Background Sensitization to indoor allergens, particularly to dust mites, is a strong risk factor for asthma in children and adults. Assessment of sensitization is carried out using in vivo and in vitro tests to detect specific IgE antibodies.Objective To investigate IgE antibody responses to mites in patients with asthma, wheezing and/or rhinitis, using chimeric ELISA to measure specific IgE antibodies to mite allergens Der p 1 and Der p 2.Methods Specific IgE antibodies to Der p 1 and Der p 2 were quantified by chimeric ELISA, and compared with IgE to Dermatophagoides pteronyssinus (Dpt) measured using the CAP system (Pharmacia). A panel of sera from 212 patients with asthma, wheezing and/or rhinitis and 11 controls was analysed.Results There was a significant correlation between IgE to Dpt measured by CAP and IgE to Der p 1 (r = 0.81, < P < 0.001), Der p 2 (r = 0.79, P < 0.001) and combined Der p 1 and Der p 2 (r = 0.86, P < 0.001). Seventy per cent of all patients had IgE to Dpt, and of those, 76.5% had IgE to Der p 1, 79.2% had IgE to Der p 2 and 83.1% had IgE to Der p 1 and Der p 2 combined. Considering the cut-off level of 2 IU/mL of IgE to either Der p 1 or Der p 2, the predictive value for a positive IgE to Dpt by CAP was greater than 95%.Conclusions The chimeric ELISA allowed accurate quantification of IgE antibodies to Dpt allergens Der p 1 and Der p 2, and it could be useful for studying immune responses to mites in patients with asthma and/or rhinitis.

sequences from TLPs.A cDNA clone was produced by RT-PCR using as a primer a sequence from the known TLP of tomato.The original N-terminal sequence of the bell pepper TLP was obtained by 5'-RACE.Sequence analysis of the cDNA clone revealed similarity to thaumatin (54,1%) and to TLPs, such as PR P23 from tomato (92,8%), Pru av 2 (44,3%), or Mal d 2 (42%).The 23 kDa protein from bell pepper was designated Cap a 1.To produce recombinant (r) Cap a 1, its His-tagged coding sequence was ligated into the plant expression vector potato virus X (PVX) pgR106.N. benthamiana plants were infected with A. tumefaciens GV 3101 harbouring the modified virus within an agrobacterial plasmid.RT-PCR was performed with RNA isolated from newly formed upper leaves one week post inoculation.Sequencing of the RT-PCR products confirmed the presence and the correct sequence of Cap a 1 mRNA transcribed from a viral subgenomic promoter.Two weeks after inoculation, plants were harvested and tested for the expression of rCap a 1.The protein was purified by Ni-NTA chromatography.In immunoblot analysis, rCap a 1 exhibited IgE-binding capacity identical to the natural protein, indicating that the recombinant protein was expressed in its correct conformation.IgE cross-reactivity with tbaumatin and other allergenic TLPs was demonstrated.

Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome using genetically engineered recombinant allergens. Over the past 10 years, the most important allergens from mites, pollens, animal dander, insects, and foods have been cloned, sequenced, and expressed. Allergens have diverse biological functions (they may be enzymes, enzyme inhibitors, lipocalins, or structural proteins). High-level expression systems have been developed to produce recombinant allergens in bacteria, yeast, or insect cells. Recombinant allergens show comparable immunoglobulin E (IgE) antibody binding to natural allergens and show excellent reactivity on skin testing and in in vitro diagnostic tests. Recombinant allergens will enable innovative new strategies for allergen immunotherapy to be developed. These include peptide-based vaccines, engineered hypoallergens with reduced reactivity for IgE antibodies, nucleotide-conjugated vaccines that promote Th1 responses, and the possibility of developing prophylactic allergen vaccines.

BACKGROUND:Dust mites are important inducers of allergic disease. Group 2 allergens are recognized as major allergens in several mite species, including Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae. No allergens have thus far been characterized on the molecular level from the dust mite Glycyphagus domesticus. OBJECTIVE:We sought to examine the cross-reactivity among group 2 allergens of G domesticus, L destructor, T putrescentiae, and D pteronyssinus. METHODS:A group 2 allergen from G domesticus, Gly d 2, was cloned and expressed as a recombinant protein. Cross-reactivity between Gly d 2 and 3 other group 2 allergens, Lep d 2, Tyr p 2, and Der p 2, was studied by using individual sera and a serum pool RAST-positive to G domesticus, L destructor, T putrescentiae, and D pteronyssinus. Recombinant allergens were used as inhibitors of IgE binding in immunoblotting experiments. Molecular modeling on the basis of the Der p 2 structure was carried out for Gly d 2, Lep d 2, and Tyr p 2. RESULTS:Two cDNAs encoding isoforms of Gly d 2 were isolated, but only the Gly d 2.02 isoform was used in this study. Sixteen of 17 subjects had IgE to Gly d 2. The protein sequence of Gly d 2 revealed 79% identity to Lep d 2 and 46% and 41% identity to Tyr p 2 and Der p 2, respectively. Extensive cross-reactivity was demonstrated among Gly d 2, Lep d 2, and Tyr p 2, but little cross-reactivity was found between these allergens and Der p 2. According to the tertiary structure of Der p 2 and 3-dimensional models of Gly d 2, Lep d 2, and Tyr p 2, differences reside mainly in surface-exposed residues. CONCLUSION:Gly d 2 showed high sequence homology to Lep d 2. Cross-reactivity was observed between Gly d 2, Lep d 2, and Tyr p 2, but only limited cross-reactivity was demonstrated between these 3 allergens and Der p 2.

Background: Mite group 2 allergens Der p 2, Der f 2, and Eur m 2 are 14-kDa proteins of unknown function that share 83% to 85% amino acid sequence identity. Isoforms of the allergens within each genus have been identified which differ by 3 or 4 amino acids, but little is known of the influence of group 2 polymorphisms on human IgE antibody binding. Objective: The purpose of this study was to investigate the importance of interspecies and isoform substitutions on murine mAb and IgE antibody binding and on the molecular structure of the group 2 allergens. Methods: Site-directed mutagenesis was used to incorporate the isoform amino acid substitutions onto the Der p 2.0101 sequence. Recombinant allergens were expressed and purified from Escherichia coli and used to evaluate antibody binding by enzyme-linked immunosorbent assay (ELISA). Molecular modeling of the tertiary structure was used to analyze structural differences between the various group 2 allergens. Results: The substitution of asparagine for aspartic acid at position 114 restored mAb binding of rDer p 2.0101; the other Der p 2 isoforms and the 3 rDer f 2 isoforms also reacted in the 2-site ELISA. The correlation of IgE binding to the Der p 2 isoforms was excellent and tended to be higher in the isoforms with the asparagine 114 substitution (r 2 = 0.87 vs r 2 = 0.95). rEur m 2.0101 bound to all mAb except 7A1; when compared with rDer p 2 for IgE binding, rEur m 2.0101 gave a correlation coefficient of r 2 = 0.68. Molecular modeling revealed that Eur m 2 and the storage mite homologs Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2. Eur m 2 has a conserved surface, whereas Lep d 2 and Tyr p 2 present most of the amino acid substitutions on this surface. Lep d 2 and Tyr p 2 did not react with mAb or with sera from patients with IgE to Dermatophagoides species. Conclusion: The isoform substitutions of rDer p 2 can be distinguished by mAb. The allergenic cross-reactivity between Der p 2, Der f 2, and Eur m 2 is a direct result of the conserved antigenic surface, whereas the lack of cross-reactivity with Lep d 2 and Tyr p 2 is a result of the multiple substitutions across this surface. (J Allergy Clin Immunol 2001;107:977-84.)

Background: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with >80% sequence identity. Isoforms within each genus have been identified which differ by 3–4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. Methods: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. Results: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r2 = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. Conclusions: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.

Monocyte-derived dendritic cells (MDDC) are potent antigen-presenting cells (APC). The APC functions of MDDC in allergy were examined. MDDC presentation of the major house dust mite allergen Derp 2 resulted in 4-12-fold higher T-cell proliferation and markedly higher IFN-gamma and IL-5 production than PBMC cultures. Comparable T-cell proliferation was obtained with 10-fold fewer MDDC than purified monocytes. MDDC cultured from adherent cells, or CD14(+), CD11b(+) or Percoll-purified monocytes were comparable in presenting soluble Ag, and in stimulating allogeneic MLR. Importantly, MDDC presentation of Der p2 resulted in both Th1 and Th2 stimulation, although MDDC are known to produce high levels of IL-12 and stimulate biased Th1 responses. The basis for the potent APC function of MDDC was further examined. MDDC were found to be highly phagocytic. Immunoprecipitation studies showed markedly elevated ICAM-1 expression but > 10-fold reduction in LFA-1 expression on MDDC compared with monocytes. Monoclonal antibody (MoAb)-blocking experiments showed that ICAM-LFA-1 interaction was essential for MDDC stimulation of Derp 2-specific T-cell proliferative responses. These results show that the use of MDDC as APC provides a simple, sensitive and versatile method for detecting T-cell responses to allergens and that the strong phagocytic capability and the increased ICAM-1 expression of MDDC contribute significantly to their Ag presentation potency.

Serum IgG PAN, IgG1-4, and IgE antibodies (Ab) specific for the house dust mite Dermatophagoides antigen (Ag) Der p 2 were measured by enzyme-linked immunosorbent assay (ELISA) in two groups of school children, age 12-14, exposed to high (Charlottesville, VA) and low (Los Alamos, NM) mite Ag levels in their environment. More than 90% of the children were found to have Der p 2-specific IgG antibodies, although the levels of both IgG PAN and IgG 1-4 Ab were substantially higher in the high exposure group (P = 0.001). In addition, there was considerable overlap between these two groups in all Ab measurements. The presence of IgG anti-Der p 2 Ab in the Los Alamos children was unexpected and suggests continuing Ag exposure, although the source of such exposure is not apparent. There is no correlation between RAST (+) and RAST (-) subjects with respect to the level of IgG Ab measured by ELISA. These results suggest that the IgG Ab response to house dust mite has been underestimated in the RAST negative population. Two twin pairs were included in this study and the divergent and varied Ab responses in these individuals indicate that factors in addition to environmental exposure and genetic susceptibility require further investigation and provide evidence for the complexity of the pathogenesis of the allergic response.

J Allergy Clin Immunol 1998;101:423-5.