Analysis of T-cell epitopes of Der f3 in Dermatophagoides farina

House dust mites (HDM) are most important indoor allergens for humans. Der f3, one of the potent allergens with allergenicity, is derived from Dermatophagoides farina (D. farinae), and exhibits strong allergenicity that was confirmed in our previous work. The current study was undertaken to determine the localization of T-cell epitope of Der f3. We initially developed the T-cell fraction from BALB/c mice sensitized with recombinant Der f3 to determine the T-cell epitopes in the murine models, and performed T cell proliferation assay with 25 synthetic overlapping peptides of Der f3. The results indicated that T-cell reactive region of murine were assigned on amino acid range 41-60, 101-120, 161-180 and 201-220, respectively. In addition, we did T-cell proliferation experiment, respectively using the 4 murine T-cell epitope peptide and the human T-cell lines from three patients allergic to mite allergens in order to verify homogenous T-cell epitopes in humans. The results indicated that the amino acid sequences of 41-60, 101-120 and 161-180 had induced T cell proliferation in humans, yet 201-220 failed to. These findings suggest that T-cell epitope in Der f3 is located in the amino acid sequences of 41-60, 101-120 and 161-180, respectively. T-cell epitope localization detected in our study may provide a basis for development of animal therapeutic model and peptide vaccine for asthma.     

Stimulatory and inhibitory epitopes in the T cell responses of mice to Der p 1

BACKGROUND: The responses of mice to the mite allergen Der p 1 have been used to study the mechanisms of allergic sensitization and the development of new types of immunotherapy. Many of the studies require a knowledge of the T cell epitopes, and because Der p 1 is polymorphic, the effect of natural amino acid substitution in the allergen. The intranasal administration of peptides containing T cell epitopes can induce a mucosal tolerance but it is not known if the major activity is limited to stimulatory peptides and if, as found for autoimmunity, some epitopes are not inhibitory. OBJECTIVE: To determine and compare the sequences of Der p 1 which contain stimulatory epitopes for the high responding H-2(b) and H-2(q) mice and the sequences which induce tolerance by intranasal administration of peptides. METHODS: T cell responses of mice immunized with Der p 1 were measured by in vitro T cell stimulation assays so an extensive study of epitope recognition and intranasal tolerance could be made. Synthetic peptides were used to examine the stimulatory and inhibitory ability of all Der p 1 sequences and to map the major H-2(b) epitope in detail. This included the effect of the common polymorphic amino acid 124 substitution found within this epitope. RESULTS: Three and two regions, respectively, were found to contain stimulatory T cell epitopes for H-2(b) and H-2(q) mice. The peptides in these regions were also the most active at inducing intranasal tolerance for the responding haplotype. The correspondence between inhibitory and stimulatory peptides was maintained for the fine mapping of the major H-2(b) epitope. This was found about a core region of 118-126 which was overlapping but separate to a consensus sequence for the binding of endogeneous peptides. Peptides with alanine at the naturally polymorphic residue 124 stimulated and inhibited responses to Der p 1 more effectively, while peptides with the valine 124 variant were immunogenic but poorly cross-reactive. CONCLUSIONS: The intranasal administration of peptides representing each of five epitopes recognized by two strains of mice were able to induce mucosal tolerance and the major tolerizing activity was limited to these epitopes. The position of the core major epitope for C57 mice, which differs from a previously predicted epitope, and its specificity for the natural alanine 124 variant is described.     

Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae

Background The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes.
Objective The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic detertninants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences.
Methods In order to identity any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs.
Results None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs.
Conclusion The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.     

T-cell epitope analysis of Mag 3, an important allergen from the house dust mite, Dermatophagoides farinae

Here we describe the detection of T-cell epitope region on the house dust mite allergen Mag 3, which has been shown to trigger T-cell proliferation in mite-allergic asthmatic patients. We first examined murine T-cell epitope using T-cell fraction prepared from recombinant Mag 3 (r-Mag 3)-primed H-2k mice. Initial proliferation assay with truncated r-Mag 3 indicated that N-terminal 113 amino acid region was required for triggering T-cell activation. Subsequent epitope scanning with synthetic overlapping peptides revealed that T-cell reactive region was assigned within amino acid range 56-75. We also explored human T-cell determinant using specific T-cells from mite-allergic patients. Intriguingly, we found that amino acid range 56-85, a portion partially overlapping with that identified in r-Mag 3-primed mice, was exclusively recognized by T-cells from different patients. Further investigation of unique T-cell epitope region found in this study would provide insight into the development of animal therapeutic model and/or peptide vaccine for asthma.     

An Epitope Delivery System for Use with Recombinant Mycobacteria

We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium vaccae. The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens. We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M. vaccae. The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice. This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles.     

Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus

T-cell epitopes of Der p II, a major allergen of Dermatophagoides pteronyssinus , were analyzed by using human T-cell clones. We tested 38 cloned T cells from two Japanese patients with allergic rhinitis, and identified at least two peptides (K33-T47 and 158-C73) as helper T-cell epitopes. The former epitope was shown to be restricted by HLA-DRB1* 1502, and the latter by HLA-DRB1* 0405, both of which are typical Japanese HLA-DR alleles, suggesting that those T-cell epitopes might be important for the onset of house-dust mite allergy in the Japanese population. We prepared 15 analog peptides of the HLA-DRB1* 1502-restricted 15-mer peptide. Of those 15 residues, five (F35, L37, A39, F41, and E42) were critical for the epitope activity, and three residues (F35, A39, and E42) seemed to be included in anchor motifs for HLA-DRB1* 1502. The epitope peptide was also recognized by HLA-DRB1* 1502-positive healthy donors; however, only allergic T cells showed Th2 functions. Antigen-presenting cells of nonallergic donors were able to activate allergic T cells to express Th2 function. This seemed to suggest that antigen recognition of T cells, as well as additional unknown factors which promote Th2, rather than Th1, responses, might be important for the onset of house-dust mite allergy.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8_153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8_153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = ? 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Low-level exposure to house dust mites stimulates T-cell responses during early childhood independent of atopy

Background The imtnune responses which underlie the expression of allergic symptotns in childhood are believed lo be initiated in infancy and early childhood. The kinetics of this response have hardly been researched. Objective To analyse, in an environment with low house dust mite (HDM) exposure levels, the relationship between house dust mite (HDM)-specific T-cell reactivity as expressed by in vitro proliferation of blood mononuclear cells. Methods The study comprised a prospective analysis of patterns of allergen-specific T-cell reactivity in a cohort of 19 children, from whom blood samples were obtained in the spring during their second and third years of life. Blood mononuclear cell cultures were established in 200 μL AIM-V serum free medium. Crude house dust mite (HDM) and purified Der p 1 and Der p 2 extracts were used at optimal concentrations, i.e. 100μg/mL for HDM and 30μg/mL for the purified allergens. Tetanus toxoid (0.5 μglrnL) and ovalbumin (10 μg/mL) served as positive controls. A clinical diagnosis of allergy was verified with skin-prick tests. Dust samples were collected from a mattress and/or carpet or sofa in homes, day care centres and day care homes. Major mite allergen levels (Der p 1/Der f 1) in dust were analysed by an enzyme linked immunosorbent assay (ELISA). Results Specific T-cell responses were seen in the majority of the children against house dust mite (crude HDM extract. Der p 1 and Der p 2). The levels of the house dust mite allergens Der p 1 and Der f I were low, i.e. < 0.68 μg/g fine dust in the homes of the children and the day care centres that they were attending. This indicates that doses of mite antigen well below the suggested sensitization threshold level of 2 μg/g dust can induce mite-specific T-cell responses in young children. None of them showed clinical reactivity to house dust mites as indicated by negative skin-prick tests. Conclusions The findings suggest that active immunological recognition of environmental allergens and the ensuing initiation of allergen-specific T-cell responses, is a normal part of the ‘education’ of the immune system in early childhood and can occur even at very low exposure levels. Priming per se does not imply clinically significant sensitivity, however.     

Crossreactivity and T-cell epitope specificity of Bet v 1-specific T cells suggest the involvement of multiple isoallergens in sensitization to birch pollen

Background Allergen-specific T lymphocytes play an important role in the pathophysiology of atopic disease. Detailed studies of their epitope-specificity and crossreactivity are required for the development of novel approaches for specific immunotherapy. Objectives The aim of the study was to characterize the fine specificity of Bet v 1-specific T cells from allergic donors. Methods Polyclonal T-cell lines (TCL) and T-cell clones (TCC), specific for Bet v 1, the major birch (Betula verrucosa) pollen allergen, were isolated from the peripheral blood of three birch-allergic patients. Their epitope-specificity was studied using overlapping synthetic peptides, and crossreactivity with other tree pollen allergens of the Fagales order was evaluated. In addition, the Bet v 1-specific TCC were studied for their phenotype and cytokine production. Results All isolated Bet v 1-specific TCC (19/21 CD4⁺, 2/21 CD8⁺) reacted with affinity purified Bet v 1, but showed different reactivities with recombinant Bet v 1 (rBet v 1), and with group 1 allergens from other Fagales species. Epitope mapping of rBet v 1-reactive TCC with synthetic peptides of Bet v 1 showed the presence of four T-cell epitopes. Polyclonal T-cell lines reacted with 13 different peptides, and displayed even broader crossreactivity with group 1 pollen allergens from other Fagales members. Conclusion This study demonstrates that apart from T-cell epitopes of rBet v 1, many other crossreactive or Bet v 1 isoallergen-specific epitopes exist. This indicates that isoallergenic variation plays an important role in the induction of Bet v 1-specific and crossreactive T-cell responsiveness to allergens.     

T-cell activation by transgenic rice seeds expressing the genetically modified Japanese cedar pollen allergens

Transgenic rice seeds that contain genetically modified Cry j 1 and Cry j 2, the two major allergens of Cryptomeria japonica (Japanese cedar; JC), have been developed as immunotherapeutic candidates for JC pollinosis. Because the transgenic rice (TG-rice) seeds express allergens containing whole amino acid sequences of Cry j 1 and Cry j 2 in the endosperm tissue (edible part of rice grain), they can potentially target all Cry j 1- and Cry j 2-specific T-cells. However, it was unknown whether antigenicity of Cry j 1 and Cry j 2 could be completely preserved in TG-rice seeds. We verified the antigenicity of TG-rice seeds to T-cells through the analysis of the proliferative responses of T-cells in Cry j 1- or Cry j 2-immunized mice or T-cell lines to TG-rice seed extract. First, four mouse strains were immunized with Cry j 1 or Cry j 2. T-cells in the immunized mice proliferated on treatment with TG-rice seed extract, but not non-transgenic wild-type rice (WT-rice) seed extract. Furthermore, T-cell lines were established from the spleen cells of the immunized mice. Each T-cell line resulted in a proliferative response to TG-rice seed extract, but not to WT-rice seed extract, suggesting that TG-rice seeds certainly express T-cell epitopes corresponding to T-cell lines. Considering the modified amino acid sequences of Cry j 1 and Cry j 2 in TG-rice seeds, the expression of specific T-cell epitopes suggested that TG-rice seeds express all possible T-cell epitope repertoires of Cry j 1 and Cry j 2.     

Mapping of T-cell epitopes of Phl p 5: evidence for crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens

BACKGROUND: Group 5 allergens represent major grass pollen allergens because of their high sensitization indices. The identification of T-cell epitopes of these allergens is a prerequisite for the design of immunotherapeutic strategies based on peptide vaccination or modified allergens with conserved T-cell epitopes. OBJECTIVE: This study was undertaken to determine T-cell epitopes on Phl p 5 major pollen allergen of timothy grass (Phleumn pratense). METHODS: T-cell lines (TCLs) and T-cell clones (TCCs), specific to Phl p 5, were established from the peripheral blood of 18 patients allergic to grass pollen. All TCCs were mapped for epitope specificities using 178 overlapping dodecapeptides representing the primary structures of two isoforms of Phl p 5 (Phl p 5a and Phl p 5b). Phenotype and cytokine production profiles of TCCs were tested. Selected TCCs were analysed for HLA class II restriction. RESULTS: A total of 82 TCCs were isolated. All TCCs displayed the helper cell (TH) phenotype. Their reactivity with two recombinant expressed isoforms of Phl p 5a and Phl p 5b was heterogeneous. The epitope specificity of the TCCs was then revealed. Nineteen T-cell epitopes could be identified on Phl p 5. Eighty-one percent of mapped TCCs recognized three T-cell reactive regions on the Phl p 5 allergen. Some TCCs were reactive with isoepitopes presenting on Phl p 5a as well as Phl p 5b. Allergen-specific stimulation induced a TH0-like type of cytokine production in 25 of 50 TCCs. Almost all TCCs secreted high concentrations of interleukin-13. CONCLUSION: Phl p 5, a major grass pollen allergen, contains several T-cell epitopes. Some epitope regions were recognized by several patients. Epitope recognition pattern could not be correlated with special HLA class II haplotypes. T-cell stimulating isoepitopes were found at corresponding regions of Phl p 5a and Phl p 5b isoforms.     

Sensitisation to the lipid-binding apolipophorin allergen Der p 14 and the peptide Mag-1

BACKGROUND: The IgE-binding peptides Mag 1 and Mag 3 and the high-molecular-weight protein M-177 have been identified as parts of the apolipophorin-like group 14 house dust mite allergen. By analogy with the homologous insect proteins, apolipophorins are hydrophobic proteins found in lipid bodies and lipid transport particles. This explains why they degrade and are poorly represented in extracts. METHODS: We have examined the T cell stimulation induced by a 341-residue recombinant Der p 14 peptide equivalent to the Mag 1 polypeptide examined by others. RESULTS: Peripheral blood mononuclear cells from both allergic and non-allergic donors responded strongly in in vitro proliferation assays to the Der p 14 peptide to induce markedly more (3)H-thymidine incorporation than Der p 2 and the release of Th2 cytokines. CONCLUSIONS: The apolipophorin-like group 14 allergens, despite their predicted hydrophobicity and lipid-binding activity, can induce high IgE responses and T cell stimulation. They appear to be important mite allergens unsuited to representation by aqueous extracts of mites.     

An immunogenetic analysis of the T-cell recognition of the major house dust mite allergen Der p 2: identification of high- and low-responder HLA-DQ alleles and localization of T-cell epitopes.

Cellular reactivity to Der p 2, a major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus, was studied in a group of 41 symptomatic HDM sensitive patients, using fresh peripheral blood mononuclear cells (PBMC) and assays of proliferation. Sixty per cent of the patients responded to Der p2, with reactivities being greater in patients with asthma as one of their clinical manifestations and also in those who had skin-test reactivity to a number of allergens. HLA-DR and -DQ serotyping was undertaken in 39 of the patients and the magnitude of T-cell proliferative responses to Der p 2 were found to be positively associated with DQ7 and negatively associated with DQ2. T-cell determinants within the Der p 2 molecule were identified by assays using a series of overlapping peptides (15- to 19-mers) spanning the entire protein. Fifty-nine per cent of the 41 HDM-sensitive patients responded to one or more of the peptides. All of the peptides were antigenic for at least one of the individuals, indicating the heterogeneity of the human repertoire reactive with Der p 2. There was a substantial variability in the number and location of epitopes recognized by T cells from the different allergic patients, the mean number per patient being 2.3 +/- 1.3 (SD). The most frequently recognized peptide was that spanning residues 111-129, being stimulatory in 66.7%, the other peptides were each recognized by between 8 to 25% of individuals. There was no correlation between the epitope recognized and the presence of particular HLA-DQ antigens.     

The structure of the mite allergen Blo t 1 explains the limited antibody cross‐reactivity to Der p 1

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro‐form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface‐exposed residues, suggests limited cross‐reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross‐reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.     

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.     

Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes

Although a T-cell response in human cytomegalovirus (HCMV)-immune individuals exists against the most abundantly expressed protein pp65 of the virus matrix, less is known about the determinants that evoke this response. The aim of the study was to identify regions within HCMV pp65 (ppUL83) that contain sequences for the cellular immune response by the use of three recombinant overlapping β-galactosidase pp65 fusion proteins (C74, C35, and C47), covering the C-terminal 265 amino acids of the entire pp65 sequence. Two T-cell epitope determinants were recognized by human lymphocytes of healthy, HCMV-seropositive, human leukocyte antigen (HLA)-typed individuals. One T-cell determinant (amino acids [aa] 303–388) was localized in the mid-region of the entire pp65 sequence and a second T-cell determinant (aa 477–561) within the C-terminal region. By fine mapping with synthetic hexadecamer peptides three T-cell epitopes were identified within these two regions: P10-I (aa 361–376) in the mid-region, P3-II (aa) 485–499), and P6-II (aa 509–524) in the C-terminal region. Inhibition studies with monoclonal antibodies to HLA class I or class II revealed a class I restricted response to peptides P10-I or P6-II, respectively. P10-I responders shared the HLA-DR11 allele and P6-II responders the -DR3 allele. Therefore, these T-cell epitopes of HCMV pp65 might be presented in association with particular HLA class II alleles. J. Med. Virol. 52:68–76, 1997. © 1997 Wiley-Liss, Inc.     

T-cell sensitization to epitopes from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei

Background Dermatophagoides pteronyssinus and Euroglyphus maynei frequently occur in house dust but little is known about primary sensitization to the less abundant E. maynei. Objective To determine the occurrence of primary sensitization to E. maynei by T-cell responses and the crossreactivity to D. pteronyssinus. Methods The proliferative response ot peripheral blood cells to overlapping peptides from Derp I and Eur m 1 were measured as well as to peptides from Der f 1, an allergen not found in the study environment. Results The most frequent and strongest responses were to Der p 1 peptides especially in the region 105–133. However, 3/17 responders to mite peptides were stimulated predominantly by Eur m 1 peptides and a further two had their highest response to an Eur m 1 peptide. There was very little crossreactivity between Der p 1 and Eur m 1 peptides and very little response to peptides from Der f 1. Conclusion E. maynei group 1 allergens are a significant source of primary T-cell sensitization and have little T-cell crossreactivity with D. pteronyssinus or D. farinae.     

Asp159 is a critical core amino acid of an IgE-binding and cross-reactive epitope of a dust mite allergen Der f 7

Der f 7 and Der p 7 are important house dust mite allergens with known structure and suggested biological function recently. However, their IgE-binding determinants remain unknown. The purpose of this study is to identify the IgE-reactive epitopes of Der f 7 and the determinants of IgE-mediated cross-reactivity between Der f 7 and Der p 7. IgE-reactive determinants were identified by immunodot blot inhibition using synthetic overlapping peptides, allergen mutants, and a Der f 7 structural model. Our results showed that synthetic peptides with sequence ¹⁵⁶SILDP¹⁶⁰ on Der f 7 bind IgE in two of the 30 asthmatic serum samples tested. Recombinant Der f 7 I157A, L158A, or D159A mutants have reduced IgE-binding activity. Inhibition experiments confirmed Asp159 as a critical core residue for IgE-binding. Among Der p 7, Der f 7 and Der f 7 mutants with single substitution between residues 156 and 160, only the D159A mutant cannot inhibit significantly IgE-binding against Der p 7. Therefore, Asp159 contributes to IgE-mediated cross-reactivity between Der f 7 and Der p 7. The structural model constructed for Der f 7 suggests that the IgE-binding epitope forms a loop-like structure on the surface of the molecule. In conclusion, Asp 159 is a critical core residue of an IgE-binding and IgE-mediated cross-reactive epitope ¹⁵⁶SILDP¹⁶⁰ of Der f 7. Results obtained from this study provide more information on molecular and structural features related to allergenicity, underlying basis of IgE cross-reactivity between allergens, and in designing safer immunotherapy.     

The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.

The T-helper response to the measles virus nucleoprotein (NP) has been studied in mice. The T-cell proliferative response was measured in lymphocytes from mice immunized with either a vaccinia measles-NP recombinant virus or a mouse neuro-adapted measles virus. A T-cell response was obtained with lymphocytes from H2d or H2k mice when stimulated with either measles virus or the NP expressed in bacteria. The response was CD4+ specific. The T-helper epitopes were mapped using truncated NP peptides. The major epitopes in both H2d and H2k mice were determined to be between amino acids 67-98. A further T-cell epitope (between amino acids 457-525) was identified when H2d mice were immunized with measles virus. Studies to quantitate the precursor cells for these epitopes confirmed that the region 67-98 of NP was immunodominant in both haplotypes immunized with the vaccinia-NP recombinant virus, whereas an additional major epitope was observed in the measles virus-infected H2d mice. The primary structure of the epitopes determined here are compared to predicted T-cell epitope motifs.