Viral Vector Based Immunotherapy for Peanut Allergy

Abstract

Food allergy (FA) is estimated to impact up to 10% of the population and is a growing health concern. FA results from a failure in the mucosal immune system to establish or maintain immunological tolerance to innocuous dietary antigens, IgE production, and the release of histamine and other mediators upon exposure to a food allergen. Of the different FAs, peanut allergy has the highest incidence of severe allergic responses, including systemic anaphylaxis. Despite the recent FDA approval of peanut oral immunotherapy and other investigational immunotherapies, a loss of protection following cessation of therapy can occur, suggesting that these therapies do not address the underlying immune response driving FA. Our lab has shown that liver-directed gene therapy with an adeno-associated virus (AAV) vector induces transgene product-specific regulatory T cells (Tregs), eradicates pre-existing pathogenic antibodies, and protects against anaphylaxis in several models, including ovalbumin induced FA. In an epicutaneous peanut allergy mouse model, the hepatic AAV co-expression of four peanut antigens Ara h1, Ara h2, Ara h3, and Ara h6 together or the single expression of Ara h3 prevented the development of a peanut allergy. Since FA patients show a reduction in Treg numbers and/or function, we believe our approach may address this unmet need.

Keywords: adeno-associated virus; food allergy; immune tolerance; liver gene transfer; peanut allergy.

Figure 1

Characterization of in-house generated peanut extract. (A) Raw values of relative Ara h1, Ara h2, Ara h3, and Ara h6 levels measured by Ara h specific ELISA (Indoor Biotechnologies) in peanut extracts from peanut flour and dry roasted peanuts at different pHs (7.2 and 8.5) and a commercial peanut extract (Stallergenes Greer). (B) Relative abundance of Ara h1, 2, 3, and 6 in peanut extracts obtained from different sources. (C) SDS-PAGE of two different peanut protein extracts and purified Ara h1, 2, 3, and 6 peanut proteins (Indoor Biotechnologies).

Figure 2

Validation of peanut allergy sensitization in the FT^+/− model and assessment of allergic responses following two separate challenges. (A) Experimental timeline followed. (B) Changes in core body temperature from baseline to 30 min post challenge. (C) Symptom score (see Table 1 for definitions) following challenge with peanut extract after sensitization and four weeks later. (D) Peanut-specific levels of IgE and (E) IgG1 measured after peanut sensitization and four weeks later. Data are presented as single data points and means ± standard deviation. Statistical testing was conducting using unpaired T-test for all the data sets excluding panel (C), which was performed using the Mann–Whitney test. ** p < 0.01, *** p > 0.001 and **** p < 0.0001.

Figure 3

AAV Ara h cocktail prevents peanut sensitization. (A) Experimental timeline followed. (B) Changes in core body temperature and (C) symptom score (see Table 1 for definitions) following challenge with peanut extract. (D) Peanut-specific levels of IgE and (E) IgG1. (F) Ara h vector genome (vg) copy numbers and (G) Ara h mRNA expression in liver samples. (H) Expression of Ara h proteins in liver and (I) plasma. Data are presented as single data points and means ± standard deviation. Statistical testing was conducting using unpaired T-test for panels (B–E), one-way ANOVA statistic test was used for panels (F–H) and Kruskal–Wallis test was used for panel (I). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

Single AAV-Ara h vectors are less effective for PE prophylaxis. (A) Experimental timeline followed. (B) Changes in core body temperature and (C) symptom score (refer to symptom score table here) following challenge with peanut extract. (D) Peanut-specific levels of IgE and (E) IgG1. (F) Representation of Ara h VG copy numbers and (G) Ara h mRNA expression in liver samples. (H) Expression of Ara h proteins in liver and (I) plasma. Data are presented as single data points and means ± standard deviation. Statistical testing was conducting using one-way ANOVA statistic test for panels (B,D–H), and Kruskal–Wallis was used for panels (C,I). * p < 0.05, ** p < 0.01, *** p <0.001 and **** p < 0.0001.

Figure 5

Increased dose of a single AAV-Ara h3 vector prophylactic effect was comparable to the AAV cocktail. (A) Experimental timeline followed. (B) Changes in core body temperature and (C) symptom score (refer to symptom score table here) following challenge with peanut extract. (D) Peanut-specific levels of IgE and (E) IgG1. (F) Representation of Ara h VG copy numbers and (G) Ara h mRNA expression in liver samples. (H) Expression of Ara h proteins in liver and (I) plasma. Data are presented as single data points and means ± standard deviation. Statistical testing was conducting using one-way ANOVA statistic test for panels (B,D,F,G), and Kruskal–Wallis test was used for panels (C,E,H,I). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.