Exploiting CD22 on Antigen-Specific B-Cells to Prevent Allergy to the Major Peanut Allergen Ara h 2

To the Editor

Oral, sublingual, and epicutaneous immunotherapies are under clinical study as potential food allergy therapies, yet the side effects, requirement for daily dosing, and lack of prolonged efficacy remain limitations in these human trials.[1, 2] Targeting the allergen-specific B-cells may limit side effects and promote long-term tolerance.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunomodulatory receptors with cell-specific expression.[3] Inhibitory Siglecs, including CD22 expressed on B-cells, use immunoreceptor tyrosine-based inhibitory motifs (ITIMs) to suppress activatory receptors, such as the B-cell Receptor (BCR). Enforcing co-localization of CD22 with the BCR, with liposomes that co-display an antigen and high affinity CD22 ligand, not only prevents B-cell activation but also induces apoptosis of the antigen-reactive B-cells, resulting in robust immunological tolerance due to depletion of the antigen-specific B-cells from the B-cell repertoire.[4] These Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) can be formulated with any antigen of choice. STALs displaying Factor VIII (FVIII) inhibit antibody responses to exogenous FVIII, preventing bleeding in FVIII^-/- mice.[4] Accordingly, STALs have the potential to prevent undesired B-cell responses and we were motivated to examine their potential for inducing immunological tolerance to a food allergen.

Peanut allergies are dominated by undesired IgE antibody responses to the 2S albumin Ara h 2 (Ah2), [5, 6] which induce degranulation of effector cells upon exposure to the antigen. We hypothesized that STALs displaying both a high affinity and selective CD22 ligand and Ah2 (Figure 1A) could be an attractive strategy to prevent sensitization and subsequent anaphylaxis to Ah2 and potentially whole peanut extract (WPE).

Figure 1.

Ah2-specific immune responses are prevented in mice administered Ah2 STALs. Schematic of Ah2 STALs (A) and experimental protocol (B); Serum levels of Ah2 sIgE (C) and Ah2 sIgG1 (D) following oral sensitization; Body temperatures (E) and symptom scores (F) after challenge with 200 μg Ah2 i.p. Lines “a” are statistically different from lines “b” (p<0.001) at 15 and 30 min; Mann-Whitney U test *p<0.05, **p<0.01, ***p<0.001

A schematic representing the experimental design is shown in Figure 1B. All animal studies were approved by the UNC IACUC and investigated under protocol # 13-216.0. Four-week old female BALB/cJ mice (Jackson Laboratories, Bar Harbor, Maine) were injected intravenously with 200 μL of 100 μM Ah2 STALs (n=8), 300 μM Ah2 STALs (n=8), 100 μM immunogenic Ah2 liposomes (n=8), or 300 μM immunogenic Ah2 liposomes (n=7). All liposomes consisted of 0.03 mol % Ah2, which amounted to a dose of 0.12 μg of Ah2 in the 100 μM group. STALs additionally consisted of 1% BPA-Neu5Gc, the high affinity and selective CD22 ligand.[4] Two weeks following infusion of STALS, a timeframe previously determined to maximize tolerance induction through STALs [4], the mice were orally-sensitized, with 2 mg WPE and 10 μg Cholera toxin (CT) weekly for three weeks followed by a boost dose of 5 mg WPE and 10 μg CT. A group of naïve mice (n=8) underwent the same protocol and were injected with PBS to determine baseline titers. Serum was collected one week later to quantify specific IgE (sIgE) and sIgG1 to Ah2, WPE, Ah1, and CT by ELISA.[6] Mice were initially challenged with 200 μg Ah2 via an intraperitoneal (i.p.) injection. One week later, mice were challenged i.p. with 750 μg WPE. To assess anaphylaxis during challenge, rectal temperatures were recorded for 30 minutes, and symptom scores were documented at 30 minutes using a 0-5 point scale where 0 represents no symptoms and 5 represents death, as described previously.[6] All methods are described in detail in the online repository material.

On day 42, prior to the challenge, Ah2 sIgE levels were significantly lower in animals injected with either 100 μM or 300 μM Ah2 STALs compared to those injected with the same dose of immunogenic controls (100 μM, p=0.0002; 300 μM, p=0.0006) (Figure 1C). Pre-treatment with Ah2 STALs also inhibited production of Ah2 sIgG1 compared to controls (100 μM, p=0.0047; 300 μM, p=0.0006) (Figure 1D). Upon challenge with 200 μg Ah2, mice pre-treated with either 100 μM or 300 μM Ah2 STALs were protected from hypothermia, an objective feature of anaphylaxis in mice, compared to mice pre-treated with immunogenic controls that had severe reactions (Figure 1E). The symptom scores, as defined previously,[6] also reflected more severe reactions in animals pre-treated with immunogenic Ah2 liposomes compared to those pre-treated with Ah2 STALs (Figure 1F; 100 μM, p=0.0126; 300 μM, p=0.0002). CT sIgE was not different amongst treatment groups (Supplemental Figure E1), indicating that tolerance induction is antigen-specific and that no intrinsic differences in an ability to mount antibody responses were present between the groups. These findings validated the results of a pilot study where 100 μM, but not 20 μM Ah2 STALs, led to significantly lower Ah2-specific IgE and IgG1. Taken together, these results suggest that Ah2 STALs induce antigen-specific tolerance toward the major peanut allergen, severely blunting Ah2 sIgE and sIgG1 levels and reactions upon i.p. challenge with Ah2.

Since Ah2 is only one of several related antigens in peanuts and sensitization was done with WPE, we also examined the impact of Ah2 STALs on sIgE levels to WPE (Figure 2A) and to another peanut allergen, Ara h 1 (Ah1). Pre-treatment with 100 μM Ah2 STALs by intravenous (i.v.) injection resulted in lower peanut-specific IgE compared to the immunogenic control mice, though differences were not statistically significant (p=0.1296). However, mice that received 300 μM Ah2 STALs (i.v.) had significantly less peanut-specific IgE than their control counterparts (p=0.0037). Interestingly, mice injected with Ah2 STALs had lower Ah1 sIgE than their controls at each dose (100 μM, p=0.0146; 300 μM, p=0.0140; Figure 2B). Extensive cross-reactivity between Ah1 and Ah2 has previously been demonstrated and is likely to account for this effect.[7, 8] Finally, these mice were challenged with 750 μg WPE and, similar to the WPE sIgE results, the body temperatures of mice pre-treated with 300 μM Ah2 STALs were significantly greater than mice that received 300 μM immunogenic controls, demonstrating that the STALs attenuated anaphylaxis to WPE (Figure 2C). Symptom scores reflected similar results with the 300 μM immunogenic control mice reacting more severely than the Ah2 STALs mice (p=0.0350). The groups treated with 100 μM Ah2 STALs were not significantly different in body temperatures or symptom scores from the immunogenic control group, suggesting a dose effect. These results demonstrate for the first time that antigen-specific B-cells for a single component can be selectively targeted to diminish an immune response to a complex mixture of several allergens.

Figure 2.

Immune responses to WPE and the peanut allergen, Ah1 in mice treated with Ah2 STALs or immunogenic Ah2 liposomes. Peanut sIgE (A) and Ah1 sIgE (B) following oral sensitization with WPE and cholera toxin. Body temperatures (C) and symptom scores (D) after i.p. challenge with 750 μg WPE. Line “a” is statistically different from line “b” (p<0.05) at 15 and 30 min; Mann-Whitney U test *p<0.05, **p<0.01

In conclusion, our data demonstrate that liposomes simultaneously targeting CD22 and the BCR specific for the major peanut allergen, Ah2, can be used to prevent sensitization to Ah2. Based on previous studies using other antigens, we hypothesize that simultaneous engagement of CD22 and the Ah2-specific BCR leads to deletion of the Ah2 B-cells. Future experiments include testing a combination of STALs that display several peanut antigens as well as using Ah2 STALs as a post-sensitization therapy. These findings provide the foundation for the development of a novel therapy for peanut allergy using a highly targeted, antigen-specific approach.

Supplementary Material

Supplemental Figure 1E. Cholera toxin-specific IgE in mice treated with Ah2 STALs or immunogenic Ah2 liposomes. Mann Whitney statistical analysis **p<0.01, ***p<0.001

Abbreviations

Ah1

Ara h 1

Ah2

Ara h 2

BCR

B-cell receptor

BPA-NeuGc

9-N-biphenylacetyl-NeuGcα2-6Galβ1-4GlcNAc

CT

Cholera toxin

Gal

Galactose

GlcNAc

N-acetylglucosamine

i.p.

Intraperitoneal

ITIM

Immunoreceptor tyrosine-based inhibitory motif

NeuGc

N-glycolylneuraminic acid

sIgE

Specific IgE

Siglec

Sialic acid-binding immunoglobulin-type lectin

STALs

Siglec-engaging Tolerance-inducing Antigenic Liposomes

WPE

Whole peanut extract