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The Journal of Immunology, 2001, 166: 6057-6065.
Copyright © 2001 by The American Association of Immunologists

Recombinant Allergens with Reduced Allergenicity but Retaining Immunogenicity of the Natural Allergens: Hybrids of Yellow Jacket and Paper Wasp Venom Allergen Antigen 5s1

Te Piao King2,*, Sui Y. Jim*, Rafael I. Monsalve*, Anne Kagey-Sobotka{dagger}, Lawrence M. Lichtenstein{dagger} and Michael D. Spangfort{ddagger}

* Rockefeller University, New York, NY 10021; {dagger} Johns Hopkins University School of Medicine, Baltimore, MD 21205; and {ddagger} ALK-Abelló, Hørsholm, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homologous venom allergen Ag 5s from the yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) have 59% sequence identity of their respective 204 and 205 amino acid residues, and they have low degrees of antigenic cross-reactivity in insect allergic patients and in animal models. Hybrids containing different segments of these two vespid Ag 5s were expressed in yeast. Circular dichroism spectroscopy suggests the hybrids to have the secondary structure of natural Ag 5. Inhibition ELISA with human and murine Abs suggests the hybrids to have the discontinuous B cell epitopes of the natural Ag 5 but with an altered epitope density. The hybrids were immunogenic in mice for B and T cell responses to both Ag 5s. The N-terminal region of Ag 5 was found to contain its dominant B cell epitope(s). Hybrids containing 10–49 residues of yellow jacket Ag 5 showed 100- to 3000-fold reduction in allergenicity when tested by histamine release assay with basophils of yellow jacket-sensitive patients. Our findings suggest that hybrids represent a useful approach to map the discontinuous B cell epitope-containing regions of proteins. They also suggest that Ag 5 hybrids may be useful immunotherapeutic reagents in man.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunotherapy by repeated s.c. injections of increasing doses of allergens into patients is one way to reduce allergic symptoms (1). Treated patients showed rapid increases in their allergen-specific IgGs and slow decreases in their specific IgEs (2). Treated patients also showed changes in their T cell cytokine profile; their IL-4 and IL-5 levels decreased and their IFN-{gamma} level increased (3). Studies have shown that immunotherapy with high doses of allergens was more effective than that with low doses for symptom reduction (1). However, effective dosages are limited by potential systemic reactions caused by the allergens.

Allergenicity depends on the interaction of a multivalent allergen with basophil or mast cell-bound IgE Abs. Therefore, allergenicity can be reduced by decreasing its B cell epitope density. Reduction of B cell epitope density of a protein can be accomplished by several approaches. One approach is by partial or complete denaturation of allergens on chemical modification (4, 5, 6, 7, 8), because the majority of B cell epitopes are of the discontinuous type, being dependent on the native conformation of proteins.

A second approach to reduce the accessibility of B cell epitopes of allergen is by polymerization on formaldehyde or glutaraldehyde treatment (9, 10, 11) or by attachment of nonimmunogenic polymers (12). One limitation of this approach is that near-complete loss of the discontinuous B cell epitopes usually occurred when allergens were modified with >100-fold reduction in allergenicity. A third approach is by site-directed mutagenesis to alter selectively the contact amino acid residues of B cell epitopes of allergens. If the key contact residues of B cell epitopes are known, this can be a useful approach (13).

T cell peptide treatment of naive or primed mice was found to down-regulate T cell responses for cat and mite allergens (14, 15). Thus, a fourth approach is the use of T cell peptides as immunotherapeutic reagents, because T cell peptides lack the discontinuous B cell epitopes of native allergens.

We report here a new approach to prepare modified allergens. It is to prepare hybrids consisting of a small portion of the guest allergen of interest and a large portion of a homologous but poorly cross-reacting host protein. The homologous host protein functions as a scaffold to maintain the native structure of the guest allergen of interest so that the conformation-dependent B cell epitopes of the guest allergen of interest are preserved in the hybrid, but at a reduced density. Homologous proteins of >30% sequence identity and of similar functions are known to have closely similar three-dimensional structures (16, 17).

Clinical studies in patients and tests with experimental animals have shown that there is limited cross-reactivity of Abs specific for the yellow jacket and paper wasp venom proteins (18, 19). These observations form the basis of the present studies. Our model guest allergen Ag 5 is Ves v 5, a yellow jacket venom protein of 23 kDa, and our homologous host allergen is Pol a 5, a paper wasp venom protein of similar size. Ves v 5 and Pol a 5 have a 59% sequence identity, as shown in the upper portion of Fig. 1Go. Both can be expressed in yeast, and the recombinant proteins were shown to have the native conformation of the natural proteins (20). The structure of Ves v 5 was solved recently by x-ray crystallography.3 The darkened regions of the Ves v 5 structure in Fig. 2Go, A and B, represent portions of the molecule chosen for preparation of hybrids.



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  		FIGURE 1. Upper portion, Amino acid sequences of Ves v 5 (V) and Pol a 5 (P). Lower portion, Schematic sequence representations of Ag 5s and hybrids. Residue numbers given for hybrids refer to those of Ves v 5.

 


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  		FIGURE 2. Front and back views of C{alpha} tracing of the structure of Ves v 5. Regions of Ves v 5 present in hybrids PV1–46 and PV156–204 are black colored-in patterns in A and B, respectively.

 
These hybrids are shown schematically in the lower portion of Fig. 1Go, and they are designated by the residue numbers of the Ves v 5 portion of the molecule. Hybrids PV1–46, PV108–155, and PV156–204 contain, respectively, the first, the third, and the last quarter of the guest Ves v 5 molecule together with portions of the host Pol a 5 molecule to complete the Ag 5 molecule. A hybrid containing the second quarter of the Ves v 5 molecule was not prepared, as this is a region of high sequence identity of Ves v 5 and Pol a 5. Hybrid PV1–155 has the opposite arrangements of the Ves v 5 and Pol a 5 fragments when compared with PV156–204. The aforementioned four hybrids were designed without considering the structural features of Ves v 5. The other five hybrids, shown in Fig. 1Go, were designed subsequently to contain segments that are located on the surface of Ves v 5, and they are of 7–32 residues in length. We report below our immunochemical findings with these hybrids of Ves v 5 and Pol a 5.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The cDNAs encoding Ag 5 hybrids

The cDNA encoding Ves v 5 of the EA series was prepared by PCR amplification of the appropriate template (19) with primers 1 and 3, found in Table IGo, and that encoding Ves v 5 of the KR series with primers 2 and 3. The cDNA encoding Pol a 5 of the EA series was similarly prepared with primers 4 and 6 of Table IGo, and that encoding Pol a 5 of the KR series with primers 5 and 6.


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  		Table I. Primers for preparation of Ves v and Pol a 5s and their hybrids1

 
PV1–46. Ves v 5 and Pol a 5 have a common peptide sequence of EH at positions 47–48 and 49–50, respectively. The nucleotide sequence encoding peptide EH in Ves v 5 is GAG CAC, which is a Bsi HKA I restriction site. That of Pol a 5 is GAG CAT, and it was mutated to a Bsi HKA I site by the PCR overlap extension method (21) using primers 7 and 8, found in Table IGo. Hybrid PV1–46-encoding cDNA was prepared by ligation of their appropriate Bsi HKA I fragments.

PV108–155. Ves v 5 and Pol a 5 have a common peptide sequence of KY at positions 106–107 and 109–110, respectively. Their KY sequence is encoded by the nucleotide sequence of AAA TAT. This nucleotide sequence was mutated to an ApoI restriciton site of AAA TTT encoding the peptide sequence of KF. The mutation of Ves v 5 or Pol a 5 cDNA was made with the primers 9 and 10, or 11 and 12, respectively, shown in Table IGo. Hybrid PV108–155-encoding cDNA was prepared by ligation of the appropriate fragments from ApoI digestion of Pol a 5 and PV1–155-encoding cDNAs.

PV1–155 and PV156–204. Ves v 5 and Pol a 5 cDNAs have a common EaeI restriction site encoding amino acid residues 154–156 and 155–157, respectively. The cDNAs encoding these two hybrids were prepared by ligation of the appropriate EaeI fragments of their parent cDNAs.

PV1–18 and PV195–204. Their cDNAs were prepared by PCR with cDNA of Pol a 5 as the template and primers 13 and 6, or primers 4 and 14, respectively, found in Table IGo.

PV1–24, PV1–32, and PV176–182. Their cDNAs were prepared by the PCR overlap extension method (21) with Ves v 5 and/or Pol a 5 cDNAs as templates and primers 15–20, found in Table IGo.

Recombinant plasmids encoding Ag 5 or hybrids

Ag 5 or hybrid-encoding cDNAs of the EA or the KR series were digested with restriction enzymes EcoRI or XhoI, and XbaI, and then inserted into similarly cut pPICZ{alpha}-A vector (Invitrogen, San Diego, CA). The recombinant plasmids were amplified in TOP10F' bacterial cells. The Ag 5-coding regions of all recombinant plasmids were confirmed by DNA sequencing. They correspond to the sequence data in GenBank with two exceptions for Ves v 5. There are two nucleotide changes at positions 579 and 587: the first change is a silent mutation of G to A substitution, and the second one of T to A substitution results in a codon change, M to K at amino acid residue 196. These two nucleotide changes may represent insect polymorphism rather than random mutations, because the Ag 5 cDNA used in this study was prepared in the same manner as it was done previously.

Molecular biology techniques used in this paper are the same as reported in our previous publication for expression of vespid Ag 5s in yeast (20).

Expression of Ag 5s and hybrids in yeast

Recombinant plasmids (1–2 µg) were linearized by cutting with the restriction enzyme SacI and then used to transform competent Pichia pastoris KM71 yeast cells (~8 x 109 cells in 40 µl of 1 M sorbitol) by electroporation. Transformed cells were diluted to 2 ml with 1 M sorbitol. They were allowed to recover at 30oC for 1 h without shaking and for 1 more h with shaking at 200 rpm. Next, 50- or 100-µl aliquots were plated out on 100-mm plates with yeast extract, peptone, dextrose, and sorbitol medium containing 1.5 mg/ml Zeocin for selection of multicopy integrants (22). Colonies were picked after a 3- to 4-day incubation and were screened by small-scale expression to find the best producing colonies. All reagents were obtained from Invitrogen (San Diego, CA).

Yeast cells from selected clones were grown in two 500-ml bottles, each containing 150 ml (pH 6.0) phosphate buffer containing yeast nitrogen base, biotin, glycerol, and histidine at 30°C with orbital shaking at 250 rpm until A600 nm of 10–12. The cells were collected by centrifugation and resuspended in 100 ml similar buffered medium containing methanol in place of glycerol. Incubation was continued at 30°C with shaking at 250 rpm for 4–6 days with a daily addition of 1 ml 50% methanol.

Ag 5s or their hybrids were purified from the culture fluid concentrate following our previously reported procedure, namely ion-exchange chromatography on SE-cellulose (Sigma, St. Louis, MO). About 70% of the main peak was pooled, desalted by reversed phase chromatography on C18 silica, and then lyophilized. Recombinant Ag 5s or hybrids were dissolved in 0.01 M ammonium acetate buffer (pH 4.6) and stored at 4°C. Their concentrations were established from their absorbance at 280 nm, using molar extinctions calculated from their tyrosine and tryptophan contents. The yields of Ag 5s or hybrids ranged from 1 to 7 mg/100 ml of 4-day cultures.

All recombinant Ag 5s or hybrids were characterized by SDS gel electrophoresis, N-terminal sequence analysis, and matrix-assisted laser desorption ionization mass spectrometry. Circular dichroism (CD)4 spectra at 0.2 mg/ml of recombinant proteins in 0.01 M acetate buffer (pH 4.6) were taken in cells of 1-mm path-length in an AVIV 62DS spectrometer (Aviv Associates, Lakewood, NJ).

Immunological studies

Ag 5- or hybrid-specific sera were collected at week 5 or later from groups of three or four female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) that had received three or more biweekly i.p. injections of 4 µg immunogen and 1 mg alum in 0.2 ml of PBS. Ag5-specific sera from ASW/sn and P/J mice strains were from a previous study (23).

Murine polyclonal Abs specific for natural Ves v 5 were isolated from BALB/c sera by affinity chromatography on Ves v 5-specific immunosorbent, and they were depleted of Pol a 5-cross-reacting Abs by passage through Pol a 5-specific immunosorbent. The immunosorbents were prepared with cyanogen bromide-activated Sepharose 2B (Pharmacia, Piscataway, NJ). Murine mAbs specific for Ves v 5 were from our earlier work (24).

ELISA was made in 96-well plates (Falcon 3911; BD Biosciences, San Jose, CA), and the wells were coated with 4 µg/ml Ag 5 in 0.05 M Tris-HCl buffer (pH 8). Bound human or mouse IgG was detected with 2 µg/ml biotinylated goat anti-human IgG ({gamma}-chain specific), or 4 µg/ml mouse IgG ({gamma}1 specific), followed with 2 µg/ml avidin-peroxidase conjugate (25). Ab concentration of sera samples was determined by comparison of their ELISA data with that of an immuno-affinity purified sample of Ves v 5-specific Ab as described above.

Proliferation assays were done in triplicates with spleen cells pooled from two to three mice 10 days after five biweekly immunizations. Spleen cells (4 x 105) were cultured with test Ag in 0.2 ml culture medium at 37°C and 5% CO2. Tritiated thymidine (1 µCi) was added on day 3, and the thymidine uptake was determined on day 4. The results were expressed as stimulation index values.

Allergenicity was determined by histamine release assay from basophils of yellow jacket-sensitive patients following challenge with Ag 5 or hybrids (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of vespid Ag 5s and their hybrids in yeast

The proteins expressed in yeast contain a secretory signal peptide. The signal peptide is linked to the expressed protein via a peptide of KR or KREAEAEF sequence. These two types of proteins are designated as the KR and the EA series, respectively. Upon secretion from the yeast cells, the signal peptide is cleaved from the secreted protein at the KR sequence (Kex 2 site) or the two EA sequences (Ste 13 site) (22). In our previous work with vespid Ag 5s of the EA series (20), we observed cleavage at the Kex 2 site but variable cleavages at the two Ste 13 sites. The recombinant proteins had the sequences of EAEAEF-, EAEF-, and EF-protein, where the EF sequence represents the EcoRI site for insertion of the cDNA into the vector. Similar findings were made in the present work, as shown by the mass spectrometric data of the six hybrids of the EA series in Table IIGo.


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  		Table II. Mass spectrometric data of recombinant vespid Ag 5s and hybrids

 
The EAEAEF sequence of recombinant Ves v 5 was found to function as a strong hapten (20). Therefore, we expressed Ves v and Pol a 5s and two hybrids of the KR series. Cleavage at the Kex 2 site would have yielded the recombinant proteins with the N-terminal sequence of the natural proteins. The expected cleavage did occur partially together with cleavages at sites 2 or 7–9 residues upstream of the Kex 2 site. This is shown by the mass spectrometric data of the proteins of the KR series in Table IIGo. The recombinant proteins of the KR series were of slightly lower yields than those of the EA series.

Two of the hybrids, EA-PV108–155 and EA-PV176–182, were secreted in such poor yield that we were not able to isolate them.

Physicochemical characterization of recombinant vespid Ag 5s and their hybrids

The recombinant proteins were characterized by SDS gel electrophoresis (Fig. 3Go). Several of them showed two closely spaced doublet bands with mobilities similar to that of natural Ves v 5. The doublet bands probably reflect the varying extents of processing at their N-terminal ends, as indicated by the mass spectrometry data in Table IIGo and by N-terminal sequencing of two hybrids, PV1–155 and PV156–204.



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  		FIGURE 3. SDS gel patterns of Ag5 hybrids. Approximately 2 µg of each sample was applied.

 
The recombinant Ag 5s and hybrids showed nearly identical CD spectra as those of the natural Ag 5s. As can be seen in Fig. 4Go, the spectra of the natural and the EA-Ves v 5 and those of EA-PV1–46, EA-PV1–155, and EA-PV156–204 showed the presence of minima at ~208 nm with a shoulder at 225 nm. These features are indicative of an ordered structure (27). Proteins with disordered structure have broad minima at 198 nm, as we observed with denatured Ves v 5 (20). Similar CD spectra were observed for Pol a 5 and all other hybrids in Table IIGo; these data are not shown.



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  		FIGURE 4. CD spectra of natural (n) and recombinant EA-Ves v 5 and recombinant EA-PV hybrids.

 
The recombinant Ag 5s and hybrids from yeast were as freely soluble in acid or basic buffers as the natural Ag 5s were. This is in contrast to the recombinant vespid Ag 5s from bacteria, which were soluble only in acidic buffer (27).

Ves v 5-specific B cell epitopes of hybrids

Ves v 5-specific B cell epitopes of hybrids were detected by the hybrid inhibition of binding of mouse IgG Abs specific for natural Ves v 5 to solid-phase Ves v 5. Both EA- and KR-Ves v 5 were tested as solid-phase Ags with similar results, and the data given below were obtained with solid-phase KR-Ves v 5. Five samples of mouse antisera were tested; three were from mice of BALB/c strains and one each from mice of ASW/sn and P/J strains. Closely similar data were obtained from sera of three groups of BALB/c mice, and the data from sera of one group are shown in Fig. 5GoA. At the highest concentration of 50 or 500 µg/ml inhibitor tested, the two N-terminal hybrids, EA-PV1–46 and EA-PV1–155, showed maximal inhibition approaching 100%, as EA- or KR-Ves v 5 did. Two other N-terminal hybrids, KR-PV1–24 and EA-PV1–32, had maximal inhibition of ~60%, and the shortest N-terminal hybrid, EA-PV1–18, had maximal inhibition of ~20%. The C-terminal hybrid EA-PV156–204 had maximal inhibition of ~15%, and the shorter C-terminal hybrid, EA-PV195–204, was not tested. The data from ASW/sn and P/J sera are given in Fig. 5Go, B and C, and their results are similar to those from BALB/c.



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  		FIGURE 5. Inhibition ELISA with mouse Abs specific for natural Ves v 5. Solid-phase Ag was KR-Ves v 5. A, Ves v 5-specific Abs isolated from BALB/c mice strain and depleted of Pol a 5-cross-reacting Abs; its concentration was 0.1 µg/ml. B and C, Antisera from ASW/sn and P/J strains were used directly at reciprocal dilutions of 2 x 105 and 5 x 104, respectively.

 
Inhibition ELISA was also made with specific IgGs from sera of six yellow jacket-sensitive patients. The data from three yellow jacket-sensitive patients are shown in Fig. 6Go, A–C. There are minor individual variations, but the results are similar to those with mouse IgGs. The inhibition data of the N-terminal hybrids in Figs. 5Go and 6Go suggest the immunodominance of the N-terminal region of Ves v 5.



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  		FIGURE 6. Inhibition ELISA with sera from yellow jacket-sensitive patients. Solid-phase Ag was KR-Ves v 5. A, B, and C, Sera from patients 8801, 9079, and DL were used at reciprocal dilutions of 20, 200, and 100, respectively.

 
The observed inhibition by the hybrids in Figs. 5Go and 6Go was not due to cross-reacting epitopes of the Pol a 5 portion of the molecule, because Pol a 5 at 500 µg/ml showed only 4–12% inhibition. The high concentrations of hybrids required for half-maximal inhibition relative to that of Ves v 5 are probably related to their epitope densities, because epitope density is known to strongly influence the affinity constant of a multivalent Ag and a bivalent Ab (28, 29). This may explain why the longest N-terminal hybrid, PV1–155, showed the lowest concentration for half-maximal inhibition, and it is followed by the next longest hybrids, PV1–46, PV1–32, PV1–24, and PV1–18.

Seventeen monoclonal mouse Abs specific for the natural Ves v 5 were tested for their binding of hybrids. ELISA showed one of these mAbs, 870201, bound EA-Ves v 5 and EA-PV1–46 with similar affinity and maximal binding (Fig. 7GoA), and it did not bind any of the other N- or C-terminal hybrids (data not shown). Four other mAbs showed greatly reduced maximal binding to EA-PV1–46 but no binding to any of the shorter N-terminal hybrids; the data for one such Ab, 870213, are given in Fig. 7GoB. Lastly, mAb 870207 in Fig. 7GoC showed greatly reduced binding to EA-PV1–32 and EA-PV1–46, but it showed moderate binding to EA-PV1–18 and EA-PV1–24. Data not shown indicated that these mAbs did not bind denatured Ag 5. Therefore, these data suggest the N-terminal hybrids have the native structure of Ves v 5.



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  		FIGURE 7. Binding of mouse Ves v 5-specific mAbs to solid-phase Ves v 5 or hybrids. A, Ab 870201; B, Ab 870213; and C, Ab 870207.

 
Immune responses to hybrids

Mice immunized with hybrids produced Abs specific for the hybrid, Pol a 5 and Ves v 5. The Ab levels of sera samples were measured before and after absorption with Pol a 5 to study their specificity for Ves v 5. These data are summarized in Table IIIGo. Mice immunized with natural EA- or KR-Ves v 5 gave nearly the same Ab responses, and only those of the KR-Ves v 5 are given in the Table. EA-PV1–46 gave a higher Ab response in set A mice than KR-PV1–46 did in set B mice. This difference may be due to the different sets of mice used. EA-PV1–18 was used in both sets of experiments, and it gave higher Ab response in set A mice than in set B mice.


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  		Table III. Murine Ab responses to vespid Ag 5s and hybrids

 
Comparison of Ab levels in the N-terminal hybrid-specific sera samples in Table IIIGo, before and after Pol a 5 absorption, indicate that 30–80% of their Abs were specific for Ves v 5 when tested on solid-phase Ves v 5, and these values were less when tested on solid-phase hybrid. The data in set A of Table IIIGo indicate that, of the three N-terminal hybrids, PV1–155 was as immunogenic as Ves v 5 was, PV1–46 was half as imunogenic, and PV1–18 was approximately one-ninth as immunogenic. The data in set B indicate that PV1–46 and PV1–32 were more immunogenic than PV1–24 and PV1–18. More importantly, the data from both sets suggest that the longer N-terminal hybrids PV1–46 and PV1–32 stimulate higher contents of Ves v 5-specific Abs and lower contents of Pol a 5-specific Abs than the two shorter hybrids, PV1–24 and PV1–18, did.

Proliferation assays were made with spleen cells from mice immunized with vespid Ag 5 or hybrid to study the specificity of T cell responses. The results summarized in Table IVGo show that the hybrids EA-PV1–46, EA-PV1–155, and EA-PV156–204 induced hybrid-specific, as well as vespid Ag 5-specific, T cell responses. The data in Table IVGo indicate that the best proliferative responses were obtained when the stimulating Ag was the immunogen. This is apparent from comparing the maximal stimulation index values at the highest Ag concentration of 100 µg/ml tested and from comparing the lowest Ag concentration required for a stimulation index value of 4.


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  		Table IV. Vespid Ag 5 or hybrid-stimulated proliferation of murine spleen cells1

 
Allergenicity of recombinant vespid Ag 5s and their hybrids in patients

Allergenicity of recombinant vespid Ag 5s and their hybrids in patients was tested by histamine release assay with basophils from ten yellow jacket-sensitive patients. The results in Table VGo are divided into two groups. The seven patients in group A were ~1000 times more sensitive to Ves v 5 than to Pol a 5, and the three patients in group B were about equally sensitive to both Ag 5s. The complete data from one patient of each group are given in Fig. 8Go.


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  		Table V. Summary of histamin release data of hybrids1

 


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  		FIGURE 8. Histamine release assay of Ves v 5, Pol a 5 and their hybrids. Data from two patients, JR (group A) and DH (group B), are shown.

 
Of the three N-terminal hybrids tested, EA-PV1–155 showed no decrease in allergnenicity. EA-PV1–46 and EA-PV1–18 showed geometric mean reductions of 126- and 583-fold, respectively, in group A patients and 0.7- and 24-fold decreases, respectively, in group B patients. The two C-terminal hybrids, EA-PV156–204 and EA-PV195–204, had reductions of 1139- and 3207-fold in group A patients, respectively, and 3- and 32-fold in group B patients, respectively.

The different extents of reduction in allergenicity of the N- and C-terminal hybrids reflect both their IgE Ab concentration and their epitope density. The inhibition ELISA data in Fig. 6Go suggest a higher concentration of human IgG Abs for the N-terminal region of Ves v 5 than those for the C-terminal region, and this is likely also the case for IgE Abs. Another contributing factor to the greater reduction in allergenicity of the C-terminal hybrid EA-PV156–204 as compared with the N-terminal hybrid EA-PV1–46 is probably due to its decreased epitope density, because the C-terminal hybrid has fewer surface-accessible residues of Ves v 5 than the N-terminal hybrid does. Similarly, the greater reduction in allergenicity of the shorter N- or C-terminal hybrids, PV1–18 or PV195–204, as compared with their respective longer ones probably also reflects the influence of epitope density.

The allergenicity of recombinant Ves v 5 from bacteria was compared with that of the natural Ves v and the recombinant Ves v 5 from yeast. In three patients tested, the recombinant protein from bacteria was ~103 times less potent than the natural protein or the recombinant protein from yeast. The data, which are not shown, confirm our previous observations that the majority of B cell epitopes are dependent on the conformation of the native allergen.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The CD spectral data in Fig. 4Go suggest that the hybrids have secondary structures that are closely similar to those of vespid Ag 5s. The inhibition data in Figs. 5Go and 6Go with Ves v 5-specific human and mouse Abs support that the hybrids have tertiary structures that are closely similar with that of Ves v 5, as data not shown indicate these Abs do not bind the denatured Ves v 5. Additional evidence came from screening with 17 monoclonal mouse IgG1 Abs specific for the natural Ves v 5; six of them bound the N-terminal hybrid PV1–46 (Fig. 7Go). The mAbs have the same specificity as the polyclonal Abs; they do not bind the denatured Ag 5. Therefore, these data together indicate that the hybrids have the native structure and they contain the discontinuous B cell epitopes of Ves v 5.

The inhibition data with polyclonal Abs and the binding data with mAbs in Figs. 5–7GoGoGo indicate that the dominant B cell epitopes of Ves v 5 are in its N-terminal region. Inspection of the structure of Ves v 5 in Fig. 2Go shows that nearly all residues in the N-terminal hybrid PV1–46 are surface accessible. This is in contrast to the C-terminal hybrid PV156–204, in which only segments of Ves v 5 are surface accessible. This difference in surface accessibility may explain the immunodominance of the N-terminal region of Ag 5. Others have shown that the entire surface of a protein is potentially antigenic, but the regions with high surface accessibility and surface protrusion are dominant (30, 31).

There is only one known way to map discontinuous epitopes. It is by x-ray crystallography of Ag-Ab complexes (32), and this requires having specific mAbs. The discontinuous epitopes of CD39 were mapped with a series of mouse-human hybrids; mouse and human CD39 molecules have 75% sequence identity, and they share limited antigenic cross-reactivity (33). These findings with CD39 and Ag 5 indicate that hybrids of two homologous proteins represent a useful alternate approach to map their discontinuous B cell epitopes. Advantages of this approach are the use of polyclonal Abs and the detection of the immunodominant regions, but this approach can map only the residues that are contiguous to each other in the peptide chain. Crystallographic studies of protein Ag-Ab complexes have shown that the contact residues of an epitope may contain as many as 17 residues on the surface of an Ag, and these residues may or may not be contiguous to each other in the peptide chain (32).

Our results with hybrid Ag 5s demonstrate that hybrid allergens can have a 100- to a 1000-fold reduction in allergenicity while retaining the immunogenicity of the natural allergens. The reduction in allergenicity of hybrids is due to a decrease of B cell epitope density. and this is suggested by the decreasing allergenicity of the N- and C-terminal PV hybrids as the Ves v 5 segment is shortened (Table VGo). As to be expected, the immunogenicity of the hybrids appeared to depend on the length of Ves v 5 segment (Table IIIGo). Our data suggest that a PV hybrid with 20–30 residues of Ves v 5 can have maximal reduction in allergenicity while still retaining adequate immunogenicity for Ves v 5.

Each of the hybrids we have studied has only a portion of the B and T cell epitopes of Ves v 5, and a mixture of appropriately chosen hybrids can, in principle, reconstitute the complete epitope library. This is in contrast to the allergens modified by the other procedures described in the introduction to this paper, as most known modified allergens lack part or all of their discontinuous B cell epitopes. It is known from studies in animals or humans that denatured allergens (4, 34) or T cell epitope peptides of allergens (35, 36, 37, 38) did not induce allergen-specific Ab responses, although they did stimulate allergen-specific T cell responses. Thus, the hybrids may represent a better way to prepare modified allergens for use as vaccines, because they can induce both cellular and Ab responses. Because the hybrids can induce immune responses to both Ves v 5 and Pol a 5 in mice, studies need to be made to discover whether such treatment with hybrids may lead to wasp sensitivity and/or protection in yellow jacket-sensitive patients. The hybrid approach, if shown to be useful, is applicable to other allergens because many allergens have sequence homology with proteins from diverse sources (39).


   Acknowledgments
 
We thank the Protein/DNA Technology Center of Rockefeller University for synthetic oligonucleotides, N-terminal sequencings, and mass spectrometric data.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants AI-17021 and AI-08270, and a grant from ALK-Abelló. Back

2 Address correspondence and reprint requests to Dr. Te Piao King, Rockefeller University, 1230 York Avenue, New York, NY 10021. Back

3 A. Henriksen, T. P. King, O. Mirza, K. Meno, H. Ipsen, M. Gajhede, and M. Spangfort. Structure of a vespid allergen, Ves v 5. Submitted for publication. Back

4 Abbreviation used in this paper: CD, circular dichroism. Back

Received for publication January 2, 2001. Accepted for publication March 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bousquet, J., R. Lockey, H. Malling. 1998. Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper. J. Allergy Clin. Immunol. 102:558.[Medline]
  2. Norman, P. S.. 1993. Modern concepts of immunotherapy. Curr. Opin. Immunol. 5:968.[Medline]
  3. Secrist, H., C. J. Chelen, Y. Wen, J. D. Marshall, D. T. Umetsu. 1993. Allergen immunotherapy decreases interleukin 4 production in CD4+ T-cells from allergic individuals. J. Exp. Med. 178:2123.[Abstract/Free Full Text]
  4. Takatsu, K., K. Ishizaka, T. P. King. 1975. Immunogenic properties of modified antigen E. III. Effect of repeated injections of modified antigen on immunocompetent cells specific for native antigen. J. Immunol. 115:1469.[Abstract/Free Full Text]
  5. Pesce, A. J., J. M. Freisheim, A. Litwin, J. G. Michael. 1990. Modulation of the immune response to allergen: phospholipase A degradation products suppress IgG and IgE response in mice. Int. Arch. Allergy Appl. Immunol. 92:88.[Medline]
  6. Smith, A. M., M. D. Chapman. 1996. Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2. Mol. Immunol. 33:399.[Medline]
  7. Takai, T., T. Ykota, M. Yasue, C. Nishiyama, T. Yuuki, A. Mori, H. Okudaira, Y. Okumura. 1997. Engineering of the major house dust mite allergen Der f 2 for allergen-specific immunotherapy. Nat. Biotechnol. 15:754.[Medline]
  8. Vrtala, S., K. Hirtenlehner, L. Vangelista, A. Pastore, H. G. Eichler, W. R. Sperr, P. Valent, C. Ebner, D. Kraft, R. Valenta. 1997. Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments. J. Clin. Invest. 99:1673.[Medline]
  9. Marsh, D. G.. 1971. Preparation and properties of allergoids derived from native pollen allergens by mild formalin treatment. Int. Arch. Allergy Appl. Immunol. 41:199.[Medline]
  10. Patterson, R., I. M. Suszko, J. J. Pruzansky, C. R. Ziess. 1973. Polymerized ragweed antigen E. II. In vivo elimination studies and reactivity with IgE antibody systems. J. Immunol. 110:1413.[Abstract/Free Full Text]
  11. Gieni, R. S., X. Yang, K. T. HayGlass. 1993. Allergen-specific modulation of cytokine synthesis patterns and IgE responses in vivo with chemically modified allergen. J. Immunol. 150:302.[Abstract]
  12. King, T. P., L. Kochoumian, N. Chiorazzi. 1979. Immunological properties of conjugates of ragweed antigen E with methoxypolyethylene glycol or a copolymer of D-glutamic acid and D-lysine. J. Exp. Med. 149:424.[Abstract/Free Full Text]
  13. Mirza, O., A. Henriksen, H. Ipsen, J. N. Larsen, M. Wissenbach, M. D. Spangfort, M. Gahjede. 2000. Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1. J. Immunol. 165:331.[Abstract/Free Full Text]
  14. Briner, T. J., M. Kuo, K. M. Keating, B. L. Rogers, J. L. Greenstein. 1993. Peripheral T-cell tolerance induced in naive and primed mice by subcutaneous injection of peptides from the major cat allergen Fel d I. Proc. Natl. Acad. Sci. USA 90:7608.[Abstract/Free Full Text]
  15. Hoyne, G. F., R. E. O’Hehir, D. C. Wraith, W. R. Thomas, J. R. Lamb. 1993. Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice. J. Exp. Med. 178:1783.[Abstract/Free Full Text]
  16. Chothia, C., A. V. Finkelstein. 1990. The classification and origins of protein folding patterns. Annu. Rev. Biochem. 59:1007.[Medline]
  17. Russell, R. B., G. J. Barton. 1994. Structural features can be unconserved in proteins with similar folds: an analysis of side-chain to side-chain contacts secondary structure and accessibility. J. Mol. Biol. 244:332.[Medline]
  18. Lichtenstein, L. M., M. D. Valentine, A. K. Sobotka. 1979. Insect allergy: the state of the art. J. Allergy Clin. Immunol. 64:5.[Medline]
  19. Lu, G., M. Villalba, M. R. Coscia, D. R. Hoffman, T. P. King. 1993. Sequence analysis and antigen cross-reactivity of a venom allergen, antigen 5, from hornets, wasps, and yellow jackets. J. Immunol. 150:2823.[Abstract]
  20. Monsalve, R. I., L. Gang, T. P. King. 1999. Recombinant venom allergen, antigen 5, of yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) by expression in bacteria or yeast. Protein Expression Purif. 16:410.[Medline]
  21. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51.[Medline]
  22. Higgins, D. R., J. M. Cregg. 1998. Pichia Protocols Humana, Totowa.
  23. King, T. P., G. Lu. 1997. Hornet venom allergen, Dol m 5: its T cell epitopes in mice and its antigenic cross-reactivity with a mammalian testis protein. J. Allergy Clin. Immunol. 99:630.[Medline]
  24. King, T. P., L. Kochoumian, T. Lam. 1987. Immunochemical observations of antigen 5, a major venom allergen of hornets, yellowjackets and wasps. Mol. Immunol. 24:857.[Medline]
  25. King, T. P., L. Kochoumian, G. Lu. 1995. Murine T and B cell responses to natural and recombinant hornet venom allergen Dol m 5.02 and its recombinant fragments. J. Immunol. 154:577.[Abstract]
  26. Colombo, M., E. M. Horowitz, A. Kagey-Sobotka, L. M. Lichtenstein. 1995. Histamine release from human basophils induced by platelet activating factor: the role of extracellular calcium, interleukin-3, and granulocyte-macrophage colony-stimulating factor. J. Allergy Clin. Immunol. 95:565.[Medline]
  27. Yang, J. T., C. S. C. Wu, H. M. Martinez. 1986. Calculation of protein conformation from circular dichroism. Methods Enzymol. 130:208.[Medline]
  28. Hornick, C. L., F. Karush. 1972. Antibody affinity. III. The role of multivalence. Immunochemistry 9:325.[Medline]
  29. Crothers, D. M., H. Metzger. 1972. The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9:341.[Medline]
  30. Newmann, M. A., C. R. Mainhart, C. P. Mallett, T. B. Lavoie, S. J. Smith-Gill. 1992. Patterns of antibody specifity during the BALB/c immune response to hen eggwhite lysozyme. J. Immunol. 149:3260.[Abstract]
  31. Novotny, J., J. Bajorath. 1996. Computational biochemstry of antibodies and T-cell receptors. Adv. Protein Chem. 49:149.[Medline]
  32. Davies, D. R., G. H. Cohen. 1996. Interaction of protein antigens with antibodies. Proc. Natl. Acad. Sci. USA 93:7.[Abstract/Free Full Text]
  33. Maliszewski, C. R., G. J. T. Delespesse, M. A. Schoenborn, R. J. Armitage, W. C. Fanslow, T. Nakajima, E. Baker, G. R. Sutherland, K. Piondexter, C. Birks, et al 1994. The CD39 lymphoid cell activation antigen: molecular cloning and structural characterization. J. Immunol. 153:3574.[Abstract]
  34. Norman, P. S., K. Ishizaka, L. M. Lichtenstein, N. F. Adkinson. 1980. Treatment of ragweed hay fever with urea-denatured antigen E. J. Allergy Clin. Immunol. 66:336.[Medline]
  35. Muller, U., C. A. Akdis, M. Fricker, M. Akdis, T. Blesken, F. Bettens, K. Blaser. 1998. Successful immunotherapy with T-cell epitope peptides of bee venom phospholipase A2 induces specific T-cell anergy in patients allergic to bee venom. J. Allergy Clin. Immunol. 101:747.[Medline]
  36. Simons, F. E., M. Imada, Y. Li, W. T. Watson, K. T. HayGlass. 1996. Fel d 1 peptides: effect on skin tests and cytokine synthesis in cat-allergic human subjects. Int. Immunol. 8:1937.[Abstract/Free Full Text]
  37. Marcotte, G. V., C. M. Braun, P. S. Norman, C. F. Nicodemus, A. Kagey-Sobotka, L. M. Lichtenstein, D. M. Essayan. 1997. Effect of peptide immunotherapy on T cell proliferation and cytokine production. J. Allergy Clin. Immunol. 99:405.
  38. Creticos, P. S., J. Hebert, G. Philip, T. A. R. S. Group. 1997. Efficacy of Allervax ragweed peptides in the treatment of ragweed-induced allergy. J. Allergy Clin. Immunol. 99:401.
  39. Larsen, J. N., H. Lowenstein. 1996. Allergen nomenclature. J. Allergy Clin. Immunol. 97:577.[Medline]



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