David C. Benjamin

Trichophyton Antigens Associated with IgE Antibodies and Delayed Type Hypersensitivity SEQUENCE HOMOLOGY TO TWO FAMILIES OF SERINE PROTEINASES

The dermatophyte fungus Trichophytonexhibits unique immunologic properties by its ability to cause both immediate and delayed type hypersensitivity. An 83-kDaTrichophyton tonsurans allergen (Tri t 4) was previously shown to elicit distinct T lymphocyte cytokine profiles in vitro. The homologous protein, Tri r 4, was cloned from aTrichophyton rubrum cDNA library, and the recombinant protein was expressed in Pichia pastoris. This 726-amino acid protein contained an arrangement of catalytic triad residues characteristic of the prolyl oligopeptidase family of serine proteinases (Ser-Asp-His). In addition, a novelTrichophyton allergen, encoding 412 amino acids, was identified by its human IgE antibody-binding activity. Sequence similarity searches showed that this allergen, designated Tri r 2, contained all of the conserved residues characteristic of the class D subtilase subfamily (41–58% overall sequence identity). Forty-two percent of subjects with immediate hypersensitivity skin test reactions to a Trichophyton extract exhibited IgE antibody binding to a recombinant glutathione S-transferase fusion protein containing the carboxyl-terminal 289 amino acids of Tri r 2. Furthermore, this antigen was capable of inducing delayed type hypersensitivity skin test reactions. Our results define two distinct antigens derived from the dermatophyte Trichophyton that serve as targets for diverse immune responses in humans.

Expression and Secondary Structure Determination by NMR Methods of the Major House Dust Mite Allergen Der p 2

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Der p 2 is composed of β-sheets and random coil. Based on long range distance constraints, a number of β-strands were aligned into two three-stranded, anti-parallel β-sheets.

Hydrogen Exchange Nuclear Magnetic Resonance Spectroscopy Mapping of Antibody Epitopes on the House Dust Mite Allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10–100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2–3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.

Distinct Human T Cell Repertoires Mediate Immediate and Delayed-Type Hypersensitivity to the Trichophyton Antigen, Tri r 2

The 29-kDa subtilase homologue, Tri r 2, derived from the dermatophyte fungus Trichophyton rubrum, exhibits unique immunologic characteristics in its ability to elicit immediate (IH) and delayed-type (DTH) hypersensitivity skin tests in different individuals. Thus, Tri r 2 provides a model for comparing the T cell repertoire in subjects with distinct immune responses to a single Ag. Recombinant Tri r 2 produced as a GST fusion protein in Escherichia coli stimulated strong in vitro lymphoproliferative responses in 10 IH and 10 DTH responders. Patterns of T cell epitope recognition were compared between skin test groups using 28 overlapping peptides (each in 12 replicate wells) derived from Tri r 2 to stimulate T lymphocyte proliferation in vitro. Peptide 5 (P5; aa 41–60) induced the strongest response in DTH subjects and showed the largest difference between DTH and IH responders in proliferation (mean standardized index, 2.22 and 0.82, respectively; p = 0.0047) and number of positive wells (81 vs 12). Responses to P5 were associated with diverse HLA haplotypes. These results showed that P5 contains an immunodominant epitope specifically associated with DTH and that this peptide is recognized in a permissive manner. Cross-validated linear discriminant analysis using T cell proliferative responses to two regions of Tri r 2 (aa 51–90 and 231–270) gave a 95% predictive accuracy for classification of subjects into IH or DTH groups. We conclude that different immune responses to Trichophyton are mediated by distinct T cell repertoires between individuals with IH and DTH reactions to Tri r 2.

Molecular Cloning of German Cockroach (Blattella germanica) Allergens

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Monoclonal antibodies to bovine serum albumin: Affinity and specificity determinations

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

Sequence Polymorphisms and Antibody Binding to the Group 2 Dust Mite Allergens

Background: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with >80% sequence identity. Isoforms within each genus have been identified which differ by 3–4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. Methods: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. Results: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r2 = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. Conclusions: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

Bone marrow irradiation chimeras in the BB rat: evidence suggesting two defects leading to diabetes and lymphopoenia

SummaryA series of bone marrow irradiation chimeras were constructed in an attempt to determine the site of the defect(s) leading to diabetes and/or lymphopoenia in the BB rat. In BB rats that were lethally irradiated and reconstituted with T-cell-depleted Wistar-Furth (WF) rat bone marrow, the incidence of diabetes was reduced, and in animals treated with WF bone marrow at < 44 days of age, the disease was completely prevented. Such animals demonstrated normal lymphocyte counts in peripheral blood, and normal lymphocyte function (as indicated by mixed lymphocyte response), but retained an abnormal T-cell subset distribution only partially improved above that of diabetes-prone BB rats. The incidence of diabetes in these irradiated chimeras was significantly reduced compared to the indicence in BB rats irradiated at the same age but reconstituted with bone marrow from BB rats. In WF rats that were lethally irradiated and reconstituted with T-cell-depleted bone marrow from overtly diabetic BB rats, no diabetes was induced. Such animals demonstrated normal lymphocyte counts in peripheral blood, normal lymphocyte function, and normal T-cell subset distributions. Overall, these results suggest two defects leading to diabetes and/or lymphopoenia in the BB rat. One of these occurs at the level of the bone marrow stem cell while the other resides in the T-cell differentiative environment.

Prevention of Diabetes in BB Rats: I. Evidence Suggesting a Requirement for Mature T Cells in Bone Marrow Inoculum of Neonatally Injected Rats

Injection of major histocompatibility complex (MHC)-compatible bone marrow cells from normal animals into neonatal BB rats resulted in a striking decrease in incidence of diabetes and restoration of concanavalin A (ConA) and mixed lymphocyte responses. However, injection of bone marrow cells pretreated with antirat thymocyte antiserum plus complement to remove mature T cells had no effect on incidence of disease, suggesting that mature T cells in the bone marrow inoculum were responsible for prevention of diabetes. Because the decreased incidence of diabetes in rats injected with untreated bone marrow appeared to be unrelated to the extent of lymphopenia in these animals, the involvement of T cells in the onset of diabetes must reflect a defect in the normal function of these cells rather than their absolute number. Approximately 50% of the W3/13+ cells in the spleens of BB rats lacked the OX-8 and W3/25 T cell subset markers. The identity of this W3/13+, OX-8−, W3/25− blank subset remains to be established. Our results, interpreted in light of studies from the other laboratories, suggest the existence of multiple abnormalities in the BB rat, including 1) the presence of T cells as effector or helper cells that augment onset of disease and 2) the absence of a regulatory T cell circuit that could prevent the disease.

Analysis of the relationship of bovine high molecular weight protein-2 and tau protein using radioimmunoassay

Abstract Microtubule-associated proteins isolated from cycle-purified bovine brain tubulin by boiling in high salt were fractionated into two samples by chromatography on A15M agarose. One sample is composed primarily of a 300 000 dalton polypeptide (HMW-2) and the other is composed of four polypeptides with apparent molecular weights between 53 000 and 68 000 (tau). Aliquots of the HMW-2 and tan samples were labelled with 125 I and used in a radioimmunoassay to determine their crossreactivity. The antiserum for the radioimmunoassay was produced in a rabbit by injection of cycle-purified bovine brain tubulin and was found to contain antibodies against HMW-2 and tau when assayed by quantitative precipitation and radioimmunoassay. The data from the quantitative precipitation experiment demonstrate that the antiserum recognizes approximately 30 antigenic domains on the HMW-2 molecule. Several radioimmunoassays were done to determine the competition between either labelled HMW-2 as the indicator molecule and unlabelled tau as the competitor or labelled tau as the indicator molecule and unlabelled HMW-2 as the competitor. In all assays less than 5% of the competitor crossreacted with antibodies to the labelled protein. The results demonstrate that our antiserum recognizes HMW-2 and tau as distinct antigenic species.