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Ambrosia artemisiifolia (Amb a 5; Ra5S) and Ambrosia trifida (Amb t 5; Ra5G) are homologous allergens purified from short and giant ragweed pollen, respectively. Allergic human sera and hyperimmunized animal antisera directed against Amb a 5 or Amb t 5 show a high degree of species specificity, with little or no cross-reactivity between these two allergens, suggesting that the major Ab binding epitopes of Amb a 5 and Amb t 5 are distinct. Overlapping synthetic peptides derived from the allergen sequences were used to investigate the specificity of T cell responses in four strains of mice, BALB/c (H-2d), CBA (H-2k), C57BL/6 (H-2b), and A/J (H-2a). All four strains of mice responded to purified Amb a 5 and Amb t 5. Cross-reactivity was found at the T cell level between Amb a 5 and Amb t 5 in T cells from BALB/c, A/J, and CBA mice, but not in T cells from C57BL/6 mice. A T cell epitope from Amb a 5, residues 27-36 (PWQVVCYESS), was mapped using T cell hybridomas from BALB/c mice. A T cell epitope in Amb t 5 was mapped in the same strain to residues 24-34 (KYCVCYDSKAI). Disulfide bonds in Amb a 5 and Amb t 5 were found to be involved in T cell reactivity. Conversion of disulfide bridges into free sulfhydryl (SH) forms was required for the response of T cell hybridomas to peptide t5 (residues 27-40) from Amb t 5. Reduction of peptide a4 (residues 21-37) from Amb a 5 was essential for inducing the cross-reactivity observed with Amb t 5-specific T cell hybridomas. It is concluded that free sulfhydryl groups play a major role in the T cell recognition of cross-reactivity T cell epitopes within these related allergens.

The complete primary structure of Fel d I2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

House dust mites are widely recognized as major factors involved in the triggering of allergic diseases such as asthma. It is now apparent that the group III allergens of the Dermatophagoides mite species may play a significant role in a number of house dust mite allergic cases. Natural Der p III was isolated by gel filtration of salt precipitated Dermatophagoides pteronyssinus extract and as reported previously ran as a doublet of Mr 28 and 30 K on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Natural Der fIII was isolated by affinity purification with the 5A12 monoclonal antibody. Amino acid sequence data was generated for both these proteins which was used to construct DNA probes to screen a Dermatophagoides pteronyssinus cDNA library by hybridization and resulted in the isolation of a recombinant Der p III cDNA clone, P3WS1. The 1059 bp cDNA fragment included a 786 bp open reading frame which encodes a pre-pro region of 29 amino acids and a mature protein of 232 amino acids with a calculated Mr 24,985. A search of the BLAST protein database has confirmed that the Der pIII P3WS1 clone is approximately 50% homologous with other trypsin proteins. We have confirmed with both our natural protein sequence and the P3WS1 amino acid sequence data that the group III allergens are trypsin-like proteins.

To delineate the relationship between the structural conformation and the stability of an allergen and its antigenicity, we have chosen the major allergen from cat dander, Fel dI. From protein sequence analysis data we have examined the structure of the naturally occurring Fel dI and we have found it to exist as an anti-parallel heterodimer. We have used ELISA, RAST, Western blot and histamine release techniques to compare the IgE reactivity of a set of cat allergic patient samples to purified, native Fel dI and the E. coli expressed chains 1 and 2. Results from these studies demonstrate a significant level of IgE reactivity to all forms when examined for direct binding. However, both blot and ELISA competition assays show a much higher reactivity to Fel dI in solution compared to the separate recombinant chains and this is supported by the histamine release data. Although native Fel dI chain 2 contains an N-linked carbohydrate moiety, this does not seem to play a role in the reactivity of IgE to chain 2. Denaturation of Fel dI with alkali conditions leads to a dramatic decrease in IgE reactivity, even though measurable changes to the backbone structure of the protein are minimal. One proposed explanation is that both chains possess a core region determined by their primary structures and that the major IgE epitopes are dependent upon them. The relative reactivity amongst these allergen forms varied with the method of analysis, implying that the conformational requirements for IgE antibody binding are best studied by the application of more than one experimental protocol. Results from these qualitative analyses afford insight into the allergenicity of this exceptionally stable cat pelt protein.

T cells control the majority of antigen-specific immune responses. Therefore, influencing the activation of the T-cell response in order to modify immune responsiveness is an obvious therapeutic goal. We have used a mouse model of response to Fel d I, the major cat protein allergen in humans, to explore the ability of peptides derived from Fel d I to inhibit T-cell-dependent immune responses to the peptides themselves and to larger polypeptides. T cells from B6CBAF1 mice respond to the Fel d I peptide IPC-2 after challenge with IPC-2. However, subcutaneous tolerization with IPC-2 prevents this response as measured by production of interleukins 2 and 4 and interferon gamma. Fel d I immunization of B6D2F1 mice results in T-cell responses primarily to one peptide derived from Fel d I. Injecting this peptide in soluble form inhibits T-cell activation (as measured by interleukin 2 production) and antibody production in Fel d I-primed animals when they are subsequently challenged with peptide in adjuvant. Most of the cat-allergic human T-cell response to Fel d I is specific for two peptides on one of its two chains. Immunization of B6CBAF1 mice with recombinant Fel d I chain 1 results in T-cell responses to the same peptides. Subcutaneous administration of these two peptides, which contain some, but not all, of the T-cell epitopes from Fel d I chain I, decreases the T-cell response to the entire recombinant Fel d I chain 1. The ability to tolerize T-cell responses with subcutaneous injections suggests a practical approach to treating human diseases with peptides containing T-cell epitopes.

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547-2552; Griffith et al. (1991a), Int. Archs Allergy appl. Immun. 96, 296-304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti-Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb aI.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pI in the 5.5-5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5-5.2 range detected by an anti-Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb aI.1 and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.1. These data suggest that recombinant Amb a I.1 and Amb a II, isolated in a denatured form, differ significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

This study describes the properties of the two recombinantly expressed polypeptide chains of Fel d I, the major allergen produced by the domestic cat (Felis domesticus). An inframe linker encoding polyhistidine has been added to the 5' ends of the Fel d I chains 1 and 2 cDNAs to facilitate purification using Ni2+ ion affinity chromatography. This method provides high yields in a single step of rchain 1 and rchain 2 of Fel d I with a > 90% level of purity. Polymerase chain reaction (PCR) methods were used to introduce a thrombin cleavage site (LVPR decreases GS) at the N-terminus of both chains. Thrombin cleavage of rchain 1 and rchain 2 followed by HPLC purification of the cleavage products allowed the isolation of each recombinant chain with only two additional residuals (GS) at the N-terminus of the native sequence. Amino acid sequencing analysis of the N-terminus and mass spectrometry of these polypeptides demonstrated that they are highly pure and full-length. Direct ELISA assays showed that IgE from cat-allergic patients binds to both rchain 1 and rchain 2 of Fel d I, demonstrating that both these chains contribute to the allergenicity of this heterodimeric protein. An examination of the reactivity of T cells derived from cat-allergic patients revealed that both polypeptide chains contribute to the T cell response to this allergen. Consequently, it is concluded that the immunological response to Fel d I is composed of a reaction at both the B and T cell level to each of the two chains that constitute the native allergen.

Recombinant proteins expressed in E. coli are often sequestered into inclusion bodies and require the use of denaturing agents in order to solubilize them. The recombinant form of Amb a I, the major allergen from short ragweed pollen, is one such protein. In some cases solubility can be maintained after the removal of the denaturing agent, particularly if the protein can be folded into its native conformation. However, not all proteins refold readily and after the removal of the denaturing agent the proteins will reaggregate and/or precipitate. In the case of Amb a I, the recombinant protein stays in solution at low concentrations but aggregates with itself and other proteins. The recombinant Amb a I is not expressed at high levels and may be toxic to E. coli. Therefore, isolation from a complex mixture of E. coli proteins was necessary. Monoclonal antibodies which recognize the denatured form of Amb a I were available, allowing for immunoaffinity purification. However, because the protein was not monomeric, this chromatographic technique did not provide an improvement in the purity level when run in normal buffer solutions. Analysis of one monoclonal antibody's stability to urea indicated it could tolerate the presence of 2 M urea and recover full activity. Use of this antibody as an immunoaffinity reagent in a column run in 2 M urea, which minimized aggregation of the E. coli produced proteins, gave a high degree of purification of recombinant Amb a I in one step. This illustrates the potential for the use of denaturing and other solubilizing agents in immunoaffinity chromatography of recombinant proteins.

The genes encoding chain 1 (Ch1) and chain 2 (Ch2) of the major allergen of the domestic cat, Felis domesticus I, have been analyzed by genomic cloning and by polymerase chain reaction (PCR). Ch1 has two potential leader sequences, designated A and B. Analysis of a genomic clone encoding Ch1 demonstrated that one structural gene contains sequences corresponding to both leaders, which utilize different Met start codons. PCR analysis showed that genes encoding Ch1 and Ch2 are co-expressed in both the salivary glands and the skin, and that leader sequence A of Ch1 is utilized preferentially in both tissues. Ch2 was shown to have two dominant forms that are differentially expressed in the aforementioned tissues. The long form (Ch2L), composed of 92 amino acids (aa), is preferentially expressed in the salivary glands, while the short form (Ch2S), composed of 90 aa, is preferentially expressed in the skin. There is minor sequence polymorphism in both forms of Ch2. A genomic clone for Ch2 only contained sequences for Ch2S, suggesting that Ch2L is encoded by an exon not contained within this genomic clone.

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

This study defines the complete primary structure of Amb alpha II, an important allergen produced by short ragweed (Ambrosia artemisiifolia). The deduced amino acid sequence derived from the cDNA indicates that Amb alpha II shares approximately 65% sequence identity with the Amb alpha I multigene family of allergens. Full-length cDNA encoding Amb alpha I.1 and Amb alpha II have been expressed in E. coli and purified. An in-frame linker encoding polyhistidine has been added to the 5' end of the cDNA to facilitate purification using Ni2+ ion affinity chromatography, yielding greater than 90% pure recombinant protein in a single step. T cells from patients allergic to ragweed proliferate in response to pollen extract as well as purified recombinant Amb alpha I.1 and Amb alpha II. T cell lines established using either Amb alpha I.1 or II as the stimulating Ag exhibit a high level of cross-reactivity to both proteins. This result is entirely consistent with the extensive primary sequence identity shared by these two proteins. These data suggest that allergic humans recognize shared T cell epitopes on these two related molecules.

The relationship between the structure and abundance of an inhaled protein and its potential for causing an allergic response is unknown. This study analyzes Amb a I, a family of related proteins formerly known as Ag E, that comprise the major allergens of short ragweed (Ambrosia artemisiifolia). T cells isolated from ragweed allergic patients were shown to proliferate in response to purified Amb a I.1 protein from pollen in in vitro secondary cultures, demonstrating the presence of T cell stimulatory epitopes in Amb a I.1. Three recombinant forms of Amb a I (Amb a I.1, Amb a I.2, and Amb a I.3) obtained as cDNA derived from pollen mRNA were expressed in bacteria. All three recombinant forms were shown to be specifically recognized by pooled ragweed-allergic human IgE on immunoblots, confirming these gene products are important allergens. An examination of immunoblots probed with sera derived from allergic patients revealed a variation in IgE binding specificity. A minority of patients' IgE exclusively reacted with recombinant Amb a I.1, whereas most patients' IgE reacted with Amb a I.1 as well as Amb a I.2 and Amb a I.3 proteins. A detailed examination of the reactivity of T cells derived from 12 allergic patients to these recombinant Amb a I forms revealed that these allergens are all capable of stimulating T cell proliferation in in vitro assays. It is concluded that the allergic response to ragweed pollen in most allergic patients is composed of a reaction to multiple related Amb a I proteins at both the B and T cell levels.

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.

Two of the major allergens in the pollen of short ragweed are Amb a I and Amb a II (formerly antigen E and antigen K, respectively). The genes for Amb a I and Amb a II have recently been cloned, and it was shown that Amb a I is a family of proteins with at least three distinct polymorphic family members. This study addresses the number of individual Amb a I and Amb a II family members, the polymorphism in each family member and the expression of these genes in ragweed plants from different geographical locations. This work led to the cloning and characterization of a fourth Amb a I family member, designated Amb a I.4.

IgE- and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fel d) I. Recombinant chain 1 and chain 2 were coupled to sepharose and tested in IgE- and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE- and IgG4 binding was found. The fraction of Fel d I-specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for IgG4 (P = 005). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P = 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Fel d I. compared with IgG4. [ABSTRACT FROM AUTHOR]