Cloning of the patatin-like latex allergen Hev b 7, its expression in the yeast Pichia pastoris and its immunological characterization

The 43-kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N-terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex-allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 micrograms/ml in skin prick tests produced wheal-and-flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed.     

Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis)

BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.     

PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8)

BACKGROUND: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14-kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE-binding reactivity. METHODS: A profilin-specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose-binding protein (MBP) in E. coli. The IgE-binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health-care workers with latex allergy and 17 sera from latex-sensitive spina bifida patients. RESULTS: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2). Analysis by the CAP system revealed the presence of rHev b 8-specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health-care workers. Two subjects of the latter group with rHev b 8-specific IgE showed negative results in the skin prick tests with tree-pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE-binding epitopes on the Hev b 8-molecule which do not cross-react with birch profilin. Immunoblot inhibition assays using MBP-rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract. IgE binding to the native latex profilin could be completely inhibited by the MBP-rHev b 8. CONCLUSIONS: Latex profilin represents a minor allergen in NRL and may have IgE-binding epitopes different from Bet v 2.     

Hev b 8, the Hevea brasiliensis latex profilin, is a cross-reactive allergen of latex, plant foods and pollen

BACKGROUND: Plant profilins are important pan-allergens. They are responsible for a significant percentage of pollen-related allergies. Limited information is available about their involvement in the latex-fruit syndrome and the cross-reactivities between latex and pollen. We aimed to clone and express the Hevea brasiliensis latex profilin to investigate its allergological significance and serological cross-reactivities to profilins from plant foods and pollens. METHODS: A DNA complementary to messenger RNA (cDNA) coding for the Hevea latex profilin, Hev b 8, was amplified by polymerase chain reaction from latex RNA. Recombinant (r)Hev b 8 was produced in Escherichia coli and used to screen sera from 50 latex- allergic health care workers (HCWs) with well-documented histories of food and pollen allergy and 34 latex-allergic spina bifida (SB) patients. The cross-reactivity of natural Hev b 8 and rHev b 8 with other plant profilins was determined by ELISA inhibition assays. A three-dimensional homology model of Hev b 8 was constructed based on known profilin structures. RESULTS: The cDNA of Hev b 8 encoded a protein of 131 amino acids with a predicted molecular mass of 14 kD. Twelve of the 50 HCWs and 2 of the 34 SB patients were sensitized to Hev b 8. All Hev b 8-sensitized patients showed allergic symptoms to pollen or plant foods. Cross-reactivities between profilins of latex, pollen and plant food were illustrated by their ability to inhibit IgE binding to rHev b 8. Homology modeling of Hev b 8 yielded a structure highly similar to Bet v 2, the birch pollen profilin, with the most distinct differences located at the N-terminus. CONCLUSIONS: We conclude that primary sensitization to latex profilin in the majority of cases takes place via pollen or food profilins. Additionally, pollinosis and food-allergic patients with profilin-specific IgE can be at risk of developing latex allergy.     

Specific monoclonal antibodies and human immunoglobulin E show that Hev b 5 is an abundant allergen in high protein powdered latex gloves

BACKGROUND: Hev b 5 is a major latex allergen recognized predominantly by latex-allergic health care workers (HCWs). Recombinant Hev b 5 (rHev b 5) was previously expressed as a fusion protein with maltose binding protein (MBP), itself an immunogenic molecule; therefore non-fusion rHev b 5 is desirable. Moreover, standardized immunological assays for the detection of Hev b 5 are currently lacking and may have important implications for both allergen avoidance and diagnosis in latex allergy. OBJECTIVES: To generate and use Hev b 5-specific mAbs to determine the relative abundance of Hev b 5 in different latex extracts, correlating this with the IgE reactivity of latex-allergic HCWs and to produce non-fusion rHev b 5. METHODS: For the production of mAbs, mice were immunized with rHev b 5/MBP fusion protein and mAbs selected with rHev b 5/MBP but not MBP reactivity. The mAb reactivity was compared with polyclonal IgE from latex-allergic HCWs using direct and inhibition ELISA and immunoblot assays. Recombinant Hev b 5 was expressed and purified in the pPROEX-HTa bacterial expression system. RESULTS: Four Hev b 5-specific mAbs were produced. Immunoblotting and ELISA using the mAbs indicate abundant Hev b 5 in high protein powdered latex glove extracts as compared with crude latex sap extracts. High quality surgical gloves with no detectable protein have no detectable Hev b 5. Inhibition ELISAs using serum IgE from latex-allergic HCWs and Hev b 5-specific mAbs gave strong correlation. Non-fusion recombinant Hev b 5 was successfully expressed and purified, showing reactivity with both the Hev b 5-specific mAbs and serum IgE of latex-allergic HCWs. CONCLUSION: Hev b 5-specific mAbs and human IgE from latex-allergic HCWs demonstrate the greater content of Hev b 5 in high protein powdered glove extracts. This may explain the observed higher frequency of sensitization to this allergen in HCWs.     

Recombinant Hev b 1: large-scale production and immunological characterization

BACKGROUND: Hev b 1 represents one of the most important allergens in Hevea brasiliensis latex. It is difficult to get an appropriate amount of native Hev b 1 (nHev b 1) for research purposes. OBJECTIVE: The aim of this study was to produce sufficient amounts of Hev b 1 by recombinant methods to prove its suitability for latex allergy diagnostics. METHODS: We isolated total RNA of Hevea brasiliensis leaves and synthesized cDNA by RT PCR. Recombinant Hev b 1 (rHev b 1) as well as three fragments (amino acid residues 29-137, 48-137, 78-137) were subcloned and expressed as fusion proteins with Maltose-binding protein (MBP) in Escherichia coli. The MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, histamine release test and immunoblots with human sera from spina bifida patients as well as from health care workers with latex allergy and monoclonal antibodies. RESULTS: Histamine release test and immunoblots revealed the high allergenicity of the MBP-rHev b 1 construct. By the CAP method, 54 out of 58 serum samples (93%) from latex-sensitized spina bifida patients previously showing immunoglobulin (Ig) E to nHev b 1 exhibited IgE-binding to rHev b 1. Among 71 latex-allergic health care workers tested, 16 (22.5%) had IgE antibodies to rHev b 1. The analysis of the fusion proteins carrying rHev b 1 fragments revealed that the loss of the N-terminal 28 amino acid residues did not affect IgE-binding. In contrast, the lack of the first 47 amino acid residues led to decreased IgE-binding reactivity in two out of four sera tested, whereas the absence of the N-terminal 77 residues abolished IgE-binding in these two sera. CONCLUSION: The MBP-rHev b 1 fusion protein exhibits a corresponding IgE-binding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 for both in vitro diagnostics and research purposes.     

Induction of mucosal tolerance with recombinant Hev b 1 and recombinant Hev b 3 for prevention of latex allergy in BALB/c mice

The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure to latex products, such as health-care workers (HCW) and patients with spina bifida (SB). Treatment of latex allergy seems problematic as preventive measures, such as allergen avoidance, are not always possible and conventional immunotherapy with standardized latex extracts is not performed routinely. Thus, the aim of the present study was to establish a mouse model of latex allergy using two major latex allergens for HCWs and SB patients, Hev b 1 and Hev b 3, for sensitization. Prophylactic measures on the basis of mucosal tolerance induction with the recombinant allergens were tested in this model. Female BALB/c mice immunized intraperitoneally with recombinant (r)Hev b 1 or rHev b 3 displayed strong immune responses in vivo and in vitro. Intranasal treatment with rHev b 1 and rHev b 3 prior to sensitization led to reduced allergen-specific IgG1/IgE levels and significantly suppressed allergen-induced basophil degranulation. Moreover, lymphocyte proliferation and cytokine production (IL-4, IL-5, IFN-gamma) in vitro were significantly suppressed after pretreatment with both allergens. Suppressive cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-beta, remained unchanged after the intranasal pretreatment, indicating mechanism of anergy rather than active immunosuppression. Taken together, these results suggest that mucosal tolerance induction with recombinant allergens could present a promising prevention strategy against latex allergy.     

Hev b 7 is a Hevea brasiliensis protein associated with latex allergy in children with spina bifida

BACKGROUND: In addition to their disease-associated handicaps, patients with spina bifida (SB) are at high risk of developing latex allergy. Individuals with SB represent a special group of latex-allergic patients, inasmuch as their IgE-binding patterns differ from those of other populations of latex-allergic individuals. Two allergens strongly associated with latex allergy in patients with SB--Hev b 1 and Hev b 3--have already been identified. OBJECTIVE: We intended to identify a predominant IgE-binding band--in addition to Hev b 1 and 3--at 43 kDa in a study population of 38 latex-allergic (IgE antibodies to latex and symptoms on provocation with latex gloves) and 15 latex-sensitized (IgE antibodies to latex but no symptoms on provocation) children with SB (mean age, 12.3 years) and to determine its frequency of recognition. METHODS: Sera of latex-sensitized or latex-allergic patients with SB were tested on latex C extract containing natural Hev b 1, Hev b 3, and Hev b 7 and with the recombinant 43-kDa Hev b 7 in immunoblot and inhibition studies. RESULTS: Natural Hev b 1 was recognized by 82% and natural Hev b 3 by 79% of the latex-allergic children with SB. In addition to some other proteins, 15 (39.5%) of 38 latex-allergic and 2 (13%) of 5 latex-sensitized children with SB revealed IgE binding to a 43-kDa band in the latex protein extract. We identified this 43-kDa IgE-binding band as natural Hev b 7 by immunoblotting and inhibition experiments using recombinant Hev b 7. CONCLUSION: From these data, we conclude that Hev b 7, the patatin-like Hevea latex protein, is the third SB-associated latex allergen. Future immunotherapy for latex-allergic individuals with SB will have to include Hev b 7 in addition to Hev b 1 and Hev b 3.     

Identification of a Hevea brasiliensis latex manganese superoxide dismutase (Hev b 10) as a cross-reactive allergen.

BACKGROUND: Cross-reactive allergens play an increasingly important role in latex allergy in complicating both the diagnosis and time course of allergic symptoms. Manganese superoxide dismutase (MnSOD), a ubiquitous protein of prokaryotic and eukaryotic organisms, was described as a cross-reactive allergen in Aspergillus fumigatus. Little information is available on the importance of this pan-allergen in Hevea brasiliensis latex. The aim of this study was to clone and express MnSOD from H. brasiliensis latex, and to obtain the soluble and immunologically active recombinant allergen for diagnosis of latex allergy and to investigate possible cross-reactivities with the structurally related A. fumigatus and human MnSODs. METHODS: A complementary DNA coding for Hevea latex MnSOD was amplified by PCR. The recombinant protein was produced in Escherichia coli with an N-terminal hexahistidyl tag. Enzymatic activity of the recombinant protein was determined using an enzyme assay for SODs. IgE immunoblotting and IgE inhibition assays were performed to characterize the recombinant allergen and its cross-reactivity. RESULTS: A Hevea latex MnSOD consisting of 206 amino acid residues was cloned and expressed in E. coli. The allergen was designated Hev b 10. The recombinant protein was enzymatically active, indicating the correct folding of the protein. In immunoblots, latex- as well as A. fumigatus-allergic patients revealed IgE binding to recombinant (r)Hev b 10. Cross-reactivity to Asp f 6, the MnSOD from A. fumigatus, and human MnSOD was determined by inhibition of IgE binding to these MnSODs by rHev b 10. CONCLUSIONS: Hev b 10 is a new cross-reactive allergen of H. brasiliensis which belongs to the 'latex-mold' group of latex allergens. Furthermore, it is a candidate for primary sensitization in patients allergic to the pan-allergen MnSOD.     

IgE reactivity to latex allergens among sensitized healthcare workers before and after immunotherapy with latex

BACKGROUND: New IgE sensitizations to proteins in allergen extracts have been shown to occur during allergen-specific immunotherapy (IT). METHODS: Twenty-four healthcare workers (HCWs) -- patients included in a latex IT study -- were analysed, 16 in active treatment and eight in placebo. Sera were obtained at baseline and after 6 months of IT and analysed with immunoblotting and CAP System with eight single recombinant latex allergens (rHev b 1, 3, 5, 6.01, 8, 9, 10, 11, and a mix of rHev b1, 5, 6.01 and 8). RESULTS: After IT with latex, three patients in the active treatment group had new IgE sensitizations, one to Hev b 5, one to Hev b 11 and another to Hev b 6.01. No other significant variation in mean of specific IgE to latex or recombinant allergens were observed in patients who received placebo or active treatment. A significant (P = 0.012) negative correlation (-0.72) was observed between maximal tolerated dose and specific IgE to Hev b 6.01 at baseline. After IT, immunoblot analysis demonstrated a significant increase in IgE binding in a band of approximately 22 kDa (P = 0.032) that may correspond to Hev b 6.01. New or more intense bands appeared in seven patients of the active group, while in three subjects a reduction was observed. CONCLUSIONS: Hev b 6.01 seems to be the most relevant latex allergen in HCWs. New or more intense IgE binding to latex allergenic components occurs during latex immunotherapy. However, the levels of specific IgE against these new components are low and do not seem to have clinical relevance.     

Component‐resolved diagnosis from latex allergy by microarray

Background A positive specific IgE (sIgE) result for latex does not always mirror the clinical situation and is frequently found in individuals without overt latex allergy. Objective We sought to investigate the potential of component‐resolved diagnosis (CRD) of latex allergy by microarray and to assess whether the technique allows discriminating genuine allergy from asymptomatic sensitization. Methods Twenty‐six healthy controls without a history of latex allergy with a negative latex sIgE and skin test, 22 latex‐allergic patients with a compelling history of latex allergy with a positive latex sIgE and prick test and 20 latex‐sensitized individuals with a frequent asymptomatic exposure to natural rubber latex‐containing devices with a negative latex skin test but a positive sIgE were also included. CRD was performed with the ImmunoCAP ISAC microarray and traditional singleplexed ImmunoCAP. Results In all patients, the diagnosis of latex allergy could be established by the combination of recombinant latex components present on the microarray (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02). Over three‐quarters of our patients were sensitized for Hev b 5 and/or Hev b 6.02. Some patients also displayed reactivity for Hev b 1 and/or Hev b 3. In contrast, none of the individuals sensitized to natural rubber latex or control individuals demonstrated IgE reactivity for rHev b 1, rHev b 3, rHev b 5 or rHev b 6.02. Three‐quarters of the patients sensitized to latex displayed a positive microarray result for recombinant latex profilin (rHev b 8). In contrast to the results obtained by traditional ImmunoCAP for bromelain, almost no sensitization for cross‐reactive carbohydrates was demonstrated by bromelain spotted on the microarray. CRD by traditional singleplexed ImmunoCAP showed highly comparable results. Conclusion CRD by microarray is a reliable tool for diagnosing latex allergy. In addition, the technique allows discrimination between genuine allergy and sensitization. CRD by microarray can improve the diagnosis of IgE‐mediated latex allergy by discriminating between genuine allergy and sensitization. CRD by microarray is a reliable tool to diagnose latex allergy. In addition, the technique allows discrimination between a genuine allergy and simple sensitization. Cite this as: D. G. Ebo, M. M. Hagendorens, K. J. De Knop, M. M. Verweij, C. H. Bridts, L. S. De Clerck and W. J. Stevens, Clinical & Experimental Allergy, 2010 (40) 348– 358.     

Assessment of profilin as an allergen for latex-sensitized patients

BACKGROUND: The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the clinical significance of this molecule as an allergen for latex-allergic patients is not clear. We studied the allergenic relevance of isolated latex natural and recombinant profilin, by in vivo and in vitro techniques, in two populations of spina bifida children (SB) and adults allergic to latex (AL). METHODS: Profilin is present in small amounts in latex extracts obtained from low ammoniated (LA) natural latex. Its purification by affinity chromatography resulted difficult due to Heb v 1 unspecific binding. Therefore a method was developed to obtain natural profilin from natural latex, combining affinity chromatography (PLP, poly-L-proline Sepharose column) and previous ammonium sulfate fractionation. Alternatively, latex c-serum containing a low amount of Hev b 1 and a relatively higher profilin content could be used. Recombinant latex profilin isoform (rHev b 8) was cloned by PCR amplification. The entire coding region of Hev b 8 was subcloned into the expression vector pKN172 and a non-fusion form of Hev b 8 was expressed in Escherichia coli BL21 (DE3). Purified recombinant protein was obtained after a single passage through PLP-Sepharose column. RESULTS: Natural and recombinant purified Hev b 8 were tested cutaneously by intradermoreaction (ID) in 17 SB and 14 AL patients. They were positive in 15 SB and 14 AL patients. No wheals were produced when tested in nonatopic control patients. Only 42% of sera from latex-allergic patients revealed specific IgE titers of class 1 or higher by enzyme immunoassay and only 39% of them exhibited IgE binding by SDS-PAGE immunoblotting with any natural or recombinant Hev b 8 forms. CONCLUSION: It seems that profilin is a relevant allergen for both groups of patients from a frequency point of view, but with scarce presence in natural latex extracts and raw sources, with a subsequent low IgE induction capacity.     

Natural rubber latex and chestnut allergy: cross‐reactivity or co‐sensitization?

BACKGROUND: Chestnut and natural rubber latex (NRL) allergy are often associated in the latex-fruit syndrome. AIM OF THE STUDY: To establish whether the concurrent NRL and chestnut IgE antibody reactivity are the results of co-sensitization or cross-reactivity. METHODS: Sera from 19 patients with chestnut- and NRL-specific IgE were selected and tested for reactivity with recombinant (r) latex allergens. Cross-reactivity was explored by IgE-inhibition experiments using chestnut or NRL allergens as solid phase on ImmunoCAP. RESULTS: IgE-antibodies were detected to rHev b 6.01 (prohevein) in 58% of the sera, to rHev b 5 in 32%, to rHev b 12 in four of 13 sera, to rHev b 7.02 and rHev b 11 in four, and to rHev b 1 in two of 19 sera. rHev b 8-IgE antibodies were found in nine sera (47%), whereas six displayed mono-sensitization to rHev b 8 with regard to our test panel. Three of 16 sera showed IgE to cross-reactive carbohydrate determinants. In most sera recognizing rHev b 5 and/or rHev b 6.01 as major allergens the IgE-reactivity to NRL remained unaffected by chestnut extract and chestnut-IgE remained unaffected by NRL extract. Conversely, in sera with rHev b 8 as dominant allergen IgE-binding to NRL was nearly completely inhibited by chestnut and vice versa. IgE-binding to rHev b 8 was abolished by chestnut extract. CONCLUSIONS: Although patients have concomitant IgE antibody reactivity to chestnut and NRL, cross-reactivity could be demonstrated mainly in those patients with IgE to Hev b 8 (profilin) from NRL.     

Unique and shared IgE epitopes of Hev b 1 and Hev b 3 in latex allergy

Of the several latex proteins cloned and expressed, the rubber elongation factor, Hev b 1, and the closely related Hev b 3, represent two major allergens associated with latex allergy. Although both allergens demonstrated IgE binding with sera from latex allergic patients, it was not known whether these two molecules shared any epitopes. Hence, in the present study using health care workers (HCW) and spina bifida (SB) patients with latex allergy, we investigated the IgE binding epitopes in Hev b 1 and Hev b 3. Recombinant Hev b 1 and Hev b 3 were expressed in a prokaryotic expression system, while overlapping decapeptides of Hev b 1 and Hev b 3 were synthesized on derivatized cellulose membrane. Eight IgE binding epitopes for Hev b 1 and eleven for Hev b 3 were identified using sera from latex allergic patients with SB. On further analysis of synthetic peptides encompassing these epitopes, similar IgE antibody reactivity was demonstrated with three Hev b 1 epitopes b1E3, b1E5, b1E6 and two Hev b 3 epitopes; b3E10 and b3E 11. For Hev b 1, a unique IgE binding epitope was identified in the region of amino acid residues 16-25. In competitive ELISA, peptides bIE2 and bIE4 together inhibited 58% of IgE binding of Hev b 1, while b3E5 showed 22% inhibition in the IgE binding of Hev b 3. The results of the present study suggest that the understanding of linear and conformational IgE epitopes in the major latex allergens may provide better insight into the structure-function relationship of the allergens, and may lead to the development of better patient care and management strategies in latex allergy.     

Percutaneous reactivity to natural rubber latex proteins persists in health-care workers following avoidance of natural rubber latex

BACKGROUND: Long-term avoidance of natural rubber latex [Hevea brasiliensis (Hev b)] is currently recommended for health-care workers (HCWs) with established natural rubber latex (NRL) allergy. Percutaneous sensitivity to eight Hev b NRL allergens was evaluated in HCWs in 2000. To date, no studies have evaluated the longitudinal effects of NRL avoidance on percutaneous sensitivity to NRL allergens. OBJECTIVE: The aims of this study were to evaluate changes in percutaneous reactivity to non-ammoniated latex (NAL) and NRL allergens in HCWs 5 years after a recommendation to avoid NRL and to evaluate factors that predict the persistence of in vivo sensitivity to NAL and NRL allergens. METHODS: Skin prick testing was performed with NAL, seven NRL allergens (Hev b 1, 2, 3, 4, 6.01, 7.01, and 13), and recombinant Hev b 5 (rHev b 5) in 34 HCWs who were initially evaluated in 2000 for occupationally related NRL allergy. Serial 10-fold dilutions of NAL and NRL allergens were employed in skin testing. Sera from the HCWs were assayed for latex and enhanced latex (rHev b 5-enriched allergosorbent)-specific IgE antibodies using the ImmunoCAP assay. RESULTS: The prevalence of work-related symptoms significantly decreased between 2000 and 2005 with avoidance of NRL (P<0.05). A >/=100-fold reduction in percutaneous sensitivity to Hev b 2 and Hev b 7 was less likely in those with prior history of systemic reactions to NRL (P=0.0053), reported history of reaction to cross-reactive foods (P=0.014), continued local reactions to NRL gloves (P<0.0001), or high NRL glove exposure since the initial study (P=0.0075). The diagnostic sensitivity and specificity of the latex-specific IgE serology was 54% and 87.5%, respectively, in comparison with NAL skin tests. The addition of rHev b 5 to the ImmunoCAP (enhanced latex) allergosorbent altered the diagnostic sensitivity and specificity of the ImmunoCAP to 77% and 75%, respectively. CONCLUSION: While symptoms may resolve quickly with NRL avoidance therapy, detectable IgE indicating continued sensitization remains beyond 5 years, and thus continued avoidance of NRL should be recommended.     

The hevein domain of the major latex-glove allergen Hev b 6.01 contains dominant T cell reactive sites

BACKGROUND: Sensitization to natural rubber latex (Hevea brasiliensis) is a major cause of occupational asthma and rhinitis affecting frequent latex-glove users. Hev b 6.01, a known major latex allergen, is cleaved naturally into hevein (4.7 kDa) and a C-terminal fragment (14 kDa). Hevein is an abundant protein in latex-glove extracts. As the immune response to allergens is initiated by activation of allergen-specific CD4(+) T cells, identification of dominant T cell epitopes is crucial for the development of specific immunotherapy. OBJECTIVE: To identify dominant T cell epitopes of Hev b 6.01 in latex-allergic glove users. METHODS: Ten latex-allergic frequent glove users and six non-latex-allergic atopic control subjects were selected, based on clinical symptoms and positive latex-specific serum IgE. Serum IgE reactivity to glove extract and recombinant Hev b 6.01 (rHev b 6.01) were analysed by ELISA. Latex-specific short-term oligoclonal T cell lines were generated from peripheral blood of latex-allergic subjects. These lines were tested for proliferative responses to overlapping 20-mer peptides of the Hev b 6.01 molecule. CD4(+) T cell intracellular cytokines, IL-4 and IFN-gamma were assessed following stimulation with immobilized anti-CD3 in the presence of IL-2. RESULTS: All ten of the latex-allergic patients showed serum IgE binding to glove extract while eight of these also showed IgE binding to rHev b 6.01 by ELISA. Western blotting confirmed reactivity with rHev b 6.01 at around 20 kDa. T cell proliferation assays showed that latex-specific T cell lines from all subjects responded to one or more peptides, with greatest frequency of reactivity to peptides Hev b 6.01 p(10-29) and Hev b 6.01 p(19-38) in the hevein domain. An allergic-type cytokine profile with considerable IL-4 in addition to IFN-gamma was evident from intracellular cytokine staining. CONCLUSION: Hevein is an important T cell as well as B cell immunogen and contains dominant T cell reactive sites.     

Skin prick test reactivity to recombinant latex allergens

BACKGROUND: Allergy to latex has become a serious and increasingly common health problem, particularly for healthcare workers and patients who undergo frequent surgical procedures. Testing for latex allergy currently involves in vitro tests and skin prick testing using crude preparations of natural rubber latex (NRL). To date, 10 latex proteins have received designation as allergens (Hev b 1 to Hev b 10) and, except for Hev b 4, have been cloned as recombinant proteins. Our aim was to compare the skin prick test (SPT) reactivity of six recombinant latex allergens with SPT reactivity to natural rubber latex proteins in known latex-allergic individuals. METHODS: Six recombinant proteins were expressed in Escherichia coli, and tested as the intact fusion proteins (Hev b 2, 5, 6, 8) or as purified proteins (Hev b 3 and 7). SPT with the six recombinant latex allergens was performed using 10-fold serial dilutions on 31 latex-allergic subjects to determine the level of reactivity to each recombinant allergen. Latex-specific IgE was determined using the AlaSTAT assay. RESULTS: All six recombinant allergens were reactive by SPT in at least 1 latex-allergic patient but not in any of the control patients. The frequency of sensitization to the various recombinant allergens was similar to previous studies using the native proteins isolated from NRL. The minimal level of protein for a positive skin test was 70 pg/ml for NRL and 1 ng/ml for one recombinant allergen (Hev b 7). In our patients, the use of a combination of recombinant latex allergens Hev b 5, 6 and 7 diagnosed latex allergy with 93% sensitivity and 100% specificity. CONCLUSION: Recombinant latex allergens are clinically reactive, can be produced in a standardized manner, and could potentially provide safe, sensitive and specific reagents for the diagnosis of latex allergy.     

In vivo sensitization to purified Hevea brasiliensis proteins in health care workers sensitized to natural rubber latex

BACKGROUND: Thirteen proteins of natural rubber latex (Hevea brasiliensis) known to bind human IgE have been isolated and characterized as Hev b allergens. However, the in vivo importance of native Hev b allergens has not been defined in health care workers (HCWs) with natural rubber latex (NRL) allergy. OBJECTIVES: The principal aim of this study was to identify the major in vivo Hev b allergens in HCWs with NRL allergy confirmed by percutaneous sensitivity to nonammoniated latex (NAL). METHODS: Skin prick testing was performed with 7 (native) proteins purified from NAL (Hev b 1, 2, 3, 4, 6.01, 7.01, and a newly described Hev b 13) and recombinant Hev b 5 in 62 HCWs with histories of NRL allergy (group 1) confirmed by percutaneous reactivity to NAL and in 49 atopic HCWs without NRL allergy (group 2). Serial 10-fold concentrations of Hev b proteins (5 x 10(-5) microg/mL to 50 microg/mL) were tested; serum samples of subjects were assayed for serum specific IgE by immunoassays. RESULTS: Hev b 2, Hev b 5, Hev b 6.01, and Hev b 13 produced skin reactions in more than 60% of group 1 subjects, with Hev b 1, 3, 4, and 7.01 eliciting reactions in less than 50%. Only 1 of 49 group 2 workers reacted to a single Hev b antigen (Hev b 13). Specificity of 7 Hev b allergens was 100% and 98% for Hev b 13 in identifying workers with confirmed NRL allergy. Specific IgE by AlaSTAT and CAP immunoassays was elevated in 40 of 60 (67%) and 33 of 62 (53%) of NAL-reactive workers and produced false-positive test results in 4 of 49 (8%) and 3 of 48 (6%) group 2 subjects, respectively. CONCLUSION: Hev b 2, 5, and 6.01 are major in vivo allergens and Hev b 13 is a new major in vivo allergen among HCWs with allergy to NRL.