Isoforms of Group 1 Allergens from a Tropical/Subtropical Para Grass (Urochloa mutica) Display Different Levels of IgE Reactivity and Cross-reactivity.

Summary: Group 1 grass pollen allergens, or beta-expansins, are the most important major allergens from tropical/subtropical grasses. This study aimed to investigate the sequence similarity, and immunoreactivity of group 1 allergens from Para grass (Urochloa mutica). Three isoforms (Uro m 1.01, Uro m 1.02, and Uro m 1.03) were cloned from cDNA of Para grass pollen. The acidic-neutral isoforms rUro m 1.01 and rUro m 1.02 could effectively inhibited beta-expansins in pollen extract of Bermuda and Johnson grasses, suggesting that these isoforms could be major cross-reacting allergens among these grasses. In contrast, the basic isoform rUro m 1.03 had limited IgE reactivity. Thus, group 1 allergens both acidic-neutral and basic isoforms could have markedly different IgE reactivity.

The minor house dust mite allergen Der p 13 is a fatty acid-binding protein and an activator of a TLR2-mediated innate immune response.

BACKGROUND: The house dust mite (HDM) allergen Der p 13 could be a lipid-binding protein able to activate key innate signaling pathways in the initiation of the allergic response. We investigated the IgE reactivity of recombinant Der p 13 (rDer p 13), its lipid-binding activities, and its capacity to stimulate airway epithelium cells. METHODS: Purified rDer p 13 was characterized by mass spectrometry, circular dichroism, fluorescence-based lipid-binding assays, and in silico structural prediction. IgE-binding activity and allergenic potential of Der p 13 were examined by ELISA, basophil degranulation assays, and in vitro airway epithelial cell activation assays. RESULTS: Protein modeling and biophysical analysis indicated that Der p 13 adopts a ß-barrel structure with a predominately apolar pocket representing a potential binding site for hydrophobic ligands. Fluorescent lipid-binding assays confirmed that the protein is highly selective for ligands and that it binds a fatty acid with a dissociation constant typical of lipid transporter proteins. The low IgE-binding frequency (7%, n = 224) in Thai HDM-allergic patients as well as the limited propensity to activate basophil degranulation classifies Der p 13 as a minor HDM allergen. Nevertheless, the protein with its presumptively associated lipid(s) triggered the production of IL-8 and GM-CSF in respiratory epithelial cells through a TLR2-, MyD88-, NF-kB-, and MAPK-dependent signaling pathway. CONCLUSIONS: Although a minor allergen, Der p 13 may, through its lipid-binding capacity, play a role in the initiation of the HDM-allergic response through TLR2 activation.

Structural and IgE binding analyses of recombinant Der p 2 expressed from the hosts Escherichia coli and Pichia pastoris.

BACKGROUND: The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared. METHODS: Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition. RESULTS: CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2. CONCLUSION: Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure.

Production and immunogenicity of hypoallergenic codon-optimized DNA vaccine encoding mature Der p 1 allergen.

BACKGROUND: Genetic vaccination with plasmid DNA encoding allergens is a promising potential approach for the treatment or prevention of allergy. Nonetheless, because the allergens expressed can display immunoglobulin (Ig) E reactivity, methods to deliver hypoallergenic variants can minimize the risk of type 2 helper (T(H)2) cell priming after DNA immunization. METHODS: A humanized synthetic gene encoding mature Dermatophagoides pteronyssinus group 1 (Der p 1) allergen was cloned into the pHIS expression vector carrying unmethylated CpG 2006 (CpG 2006) motif but devoid of signal sequence. The immunogenicity of this DNA construct was compared in naïve mice with that of recombinant ProDer p 1 protein adjuvanted with alum. RESULTS: Codon optimization of the cDNA encoding mature Der p 1 markedly improved allergen expression. Mature Der p 1, expressed intracellularly in Human Embryonic Kidney 293 cells (HEK 293 cells) transfected with codon-optimized Der p 1 cDNA (pHIS-mHuDer p 1), was shown to be hypoallergenic as it displayed no IgE reactivity. Intradermal vaccinations of naïve Balb/C mice with pHIS-mHuDer p 1 elicited an allergen-specific T(H)1 response characterized by the production of specific IgG2a, a very low amount of specific IgG1, and no specific IgE. Lipoplex formulation with cationic liposome composed of lecithin, N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) and cholesterol not only accelerated the induction of T(H)1 response but also increased its intensity. CONCLUSION: A codon-optimized DNA vaccine encoding mature Der p 1 in a lipoplex formulation could represent a promising hypoallergenic vaccine candidate for safer immunotherapy against house dust mite allergy.

Specific allergy to Penaeus monodon (seawater shrimp) or Macrobrachium rosenbergii (freshwater shrimp) in shrimp-allergic children.

BACKGROUND: Allergy to specific shrimp species has not been studied systematically by oral challenges. A comparison of allergy to different shrimp species, especially seawater or freshwater varieties treatment, would be useful in testing shrimp-allergic subjects. OBJECTIVE: The aim of the study was to identify cases of specific allergy to seawater shrimp, Penaeus monodon (Pm), or freshwater shrimp, Macrobrachium rosenbergii (Mr), among shrimp-allergic children. Comparisons of skin tests using commercial and crude shrimp extracts plus the prick-to-prick (PTP) method were investigated. METHODS: Sixty-eight children with a history of shrimp allergy and skin tests positive to shrimp were orally challenged to both shrimp species. Reactivity to skin prick tests using extracts of Pm (PmSPT), Mr (MrSPT), commercial shrimp (ComSPT), and PTP tests (PmPTP, MrPTP) was compared. RESULTS: Food challenges identified specific allergy to Pm and Mr in 17.65% and 23.53% of the subjects, respectively. Positive and negative challenges to both shrimp species were found in 47.06% and 11.76% of the subjects, respectively. Correlations between the mean weal diameter (MWD) from ComSPT-PmSPT, ComSPT-PmPTP, ComSPT-MrPTP, PmSPT-PmPTP and MrSPT-MrPTP, but not ComSPT-MrSPT, were observed. The MWD from PmSPT and PmPTP were significantly larger in patients with positive than negative challenges to P. monodon (P<0.05). There was a trend that MWD from MrSPT were larger in patients with positive than negative challenges to M. rosenbergii (P=0.058). In the Pm allergy group, PmSPT with an MWD of 30 mm provided 80% predictive probability for positive challenges. PmPTP and ComSPT with an MWD of 22.5 and 20 mm provided 95% predictive probability, respectively. In the Mr allergy group, MrSPT with an MWD of 30 mm provided 95% predictive probability. CONCLUSION: Specific allergy to Pm or Mr was confirmed by food challenges. SPT using crude extracts and the PTP test are useful tools for screening shrimp sensitization before a food challenge. The predictive probability of SPT is helpful where a food challenge is not feasible.

Genetic polymorphisms of major house dust mite allergens.

BACKGROUND: Polymorphic sequence substitutions in the major mite allergens can markedly affect immunoglobulin E binding and T cell responses, but there are few studies on environmental isolates from Dermatophagoides pteronyssinus and none for D. farinae. OBJECTIVE: To determine the sequence variation of the group 1 and 2 allergens from environmental D. pteronyssinus and D. farinae. METHODS: RNA from each species was isolated from homes in Bangkok and the sequence of Der p 1, Der p 2, Der f 1, and Der f 2 determined from cDNA produced by high fidelity polymerase chain reactions. RESULTS: The enlarged data set revealed preferred amino acid substitutions in residues 19, 81, and 215 of Der p 1 as well as sporadic changes. Der p 2 showed frequent variations with clusters of amino acid substitutions, but the canonical Der p 2.0101 was not found in any of 17 sequences. Der f 2 showed variants with clusters of substitutions similar to Der p 2 but in different amino acid positions and without any structural concordance. Der f 1 in contrast to the other allergens had few amino acid sequence substitutions. CONCLUSIONS: The sequence information on variants provides data important for the optimal design of allergen formulations and useful for the genetic engineering and structure-function analyses of the major allergens.