Jay E. Slater

Identification, Cloning, and Sequence of a Major Allergen (Hev b 5) from Natural Rubber Latex (Hevea brasiliensis)

Proteins in commercial latex products, derived from the rubber tree Hevea brasiliensis, cause anaphylaxis in susceptible individuals, especially health care workers and children with spina bifida. To identify latex allergens, we utilized IgE from the serum of a latex-allergic health care worker to screen a cDNA library from Hevea latex. The identified cDNA clone, cDNA Hev b 5, encodes an open reading frame of 163 peptide residues. Hybridization analysis of cDNA Hev b 5 with RNA extracted from Hevea tissue indicates that the full-length transcript is about 1000 bases. The nucleotide and deduced protein sequences have significant homology to sequences from kiwi and potato, which are known to cause allergic reactions in some latex-allergic patients. Fifty-six percent of spina bifida patients and 92% of health care workers with latex allergy have IgE specific to the protein encoded by cDNA Hev b 5. A monoclonal antibody raised from a mouse immunized with Hev b 5 binds to a protein in Hevea latex with an Mr identical to that of the expressed and cleaved recombinant protein. Taken together, these results establish that the antigen Hev b 5 contains a major epitope for IgE-mediated reactions to H. brasiliensis latex products.

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.

Human IgE-binding epitopes of the latex allergen Hev b 5

BACKGROUNDnHev b 5 is an acidic protein (isoelectric point, 3.5) rich in glutamic acid with 9 repeated amino acid (AA) sequences of XEEX or XEEEX. Although its function in Hevea brasiliensis is unknown, Hev b 5 has been identified as a major latex allergen. Immunoblot inhibition studies suggest Hev b 5 exists as multiple isoforms or contains a common epitope found in several other proteins.nnnOBJECTIVEnThe purpose of this study was to further characterize Hev b 5 and to identify linear IgE-binding epitopes.nnnMETHODSnOctapeptides spanning the entire Hev b 5 protein were synthesized on a derivatized cellulose membrane. The membrane was reacted with sera pooled from health care workers allergic to latex or rabbits immunized with latex proteins. B-cell epitopes were identified by subsequent incubations with the appropriate secondary antibodies and detected by using chemifluorescence.nnnRESULTSnSera from patients allergic to latex recognized 6 IgE-binding regions located throughout the molecule. Two epitopes (2 and 4) had the common AA sequence of KTEEP. Epitopes 3 and 5 had a similar AA sequence of EEXXA, where X was P, T, or K. Epitopes 1 and 6 appeared to be unrelated to the other epitopes. Database analysis could not identify other proteins with similar sequences. Neither of the XEEEX sequences bound IgE. Control sera failed to react to any peptides.nnnCONCLUSIONSnHev b 5 exists as multiple isoforms, but only small amounts are present in the nonammoniated latex preparations, such as those used for diagnostic tests, and this may help to explain the relatively poor sensitivity of some in vitro tests.

The latex allergen Hev b 5 transcript is widely distributed after subcutaneous injection in BALB/c mice of its DNA vaccine ☆ ☆☆ ★ ★★

BACKGROUNDnDNA vaccines reduce IgE responses to selected allergens, but severe reactions to the expressed antigen may limit the usefulness of the technique in allergen immunotherapy.nnnOBJECTIVEnWe sought to determine the extent of spread of an injected DNA vaccine in mice.nnnMETHODSnWe placed the gene encoding the potent Hevea latex allergen Hev b 5 in a mammalian expression vector and injected this DNA vaccine subcutaneously into BALB/c mice. At several times after injection, the presence of Hev b 5 transcript was determined in multiple tissues by RT-PCR. The identity of the amplification product was confirmed by Southern hybridization and restriction analyses.nnnRESULTSnHev b 5 RNA appeared at the injection site and in the lymph nodes, spleen, and lungs within 1 day after injection and persisted for at least 14 days. Hev b 5 RNA was also identified in the blood and tongue 14 days after injection. Antibody and cell-mediated responses to Hev b 5 were also noted in the immunized animals at later time points. As expected, animals injected with the identical plasmid containing the Hev b 5 DNA in the antisense orientation mounted no immune response to Hev b 5.nnnCONCLUSIONSnThe rapid and widespread appearance of the Hev b 5 transcript in the injected mice confirms that DNA is translocated from the injection site, transcribed, and expressed in immune and nonimmune tissues after injection. Controlling the extent and degree of expression in specific target tissues may allow therapeutic DNA vaccination with plasmids that encode potentially toxic allergens.

Lipopolysaccharide augments IgG and IgE responses of mice to the latex allergen Hev b 5.

BACKGROUNDnLPS is a common contaminant in the health care environment and in latex examination gloves.nnnOBJECTIVEnWe sought to investigate the role of LPS in enhancing the immune responses of mice to inhaled latex allergen.nnnMETHODSnAs our model allergen, we used a fusion protein containing the potent latex allergen Hev b 5. BALB/c mice were lightly anesthetized and given repeated intranasal doses of saline, LPS, and/or Hev b 5. The doses were given in 2 courses separated by a 6-week period, with the first course consisting of 6 doses and the second consisting of 3 doses.nnnRESULTSnAfter the first set of immunizations, mice given Hev b 5 alone had no detectable IgG1 or IgE responses to Hev b 5, whereas mice given the antigen along with LPS had significant responses (IgG1, 0.73 U +/- 0.05; IgE, 0.88 U +/- 0.2). No enhancement of specific IgG2a was observed. A stimulatory effect of LPS on all 3 immunoglobulin types was apparent after the second course. Lymphocytes from mice immunized with LPS and Hev b 5 had increased proliferation to Hev b 5 and its fusion partner.nnnCONCLUSIONSnLPS may be an important immunoadjuvant for the development of allergic reactions to latex protein allergens.

Latex Allergy in Children with Spina bifida

We studied the prevalence of latex-specific IgE among the children in our myelomeningocele clinic and several groups of controls using skin tests, a commercially available ELISA and an in-house RAST. Thirty-nine of 83 (47%) children with myelomeningocele had antibodies directed against latex as did 6 of 40 (15.7%) chronically ill controls, 4 of 105 (3.8%) medical controls and 2 of 75 (2.7%) well controls. Within each study group the likelihood of a positive skin test increased with the number of operations the subject had undergone. Children with myelomeningocele were much more likely to have antibodies to latex than were chronically ill controls with similar surgical histories. A retrospective chart review of 18 years and a total of 646 operations disclosed only one episode of intraoperative anaphylaxis which appeared to be related to latex within our study group.

Affinity purification of latex antigens

Latex extracts are complex mixtures of antigenic peptides. We attempted to raise monoclonal antibodies to latex and to use these antibodies to purify latex antigens. A monoclonal antibody, CRI-C, was raised by standard techniques. Peptides of nonammoniated latex (NAL) and ammoniated latex were electrophoretically separated and transferred for immunoblots. CRI-C was covalently attached to an agarose column. NAL was passed over the column, and purified antigen was then eluted. The eluate was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and RAST inhibition with sera from health care workers and children with spina bifida. CRI-C recognized a single band in ammoniated latex immunoblots and several distinct bands in NAL. The affinity-purified antigen of CRI-C (C-Ag) had multiple bands of less than 20 kd and was 3.9 times more potent in RAST inhibition than NAL when sera from patients with spina bifida were used. However, when health care workers sera were used, there was no significant difference in the inhibitory potency of NAL and C-Ag. CRI-C appears to recognize a distinct and important epitope in the IgE immune response to latex of patients with spina bifida.

Murine B-cell and T-cell epitopes of the allergen Hev b 5 from natural rubber latex.

Hev b 5, a proline-rich acidic protein with a predominantly random secondary structure, is a major allergen in natural rubber latex and a candidate for specific immunotherapy of latex allergy. As a first step in the identification of candidate peptides for immunotherapy, we have begun to identify the B-cell and T-cell epitopes of Hev b 5 in BALB/c mice. The mice were immunized with a Hev b 5 fusion protein. The B-cell epitopes were determined by the SPOTS method using overlapping octamers or by ELISA inhibition using a series of overlapping 20-mers. The T-cell epitopes were determined by the proliferation and cytokine release of splenocytes cultured in the presence of the 20-mers. Potential antibody binding regions included residues in regions 1-38, 55-74, 109-128 and 132-151. Examination of the binding sequences for common motifs suggested enhanced antibody binding to the KXEE or KEXE sequences, where X is empty, threonine or alanine. Splenocyte stimulation and cytokine release suggest T-cell epitopes with the regions 1-20, 37-56, 73-101 and 109-146. Since they may contain major T-cell epitopes but do not exhibit significant antibody binding, peptide regions 38-48 and 75-101 are candidates for specific immunotherapy to Hev b 5 in the BALB/c mouse model.

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

Background German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency. Objective Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts. Methods Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA. Results Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target. Conclusions An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.