Genetic engineering of trimers of hypoallergenic fragments of the major birch pollen allergen, Bet v 1, for allergy vaccination

Abstract

An immunotherapy trial performed in allergic patients with hypoallergenic recombinant fragments, comprising aa 1-74 and 75-160 of the major birch pollen allergen, Bet v 1, has indicated that the induction of allergen-specific IgG responses may be an important mechanism of this treatment. To investigate whether the immunogenicity of the rBet v 1 fragments can be increased, recombinant trimers of the fragments were produced. For this purpose, DNA trimers of rBet v 1 aa 1-74 as well as of rBet v 1 aa 75-160 were subcloned into expression plasmid pET 17b, expressed in Escherichia coli and purified. The fragments as well as the fragment trimers showed a reduced IgE-binding capacity and allergenic activity compared to rBet v 1 wildtype when tested in allergic patients. Both rBet v 1 aa 75-160 monomer and trimer induced high titers of allergen-specific IgG1 Abs in mice. Interestingly, rBet v 1 aa 1-74 trimer induced a much higher IgG(1) response to rBet v 1 than rBet v 1 aa 1-74 monomer. Consequently, IgG Abs induced with the rBet v 1 aa 1-74 trimer inhibited birch pollen allergic patients' IgE-binding 10-fold more efficiently than IgG Abs induced with the monomer. Our data show that the immunogenicity of allergy vaccines can be increased by oligomerization.

Fig. 1

(A) Overview of rBet v 1 derivatives. Recombinant Bet v 1 consists of 160 amino acids including the first methionine. Two hypoallergenic fragments comprise aa 1–74 and aa 75–160. A recombinant trimer of fragment aa 1–74 consists of three fragments of which the first two N-terminal fragments are linked by the spacer LVP and the two C-terminal fragments by the spacer PLV. A recombinant trimer of fragment aa 75–160 has been constructed in the same way. (B) Expression and purification of rBet v 1 and rBet v 1-derivatives. rBet v 1 and rBet v 1-derivatives were expressed in E. coli and purified to homogeneity by ion-exchange chromatography. Aliquots of 3 μg of each protein were analyzed by SDS-PAGE and Coomassie blue staining. In lane M, a molecular weight marker was loaded. Molecular weights are indicated in kDa on the left margin.

Fig. 2

IgE-binding capacity of rBet v 1 and rBet v 1-derivatives. Sera from 26 birch pollen allergic patients (lanes 1–26) and from a non-allergic individual (lane N) were tested for IgE-reactivity with dot-blotted rBet v 1, rBet v 1 aa 1–74, rBet v 1 aa 75–160, rBet v 1 aa 1–74 trimer, rBet v 1 aa 75–160 trimer and BSA. Bound IgE-antibodies were detected with ¹²⁵I-labeled anti-human IgE antibodies.

Fig. 3

Reduced allergenic activity of rBet v 1 derivatives compared to rBet v 1, as measured by CD203c expression. Blood samples from 10 birch pollen allergic patients (patients 31–40) were exposed to rBet v 1, an equimolar mixture of rBet v 1 aa 1–74 trimer and rBet v 1 aa 75–160 trimer (F1trimer + F2trimer) (0.5–0.00005 nM), anti-IgE (1 μg/ml) or buffer (0) (x-axes). Up-regulation of CD203c expression is expressed as stimulation index (SI) (y-axes).

Fig. 4

Bet v 1-specific IgG₁ antibodies induced by immunization of mice with hypoallergenic rBet v 1-derivatives. Groups of 5 mice each were immunized monthly with 5 μg of rBet v 1 aa 1–74 or rBet v 1 aa 1–74 trimer (A), rBet v 1 aa 75–160 or rBet v 1 aa 75–160 trimer (B). Blood samples were taken shortly before each immunization and tested for IgG₁-reactivity with rBet v 1-wildtype in an ELISA assay. On the x-axis, the preimmune sera (lanes 0) and the immune sera (lanes I–V) are shown. The optical densities (OD) corresponding to the amount of bound antibodies are displayed on the y-axis. Data are expressed as mean ± SD. Statistically significant differences of antibody reactivity (p < 0.5) between mouse-groups are indicated for the different blood samples.