IgE-Associated IGHV Genes from Venom and Peanut Allergic Individuals Lack Mutational Evidence of Antigen Selection

Antigen selection of B cells within the germinal center reaction generally leads to the accumulation of replacement mutations in the complementarity-determining regions (CDRs) of immunoglobulin genes. Studies of mutations in IgE-associated VDJ gene sequences have cast doubt on the role of antigen selection in the evolution of the human IgE response, and it may be that selection for high affinity antibodies is a feature of some but not all allergic diseases. The severity of IgE-mediated anaphylaxis is such that it could result from higher affinity IgE antibodies. We therefore investigated IGHV mutations in IgE-associated sequences derived from ten individuals with a history of anaphylactic reactions to bee or wasp venom or peanut allergens. IgG sequences, which more certainly experience antigen selection, served as a control dataset. A total of 6025 unique IgE and 5396 unique IgG sequences were generated using high throughput 454 pyrosequencing. The proportion of replacement mutations seen in the CDRs of the IgG dataset was significantly higher than that of the IgE dataset, and the IgE sequences showed little evidence of antigen selection. To exclude the possibility that 454 errors had compromised analysis, rigorous filtering of the datasets led to datasets of 90 core IgE sequences and 411 IgG sequences. These sequences were present as both forward and reverse reads, and so were most unlikely to include sequencing errors. The filtered datasets confirmed that antigen selection plays a greater role in the evolution of IgG sequences than of IgE sequences derived from the study participants.

IgE+ B cells are scarce, but allergen-specific B cells with a memory phenotype circulate in patients with allergic rhinitis

Background Despite the critical role of immunoglobulin E (IgE) in allergy, circulating IgE+ B cells are scarce. Here, we describe in patients with allergic rhinitis B cells with a memory phenotype responding to a prototypic aeroallergen. Methods Fifteen allergic rhinitis patients with grass pollen allergy and 13 control subjects were examined. Blood mononuclear cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) were cultured with Bahia grass pollen. Proliferation and phenotype were assessed by multicolour flow cytometry. Results In blood of allergic rhinitis patients with high serum IgE to grass pollen, most IgEhi cells were CD123+ HLA-DR- basophils, with IgE for the major pollen allergen (Pas n 1). Both B and T cells from pollen-allergic donors showed higher proliferation to grass pollen than nonallergic donors (P = 0.002, and 0.010, respectively), whereas responses to vaccine antigens and mitogen did not differ between groups. Allergen-driven B cells that divided rapidly (CD19mid CD3- CFSElo) showed higher CD27 (P = 0.008) and lower CD19 (P = 0.004) and CD20 (P = 0.004) expression than B cells that were slow to respond to allergen (CD19hi CD3- CFSEmid). Moreover, rapidly dividing allergen-driven B cells (CD19mid CFSElo CD27hi) showed higher expression of the plasmablast marker CD38 compared with B cells (CD19hi CFSEmid CD27lo) that were slow to divide. Conclusion Patients with pollen allergy but not control donors have a population of circulating allergen-specific B cells with the phenotype and functional properties of adaptive memory B-cell responses. These cells could provide precursors for allergen-specific IgE production upon allergen re-exposure. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Induction of a Th1 immune response and suppression of IgE via immunotherapy with a recombinant hybrid molecule encapsulated in liposome-protamine-DNA nanoparticles in a model of experimental allergy

Liposome-protamine-DNA nanoparticles (LPD) are safe, effective, and non-toxic adjuvants that induce Th1-like immune responses. We hypothesized that encapsulation of allergens into liposomes could be an appropriate option for immunotherapy. The present study evaluated the immunotherapeutic potential of a recombinant hybrid molecule (rHM) encapsulated in LPD nanoparticles in a murine model of Chenopodium album allergy. BALB/c mice were sensitized with the allergen in alum, and the immunotherapy procedure was performed by subcutaneous injections of LPD-rHM, rHM, or empty LPD at weekly intervals. Sensitized mice developed a Th2-biased immune response characterized by strong specific IgG1 and IgE production, IL-4, and the transcription factor GATA3 in spleen cell cultures. Treatment with the LPD-rHM resulted in a reduction in IgE and a marked increase in IgG2a. The LPD-rHM induced allergen-specific responses with relatively high interferon-gamma production, as well as expression of the transcription factor T-bet in stimulated splenocytes. In addition, lymphoproliferative responses were higher in the LPD-rHM-treated mice than in the other groups. Removal of the nanoparticles from the rHM resulted in a decrease in the allergen's immunogenicity. These results indicate that the rHM complexed with LPD nanoparticles has a marked suppressive effect on the allergic response and caused a shift toward a Th1 pathway.

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics

Background Pollens of subtropical grasses, Bahia (Paspalum notatum), Johnson (Sorghum halepense), and Bermuda (Cynodon dactylon), are common causes of respiratory allergies in subtropical regions worldwide. Objective To evaluate IgE cross-reactivity of grass pollen (GP) found in subtropical and temperate areas. Methods Case and control serum samples from 83 individuals from the subtropical region of Queensland were tested for IgE reactivity with GP extracts by enzyme-linked immunosorbent assay. A randomly sampled subset of 21 serum samples from patients with subtropical GP allergy were examined by ImmunoCAP and cross-inhibition assays. Results Fifty-four patients with allergic rhinitis and GP allergy had higher IgE reactivity with P notatum and C dactylon than with a mixture of 5 temperate GPs. For 90% of 21 GP allergic serum samples, P notatum, S halepense, or C dactylon specific IgE concentrations were higher than temperate GP specific IgE, and GP specific IgE had higher correlations of subtropical GP (r = 0.771-0.950) than temperate GP (r = 0.317-0.677). In most patients (71%-100%), IgE with P notatum, S halepense, or C dactylon GPs was inhibited better by subtropical GP than temperate GP. When the temperate GP mixture achieved 50% inhibition of IgE with subtropical GP, there was a 39- to 67-fold difference in concentrations giving 50% inhibition and significant differences in maximum inhibition for S halepense and P notatum GP relative to temperate GP. Conclusion Patients living in a subtropical region had species specific IgE recognition of subtropical GP. Most GP allergic patients in Queensland would benefit from allergen specific immunotherapy with a standardized content of subtropical GP allergens.

An immunodiagnostic assay for quantitation of specific ige to the major pollen allergen component, pas n 1, of the subtropical bahia grass

Background Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-Atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-Allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-Assay coefficient of variation for the BaGP-Allergic serum pool was 6.92%. Conclusions Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

The enigma of IgE+ B-cell memory in human subjects

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source. © 2013 American Academy of Allergy, Asthma & Immunology.

Allergen capture by IgE and IgG enhanced cell-binding by allergen multimers: How complex is it?

Allergic diseases are the most common chronic disease of the western world, costing $7.8 billion per year in lost productivity and medical care in Australia alone.1 IgE is central to the immunopathogenesis of allergic diseases and important advances are now being made on multiple fronts of IgE research. In particular, two groups independently invested in the generation of IgE reporter mice to address the vexing question of the route of development of the elusive IgE+ B cell.2, 3 Two new anti-IgE mAb targeting membrane IgE and cell-bound IgE have the potential to deplete the cellular source of IgE.4, 5 These could be candidates for alternative anti-IgE treatment options with advantages over current anti-IgE therapy (OmalizumAb), which depletes free serum IgE. Researchers are still intrigued by the modes of interaction of IgE with allergen, and with both its receptors; the high affinity FcεR1 on mast cells and basophils, and the low affinity, C-type lectin, IgE receptor, CD23,6 on B cells and monocytes (Figure 1a and b). A new approach to the study of the complexity of these interactions was recently reported by Reginald et al.7 on page 167 of this issue.

IgE cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens

Background The subtropical Bahia grass (Paspalum notatum) is an important source of pollen allergens with an extended season of pollination and wide distribution in warmer climates. The immunological relationship between pollen allergens of Bahia grass and temperate grasses is unresolved. Methods Serum IgE reactivity of grass pollen-allergic patients with Bahia, Ryegrass and Bermuda grass pollen extracts and their purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA, inhibition ELISA, basophil activation by flow cytometry and molecular modeling. Results Differences in antibody recognition of allergenic components between Bahia grass and Ryegrass pollen were observed by immunoblotting. Eight grass pollen-allergic patients from a temperate region showed greater serum IgE reactivity with Ryegrass pollen than Bahia grass by ELISA. For seven of these sera, Ryegrass pollen inhibited IgE reactivity with Bahia grass pollen but not the converse. For 51 sera from grass pollen-allergic patients in this temperate region, IgE reactivity with Lol p 1 was greater than Pas n 1 or Cyn d 1. IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1, but Pas n 1 IgE reactivity was inhibited by Lol p 1. Two group 1 grass pollen allergen-specific mAb distinguished between temperate and subtropical grass pollens. Basophil activation for three patients tested was greater by Ryegrass pollen than Bahia or Bermuda grass, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, two patients from a subtropical region had higher serum IgE reactivity with Bahia grass pollen than Ryegrass and Bahia grass pollen inhibited IgE reactivity with Ryegrass. A structural model of Pas n 1 showed amino acids implicated in IgE epitopes of other group 1 allergens were juxtaposed on the surface. Conclusion Allergens from subtropical Bahia grass pollen, including Pas n 1, share antigenic determinants with temperate grass pollen allergens, but patients exhibit higher serum IgE reactivity to their locally predominant grass pollen. Basophil activation by Bahia grass pollen and Pas n 1 in patients from a temperate climate indicates clinically relevant cross-sensitization between temperate and subtropical grass pollens.

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass

Background: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. Methods: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. Results: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. Conclusions: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in' the grass pollen season.

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents. © 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.