Carbohydrate-based particles: a new adjuvant for allergen-specific immunotherapy

Abstract

The occurrence of systemic anaphylactic side-effects in the course of allergen-specific immunotherapy has been strongly reduced by the adsorption of allergens to aluminium hydroxide, the most frequently used adjuvant in humans. Using the major timothy grass pollen allergen, Phl p 5b, in its recombinant form for immunization of mice, we demonstrate that carbohydrate-based particles (CBP) exhibit several potential advantages over aluminium-hydroxide as adjuvant for immunotherapy. Similar to alum-bound rPhl p 5b, CBP-bound rPhl p 5b induced a stronger antibody and cytokine response than unbound rPhl p 5b after subcutaneous injection in mice. The antibodies induced by CBP-bound rPhl p 5b, exhibited potentially beneficial activities as they cross-reacted with group 5 allergens from five other grass species and inhibited the binding of grass pollen allergic patients IgE to Phl p 5b. Alum-bound rPhl p 5b induced a preferential allergen-specific Th2-response characterized by high immunoglobulin G1 (IgG1) antibody levels and elevated interleukin (IL)-4 and IL-5 production in cultured splenocytes. By contrast, CBP-bound rPhl p 5b, but not rPhl p 5b alone or coadministered with CBP, induced a mixed allergen-specific T helper 1 (Th1)/Th2 immune response characterized by the additional production of allergen-specific IgG2a/b antibody responses and elevated interferon-gamma production. Conjugation of rPhl p 5b to CBP yielded a stable vaccine formulation with preserved immunogenic features of the allergen and, in contrast to alum, induced no granulomatous tissue reactions. Based on these results, CBP is suggested as a potentially useful adjuvant for specific immunotherapy of IgE-mediated allergies.

Figure 1

IgG subclass responses of mice to ELISA plate-bound rPhl p 5b. The optical density values (OD 405 nm) displayed on the y-axis correspond to the levels of Phl p 5b-specific IgG1, IgG2, and IgG3 antibodies in the sera of the four mouse groups (1: CBP-p5; 2: alum-p5; 3: CBP + p5; 4: p5). Results are displayed for preimmune sera (Pre-imm.), the first (1st bl.) and second (2nd bl.) bleeding as box plots where 50% of the values are within the boxes and nonoutliers between the bars. Filled squares denote median values and open circles outliers of each group, respectively. Kruskal–Wallis anova: *P < 0·05; **P < 0·01.

Figure 2

In vitro cytokine production in spleen cell cultures. IFN-γ, IL-5 and IL-4 levels were measured in supernatants of antigen-stimulated spleen cells of each group of mice (CBP- p5:group 1; alum-p5: group 2; CBP + p5: group 3; p5: group 4; alum: group 5; CBP: group 6). In the plot 50% of the values are within the boxes and non-outliers between the bars. Filled squares denote median values, open circles outliers and stars extremes of each group, respectively. Kruskal–Wallis anova, *P < 0·05; ***P < 0·001.

Figure 3

Histopathological analysis of injection sites. On the left side representative skin sections (10-fold magnification) from CBP-p5 (a), alum-p5 (b), p5 (c), alum (d) and CBP (e) immunized mice are shown. The arrows indicate the margins of the granuloma at the injection sites. On the right side sections close ups (100-fold magnification) of the tissue reactions are displayed.

Figure 4

Cross-reactivity of IgG antibodies from CBP-p5-immunized mice (n = 5) with natural group 5 allergens from various grass species. Optical density values (y-axis, OD 405 nm) corresponding to the serum IgG antibody levels to pollen extracts from nine grass species (Phleum pratense, Lolium perenne, Poa pratensis, Anthoxantum odoratum, Triticum sativum, Avena sativa, Cynodon dactylon, Zea mays, Phragmites antralis) are displayed for sera from five mice collected before (P) the immunizations and during the second bleeding (2) at the x-axis. Boxes and horizontal bars denote 50% of the values and nonoutlier range, respectively. Mean values are indicated in the boxes by horizontal bars.

Figure 5

Inhibition of grass pollen allergic patients' IgE binding to rPhl p 5b by murine sera. ELISA plate-bound rPhl p 5b was preincubated with a serum pool from CBP-p5 (Group 1) or from alum-p5 (Group 2) immunized mice. The percentage inhibition of IgE binding determined for sera from nine grass pollen allergic patients is displayed on the y-axis. Boxes and horizontal bars denote 50% of the values and non-outlier range, respectively. Mean values are indicated in the boxes by horizontal bars and open circles denote outliers.