Peptide immunotherapy for aeroallergens

Abstract

Background:

Allergen specific immunotherapy (SIT) has been used for more than a century. Researchers have been working to improve efficacy and reduce the side effects.

Objective:

We have reviewed the literature about peptides immunotherapy for inhaled allergens. The mechanism of SIT is to induce regulatory T (Treg) cells and to reduce T helper (Th)2 cells to induce class switching from IgE to IgG and induce blocking antibodies to inhibit allergen binding of IgE.

Methods:

The relevant published literatures on the peptide SIT for aeroallergens have been searched on the medline.

Results:

Modification of allergens and routes of treatment has been performed. Among them, many researchers were interested in peptide immunotherapy. T-cell epitope peptide has no IgE epitope, that is able to bind IgE, but rather induces Treg and reduces Th2 cells, which was considered an ideal therapy. Results from cellular and animal model studies have been successful. However, in clinical studies, T-cell peptide immunotherapy has failed to show efficacy and caused side effects, because of the high effective rate of placebo and the development of IgE against T-cell epitope peptides. Currently, the modifications of IgE-allergen binding by blocking antibodies are considered for successful allergen immunotherapy.

Conclusion:

Newly developed hypoallergenic B cell epitope peptides and computational identification methods hold great potential to develop new peptide immunotherapies.

Immunotherapy for allergic diseases, especially allergic diseases caused by inhaled allergens, was first reported by Noon¹ and Freeman² in 1911 on grass pollen allergies (Timothy grass [Phleum pretense], Poa trivialis, Holcus lanatus, Agropyrum caninum, Poaceae).³ Immunotherapy for hay fever has been widely used since then and has also been used to treat perennial allergic rhinitis and asthma caused by mites and animal allergens. In the 1920s, in the United States, weekly subcutaneous injections of crude allergen extracts over a period of years were used.^3,4 However, the anaphylactic response develops even after years of apparent treatment success. Recently, technologic advances have led to the development of defined reagents for immunotherapy that carry less potential for reactivity and require fewer injections.

Mechanism of Action of Immunotherapy

Immunotherapy for allergic diseases has been considered effective by inducing blocking antibodies and regulatory T (Treg) cells. Two to 4 weeks after the start of low-dose allergen immunotherapy, the number of Treg cells producing interleukin (IL) 10 and transforming growth factor (TGF) β production increased, with accompanying suppression of allergen-specific T-helper (Th) 2 cells and production of immunoglobulin E (IgE). Induction of B-cell class switching induces production of allergen-specific IgG2, IgG4, and IgA.^5–12 High-dose allergens can be administered within 1 year of immunotherapy, and the production of IL-12 and IL-27 predominates Th1 and increases the production of interferon γ, which induces IgE-producing B cells to be converted to IgG-producing cells.^13–16 Results of recent studies suggest that these changes in cytokine production are due, at least in part, to epigenetic mechanisms.

In addition, regulatory B cells, a subtype of B cells that produces IL-10, suppress T cells and dendritic cells mediated inflammation and induce immune tolerance. Allergen-specific IgG4 production was confirmed in regulatory B cells induced after immunotherapy for bee venom, and it is presumed that similar reactions occur after immunotherapy with house-dust mite and grass pollen allergens.¹⁷

Treg cells are a subtype of CD4⁺ T cells. Treg cells include natural Treg cells derived from the thymus and induced Treg derived from the periphery. The induced Treg cells control the immune response against non–self-antigens by producing TGB-β. The pathogenic mechanism of allergic rhinitis is associated with Treg cells and Th1/Th2 imbalance. In patients with allergic nasal inflammation, the production of small molecules, especially prostaglandin E₂ (PGE₂), is high, and PGE₂ activates the E prostanoid receptor (EP4) of Treg cells via the cyclic AMP (cAMP) dependent protein kinase A pathway. The PGE₂-EP4-cAMP signaling pathway is involved in patients with allergic nasal inflammation. In these patients, the induction of Treg cells is suppressed via this PGE₂-EP4-cAMP signaling pathway.¹⁸

Methylation of key genes may also indicate successful allergen specific immunotherapy (SIT). It has been reported that methylation of cytosine in the promoter region of forkhead box protein P3 (FOXP3) of Treg cells is closely associated with allergic diseases.¹⁸ Investigation of methylation of Th1-related genes interferon gamma (IFNG), RUNX family transcription factor 3 (RUNX3), and Th2 related genes interleukin 4 (IL4), interleukin 13 (IL13), CCL chemokine 17 (CCL17), and IL-2 in peripheral blood cells after house-dust SIT shows that DNA methylation increased overall, with particularly increased methylation in the promoter region of IL4.¹⁹

Problems with Immunotherapy

In the standard treatment of allergen immunotherapy, intradermal injection of a crude allergen solution is started from a low dose, and inoculation is performed twice a week to every other week. The amount of allergen is gradually increased, and the effect is seen several years later. The burden on the patient is heavy because frequent consultations are required and there is a rare but severe threat of anaphylaxis. In 2017, a European study reported systemic reactions in 90 of 4316 patients (2.1%), of which 65% were determined to be anaphylaxis.²⁰ A North American study of 54.4 million injections showed a 0.1% adverse reaction with 0.8 fatal reactions per year (0.005% life-threatening reaction).²¹ A Canadian study with 380 patients showed 0.095% systemic reaction cases of patients who required epinephrine administration.²² A crude allergen extract contains IgE epitopes and hence the possibility of inducing an anaphylactic reaction. New immunotherapies have been developed to eliminate the risk of these adverse reactions, which largely depend on the ability to identify the amino acid sequences of major allergens and to produce recombinant allergens. The development of recombinant allergens has made it possible to mass produce allergens, fragment allergens, create peptides, and mutate and delete amino acids. In addition, even the synthesis of a short peptide that can inhibit FOXP3/ Nuclear factor of activated T cells (NFAT) interaction suppressor activity of conventional Tregs in vitro is possible.²³

Development of Peptide Immunotherapy

Recombinant proteins, less allergenic homologues of allergens, have been developed to shorten the duration of allergen immunotherapy and reduce adverse effects. These include early peptide immunotherapy, recently developed B-cell epitope peptide, and T-cell epitope peptide.^24,25 (Table 1).

Table 1.

Allergen specific peptide immunotherapy

Ab = antibody; Treg = regulatory T; = IPC = Fel d 1 chain (1 homologous synthetic peptides); aa = amino acid; BM32 = a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens Phl p 1, 2, 5 and 6.

Early Peptide Immunotherapy

Allergen immunotherapy was mainly aimed at suppressing the reaction of Th2 cells, and peptide immunotherapy developed in the 1990s used allergen proteins cleaved into fragments. After that, the amino acid sequence of the allergen was identified, and a T-cell epitope peptide with low or non-reactivity to IgE was determined by using an overlapping peptide scanning. This method was used to treat birch (Betula platyphylla, Betulaceae, Bet v 1) and cat (Felis domesticus, Felidae, Fel d 1) allergies and was shown to be effective to some extent in clinical trials. However, patients treated with high concentrations of peptide showed a delayed respiratory response after inoculation, which confirmed the production of IgE antibodies against the inoculated peptide.²⁶ The effectiveness of peptide immunotherapy was questioned because equivalent responses were frequently confirmed in the placebo-inoculated group.²⁷ To solve these problems, immunotherapy that uses shorter peptides was developed.

B-Cell Epitope Peptide Immunotherapy

The B-cell epitope generally consists of 20–40 amino acids and depends on the tertiary structure of the protein. Some amino acid sequence changes or carrier proteins have been added. In many cases, noninflammatory allergen-specific immunoglobulin IgG4 is induced to act as a blocking antibody and inhibit IgE binding to the antigen.²⁵

T-Cell Epitope Peptide Immunotherapy

T-cell epitope peptides consist of linear 10–17 amino acids and may not contain IgE epitopes. These T-cell epitope peptides bind to Major histocompatibility complex (MHC) class II on antigen-presenting cells and induce Th1 and Treg reactions. Because the T-cell epitope peptide is short, it cannot bind and cross-link IgEs bound to high affinity human IgE receptor (FcεRI). The development of T-cell epitope peptide therapy was advanced because the peptides can induce Th1 and Treg without causing adverse effects. T-cell immunotherapies was considered to have a huge benefit. However, their clinical results have been mixed with limitations. T-cell epitope peptide therapy for the birch allergen Bet v 1 also showed delayed respiratory symptoms after inoculation, similar to early peptide immunotherapy.²⁸ One speculation was that the T-cell epitope stimulated the production of IgE.

In a recent study, peptides from the cat allergen Fel d 1 were used for immunotherapy with the 10 major peptides that bind to Human Leukocyte Antigen - DR isotype (HLA-DR), and the effect was observed even after 2 years without booster vaccination. However, a phase III cat allergy clinical trial revealed no significant difference from the placebo-inoculated group.^29–31 Similarly, Phase IIb mite allergy clinical trials also showed no significant difference from placebo groups.³² The reason for these results is speculated to be that successful immunotherapy needs both T-cell alterations and induction of blocking antibodies.

Hypoallergenic B-Cell Epitope Peptide Immunotherapy

A hypoallergenic B-cell epitope peptide conjugated with a nonallergenic but immunogenic carrier was shown to be hypoallergenic. N-terminal region of surface antigen domain (PreS) from hepatitis B virus was developed for grass pollen allergens, Phl p 1, Phl p 2, Phl p 5, and Phl p 6 (BM32).^33,34 PreS has the ability to provide T-cell help. Subsequent studies with BM32 showed its potential for clinical application.^35,36 In this study,³³ four injections, which included the three initial injections 3 months before the grass pollen season and a booster in the fall after the pollen season, were given during the first year. The next three injections were given before the next pollination season in the second year. A phase IIb study³³ showed an increase in IgG, IgG1, and IgG4 in the patients who were treated. Even though IgG decreased after 5 months, the application of the booster significantly restored the titers of IgG1 and IgG4. T-cell activation was reduced after the BM32 immunotherapy, and mast cell/basophil degranulation may be prevented by these IgG1 and IgG4. BM32 did not substantially modify the IgE levels compared with the baseline. The quality of life of patients with asthma during the pollination season in the second year was improved because they observed only mild reactions.^37,38

Innate Immunity and Toll-Like Receptor Stimulation

Toll-like receptors (TLR) are located on the cell surface or intracellularly and recognize pathogen-associated molecular patterns. A TLR9 stimulant by using an unmethylated cytosine and guanine separated by a phosphate (CpG) DNA motif, an agonist of TLR, coupled with the lipopolysaccharide, monophosphoryl lipid A, induced the innate immune system to lead a Th1 response. The combined use of CpG DNA and the ragweed (Ambrosia aetemisiifolia, Asteraceae) allergen Amb a 1 peptide enhances the effect of immunotherapy. Initial efficacy was observed in clinical trials, but the effectiveness was not confirmed in follow-up studies.³⁹

Potential New Peptide Immunotherapy

We have investigated allergens of mountain cedar (Juniperus ashei, Cupressaseae) pollen, which contains Jun a 1, which is closely related to Japanese cedar (Cryptomeria japonica, Taxodiaceae) and is highly homologous to its major antigen Cry j 1.^40–44 One of the monoclonal antibodies (mAb) against Jun a 1 is E58, which has unique characteristics.⁴⁵ Initial binding of E58 to Jun a 1 inhibits the binding of other anti–Jun a 1 mAbs that bind to four different epitopes.⁴⁶ When these four independent mAbs initially bind to Jun a 1, E58 can still bind to Jun a 1. From this characteristic, we hypothesized that the binding of E58 causes a change in the tertiary structure of Jun a 1 and a modification in the epitopes of the four mAbs, so that these four mAbs cannot bind. The effect of this E58 mAb was also observed in the IgE binding to Cry j 1 and the European cypress pollen allergen, Cup s 1 (Cupressus sempervirens, Cupressaseae). In an experiment that used human high-affinity receptor for IgE transgenic mice,⁴⁷ a significant reduction in symptoms was observed after nasal instillation of Jun a 1 by reacting E58 Fab with Jun a 1 (Fig. 1). Because Jun a 1 belongs to the pectate lyase family of proteins, and this family of allergens includes allergens from grass, weed, and fungi, in addition to conifer pollen, E58 has the potential to be used widely for the treatment of these allergens. We are currently elucidating the tertiary structure of the E58 epitope that induces antibodies with better suppression of the binding of patient serum IgE to allergens and future clinical applications.

Figure 1.

A significant reduction in symptoms was observed after the preincubation of Jun a 1 with E58 Fab.

Future Development of New Peptide Immunotherapies

Instead of biologic experiments, computational identification methods have been proposed to analyze the characteristics of peptides that may help in the design of novel peptides for immunotherapy.⁴⁸ The SDAP webserver⁴⁹ (https://fermi.utmb.edu/ University of Texas Medical Branch, Galveston, TX) provides rapid, validated ways to define regions in known allergens that are similar (low property distance) to a linear IgE binding peptide. Mapping these on the parent or similar allergens reveals conformational epitopes that may be the basis for IgE binding. Other epitope algorithms and webservers for predicting linear and conformational IgE epitopes are listed in Table 2, whereby a full comparison of their results is beyond the scope of this review.

Table 2.

Algorithms and webservers for predicting IgE epitopes

Physical-chemical property motifs can be used to discriminate allergens from nonallergens and may help in limiting the size of allergen peptides for immunotherapy.⁵⁰ Motif scores for Bet v 1 from birch pollen family, which cross-react with homologous fruit and nut allergens, correlated better than global sequence similarities with clinically observed cross-reactivities among those allergens.⁵⁰ The physical-chemical property motifs also discriminated allergenic pectate lyases, including Jun a 1 from mountain cedar pollen, from pectate lyases in the human microbiome, most likely nonallergens.⁵⁰ The latter lacked key motifs characteristic of the known allergens, some of which correlate with known IgE-binding sites.

Another approach is to use computational methods to make sense of large amounts of experimental data to identify motifs and sensitizing features of allergenic proteins. For example, an iterative motif-finding algorithm was applied to data obtained from 3142 serum samples, each tested against 103 highly purified natural or recombinant allergens, which revealed areas for potential cross-reactivity and sensitization.⁵¹ Suprun et al.^52,53 recently reported that they successfully identified IgE epitopes and experimentally validated them for egg and peanut allergens. Identifying regions of allergens that are specific IgE epitopes or particular areas for sensitization can aid in designing hypoallergenic peptide immunotherapies that can be applied with a larger amount of peptides in shorter time periods without risking severe adverse effects.

CONCLUSION

Extensive efforts have been made to improve immunotherapy for aeroallergens for >100 years. T-cell epitope peptide immunotherapy was once considered highly effective without causing severe adverse effects. However, clinical studies failed to show the efficacy and safety of T-cell epitope peptide therapy. Currently, inducing both blocking IgG antibodies and Treg cells is considered essential for successful immunotherapy. Clinical studies of a hypoallergenic B-cell epitope peptide vaccines are ongoing. A better understanding of antibodies that can block allergen binding to IgE and computational methods to analyze large experimental data sets should lead to developing effective and safer peptide immunotherapies against aeroallergens.