A nonallergenic birch pollen allergy vaccine consisting of hepatitis PreS-fused Bet v 1 peptides focuses blocking IgG toward IgE epitopes and shifts immune responses to a tolerogenic and Th1 phenotype

Abstract

Allergen-specific immunotherapy is the only allergen-specific and disease-modifying treatment for allergy. The construction and characterization of a vaccine for birch pollen allergy is reported. Two nonallergenic peptides, PA and PB, derived from the IgE-reactive areas of the major birch pollen allergen Bet v 1 were fused to the hepatitis B surface protein, PreS, in four recombinant fusion proteins containing different numbers and combinations of the peptides. Fusion proteins expressed in Escherichia coli and purified to homogeneity showed a lack of IgE reactivity and allergenic activity when tested with sera and basophils from patients allergic to birch pollen. Compared to Bet v 1 allergen, peptides PA and PB showed reduced T cell activation in PBMCs from allergic patients, whereas PreS fusion proteins induced less IL-5 and more IL-10 and IFN-γ. Immunization of rabbits with the fusion proteins, in particular with a PreS fusion protein 2PAPB-PreS, containing two copies of each peptide, induced high levels of IgG Abs against the major IgE-reactive site on Bet v 1 and related allergens. These IgG Abs inhibited allergic patients' IgE binding to Bet v 1 better than did IgG induced by immunization with complete Bet v 1. Furthermore, 2PAPB-PreS-induced IgG inhibited Bet v 1-induced basophil activation in allergic patients and CD23-facilitated allergen presentation. Our study exemplifies novel beneficial features for a PreS carrier-based peptide vaccine for birch pollen, which, in addition to the established reduction in allergenic activity, include the enhanced focusing of blocking Ab responses toward IgE epitopes, immunomodulatory activity, and reduction of CD23-facilitated allergen presentation.

FIGURE 1. Scheme of the construction of rPreS fusion proteins for vaccination against birch pollen allergy.

(A) Amino acid sequence of Bet v 1a. Peptide PA (light gray box) and peptide PB (dark gray box), which are part of the recombinant fusion proteins, as well as Bet v 1–derived peptides P1′– P6′ used for epitope-mapping experiments, are indicated. (B) Illustration of the four rPreS fusion proteins (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS). Peptide A (PA: light gray), peptide B (PB: dark gray), the carrier protein PreS (white), and the C-terminal hexahistidine tags (6xHis: black) are indicated.

FIGURE 2. Characterization of recombinant fusion proteins.

(A) Coomassie-stained SDS-PAGE showing purified rPreS fusion proteins and rPreS. (B) Nitrocellulose-dotted PreS fusion proteins and PreS probed with rabbit anti-PreS Ig (lane 1), rabbit preimmune Ig (lane 2), or buffer alone (lane 3) were reacted with ¹²⁵I-labeled goat anti-rabbit Ig. In addition, dotted proteins were probed with mAbs specific for Bet v 1–derived peptide P2′ (mAb2) (lane 4), Bet v 1–derived peptide P4′ (mAb12) (lane 5), or buffer alone (lane 6) and reacted with ¹²⁵I-labeled rabbit anti-mouse Ig.

FIGURE 3. IgE reactivity of rBet v 1 and of recombinant fusion proteins.

Box plots showing cpm values (y-axis) corresponding to bound IgE Abs (x-axis) for sera from 47 patients allergic to birch pollen.

FIGURE 4. Reduction in allergenic activity of PreS fusion proteins, as shown in basophil-activation tests.

Blood samples from six patients allergic to birch pollen [A1 (A), A13 (B), A22 (C), A23 (D), A24 (E), and A25 (F)] were exposed to increasing concentrations (0.00001–0.1 μg/ml) of Ags, anti-IgE, or buffer alone. Upregulation of CD203c expression is displayed as SI. Means of triplicate measurements are shown, and SD are indicated. The mean fluorescence intensities of unstimulated cells were as follows: A1: 119; A13: 141.7; A22: 112.1; A23: 242; A24: 135; and A25: 183.

FIGURE 5. Lymphocyte proliferation and cytokine responses of PBMCs from patients allergic to birch pollen.

PBMCs from six patients allergic to birch pollen were stimulated with equimolar amounts of rBet v 1, PA, PB, PreS fusion proteins, or PreS. Box plots of SI (A) and cytokines (B, C), for which 50% of the values are within the boxes; extremes and outliers are indicated by asterisks and circles, respectively. Horizontal lines represent median values. The mean cpm of unstimulated cells for the patients analyzed were A1: 1027.2; A20: 4674.3; A21: 2779.0; A25: 2066.8; A26: 9165.5; and A27: 748.0. *p < 0.05.

FIGURE 6. Titers and specificities of Abs raised by immunization of rabbits with rBet v 1 and PreS fusion proteins.

(A) Bet v 1–specific IgG levels (y-axes: OD values) for different serum dilutions of rabbits that were immunized with Ags (2PA-PreS, 2PB-PreS, 4PA-PreS, 2PAPB-PreS, rBet v 1) adsorbed to aluminum hydroxide (Alum) (left panel) or CFA (right panel). (B) IgG levels (y-axis: mean OD values) of the anti-sera shown in (A) for Bet v 1 and for six Bet v 1–derived peptides (P1′–P6′). (C) Sequence alignment of Bet v 1 and Bet v 1–homologous allergens from alder (Aln g 1), hazel (Cor a 1), and apple (Mal d 1). Identical amino acids are indicated by dots, and gaps are represented by dashes. Numbers of amino acids and percentage of sequence identity with Bet v 1 are shown to the right. Peptides A (PA: light gray box) and B (PB: dark gray box) are indicated. (D) IgG levels (y-axis: mean OD values) of the preimmune and immune sera shown in (A) for Bet v 1, Aln g 1, Cor a 1, and Mal d 1 (x-axis).

FIGURE 7. Inhibition of allergic patients’ IgE binding to Bet v 1 by anti–2PAPB-PreS and anti–rBet v 1 Abs.

The percentage inhibition of IgE binding to rBet v 1 obtained with anti–2PAPB-PreS, anti–rBet v 1, or anti–PreS rabbit IgG Abs was determined for sera from 20 patients allergic to birch pollen. Data are displayed as box plots, for which 50% of the values are within the boxes; horizontal lines indicate the median values. Significant differences in inhibition, calculated using a Student t test, are indicated.

FIGURE 8. Inhibition of basophil activation in allergic patients by anti–2PAPB-PreS and anti–rBet v 1 Abs.

Increasing concentrations (0.0000132–0.01 μg/ml) of rBet v 1 in the presence of anti–2PAPB-PreS, anti–rBet v 1, or rabbit preimmune Ig were exposed to blood samples of three patients allergic to birch pollen [A13 (A), A22 (B), A24 (C)]. Anti-IgE Abs or buffer alone were used as controls (Co). Upregulation of CD203c expression is displayed as SI. Means of triplicate measurements are shown, and SD are indicated. The mean fluorescence intensity of unstimulated cells were as follows: A13: 126.3; A22: 127.7; A24: 99.3.

FIGURE 9. Reduction in binding of Bet v 1–IgE complexes to CD23-expressing B cells by anti–2PAPB-PreS and anti–Bet v 1 Ig.

Percentage of relative binding of IgE to B cells after the addition of pre- or postimmune Ig (A) or after the addition of anti-preS Ig (B). The percentage reduction achieved with post-Ig are indicated. SD of triplicate measurements are shown.