Recombinant Hypoallergenic Cat Allergy Vaccines

Abstract

Background: Molecular forms of allergen-specific immunotherapy (AIT) for cat allergy are needed. Fel d 1, Fel d 4, and Fel d 7 are the most important cat allergens.

Methods: IgE epitopes of Fel d 4 and Fel d 7 were mapped by blocking allergic patients' IgE binding with allergen peptide-specific antisera. Five recombinant fusion proteins (PreS-Cat 1-PreS-Cat 5) each containing hepatitis B virus (HBV)-derived PreS as an immunological carrier and non-allergenic peptides from the IgE binding sites of Fel d 1, Fel d 4, and Fel d 7 were expressed in Escherichia coli, purified, and characterized by mass spectrometry, circular dichroism (CD), and size exclusion chromatography. ImmunoCAP and basophil activation experiments demonstrated the hypoallergenic activity of PreS-Cat 1-5. The ability of PreS-Cat 1-5 to induce IgE-blocking antibodies in rabbits was compared to three licensed allergen extract-based AIT vaccines. PreS-Cat 1-5-specific IgG antibodies were tested for inhibition of allergen-specific IgE binding and specific basophil activation. T cell activation and induction of specific cytokine secretion by PreS-Cat proteins were compared with cat allergens in PBMC cultures.

Results: Recombinant hypoallergenic, biochemically and structurally defined PreS-Cat 1-5 were obtained. Two subcutaneous immunizations of rabbits with PreS-Cat 1-5 induced equal (Fel d 1) or better (Fel d 4 and Fel d 7) antibodies (PreS-Cat 5 > PreS-Cat 1 > PreS-Cat 3) blocking allergic patients' IgE binding to cat allergens than six to fifteen immunizations with allergen extract-based vaccines. PreS-Cat-specific antibodies strongly inhibited specific basophil activation. PreS-Cat 5 > PreS-Cat 1 induced significantly more IL-10 in cultured PBMCs from cat allergic patients than cat allergens.

Conclusions: PreS-Cat 5 and PreS-Cat 1 are highly promising molecular vaccine candidates for AIT of cat allergy, combining Fel d 1-, Fel d 4-, and Fel d 7-peptides in single PreS fusion proteins.

Keywords: allergen; allergen‐specific immunotherapy (AIT); allergy; cat allergy; molecular allergy vaccine.

FIGURE 1

Hypoallergenic peptides (Table S2) used for the construction of the cat vaccine candidates are highlighted in the ribbon (left) and surface representation (right) of the three dimensional structures of Fel d 1 (PDB: 1PUO), Fel d 4 (PDB: 8AMC), and Fel d 7 (PDB: 8EPV) using the PyMOL software (A). NT and CT mean the N‐terminus and the C‐terminus, respectively. Remaining parts of chain 1 and chain 2 are indicated in dark and light gray, respectively. (B) Schemes of the PreS fusion proteins (PreS‐Cat 1—PreS‐Cat 5) containing allergen‐derived peptides in different order and location.

FIGURE 2

Lower IgE binding capacity of PreS‐Cat 1–5 as compared to Fel d 1 + Fel d 4 + Fel d 7 (Α). Shown are the percentages of reduction of IgE (y‐axes) when quantitative IgE levels measured by ImmunoCAP specific for PreS‐Cat 1—PreS‐Cat 5 (x‐axes) were compared with IgE levels specific for a mix of Fel d 1, Fel d 4, and Fel d 7 in the numbers of patients indicated below. (B) Comparison of the allergenic activity of different concentrations of PreS‐Cat 1–5 (x‐axes) with an equimolar mix of Fel d 1 + Fel d 4 + Fel d 7 regarding basophil activation in cat‐allergic patients (patients 3, 6, 10, 12, 14, 15, 16, 17, 18, or 19). β‐hexosaminidase releases are expressed as percentages of total mediator contents of cells (y‐axes). The horizontal dashed lines correspond to the percentages of releases observed with medium alone (negative controls).

FIGURE 3

Schedules of immunization of rabbits with PreS‐Cat 1–5 or allergen extract‐based vaccines (Alutard, Tyro‐SIT, and Clustoid). Time points of injections and blood sampling are indicated (x‐axis), and the total number of injections are displayed for each vaccine.

FIGURE 4

Allergen‐specific IgG levels induced by immunization with PreS‐Cat 1–5 (left panels) or allergen extract‐based vaccines (Alutard, Tyro‐SIT, and Clustoid) (right panels). (A) Shown are OD values corresponding to IgG levels specific for Fel d 1, Fel d 4, and Fel d 7 determined by ELISA for different dilutions of immune sera obtained 4 weeks after the last injection or of a 1:500 dilution of the pre‐immune serum (y‐axes). (B) Comparison of allergic patients' (n = 11) IgE binding to Fel d 1 (OD log₁₀) (y‐axis) after pre‐incubation of Fel d 1 with rabbit antibodies obtained after immunization with PreS‐Cat 1–5 or corresponding pre‐immune sera (x‐axis). p‐values < 0.05 were considered significant. *p‐value < 0.05, **p‐value ≤ 0.001, ns—not significant.

FIGURE 5

Comparison of allergic patients' IgE binding to (A) Fel d 1 (patients n = 22), (B) Fel d 4 (patients n = 6), or (C) Fel d 7 (patients [n = 7]) (OD log₁₀) (y‐axis) after pre‐incubation of allergens with rabbit antibodies obtained after immunization with PreS‐Cat 1–5, Alutard, Tyro‐SIT, Clustoid, or corresponding pre‐immune sera (x‐axis). Mean percentages of inhibition of IgE binding are indicated. p‐values < 0.05 were considered significant. *p‐value < 0.05, **p‐value ≤ 0.001, ***p‐value ≤ 0.0001, ns—not significant.

FIGURE 6

Induction of cytokine production in PBMCs of cat allergic patients (n = 20) (y‐axes: Cytokine concentrations; box‐and‐whisker plots showing minimum, quartiles, median, and maximum values) by allergen mix (Fel d 1, Fel d 4, Fel d 7, and PreS), PreS‐Cat 1, PreS‐Cat 5, PreS, or medium alone (x‐axes). Results are presented as bar diagrams with individual data points. p‐values < 0.05 were considered significant. *p‐value < 0.05, **p‐value ≤ 0.001, ***p‐value ≤ 0.0001.