Zo-Nan Chang

Isolation and characterization of a novel 98-kd Dermatophagoides farinae mite allergen

BACKGROUND Exposure to allergens from house dust mites is a significant cause of immediate hypersensitivity. Thus far, the active mite allergens defined are low molecular weight (MW) proteins or glycoproteins. However, other important mite allergens remain to be investigated. In this study a high MW mite antigen with a high IgE-binding activity was characterized. METHODS An anti-Dermatophagoides farinae (Df) monoclonal antibody, mAb642, which recognized a 98-kd allergenic mite protein, was used for affinity chromatography. The purified Df642 was characterized biochemically and immunologically. RESULTS Competitive ELISA demonstrated that mAb642 was inhibited by the interaction between serum IgE from allergic patients and Df642 antigen in a dose-dependent fashion. The IgE reactivity to both 98-kd and 92-kd components was removed or diminished by preincubation of asthmatic sera with Df642-coated CNBr-activated cellulose-4B gel. Two-dimensional immunoblot analysis revealed that there are at least 4 isoforms of Df642 that represent a minor component in the crude mite extract. The allergenicity of Df642 was assayed by IgE immunoassay with a large panel of 67 sera from asthmatic patients with positive skin reactions, and Df 642 showed positive IgE reactivity with more than 80% of the sera tested. Thus it should be classified as an important allergen. In addition, amino acid sequence analysis revealed that Df642 shares more than 50% homology with paramyosin from invertebrates. CONCLUSION We have identified and characterized a 98-kd house dust mite allergen that showed greater than 80% IgE reactivity with sera from patients allergic to mites. This is the first high MW allergen characterized to date, and it shares high sequence homology with paramyosins in invertebrates.

Induction of specific Th1 responses and suppression of IgE antibody formation by vaccination with plasmid DNA encoding Der f 11

DNA vaccines encoding low-molecular-weight allergens have been used to prevent IgE responses. A high-molecular-weight mite allergen Der f 11 that was hardly to be purified for immunotherapy was used to develop a DNA vaccine here. Vaccination of mice with plasmid DNA encoding Df11 (pDf11) induced Th1 responses characterized by IgG2a responses and spleen cell secretion of IFN-gamma. In contrast, sensitization with recombinant Der f 11 (rDf11) and alum induced Th2 responses characterized by IgE responses and spleen cell secretion of IL-4 and IL-5. Vaccination with pDf11 prevented the induction of IgE responses. Moreover, it could inhibit on-going IgE responses. The debate whether CD4+ or CD8+ T cells were the regulatory cells to inhibit IgE responses by DNA vaccination was also examined. First, sensitization of pDf11-vaccinated mice after depletion of CD8+ T cells still showed suppression of IgE responses. Secondly, adoptive transfer of either CD4- or CD8-depleted spleen cells from pDf11-vaccinated mice suppressed IgE responses. In conclusion, this is the first report to confirm the therapeutic effect of a DNA vaccine encoding a strong allergen on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulation of IgE responses by pDf11.

Use of monoclonal antibodies to isolate and characterize Cyn dI, the major allergen of Bermuda grass pollen

BACKGROUND Cyn d I has been found to be the major allergen of Bermuda grass (Cynodon dactylon) pollen, but its exact nature remains to be clarified. METHODS Cyn d I, the major allergen of Bermuda grass (Cynodon dactylon) pollen, was purified by monoclonal antibody (MoAb) affinity chromatography, and its biochemical and immunologic properties were characterized. Anti-Cyn d I MoAb 4-37, which recognizes all of the isoallergens of Cyn d I, was chosen as the immunosorbent. RESULTS The purified protein has an amino acid composition similar to that of the group I allergens of other grass pollens. It appears as a single 34 kd band or as a mixture of 34 and 29 kd polypeptides in sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The hydrophobicity of these two polypeptides is similar because they have the same retention time on a C18 reverse-phase column when a trifluoroacetic acid/H2O/CH3CN buffer system is used. The N-terminal amino acid sequence of the 34 kd component has a 60% homology with residues of 1-25 of Lol p I, whereas that of the 29 kd component has a 68% homology with residues 31-68 of Lol p I. In addition, this 29 kd polypeptide can be recognized by another anti-Cyn d I MoAb 1-61. CONCLUSIONS These results suggest that the 29 kd component is derived from Cyn d I. In spite of the similarity in the amino acid composition between Cyn d I and group I allergens of other grass pollens, none of our four anti-Cyn d I MoAbs cross-reacted with 10 other grass pollens tested, including ryegrass pollen. Despite biochemical similarity with other group I allergens, the B-cell epitopes on Cyn d I are different from those on other grass pollens.

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea‐denatured OVA (UD‐OVA) and carboxymethylated UD‐OVA (CM‐OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed‐type hypersensitivity (DTH) responses to OVA were not suppressed by CM‐OVA fed prior to or after immunization with OVA in complete Freunds adjuvant (CFA). When CM‐OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM‐OVA were orally tolerized by OVA, but not by UD‐OVA or CM‐OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM‐OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM‐OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma‐globulin (HGG), a well‐known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.

Characterization of the isoforms of the group I allergen of Cynodon dactylon

BACKGROUND The group I allergen of Cynodon dactylon, Cyn d I, was found to consist of four to 10 isoforms. METHODS We studied the isoforms with the use of two-dimensional gel electrophoresis. The antigenic difference of the isoforms was evaluated by radioimmunoprecipitation with monoclonal antibodies (MAbs). The acidic isoforms and the basic and neutral isoforms were further isolated by MAb-affinity chromatography for RAST and competitive RAST. In addition, the N-terminal sequence was evaluated by microsequencing. RESULTS A total of 11 isoforms were found in Cyn d I in extracts prepared from different sources of Bermuda grass pollen (BGP). They were either acidic (Cyn d I-A, I-B, I-C, I-D, I-E, I-F, I-G, I-H, and I-I), neutral (Cyn d I-X), or basic (Cyn d I-J). Cyn d I-G, with an isoelectric point of approximately 6.4, was constantly present in all the pollen preparations, whereas the content of the basic Cyn d I-J varied from less than 5% to greater than 20%. The molecular weight of the basic and neutral isoforms were slightly lower than those of the acidic isoforms. All isoforms shared a common antigenic determinant(s) recognizable by MAb 4-37, and the basic and neutral isoforms possessed a unique antigenic determinant(s) recognizable by MAb 1-61. RAST showed that both the acidic Cyn d I and the basic and neutral Cyn d I were recognized by human IgE in the pooled sera of persons allergic to BGP. Competitive RAST showed a high crossreactivity between the acidic and the basic and neutral isoforms. A 95% sequence identity also existed between the N-terminal 20 amino acid residues of basic Cyn d I-J and the dominant acidic isoform Cyn d I-G. CONCLUSIONS The present study disclosed that basic Cyn d I-J is an important allergen and that the content of this isoform varies in different lots of BGP.

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) and isoelectric focusing(IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP‐allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP‐allergic patients. Among those human IgE‐binding molecules, Cyn d Bd35K. reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purificaiton of these allergens.

A Common Allergenic Epitope of Bermuda Grass Pollen Shared by Other Grass Pollens.

The present study disclosed the cross-reactivity between Bermuda grass pollen (BGP) and other grass pollens using monoclonal antibodies (MAbs) and polyclonal antiserum. MAb 9-13, directed against a group of minor allergens of BGP (Cyn d Bd68K, 48K, 38K) was found to cross-react with extracts of ten other grass pollens. Immunoblotting assays illustrated that MAb 9-13 cross-reacted with multiple components of most of these pollens, and the major cross-reactive components had molecular weights of 29-36 kD. The crossreactivity between BGP and Lol pI, the group I allergen of rye grass pollen, was further evaluated; Lol pI was recognized by MAb 9-13, but not by our MAbs/polyclonal antiserum against Cyn dI, the major allergen of BGP. These results suggest that the epitope recognized by MAb 9-13 is a common (C) epitope shared by Lol pI and Cyn d Bd68K, 48K, 38K, and Cyn dI does not share significant antigenicity with Lol pI. In a modified radio-allergosorbent test, IgE antibodies in the serum of BGP-allergic patients reacted mildly with C-epitope-bearing components of both BGP and rye grass pollens, and this binding could be blocked specifically by MAb 9-13. This suggests that in addition to an antigenic cross-reaction, the C epitope can also lead to an allergenic cross-reaction. Copyright 1994 S. Karger AG, Basel

Identification and characterization of epitopes on Cyn d I, the major allergen of Bermuda grass pollen.

BACKGROUND We identified three epitopes on Cyn d I by using four anti-Cyn d I monoclonal antibodies (MoAbs). METHODS In a cross-inhibition binding assay, the binding of MoAbs 1-61 and 10-7 to Cyn d I was completely blocked by each other but not by MoAbs 4-37 and 11-7; the binding of MoAb 4-37 and MoAb 11-7 to Cyn d I was inhibited by themselves but not by other MoAbs. The epitope recognized by MoAbs 1-61 and 10-7 is designated as E1, and those recognized by MoAbs 4-37 and 11-7 are designated as E2 and E3, respectively. RESULTS In a radioallergosorbent inhibition assay, we found that MoAbs 1-61 and 4-37 (1:50 diluted) can inhibit the binding of human Immunoglobulin Es to Cyn d I by more than 30%, whereas MoAb 11-7 was less efficient (reduced by only 6%). These results suggest that both E1 and E2 are major allergenic epitopes but that E3 is only a minor one. Further characterization of E1 and E2 reveals that they are labile in alkaline but resistant to acid and sodium periodate treatments. Moreover, E1 is heat-labile, but guanidine- and urea-sensitive, whereas E2 is not. Both E1 and E2 lost their antigenicity after reduction and alkylation. CONCLUSIONS Results of the present study provide important information on the physicochemical properties of major allergenic epitopes on Cyn d I, which may be useful for future development of therapeutic peptides for patients allergic to Bermuda grass pollen.

Induction of Specific Th1 Responses and Suppression of IgE Antibody Formation by Vaccination with Plasmid DNA Encoding Cyn d 1

Background: DNA vaccines encoding allergens have been developed to prevent or to treat specific IgE responses. Objective: To evaluate the potential preventive and therapeutic effect of DNA vaccines encoding Cyn d 1 alone or combined with different adjuvants on specific allergies. Methods: Recombinant plasmid Cyn d 1 (pCyn d 1) was constructed by insertion of Cyn d 1 cDNA into the vector pcDNA3. BALB/c mice were injected with pCyn d 1 alone or plus adjuvants such as bupivacaine, bestatin, liposome, or CpG. Control mice were treated with pcDNA3 or PBS. They were boosted 3 weeks later and then sensitized twice with recombinant Cyn d 1 and alum. Their serum antibody responses and cytokine profiles of spleen cells were studied. Adoptive transfer of spleen cells of pCyn d 1-vaccinated mice was also performed. Results: Vaccination of mice with pCyn d 1 induced Th1 responses characterized by IgG2a responses and spleen cell secretion of interferon-γ. Vaccination with pCyn d 1 not only prevented the induction of specific IgE responses but also suppressed ongoing IgE responses. The mice receiving untreated, CD4+- or CD8+-depleted spleen cells from pCyn d 1-vaccinated mice all had suppression of IgE responses. Conclusion: This study confirms the prophylactic and therapeutic effects of DNA vaccines encoding Bermuda grass pollen allergen Cyn d 1 on specific IgE responses. Both CD4+ and CD8+ T cells are crucial for the immunomodulatory effect of pCyn d 1 on specific IgE responses.

Immunoblot analysis of components of Penicillium notatum recognized by human IgE antibodies.

Components of the crude extract of Penicillium notatum recognized by human IgE antibodies (Abs) were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The allergenic components were identified with sera from 19 allergic patients and 20 blood donors. The allergen-Ab complexes were visualized by 125I-labeled monoclonal antihuman IgE and autoradiography. A total of 11 allergenic components, ranging in molecular weights (MWs) from 94,000 to 20,000 daltons, were identified. Heterogeneity in the IgE-binding patterns of the serum samples tested was also observed. However, the major allergen appears to be the component with an MW of about 68,000 daltons that was recognized by IgE Abs in 56% of the 39 sera analyzed. Furthermore, the component with an MW of about 64,000 daltons that was recognized by IgE Abs in 46% of the sera analyzed was also considered as an important allergen. Results obtained from this study will be useful in additional characterization of allergens of P. notatum and related fungal species.