Production and Use of Recombinant Profilins Amb a 8, Art v 4, Bet v 2, and Phl p 12 for Allergenic Sensitization Studies

Abstract

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

Keywords: IgE; pollen-food allergy; profilin; recombinant allergen.

Figure 1

Crystal structure of rArt v 4.0101 (PDB code: 5EM0) with modelled poly(L-Pro). The structure is shown in two different orientations with secondary structure elements colored (α-helices—teal, β-strands—purple, loops—grey). The poly(L-Pro) is shown in yellow stick representation.

Figure 2

Comparison of Amb a 8, Art v 4, Bet v 2, and Phl p 12 sequences. All isoallergens reported by the World Health Organization (WHO) and International Union of Immunological Societies (IUIS) Allergen Nomenclature Sub-committee (allergen.org) are shown. (a) Sequence alignment generated using Clustal Omega [17] and ESPript [18]. (b) Sequence identities and similarities between the profilins as calculated by SIAS (http://imed.med.ucm.es/Tools/sias.html) using the default parameters.

Figure 3

(a) Average melting temperatures (T_m in °C) for rPhl p 12.0101 with and without purification tag. Yellow and blue represent high and low melting temperatures, respectively. (b) Melting temperature (T_m in °C) differences between rPhl p 12.0101 and rAmb a 8.0101. (c) Melting temperature differences between rPhl p 12.0101 and rArt v 4.0101. (d) Melting temperature differences between rPhl p 12.0101 and rBet v 2.0101. Yellow and blue represent high and low differences in the melting temperatures, respectively. The standard deviation for the presented values was less than 1 °C for all experiments.

Figure 4

IgE ELISA results to recombinant Amb a 8.0101, Art v 4.0101, Bet v 2.0101, and Phl p 12.0101. Geometric means with 95% CI are marked.

Figure 5

Correlation between IgE ELISA (X-axis) and multiparameter immunoblot (Y-axis) results for Phl p 12 (upper panel) and Bet v 2 (lower panel).

Figure 6

Correlation between IgE binding to individual recombinant profilins. Pairwise comparison of IgE ELISA results performed with Amb a 8 (a,c,f), Art v 4 (d,e,f), Bet v 2 (b, c, e) and Phl p 12 (a,b,d) is depicted on individual charts.

Figure 7

Results of ELISA inhibition assays, in which Amb a 8.0101 (a), Art v 4.0101 (b), Bet v 2.0101 (c), or Phl p 12.0101 (d) were used in increasing concentrations. Maximum inhibition of IgE binding by different profilins in individual patients is shown. Letters represent individual patients. Patients A–F: dominant IgE binding to Art v 4.0101; G–M: dominant IgE binding to Phl p 12.0101; N–T: no dominant IgE binding.