. For all measurements quartz cuvettes with a path length of 0.1 cm were used. Far UV CD spectra were recorded at 20°C, with a band width of 1 nm, a response time of 1 sec and a data pitch of 1 nm. The measurement range was between 190–260 nm. Each spectrum represents an average of five consecutive scans.

Figure 2. Purification of Bet v 1.0401. SDS-PAGE analysis of Bet v 1.0401 purification.

Bet v 1.0401 batch A was purified from E. coli lysates using a denaturing step during production, batch B was purified from soluble bacterial lysate. Production batch A, lane 1, total bacterial lysate; lane 2, soluble bacterial lysate; lane 3, fraction after basic denaturation; lane 4, salt precipitated fraction; lane 5 hydrophobic interaction chromatography; lane 6, reversed phase chromatography. Production batch B, lane 1, total bacterial lysate; lane 2, soluble bacterial lysate; lane 3, salt precipitated pellet; lane 4, salt precipitated fraction; lane 5, hydrophobic interaction chromatography; lane 6, anion exchange chromatography.

Figure 3. Physicochemical characterization of Bet v 1.0401.

Bet v 1.0401 batches were separated by 2D gel electophoresis. Gels were coomassie-stained, numbered spots were analyzed by MS and assigned to respective charge variants (A). ANS titration curves were recorded in 10 mM sodium phosphate (pH 7.4) using excitation and emission wavelengths of 370 and 450 nm, respectively. Solid lines represent nonlinear regressions of the experimental data (B). Circular dichroism spectra are presented as mean residue molar ellipticity [Θ]_MRW at a given wavelength and baseline corrected. Bet v 1.0101 was used as reference for Bet v 1-like fold (C). Aggregation behaviour of Bet v 1.0401 batches was investigated by dynamic light scattering (DLS) in aqueous solution at a concentration of 1 mg/ml (D) and by online HPSEC-light scattering analysis operated at 0.5 ml/min in 0.1 M sodium phosphate pH 6.5, 150 mM sodium chloride (E). Analysis of Bet v 1.0401 batch A with DLS showed higher aggregation tendency (66% RH of 2.6±0.3 nm and 34% RH of 16±8 nm) compared to batch B (98% RH of 2.1±0.3 nm and 2% RH of 16±10 nm) (D). In online HPSEC-light scattering of batch A, molecular weight values of 70–80 kDa and 17 kDa were determined from refractive index and right-angle light scattering signals from peaks of oligomeric and monomeric Bet v 1.0401 eluting at retention times (vret) of 9.1 and 10.3 ml, respectively. For batch B one peak at vret of 10.3 ml with a molecular weight of 17 kDa was detected.

Figure 4. Allergenic activity of Bet v 1.0401.

IgE binding properties of Bet v 1.0401 batches were tested by ELISA. Results are presented as mean OD values of triplicate measurements after background subtraction. Sera of 13 birch pollen-allergic individuals were tested. Data are presented as median OD405/492 units, 10^th, 25^th, 75^th, and 90^th percentiles as vertical boxes with error bars. P-values were calculated by paired-samples t-test (**P<.01) (A). Allergenic activity of Bet v 1.0401 batches was assessed in huFcεRI-transfected RBL-2H3 cells sensitized with serum IgE from birch pollen-allergic patients (n = 8). Protein concentrations (ng/ml) to obtain 50% of maximum degranulation were determined for the respective antigens. P-values were calculated by paired-samples t-test (*P<.05). In addition, protein concentrations (ng/ml) to obtain maximal mediator release was determined for the respective antigens, as indicated in the upper right corner of the figure grey bars. As negative control RBL cells were loaded with normal human serum (n = 3) and activated with the respective antigens as indicated by white bars. All data are presented as median, 10^th, 25^th, 75^th, and 90^th percentiles as vertical boxes with error bars (B).