- Email: [email protected]
B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom
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low KIT D816V allele burden that can be detected using only a highly sensitive method,4 as also demonstrated by the low levels detected in our 4 cases. The analytical sensitivity of the mutation analysis performed in the present study depends on the DNA concentration of the individual samples and was typically in the range of 0.001% to 0.03% mutation-positive alleles, as previously described in detail.3,4 The 109 patients included in our study who tested negative for the KIT D816V mutation in PB were all examined clinically in our center and none had clear indications for mastocytosis based on skin examination, clinical symptoms, and s-tryptase level. Suspected elicitors among these 109 mutation-negative patients were diverse including, for example, insects, drugs, foods, exercise, and apparent idiopathic cases, and current investigations including provocation tests are ongoing in this group. A total of 29 of these patients had anaphylaxis elicited by an insect of which 5 patients lacked any skin symptoms during the acute episode whereas 22 patients had urticaria and/or angioedema and 2 patients had flushing or pruritus. A final exclusion of SM in this group would demand a BM examination, and it thus cannot be excluded that cases of SM may have been missed in this group—and especially among the patients without skin symptoms during the anaphylactic episode—because of aforementioned reasons. However, on the basis of previous studies from our group that included a group of negative controls, ie, patients with elevated s-tryptase level, in whom SM was excluded after thorough BM investigation, we find this unlikely to be the case4 and following this we do presently not perform a BM investigation in patients with anaphylaxis if KITD816V is not detected or UP is not found, unless other signs strongly suggestive of SM are present (ie, markedly elevated/rising basal s-tryptase level, recurring idiopathic anaphylaxis, early onset osteoporosis). In conclusion, we here present data on 4 adult patients with anaphylaxis in whom a positive KIT D816V mutation in PB subsequently led to a diagnosis of SM in spite of normal or low basal s-tryptase level and absent or inconspicuous UP skin lesions. Except for absent skin symptoms during the anaphylactic episode, the 4 patients lacked any other clinical findings that might have been a clue to the diagnosis. The use of sensitive KIT D816V mutation analysis of PB as a diagnostic test in mastocytosis thereby clearly shows its clinical utility in patients with anaphylaxis. Further studies in a larger cohort of patients with fully characterized anaphylaxis are needed to fully interpret the value of performing the test indiscriminately in all patients with anaphylaxis as opposed to testing selected patients only on the basis of clinical or laboratory discriminators indicating mastocytosis, also given the low rate of positive results (3.5%) found in the present study. However, the method used for KIT D816Vanalysis in this study has been demonstrated to have a very high diagnostic sensitivity and specificity and is furthermore quick, simple, and cheap (reagents
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Thomas Kristensen, PhDd* Charlotte G. Mortz, MD, PhDa,b* On behalf of the Mastocytosis Centre Odense University Hospital (MastOUH) and Odense Research Centre for Anaphylaxis From athe Department of Dermatology and Allergy Centre, cthe Department of Hematology, and dthe Department of Pathology, Odense University Hospital, Odense, Denmark; bthe Institute of Clinical Research, University of Southern Denmark, Odense, Denmark; and ethe Department of Dermatology and Allergy, Interdisciplinary Mastocytosis Center Charite, Charite - Universit€atsmedizin Berlin, Germany. E-mail: [email protected]. *These authors contributed equally to this work. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Metcalfe DD. Mast cells and mastocytosis. Blood 2008;112:946-56. 2. Valent P, Akin C, Escribano L, Fodinger M, Hartmann K, Brockow K, et al. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest 2007;37:435-53. 3. Kristensen T, Vestergaard H, Moller MB. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay. J Mol Diagn 2011;13:180-8. 4. Kristensen T, Vestergaard H, Bindslev-Jensen C, Moller MB, Broesby-Olsen S. Mastocytosis Centre, Odense University Hospital (MastOUH). Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis. Am J Hematol 2014;89:493-8. 5. Simons FE, Ardusso LR, Bilo MB, El-Gamal YM, Ledford DK, Ring J, et al. World allergy organization guidelines for the assessment and management of anaphylaxis. World Allergy Organ J 2011;4:13-37. 6. Sampson HA. Anaphylaxis and emergency treatment. Pediatrics 2003;111:1601-8. 7. Stoevesandt J, Hain J, Kerstan A, Trautmann A. Over- and underestimated parameters in severe Hymenoptera venom-induced anaphylaxis: cardiovascular medication and absence of urticaria/angioedema. J Allergy Clin Immunol 2012; 130:698-704.e1. 8. Alvarez-Twose I, Gonzalez-de-Olano D, Sanchez-Munoz L, Matito A, JaraAcevedo M, Teodosio C, et al. Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms. Int Arch Allergy Immunol 2012; 157:275-80. 9. Oude Elberink JN, de Monchy JG, Kors JW, van Doormaal JJ, Dubois AE. Fatal anaphylaxis after a yellow jacket sting, despite venom immunotherapy, in two patients with mastocytosis. J Allergy Clin Immunol 1997; 99:153-4. Available online August 1, 2014. http://dx.doi.org/10.1016/j.jaci.2014.06.031
B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom To the Editor: Vespid venoms contain a variety of proteins, such as phospholipase A1, hyaluronidase, and antigen-5, which are often associated with allergic responses in humans.1-5 New vaccination strategies are focusing on antigen-5,6 which has been isolated from the venoms of all clinically relevant species of social wasps.1,3,7 The social wasp Polybia paulista causes hundreds of stinging accidents of medical importance each year in Brazil.8 Antigen-5 has been reported to be the primary allergen present in the venom of this social wasp,7 being a good candidate for studying the IgE-binding B-cell linear epitopes involved in wasp venom allergy. To identify the B-cell linear epitopes present in the full-length allergen, the 66 overlapping synthetic peptides corresponding to the complete sequence of P paulista antigen-5 were assayed against the pool of 5 serum samples from patients allergic to P paulista venom (see Table E1 in this article’s
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FIG 1. B-cell linear epitopes mapping along the primary sequence of the P paulista venom antigen-5 detected in the SPOT synthesis assay with the sera of patients who were sensitive to the venom of the wasp P paulista. IgG-reactive peptides (A); IgE-reactive peptides (B); the smallest peptide sequence corresponding to the B-cell linear epitope of antigen-5 that is immunoreactive to human IgE (C); P paulista venom antigen-5 primary sequence (D) showing regions of IgG reactivity (black bars), IgE reactivity (grey bars), and the identification of the smallest peptide sequence corresponding to the B-cell linear epitope that was immunoreactive to human IgE.
FIG 2. Three-dimensional mapping of B-cell linear epitopes of antigen-5 that were reactive to human IgG shown in yellow: epitope 1 (A), epitope 2 (B), epitope 3 (C), epitope 4 (D), epitope 5 (E), epitope 6 (F), epitope 7 (G), epitope 8 (H), and epitope 9 (I); epitope 7 is also reactive to human IgE. The elements of secondary structures shown in blue correspond to helices; those shown in red and green correspond to beta-sheets and loops, respectively; meanwhile, the regions represented in yellow correspond to the spatial location of the linear epitopes for human IgG and/or IgE.
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Online Repository at www.jacionline.org). These peptides were synthesized in solid phase using the SPOT method with overlaps of 11 amino acid residues with an offset of 3 residues; these sequences were designated A1 to A24, B1 to B24, and C1 to C18, and a control sequence from a bacterial antigen specific for human IgG was synthesized and assigned as C20 and C21 (see Tables E2 and E3 in the Online Repository at www.jacionline.org). These peptides were assessed for reactivity with human IgG and human IgE as described elsewhere9; the spot images of these assays are shown in Fig 1. The following 9 antigenic determinants were reactive with human IgG (Fig 1, A): epitope 1 (A15-A17, VDEHNRFRQ), epitope 2 (A19-A20, VAQGLETRGNP), epitope 3 (B3-B4, WNDELAYIAQV), epitope 4 (B11-B12, RNTAQYQVGQN), epitope 5 (B20-B22, WENEVKDF), epitope 6 (C1-C3, KENFAKVGHYT), epitope 7 (C5, VGHYTQVVWAKTKE), epitope 8 (C10-C11, SIKYIEKGMKS) and epitope 9 (C17-C18, GNVL GAQIYEI). The results of Fig 1, B show that epitope 7 (C5, VGHYTQVVWAKTKE) is reactive both to human IgG and human IgE. The analyses of the chemiluminescent signal intensities of the spot images generated by coupling the human IgEs and IgGs to the B-cell linear epitopes of antigen-5 are shown in Figs E1 and E2 (available in the Online Repository at www. jacionline.org), respectively. For further confirmation of these epitopes, the corresponding peptides were synthesized and submitted to indirect ELISA assay. Epitopes 1 to 9 were recognized by human IgG, while Epitope 7 was also recognized by human IgE (Fig E3, A and B). Epitope 7 corresponds to the linear sequence observed for C5 (VGHYTQVVWAKTKE) in Fig 1, B. Thus, it was considered to identify the smallest peptide sequence corresponding to the epitope for human IgE. The sequence KENFAKVGHYT was reactive with human IgG (epitope 6), and this sequence partially overlapped with the sequence of epitope 7 (corresponding to C5 in Fig 1, C), with the sequence of VGHYT shared by the peptides. The peptide sequence VGHYTQVVWAKTKE (peptide C5.1 in Fig 1, C) was re-synthesized using the SPOT strategy as the following 3 smaller peptides: VGHYTQVV, TQVVWAKTKE, and WAKTKE (peptides C5.2, C5.3, and C5.4, respectively, in Fig 1, C). These peptides were then assayed against a pool of serum samples from 5 patients allergic to P paulista venom using an alkaline-phosphatase-conjugated secondary antibody against human IgE. The results of the SPOT image intensities are shown in Fig 1, C, suggesting that the first 8 residues of epitope 7 (VGHYTQVV) are essential for the interaction between antigen-5 and IgG, and that the other 6 residues (WAKTKE) are critical for the interaction between antigen-5 and IgE. Fig 1, D shows the positions of the B-cell linear epitopes for human IgG and IgE along the primary sequence of the P paulista venom antigen-5. The 3-dimensional structure of P paulista venom antigen-5 was built through molecular modeling7 to map the B-cell linear epitopes onto its structure. Fig 2 shows that the sequence WAKTKE is located in a loop area on the surface of the protein, where it is accessible to the IgE antibody. These conserved surfaces were proposed to represent the major B-cell-binding epitopes and could be used as model peptides for engineering vaccines for allergen immunotherapy.10 The results shown in Fig E4 (available in the Online Repository at www.jacionline.org) suggest that the linear epitopes that were experimentally identified in the present investigation are conserved in all known antigen-5 homologs
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from other species of social wasps. The skin prick tests of the 9 epitopes in the 5 patients revealed that only epitope 7 caused positive results, indicating that only this epitope caused an IgE-mediated reaction. Immunotherapy with synthetic peptides corresponding to B-cell epitopes of antigen-5 may provide a safe and effective strategy without allergic adverse effects to patients. All of the analyzed epitopes were synthesized as linear peptides and assayed for mast cell degranulation, hemolysis, and chemotactic activities against mammalian cells; none of them showed any significant biological activity in these assays (see Fig E5 in this article’s Online Repository at www.jacionline.org). Therefore, these epitopes are potential candidates for developing vaccines, diagnostic tests, and immunotherapies against allergy caused by P paulista wasp venom. Taken together, these results could contribute to the improvement of the immunotherapy and diagnosis of allergies to social wasp venoms and contribute to the design of specific vaccines against these allergies. Jos e Roberto Aparecido dos Santos-Pinto, PhDa,d Lucilene Delazari dos Santos, PhDc,d Helen Andrade Arcuri, PhDd,e Anally Ribeiro da Silva Menegasso, MSca,d Paloma Napole~ ao P^ ego, MScb Keity Souza Santos, PhDd,e F abio Morato Castro, MD, PhDd,e Jorge Elias Kalil, MD, PhDd,e Salvatore Giovanni De-Simone, PhDb Mario Sergio Palma, PhDa,d From athe Institute of Biosciences of Rio Claro, University of S~ao Paulo State (UNESP), Rio Claro, Brazil; bthe Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Brazil; cthe CEVAP University of S~ao Paulo State (UNESP), Botucatu, Brazil; dINCT, S~ao Paulo, Brazil; and ethe Discipline of Allergy and Immunology (HC/Incor/FMUSP) S~ao Paulo, Brazil. E-mail: [email protected]. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)/INCT-iii and the BIOprospecTA/FAPESP program (Proc. 2011/51684-1). M.S.P., S.G.D.-S., and J.E.K. are researchers from the National Research Council of Brazil-CNPq, and J.R.A.d.S.-P. was a PhD student fellow from FAPESP. We thank the Programa de Desenvolvimento Tecnologico em Insumos para a Saude-PDTIS/FIOCRUZ-RJ for allowing access to the facility required for the SPOT synthesis technique. Disclosure of potential conflict of interest: The authors have received research support from the S~ao Paulo State Research Foundation and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico.
REFERENCES 1. Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol 2006;30: 109-28. 2. M€uller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m1 and Ves v5. Allergy 2009;64:543-8. 3. Santos LD, Santos KS, Pinto JR, Dias NB, Souza BM, Santos MF, et al. Profiling the proteome of the venom from the social wasp Polybia paulista: A clue to understand the envenoming mechanism. J Proteome Res 2010;9:3867-77. 4. Santos-Pinto JR, Fox EG, Saidemberg DM, Santos LD, Menegasso AR, Costa-Manso E, et al. Proteomic view of the venom from the fire ant Solenopsis invicta Buren. J Proteome Res 2012;11:4643-53. 5. Hoffman DR. Allergens in Hymenoptera venom XIV: IgE binding activities of venom proteins from three species of vespids. J Allergy Clin Immunol 1985;75: 606-10. 6. Winkler B, Bolwig C, Seppala U, Spangfort MD, Ebner C, Wiedermann U. Allergen-specific immunosuppression by mucosal treatment with recombinant Ves v 5, a major allergen of Vespula vulgaris venom, in a murine model of wasp venom allergy. Immunology 2003;110:376-85. 7. Santos-Pinto JR, Santos LD, Arcuri HA, Castro FM, Kalil JE, Palma MS. Using proteomic strategies for sequencing and post-translational modifications
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assignment of antigen-5, a major allergen from the venom of the social wasp Polybia paulista. J Proteome Res 2014;13:855-65. 8. Castro FF, Palma MS. Alergia a venenos de insetos[Allergy to insect venoms]]. Barueri, Brazil: Manole; 2009. 9. De-Simone SG, Napole~ao-P^ego P, Teixeira-Pinto LA, Melgarejo AR, Aguiar AS, Provance DW Jr. IgE and IgG epitope mapping by microarray peptide immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin. Toxicon 2014;78:83-93. 10. Mittermann I, Zidarn M, Silar M, Markovic-Housley Z, Aberer W, Korosec P, et al. Recombinant allergen-based IgE testing to distinguish bee and wasp allergy. J Allergy Clin Immunol 2010;125:1300-7.Appendix Available online August 13, 2014. http://dx.doi.org/10.1016/j.jaci.2014.07.006
Bacterial metabolites of diet-derived lignans and isoflavones inversely associate with asthma and wheezing To the Editor: Lignans and isoflavones are plant-derived chemicals with potent anti-inflammatory and antioxidant effects.1 Lignans are ubiquitous in Western diets, with flaxseeds containing the greatest amounts, while isoflavones are most abundant in soybeans (see Table E1 in this article’s Online Repository at www.jacionline.org). The end product(s) of gut microbial metabolism of lignans is enterolactone2; metabolism of the isoflavone daidzein gives rise to equol and O-desmethylangolensin (O-DMA). Human studies demonstrate inverse associations between lignans and isoflavones and hormone-dependent cancers, cardiovascular disease, and osteoporosis,1 and improvements in asthma control are reported in those with high soy consumption.3 Experimental studies suggest that bacterial metabolites of isoflavones attenuate allergic airway inflammation4; however, similar studies are unavailable for enterolactone. Prospective human studies demonstrate that microbial exposures are important for asthma development and control,5 but the mechanism of this association is unclear. Given their anti-inflammatory and antioxidative properties, we hypothesized that bacterial metabolites of lignans and isoflavones may mediate the beneficial effects of bacteria on asthma and lower airway disease. To provide evidence supporting our hypothesis, we conducted a cross-sectional population-based study using data from the National Health and Nutrition Examination Survey (NHANES), which collected urinary levels of lignan and isoflavone metabolites and medical history of asthma and wheeze.
Data were obtained from the 2003-2004, 2005-2006, 20072008, and 2009-2010 NHANES. A total of 9633 subjects aged 6 to 85 years had complete data for the primary analyses of the association between urinary levels of lignan and isoflavone metabolites (eg, enterolactone, equol, and O-DMA) and physician-diagnosed current asthma and self-reported nonasthmatic wheeze, adjusting for age, sex, race/ethnicity, log-transformed urinary creatinine level, poverty index ratio (PIR), and body mass index (BMI). We performed 3 different sensitivity analyses: (1) adjusting for smoke exposure (we adjusted for the presence of household smoke exposure among subjects <20 years old and never, former, or current smoking _20 years old); (2) adjusting for plant status among subjects > intake (using total dietary fiber intake); and (3) stratifying each analysis by atopic status (presence or absence of allergen-specific IgE level, available in 2005-2006 only). Statistical analyses were performed with STATA12.0 (StataCorp, College Station, Tex) using the sampling and weighting variables provided by NHANES. For additional details, see this article’s Methods section in the Online Repository at www.jacionline.org. Participants reporting current asthma were more likely to be female, younger, African American, aeroallergen sensitized, and have a PIR of less than 1. Those reporting nonasthmatic wheeze were more likely to be older and Caucasian. Current asthmatic subjects were more likely to report being former smokers, and those with nonasthmatic wheeze were more likely to be current smokers or have household smoke exposure. Asthma and wheeze status was positively associated with BMI (see Table E2 in this article’s Online Repository at www.jacionline.org). Urinary levels of bacterial metabolites by tertile are presented in Table E3 in this article’s Online Repository at www.jacionline.org. These levels varied by race/ethnicity, PIR, age, BMI category, and tobacco exposure status (see Tables E4-E8 in this article’s Online Repository at www.jacionline.org). NHANES participants were selected at random for urinary lignan and isoflavone analysis. The sample with data available on urinary bacterial metabolite measurements was similar to the general NHANES population, although more adults (82% vs 72%, P < .001) and more subjects with BMI greater than 30 (29% vs 26%, P < .001) composed the subsample (see Table E9 in this article’s Online Repository at www.jacionline.org).
TABLE I. ORs (and 95% CI) for current asthma and nonasthmatic wheeze by level of urinary metabolites of lignans and isoflavones Current asthma Metabolite
Enterolactone
O-DMA
Equol
Nonasthmatic wheeze
Tertile
Crude OR
Adjusted OR*
Crude OR
Adjusted OR*
1 2 3 Test for trend (P) 1 2 3 Test for trend (P) 1 2 3 Test for trend (P)
1.0 [REF] 0.59 (0.47-0.74) 0.63 (0.52-0.76) <.001 1.0 [REF] 1.03 (0.86-1.23) 0.94 (0.77-1.23) .82 1.0 [REF] 0.97 (0.79-1.20) 1.08 (0.90-1.30) .39
1.0 [REF] 0.61 (0.48-0.78) 0.69 (0.56-0.85) <.001 1.0 [REF] 1.06 (0.88-1.28) 1.00 (0.80-1.26) .98 1.0 [REF] 1.01 (0.81-1.26) 1.15 (0.93-1.43) .18
1.0 [REF] 0.64 (0.48-0.86) 0.52 (0.34-0.65) <.001 1.0 [REF] 0.79 (0.63-1.00) 0.63 (0.51-0.79) <.001 1.0 [REF] 1.15 (0.94-1.41) 0.94 (0.76-1.16) .56
1.0 [REF] 0.67 (0.49-0.90) 0.53 (0.42-0.67) <.001 1.0 [REF] 0.79 (0.62-1.00) 0.64 (0.52-0.80) <.001 1.0 [REF] 1.12 (0.91-1.40) 0.95 (0.76-1.18) .57
Values in boldface are statistically significant (P < .05). *Adjusted for age, sex, race/ethnicity, log-transformed urinary creatinine, PIR, and BMI.
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FIG E1. Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitope 7 of P paulista antigen-5 reactive to human IgE. C5.1-VGHYTQVVWAKTKE, C5.2-VGHYTQVV, C5.3-TQVVWAKTKE, C5.4-WAKTKE and Control (-).
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FIG E2. Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgG.
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FIG E3. Evaluating the potential epitopes of antigen-5 by indirect ELISA with a pool of sera from patients allergic to P paulista venom. Peptide reactivity with human IgG (A); peptide reactivity with human IgE (B).
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FIG E4. Alignment of the sequences of known antigen-5 homologs found in the venoms of different species of social wasps: POLPI-Polybia paulista; POLEX-Polistes exclamans; POLAN-Polistes annularis; POLFU-Polistes fuscatus; POLGA-Polistes gallicus; POLDO-Polistes dominula; VESVU-Vespula vulgaris; VESGE-Vespula germanica; VESMC-Vespula maculifrons; VESPE-Vespula pensylvanica; VESSQ-Vespula squamosa. The conserved B-cell epitopes identified in P paulista antigen-5 are conserved in all these sequences and are shown inside the rectangles indicating epitopes 1 to 9.
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FIG E5. Biological activities assay for the peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgE (epitope 7) and immunoreactive to human IgG (epitopes 1 to 9). Degranulation activity in rat peritoneal mast cells (A); the activity was determined by measuring the release of the granule marker, b-D-glucosaminidase, which co-localizes with histamine. The values for b-D-glucosaminidase released in the medium were expressed in the percentage of total enzyme activity. Hemolytic activity in washed rat red blood cells (B); the absorbance measured at 540 nm from lysed washed rat red blood cells in presence of 1% (v/v) Triton X-100 (Sigma, St Louis, Mo) was considered as 100%. Chemotaxis of polymorphonucleated leukocytes (C). The results were compared with the activities measured for the standard mast cell degranulating and hemolytic activity peptide melittin and compared with the activities measured for the standard chemotactic activity peptide protonectin 1-6. Data are represented as means 6 SDs (n 5 5).
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TABLE E1. Clinical features of patients with allergy to the venom of the social wasp Polybia paulista Skin prick tests (wheal size in mm2) Symptoms Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope Sex/age for P paulista SPT for 1 2 3 4 5 6 7 8 9 Histamine* Patients (y) venom other venoms Saline
1
M/23
2
M/42
3
F/35
4
M/52
5
F/47
Anaphylaxis
Negative for HB and Pol SS (Ae 1 U) Negative for HB and Pol Anaphylaxis Negative for HB and Pol Anaphylaxis Negative for HB and Pol SS (Ae 1 U) Negative for HB and Pol
2
3
4
2
3
3
1
46
3
1
33
3
1
1
2
2
1
3
34
2
2
32
2
2
1
1
1
3
2
47
1
2
35
2
1
1
2
3
1
3
53
3
1
29
3
2
2
2
1
2
3
32
2
2
27
Ae, Angioedema; F, female; HB, honeybees; M, male; Pol, Polistes species; SPT, skin prick test; SS, skin symptoms; U, generalized urticaria. *Histamine concentration: 2.5 ng/mL.
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TABLE E2. Peptide library of the antigen-5 sequences used in the SPOT synthesis assay with the sera of patients allergic to venom of the wasp P paulista Position in SPOT synthesis assay
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
Peptide sequences
Amino acid position
Position in SPOT synthesis assay
Peptide sequences
Amino acid position
NKYCNIKCSKVAHT CNIKCSKVAHTVCQ KCSKVAHTVCQTGE KVAHTVCQTGESTK HTVCQTGESTKPSS CQTGESTKPSSKNC GESTKPSSKNCAKV TKPSSKNCAKVSIT SSKNCAKVSITSVG NCAKVSITSVGVTE KVSITSVGVTEEEK ITSVGVTEEEKKLI VGVTEEEKKLIVDE TEEEKKLIVDEHNR EKKLIVDEHNRFRQ LIVDEHNRFRQKVA DEHNRFRQKVAQGL NRFRQKVAQGLETR RQKVAQGLETRGNP VAQGLETRGNPGPQ GLETRGNPGPQPAA TRGNPGPQPAASDM NPGPQPAASDMNNL PQPAASDMNNLVWN AASDMNNLVWNDEL DMNNLVWNDELAYI NLVWNDELAYIAQV WNDELAYIAQVWAS ELAYIAQVWASQCQ YIAQVWASQCQFFV QVWASQCQFFVHDK ASQCQFFVHDKCRN CQFFVHDKCRNTAQ FVHDKCRNTAQYQV DKCRNTAQYQVGQN RNTAQYQVGQNIAY
1-14 4-17 7-20 10-23 13-26 16-29 19-32 22-35 25-38 28-41 31-44 34-47 37-50 40-53 43-56 46-59 49-62 52-65 55-68 58-71 61-74 64-77 67-80 70-83 73-86 76-89 79-92 82-95 85-98 88-101 91-104 94-107 97-110 100-113 103-116 106-119
B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18
AQYQVGQNIAYSAS QVGQNIAYSASTAA QNIAYSASTAAYPG AYSASTAAYPGVVK ASTAAYPGVVKLIV AAYPGVVKLIVLWE PGVVKLIVLWENEV VKLIVLWENEVKDF IVLWENEVKDFNYN WENEVKDFNYNTGI EVKDFNYNTGITKE DFNYNTGITKENFA YNTGITKENFAKVG GITKENFAKVGHYT KENFAKVGHYTQVV FAKVGHYTQVVWAK VGHYTQVVWAKTKE YTQVVWAKTKEVGC VVWAKTKEVGCGSI AKTKEVGCGSIKYI KEVGCGSIKYIEKG GCGSIKYIEKGMKS SIKYIEKGMKSHYL YIEKGMKSHYLVCN KGMKSHYLVCNYGP KSHYLVCNYGPAGN YLVCNYGPAGNVLG CNYGPAGNVLGAQI GPAGNVLGAQIYEI PAGNVLGAQIYEIK
109-122 112-125 115-128 118-131 121-134 124-137 127-140 130-143 133-146 136-149 139-152 142-155 145-158 148-161 151-164 154-167 157-170 160-173 163-176 166-179 169-182 172-185 175-188 178-191 181-194 184-197 187-200 190-203 193-206 194-207
Control sequences C20 C21
GYPKDGNAFNNLD GYPKDGNAFNNLD
Sixty-six overlapping peptides corresponding to the complete sequence of antigen-5 were synthesized in a solid phase using the SPOT method. The peptides were sequenced with 11 overlapping amino acid residues and an offset of 3.
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TABLE E3. B-cell linear epitopes of antigen-5 identified by IgG and IgE reactivity detected in the SPOT synthesis assay with the sera of patients allergic to venom Position in SPOT synthesis assay
B-cell linear epitopes immunoreactive to human IgG 1 A15-A17 2 A19-A20 3 B3-B4 4 B11-B12 5 B20-B22 6 C1-C3 7 C5 8 C10-C11 9 C17-C18 B-cell linear epitope immunoreactive to human IgE 7 C5
Peptide sequences
Amino acid position
No. residues
VDEHNRFRQ VAQGLETRGNP WNDELAYIAQV RNTAQYQVGQN WENEVKDF KENFAKVGHYT VGHYTQVVWAKTKE SIKYIEKGMKS GNVLGAQIYEI
48-56 58-68 82-92 106-116 136-143 151-161 157-170 175-185 196-206
9 11 11 11 8 11 14 11 11
VGHYTQVVWAKTKE
157-170
14
low KIT D816V allele burden that can be detected using only a highly sensitive method,4 as also demonstrated by the low levels detected in our 4 cases. The analytical sensitivity of the mutation analysis performed in the present study depends on the DNA concentration of the individual samples and was typically in the range of 0.001% to 0.03% mutation-positive alleles, as previously described in detail.3,4 The 109 patients included in our study who tested negative for the KIT D816V mutation in PB were all examined clinically in our center and none had clear indications for mastocytosis based on skin examination, clinical symptoms, and s-tryptase level. Suspected elicitors among these 109 mutation-negative patients were diverse including, for example, insects, drugs, foods, exercise, and apparent idiopathic cases, and current investigations including provocation tests are ongoing in this group. A total of 29 of these patients had anaphylaxis elicited by an insect of which 5 patients lacked any skin symptoms during the acute episode whereas 22 patients had urticaria and/or angioedema and 2 patients had flushing or pruritus. A final exclusion of SM in this group would demand a BM examination, and it thus cannot be excluded that cases of SM may have been missed in this group—and especially among the patients without skin symptoms during the anaphylactic episode—because of aforementioned reasons. However, on the basis of previous studies from our group that included a group of negative controls, ie, patients with elevated s-tryptase level, in whom SM was excluded after thorough BM investigation, we find this unlikely to be the case4 and following this we do presently not perform a BM investigation in patients with anaphylaxis if KITD816V is not detected or UP is not found, unless other signs strongly suggestive of SM are present (ie, markedly elevated/rising basal s-tryptase level, recurring idiopathic anaphylaxis, early onset osteoporosis). In conclusion, we here present data on 4 adult patients with anaphylaxis in whom a positive KIT D816V mutation in PB subsequently led to a diagnosis of SM in spite of normal or low basal s-tryptase level and absent or inconspicuous UP skin lesions. Except for absent skin symptoms during the anaphylactic episode, the 4 patients lacked any other clinical findings that might have been a clue to the diagnosis. The use of sensitive KIT D816V mutation analysis of PB as a diagnostic test in mastocytosis thereby clearly shows its clinical utility in patients with anaphylaxis. Further studies in a larger cohort of patients with fully characterized anaphylaxis are needed to fully interpret the value of performing the test indiscriminately in all patients with anaphylaxis as opposed to testing selected patients only on the basis of clinical or laboratory discriminators indicating mastocytosis, also given the low rate of positive results (3.5%) found in the present study. However, the method used for KIT D816Vanalysis in this study has been demonstrated to have a very high diagnostic sensitivity and specificity and is furthermore quick, simple, and cheap (reagents
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Thomas Kristensen, PhDd* Charlotte G. Mortz, MD, PhDa,b* On behalf of the Mastocytosis Centre Odense University Hospital (MastOUH) and Odense Research Centre for Anaphylaxis From athe Department of Dermatology and Allergy Centre, cthe Department of Hematology, and dthe Department of Pathology, Odense University Hospital, Odense, Denmark; bthe Institute of Clinical Research, University of Southern Denmark, Odense, Denmark; and ethe Department of Dermatology and Allergy, Interdisciplinary Mastocytosis Center Charite, Charite - Universit€atsmedizin Berlin, Germany. E-mail: [email protected]. *These authors contributed equally to this work. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Metcalfe DD. Mast cells and mastocytosis. Blood 2008;112:946-56. 2. Valent P, Akin C, Escribano L, Fodinger M, Hartmann K, Brockow K, et al. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest 2007;37:435-53. 3. Kristensen T, Vestergaard H, Moller MB. Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay. J Mol Diagn 2011;13:180-8. 4. Kristensen T, Vestergaard H, Bindslev-Jensen C, Moller MB, Broesby-Olsen S. Mastocytosis Centre, Odense University Hospital (MastOUH). Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis. Am J Hematol 2014;89:493-8. 5. Simons FE, Ardusso LR, Bilo MB, El-Gamal YM, Ledford DK, Ring J, et al. World allergy organization guidelines for the assessment and management of anaphylaxis. World Allergy Organ J 2011;4:13-37. 6. Sampson HA. Anaphylaxis and emergency treatment. Pediatrics 2003;111:1601-8. 7. Stoevesandt J, Hain J, Kerstan A, Trautmann A. Over- and underestimated parameters in severe Hymenoptera venom-induced anaphylaxis: cardiovascular medication and absence of urticaria/angioedema. J Allergy Clin Immunol 2012; 130:698-704.e1. 8. Alvarez-Twose I, Gonzalez-de-Olano D, Sanchez-Munoz L, Matito A, JaraAcevedo M, Teodosio C, et al. Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms. Int Arch Allergy Immunol 2012; 157:275-80. 9. Oude Elberink JN, de Monchy JG, Kors JW, van Doormaal JJ, Dubois AE. Fatal anaphylaxis after a yellow jacket sting, despite venom immunotherapy, in two patients with mastocytosis. J Allergy Clin Immunol 1997; 99:153-4. Available online August 1, 2014. http://dx.doi.org/10.1016/j.jaci.2014.06.031
B-cell linear epitopes mapping of antigen-5 allergen from Polybia paulista wasp venom To the Editor: Vespid venoms contain a variety of proteins, such as phospholipase A1, hyaluronidase, and antigen-5, which are often associated with allergic responses in humans.1-5 New vaccination strategies are focusing on antigen-5,6 which has been isolated from the venoms of all clinically relevant species of social wasps.1,3,7 The social wasp Polybia paulista causes hundreds of stinging accidents of medical importance each year in Brazil.8 Antigen-5 has been reported to be the primary allergen present in the venom of this social wasp,7 being a good candidate for studying the IgE-binding B-cell linear epitopes involved in wasp venom allergy. To identify the B-cell linear epitopes present in the full-length allergen, the 66 overlapping synthetic peptides corresponding to the complete sequence of P paulista antigen-5 were assayed against the pool of 5 serum samples from patients allergic to P paulista venom (see Table E1 in this article’s
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FIG 1. B-cell linear epitopes mapping along the primary sequence of the P paulista venom antigen-5 detected in the SPOT synthesis assay with the sera of patients who were sensitive to the venom of the wasp P paulista. IgG-reactive peptides (A); IgE-reactive peptides (B); the smallest peptide sequence corresponding to the B-cell linear epitope of antigen-5 that is immunoreactive to human IgE (C); P paulista venom antigen-5 primary sequence (D) showing regions of IgG reactivity (black bars), IgE reactivity (grey bars), and the identification of the smallest peptide sequence corresponding to the B-cell linear epitope that was immunoreactive to human IgE.
FIG 2. Three-dimensional mapping of B-cell linear epitopes of antigen-5 that were reactive to human IgG shown in yellow: epitope 1 (A), epitope 2 (B), epitope 3 (C), epitope 4 (D), epitope 5 (E), epitope 6 (F), epitope 7 (G), epitope 8 (H), and epitope 9 (I); epitope 7 is also reactive to human IgE. The elements of secondary structures shown in blue correspond to helices; those shown in red and green correspond to beta-sheets and loops, respectively; meanwhile, the regions represented in yellow correspond to the spatial location of the linear epitopes for human IgG and/or IgE.
266 LETTERS TO THE EDITOR
Online Repository at www.jacionline.org). These peptides were synthesized in solid phase using the SPOT method with overlaps of 11 amino acid residues with an offset of 3 residues; these sequences were designated A1 to A24, B1 to B24, and C1 to C18, and a control sequence from a bacterial antigen specific for human IgG was synthesized and assigned as C20 and C21 (see Tables E2 and E3 in the Online Repository at www.jacionline.org). These peptides were assessed for reactivity with human IgG and human IgE as described elsewhere9; the spot images of these assays are shown in Fig 1. The following 9 antigenic determinants were reactive with human IgG (Fig 1, A): epitope 1 (A15-A17, VDEHNRFRQ), epitope 2 (A19-A20, VAQGLETRGNP), epitope 3 (B3-B4, WNDELAYIAQV), epitope 4 (B11-B12, RNTAQYQVGQN), epitope 5 (B20-B22, WENEVKDF), epitope 6 (C1-C3, KENFAKVGHYT), epitope 7 (C5, VGHYTQVVWAKTKE), epitope 8 (C10-C11, SIKYIEKGMKS) and epitope 9 (C17-C18, GNVL GAQIYEI). The results of Fig 1, B show that epitope 7 (C5, VGHYTQVVWAKTKE) is reactive both to human IgG and human IgE. The analyses of the chemiluminescent signal intensities of the spot images generated by coupling the human IgEs and IgGs to the B-cell linear epitopes of antigen-5 are shown in Figs E1 and E2 (available in the Online Repository at www. jacionline.org), respectively. For further confirmation of these epitopes, the corresponding peptides were synthesized and submitted to indirect ELISA assay. Epitopes 1 to 9 were recognized by human IgG, while Epitope 7 was also recognized by human IgE (Fig E3, A and B). Epitope 7 corresponds to the linear sequence observed for C5 (VGHYTQVVWAKTKE) in Fig 1, B. Thus, it was considered to identify the smallest peptide sequence corresponding to the epitope for human IgE. The sequence KENFAKVGHYT was reactive with human IgG (epitope 6), and this sequence partially overlapped with the sequence of epitope 7 (corresponding to C5 in Fig 1, C), with the sequence of VGHYT shared by the peptides. The peptide sequence VGHYTQVVWAKTKE (peptide C5.1 in Fig 1, C) was re-synthesized using the SPOT strategy as the following 3 smaller peptides: VGHYTQVV, TQVVWAKTKE, and WAKTKE (peptides C5.2, C5.3, and C5.4, respectively, in Fig 1, C). These peptides were then assayed against a pool of serum samples from 5 patients allergic to P paulista venom using an alkaline-phosphatase-conjugated secondary antibody against human IgE. The results of the SPOT image intensities are shown in Fig 1, C, suggesting that the first 8 residues of epitope 7 (VGHYTQVV) are essential for the interaction between antigen-5 and IgG, and that the other 6 residues (WAKTKE) are critical for the interaction between antigen-5 and IgE. Fig 1, D shows the positions of the B-cell linear epitopes for human IgG and IgE along the primary sequence of the P paulista venom antigen-5. The 3-dimensional structure of P paulista venom antigen-5 was built through molecular modeling7 to map the B-cell linear epitopes onto its structure. Fig 2 shows that the sequence WAKTKE is located in a loop area on the surface of the protein, where it is accessible to the IgE antibody. These conserved surfaces were proposed to represent the major B-cell-binding epitopes and could be used as model peptides for engineering vaccines for allergen immunotherapy.10 The results shown in Fig E4 (available in the Online Repository at www.jacionline.org) suggest that the linear epitopes that were experimentally identified in the present investigation are conserved in all known antigen-5 homologs
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from other species of social wasps. The skin prick tests of the 9 epitopes in the 5 patients revealed that only epitope 7 caused positive results, indicating that only this epitope caused an IgE-mediated reaction. Immunotherapy with synthetic peptides corresponding to B-cell epitopes of antigen-5 may provide a safe and effective strategy without allergic adverse effects to patients. All of the analyzed epitopes were synthesized as linear peptides and assayed for mast cell degranulation, hemolysis, and chemotactic activities against mammalian cells; none of them showed any significant biological activity in these assays (see Fig E5 in this article’s Online Repository at www.jacionline.org). Therefore, these epitopes are potential candidates for developing vaccines, diagnostic tests, and immunotherapies against allergy caused by P paulista wasp venom. Taken together, these results could contribute to the improvement of the immunotherapy and diagnosis of allergies to social wasp venoms and contribute to the design of specific vaccines against these allergies. Jos e Roberto Aparecido dos Santos-Pinto, PhDa,d Lucilene Delazari dos Santos, PhDc,d Helen Andrade Arcuri, PhDd,e Anally Ribeiro da Silva Menegasso, MSca,d Paloma Napole~ ao P^ ego, MScb Keity Souza Santos, PhDd,e F abio Morato Castro, MD, PhDd,e Jorge Elias Kalil, MD, PhDd,e Salvatore Giovanni De-Simone, PhDb Mario Sergio Palma, PhDa,d From athe Institute of Biosciences of Rio Claro, University of S~ao Paulo State (UNESP), Rio Claro, Brazil; bthe Oswaldo Cruz Institute/FIOCRUZ, Rio de Janeiro, Brazil; cthe CEVAP University of S~ao Paulo State (UNESP), Botucatu, Brazil; dINCT, S~ao Paulo, Brazil; and ethe Discipline of Allergy and Immunology (HC/Incor/FMUSP) S~ao Paulo, Brazil. E-mail: [email protected]. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq)/INCT-iii and the BIOprospecTA/FAPESP program (Proc. 2011/51684-1). M.S.P., S.G.D.-S., and J.E.K. are researchers from the National Research Council of Brazil-CNPq, and J.R.A.d.S.-P. was a PhD student fellow from FAPESP. We thank the Programa de Desenvolvimento Tecnologico em Insumos para a Saude-PDTIS/FIOCRUZ-RJ for allowing access to the facility required for the SPOT synthesis technique. Disclosure of potential conflict of interest: The authors have received research support from the S~ao Paulo State Research Foundation and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico.
REFERENCES 1. Hoffman DR. Hymenoptera venom allergens. Clin Rev Allergy Immunol 2006;30: 109-28. 2. M€uller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m1 and Ves v5. Allergy 2009;64:543-8. 3. Santos LD, Santos KS, Pinto JR, Dias NB, Souza BM, Santos MF, et al. Profiling the proteome of the venom from the social wasp Polybia paulista: A clue to understand the envenoming mechanism. J Proteome Res 2010;9:3867-77. 4. Santos-Pinto JR, Fox EG, Saidemberg DM, Santos LD, Menegasso AR, Costa-Manso E, et al. Proteomic view of the venom from the fire ant Solenopsis invicta Buren. J Proteome Res 2012;11:4643-53. 5. Hoffman DR. Allergens in Hymenoptera venom XIV: IgE binding activities of venom proteins from three species of vespids. J Allergy Clin Immunol 1985;75: 606-10. 6. Winkler B, Bolwig C, Seppala U, Spangfort MD, Ebner C, Wiedermann U. Allergen-specific immunosuppression by mucosal treatment with recombinant Ves v 5, a major allergen of Vespula vulgaris venom, in a murine model of wasp venom allergy. Immunology 2003;110:376-85. 7. Santos-Pinto JR, Santos LD, Arcuri HA, Castro FM, Kalil JE, Palma MS. Using proteomic strategies for sequencing and post-translational modifications
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assignment of antigen-5, a major allergen from the venom of the social wasp Polybia paulista. J Proteome Res 2014;13:855-65. 8. Castro FF, Palma MS. Alergia a venenos de insetos[Allergy to insect venoms]]. Barueri, Brazil: Manole; 2009. 9. De-Simone SG, Napole~ao-P^ego P, Teixeira-Pinto LA, Melgarejo AR, Aguiar AS, Provance DW Jr. IgE and IgG epitope mapping by microarray peptide immunoassay reveals the importance and diversity of the immune response to the IgG3 equine immunoglobulin. Toxicon 2014;78:83-93. 10. Mittermann I, Zidarn M, Silar M, Markovic-Housley Z, Aberer W, Korosec P, et al. Recombinant allergen-based IgE testing to distinguish bee and wasp allergy. J Allergy Clin Immunol 2010;125:1300-7.Appendix Available online August 13, 2014. http://dx.doi.org/10.1016/j.jaci.2014.07.006
Bacterial metabolites of diet-derived lignans and isoflavones inversely associate with asthma and wheezing To the Editor: Lignans and isoflavones are plant-derived chemicals with potent anti-inflammatory and antioxidant effects.1 Lignans are ubiquitous in Western diets, with flaxseeds containing the greatest amounts, while isoflavones are most abundant in soybeans (see Table E1 in this article’s Online Repository at www.jacionline.org). The end product(s) of gut microbial metabolism of lignans is enterolactone2; metabolism of the isoflavone daidzein gives rise to equol and O-desmethylangolensin (O-DMA). Human studies demonstrate inverse associations between lignans and isoflavones and hormone-dependent cancers, cardiovascular disease, and osteoporosis,1 and improvements in asthma control are reported in those with high soy consumption.3 Experimental studies suggest that bacterial metabolites of isoflavones attenuate allergic airway inflammation4; however, similar studies are unavailable for enterolactone. Prospective human studies demonstrate that microbial exposures are important for asthma development and control,5 but the mechanism of this association is unclear. Given their anti-inflammatory and antioxidative properties, we hypothesized that bacterial metabolites of lignans and isoflavones may mediate the beneficial effects of bacteria on asthma and lower airway disease. To provide evidence supporting our hypothesis, we conducted a cross-sectional population-based study using data from the National Health and Nutrition Examination Survey (NHANES), which collected urinary levels of lignan and isoflavone metabolites and medical history of asthma and wheeze.
Data were obtained from the 2003-2004, 2005-2006, 20072008, and 2009-2010 NHANES. A total of 9633 subjects aged 6 to 85 years had complete data for the primary analyses of the association between urinary levels of lignan and isoflavone metabolites (eg, enterolactone, equol, and O-DMA) and physician-diagnosed current asthma and self-reported nonasthmatic wheeze, adjusting for age, sex, race/ethnicity, log-transformed urinary creatinine level, poverty index ratio (PIR), and body mass index (BMI). We performed 3 different sensitivity analyses: (1) adjusting for smoke exposure (we adjusted for the presence of household smoke exposure among subjects <20 years old and never, former, or current smoking _20 years old); (2) adjusting for plant status among subjects > intake (using total dietary fiber intake); and (3) stratifying each analysis by atopic status (presence or absence of allergen-specific IgE level, available in 2005-2006 only). Statistical analyses were performed with STATA12.0 (StataCorp, College Station, Tex) using the sampling and weighting variables provided by NHANES. For additional details, see this article’s Methods section in the Online Repository at www.jacionline.org. Participants reporting current asthma were more likely to be female, younger, African American, aeroallergen sensitized, and have a PIR of less than 1. Those reporting nonasthmatic wheeze were more likely to be older and Caucasian. Current asthmatic subjects were more likely to report being former smokers, and those with nonasthmatic wheeze were more likely to be current smokers or have household smoke exposure. Asthma and wheeze status was positively associated with BMI (see Table E2 in this article’s Online Repository at www.jacionline.org). Urinary levels of bacterial metabolites by tertile are presented in Table E3 in this article’s Online Repository at www.jacionline.org. These levels varied by race/ethnicity, PIR, age, BMI category, and tobacco exposure status (see Tables E4-E8 in this article’s Online Repository at www.jacionline.org). NHANES participants were selected at random for urinary lignan and isoflavone analysis. The sample with data available on urinary bacterial metabolite measurements was similar to the general NHANES population, although more adults (82% vs 72%, P < .001) and more subjects with BMI greater than 30 (29% vs 26%, P < .001) composed the subsample (see Table E9 in this article’s Online Repository at www.jacionline.org).
TABLE I. ORs (and 95% CI) for current asthma and nonasthmatic wheeze by level of urinary metabolites of lignans and isoflavones Current asthma Metabolite
Enterolactone
O-DMA
Equol
Nonasthmatic wheeze
Tertile
Crude OR
Adjusted OR*
Crude OR
Adjusted OR*
1 2 3 Test for trend (P) 1 2 3 Test for trend (P) 1 2 3 Test for trend (P)
1.0 [REF] 0.59 (0.47-0.74) 0.63 (0.52-0.76) <.001 1.0 [REF] 1.03 (0.86-1.23) 0.94 (0.77-1.23) .82 1.0 [REF] 0.97 (0.79-1.20) 1.08 (0.90-1.30) .39
1.0 [REF] 0.61 (0.48-0.78) 0.69 (0.56-0.85) <.001 1.0 [REF] 1.06 (0.88-1.28) 1.00 (0.80-1.26) .98 1.0 [REF] 1.01 (0.81-1.26) 1.15 (0.93-1.43) .18
1.0 [REF] 0.64 (0.48-0.86) 0.52 (0.34-0.65) <.001 1.0 [REF] 0.79 (0.63-1.00) 0.63 (0.51-0.79) <.001 1.0 [REF] 1.15 (0.94-1.41) 0.94 (0.76-1.16) .56
1.0 [REF] 0.67 (0.49-0.90) 0.53 (0.42-0.67) <.001 1.0 [REF] 0.79 (0.62-1.00) 0.64 (0.52-0.80) <.001 1.0 [REF] 1.12 (0.91-1.40) 0.95 (0.76-1.18) .57
Values in boldface are statistically significant (P < .05). *Adjusted for age, sex, race/ethnicity, log-transformed urinary creatinine, PIR, and BMI.
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FIG E1. Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitope 7 of P paulista antigen-5 reactive to human IgE. C5.1-VGHYTQVVWAKTKE, C5.2-VGHYTQVV, C5.3-TQVVWAKTKE, C5.4-WAKTKE and Control (-).
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FIG E2. Intensity analysis of the reactivity of peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgG.
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FIG E3. Evaluating the potential epitopes of antigen-5 by indirect ELISA with a pool of sera from patients allergic to P paulista venom. Peptide reactivity with human IgG (A); peptide reactivity with human IgE (B).
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FIG E4. Alignment of the sequences of known antigen-5 homologs found in the venoms of different species of social wasps: POLPI-Polybia paulista; POLEX-Polistes exclamans; POLAN-Polistes annularis; POLFU-Polistes fuscatus; POLGA-Polistes gallicus; POLDO-Polistes dominula; VESVU-Vespula vulgaris; VESGE-Vespula germanica; VESMC-Vespula maculifrons; VESPE-Vespula pensylvanica; VESSQ-Vespula squamosa. The conserved B-cell epitopes identified in P paulista antigen-5 are conserved in all these sequences and are shown inside the rectangles indicating epitopes 1 to 9.
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FIG E5. Biological activities assay for the peptide sequences corresponding to B-cell linear epitopes of antigen-5 immunoreactive to human IgE (epitope 7) and immunoreactive to human IgG (epitopes 1 to 9). Degranulation activity in rat peritoneal mast cells (A); the activity was determined by measuring the release of the granule marker, b-D-glucosaminidase, which co-localizes with histamine. The values for b-D-glucosaminidase released in the medium were expressed in the percentage of total enzyme activity. Hemolytic activity in washed rat red blood cells (B); the absorbance measured at 540 nm from lysed washed rat red blood cells in presence of 1% (v/v) Triton X-100 (Sigma, St Louis, Mo) was considered as 100%. Chemotaxis of polymorphonucleated leukocytes (C). The results were compared with the activities measured for the standard mast cell degranulating and hemolytic activity peptide melittin and compared with the activities measured for the standard chemotactic activity peptide protonectin 1-6. Data are represented as means 6 SDs (n 5 5).
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TABLE E1. Clinical features of patients with allergy to the venom of the social wasp Polybia paulista Skin prick tests (wheal size in mm2) Symptoms Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope Epitope Sex/age for P paulista SPT for 1 2 3 4 5 6 7 8 9 Histamine* Patients (y) venom other venoms Saline
1
M/23
2
M/42
3
F/35
4
M/52
5
F/47
Anaphylaxis
Negative for HB and Pol SS (Ae 1 U) Negative for HB and Pol Anaphylaxis Negative for HB and Pol Anaphylaxis Negative for HB and Pol SS (Ae 1 U) Negative for HB and Pol
2
3
4
2
3
3
1
46
3
1
33
3
1
1
2
2
1
3
34
2
2
32
2
2
1
1
1
3
2
47
1
2
35
2
1
1
2
3
1
3
53
3
1
29
3
2
2
2
1
2
3
32
2
2
27
Ae, Angioedema; F, female; HB, honeybees; M, male; Pol, Polistes species; SPT, skin prick test; SS, skin symptoms; U, generalized urticaria. *Histamine concentration: 2.5 ng/mL.
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TABLE E2. Peptide library of the antigen-5 sequences used in the SPOT synthesis assay with the sera of patients allergic to venom of the wasp P paulista Position in SPOT synthesis assay
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
Peptide sequences
Amino acid position
Position in SPOT synthesis assay
Peptide sequences
Amino acid position
NKYCNIKCSKVAHT CNIKCSKVAHTVCQ KCSKVAHTVCQTGE KVAHTVCQTGESTK HTVCQTGESTKPSS CQTGESTKPSSKNC GESTKPSSKNCAKV TKPSSKNCAKVSIT SSKNCAKVSITSVG NCAKVSITSVGVTE KVSITSVGVTEEEK ITSVGVTEEEKKLI VGVTEEEKKLIVDE TEEEKKLIVDEHNR EKKLIVDEHNRFRQ LIVDEHNRFRQKVA DEHNRFRQKVAQGL NRFRQKVAQGLETR RQKVAQGLETRGNP VAQGLETRGNPGPQ GLETRGNPGPQPAA TRGNPGPQPAASDM NPGPQPAASDMNNL PQPAASDMNNLVWN AASDMNNLVWNDEL DMNNLVWNDELAYI NLVWNDELAYIAQV WNDELAYIAQVWAS ELAYIAQVWASQCQ YIAQVWASQCQFFV QVWASQCQFFVHDK ASQCQFFVHDKCRN CQFFVHDKCRNTAQ FVHDKCRNTAQYQV DKCRNTAQYQVGQN RNTAQYQVGQNIAY
1-14 4-17 7-20 10-23 13-26 16-29 19-32 22-35 25-38 28-41 31-44 34-47 37-50 40-53 43-56 46-59 49-62 52-65 55-68 58-71 61-74 64-77 67-80 70-83 73-86 76-89 79-92 82-95 85-98 88-101 91-104 94-107 97-110 100-113 103-116 106-119
B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18
AQYQVGQNIAYSAS QVGQNIAYSASTAA QNIAYSASTAAYPG AYSASTAAYPGVVK ASTAAYPGVVKLIV AAYPGVVKLIVLWE PGVVKLIVLWENEV VKLIVLWENEVKDF IVLWENEVKDFNYN WENEVKDFNYNTGI EVKDFNYNTGITKE DFNYNTGITKENFA YNTGITKENFAKVG GITKENFAKVGHYT KENFAKVGHYTQVV FAKVGHYTQVVWAK VGHYTQVVWAKTKE YTQVVWAKTKEVGC VVWAKTKEVGCGSI AKTKEVGCGSIKYI KEVGCGSIKYIEKG GCGSIKYIEKGMKS SIKYIEKGMKSHYL YIEKGMKSHYLVCN KGMKSHYLVCNYGP KSHYLVCNYGPAGN YLVCNYGPAGNVLG CNYGPAGNVLGAQI GPAGNVLGAQIYEI PAGNVLGAQIYEIK
109-122 112-125 115-128 118-131 121-134 124-137 127-140 130-143 133-146 136-149 139-152 142-155 145-158 148-161 151-164 154-167 157-170 160-173 163-176 166-179 169-182 172-185 175-188 178-191 181-194 184-197 187-200 190-203 193-206 194-207
Control sequences C20 C21
GYPKDGNAFNNLD GYPKDGNAFNNLD
Sixty-six overlapping peptides corresponding to the complete sequence of antigen-5 were synthesized in a solid phase using the SPOT method. The peptides were sequenced with 11 overlapping amino acid residues and an offset of 3.
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J ALLERGY CLIN IMMUNOL VOLUME 135, NUMBER 1
TABLE E3. B-cell linear epitopes of antigen-5 identified by IgG and IgE reactivity detected in the SPOT synthesis assay with the sera of patients allergic to venom Position in SPOT synthesis assay
B-cell linear epitopes immunoreactive to human IgG 1 A15-A17 2 A19-A20 3 B3-B4 4 B11-B12 5 B20-B22 6 C1-C3 7 C5 8 C10-C11 9 C17-C18 B-cell linear epitope immunoreactive to human IgE 7 C5
Peptide sequences
Amino acid position
No. residues
VDEHNRFRQ VAQGLETRGNP WNDELAYIAQV RNTAQYQVGQN WENEVKDF KENFAKVGHYT VGHYTQVVWAKTKE SIKYIEKGMKS GNVLGAQIYEI
48-56 58-68 82-92 106-116 136-143 151-161 157-170 175-185 196-206
9 11 11 11 8 11 14 11 11
VGHYTQVVWAKTKE
157-170
14