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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-1012fje. |
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* Department of Pathophysiology, Vienna General Hospital, AKH, University of Vienna, Austria; and
Institute of Chemistry, Karl-Franzens University of Graz, Austria
2Correspondence: Molecular Immunopathology Group, Department of Pathophysiology, Vienna General Hospital, AKH, Medical School, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: Rudolf.valenta{at}akh-wien.ac.at
SPECIFIC AIMS
Our aim was the production of combination vaccines to treat grass pollen allergic patients with complex sensitization patterns. Four major timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 5, Phl p 6) were expressed as recombinant hybrid molecules (rP2-P6, rP6-P2, rP5-P1) in which each component acts as a molecular scaffold to increase the immunogenicity of the other component. Whether the relevant B and T cell epitopes are present in the hybrid molecules was demonstrated with specific antibody probes by immunological competition experiments and lymphocyte proliferation experiments. Mice were immunized so as to study the immunogenicity of the hybrid molecules and to analyze whether the induced antibodies can block the binding of allergic patients’ IgE to grass pollen allergens.
PRINCIPAL FINDINGS
1. Genetic engineering of hybrid molecules consisting of immunologically unrelated grass pollen allergens that assemble most of the epitopes present in natural grass pollen extract
According to the rates of cosensitization determined in a population of 200 grass pollen allergic patients (rPhl p 1+rPhl p 5: 60%; rPhl p 2+rPhl p 6: 30%), we engineered hybrid molecules by PCR-based ‘gene-soeing’ (rP2-P6-, rP6-P2-, rP5-P1 hybrid). Analysis of the hybrids by circular dichroism spectroscopy showed that they exhibited comparable fold and secondary structure contents as equimolar mixes of their components.
IgE inhibition experiments showed that the rP2-P6 and the rP6-P2 hybrid inhibited IgE binding to each component (rPhl p 2, rPhl p 6) equally well as the single recombinant allergens. Similar results were obtained for the rP5-P1 hybrid. Using an equimolar mixture of the rP5-P1 and rP6-P2 hybrid molecules, the majority of timothy grass pollen extract-specific IgE could be adsorbed from sera of 20 grass pollen allergic patients (Table 1)
. The rP5-P1 hybrid induced a stronger lymphoproliferative response in PBMC of grass pollen allergic patients than the mix of rPhl p 5 and rPhl p 1, which was even stronger than that induced by natural timothy grass pollen extract. rP2-P6 and the rP6-P2 hybrids induced a much higher proliferative response than the rPhl p 2/rPhl p 6 protein mix (data not shown).
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2. Immunization with recombinant hybrid molecules induces stronger and earlier IgG responses than immunization with individual allergens or allergen extract
Figure 1
demonstrates that average IgG1 responses induced by the hybrid molecules to each of the individual allergens (rPhl p 1, rPhl p 2, rPhl p 5, or rPhl p 6) were higher than those obtained by immunization with the single allergen components, timothy grass pollen extract, or an equimolar mix of the four recombinant allergens (Fig. 1)
. The latter was particularly evident for Phl p 2, Phl p 6, and Phl p 1, which were poorly recognized by extract-immunized mice, whereas the hybrid molecules induced vigorous anti-Phl p 1-, anti-Phl p 2-, and anti-Phl p 6 antibody responses (Fig. 1)
.
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3. Hybrid molecules induce antibodies that block the binding of allergic patients’ IgE to grass pollen allergens
IgG antibodies induced with the rP2-P6 and the rP6-P2 hybrid molecule caused a 48%–54% inhibition of IgE binding to Phl p 2 and a 54%–67% inhibition of IgE binding to Phl p 6. By contrast, inhibition of IgE reactivity yielded by preincubation with antibodies induced with rPhl p 2 and rPhl p 6 alone was very low (0–15%). Anti-rP5-P1 antibodies caused a more than double inhibition of IgE binding to Phl p 5 (60% average inhibition) than antibodies raised against Phl p 5 alone (28%). The inhibition of IgE binding to Phl p 1 yielded with the antibodies raised against the rP5-P1 hybrid (19% average inhibition) and Phl p 1 alone (30% average inhibition) were lower. Perhaps most important, mouse antibodies raised against the rP5-P1 and rP6-P2 hybrid inhibited allergic patients’ IgE binding to timothy grass pollen extract stronger than extract-induced antibodies (average inhibition hybrids: 66%; average inhibition extract: 63%).
CONCLUSION
Specific immunotherapy is the only curative therapy approach for grass pollen allergy that uses allergen extracts prepared from grass pollen. As exemplified in Fig. 2
, grass pollen allergen extracts consist of a variety of different allergens and nonallergenic components. It has been demonstrated that different grass pollen allergens exhibit different immunogenicity (Phl p 5>Phl p 1>Phl p 6>Phl p 2) in animals and it has been observed that immunotherapy based on grass pollen allergen extracts induces highly heterogeneous immune responses in patients (Fig. 2a
). Using recombinant allergen molecules for diagnosis and treatment, it should be possible to tailor treatment according to the patient’s sensitization profile and thus avoid the induction of irrelevant or even unwanted immune responses. Treatment of allergy to complex allergen sources may, however, still be hampered by the different immunogenicity of the individual allergen molecules, that may give rise to heterogeneous immune responses even when these allergens are administered in equimolar mixtures (Fig. 2b
).
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Here we report the development of combination vaccines based on recombinant hybrid molecules consisting of four of the most important and frequently recognized grass pollen allergens (i.e., Phl p 1, Phl p 2, Phl p 5, Phl p 6). These combination vaccines should be suited to patient-tailored treatment of the majority of grass pollen allergic patients.
An unexpected finding of great importance for immunotherapy as well as vaccination in general was the strong increase of immunogenicity of unrelated allergens with poor immunogenicity (e.g., Phl p 2, Phl p 6) that was obtained by their production as hybrid molecules (Fig. 2c
).
The enhancement of immune responses in the case of the rP5-P1 hybrid molecule may be understood according to the principle of classical carrier effects. In this scenario, covalent linkage of a less immunogenic allergen (i.e., Phl p 1) with a highly immunogenic allergen (i.e., Phl p 5) containing a variety of potent T cell epitopes would explain the enhanced immunogenicity to the first allergen when it is presented in the context of the latter. On the other hand, we found that fusion of two different allergens with a low capacity to induce antibody or lymphoproliferative responses (i.e., Phl p 2, Phl p 6) leads to an almost equal enhancement of immune responses to each of the components. It seems unlikely that potent T cell epitopes or a molecular environment favoring vigorous T cell responses were created de novo in these hybrid molecules, because they did not contain relevant new sequences and the order of the allergens had no apparent influence on the enhancement of immunogenicity.
Since the hybrid molecules represented pure proteins, we consider T cell-independent carrier effects as described for monophosphoryl lipid A (MLA) as an unlikely possibility for their increased immunogenicity.
Whether the hybrid molecules induce a different repertoire of costimulatory molecules on immune cells or are processed in a different manner from the isolated allergens escapes us at this time. However, the analysis of the secondary structure of the hybrid molecules and the equimolar allergen mixes by circular dichroism demonstrates similar folds. It therefore appears unlikely that changes in secondary structure or fold may have caused profound differences in the presentation of the hybrid molecules vs. the isolated allergens.
We cannot provide a definitive answer for the mechanisms that are operative in the enhancement of immunogenicity of the hybrid molecules, but we suggest fusion of low immunogenic vaccine components against which protective immune responses are required as a generally applicable principle for immunotherapy and general vaccination. This principle may be applied when desired to induce strong protective immune responses or alternatively tolerization against independent and immunologically unrelated antigens without using a foreign carrier molecule that may give rise to unwanted immune responses. Another advantage of recombinant hybrid molecules is that they can be produced as defined molecular entities by standardized procedures, avoiding complicated chemical coupling and purification procedures.
The hybrids described in our study appear suitable for specific immunotherapy of grass pollen allergy because they contain most of the IgE epitopes and T cell epitopes present in natural grass pollen extract. Immunization of mice with the hybrid molecules induced strong and early induction of allergen-specific IgG antibody responses that strongly blocked the binding of grass pollen allergic patients’ IgE to the individual allergens and grass pollen extract. Moreover, we found that the hybrid molecules induced stronger lymphoproliferative responses in human allergen-specific mononuclear cells than the isolated allergen components.
The basic mechanisms underlying allergen-specific immunotherapy are not fully understood and there is still controversy regarding the importance of T cell and B cell epitopes for successful immunotherapy. Therefore, we constructed hybrids consisting of allergen molecules that closely resemble the immunological properties of natural grass pollen allergens. To reduce therapy-induced side effects resulting from the administration of active allergens, it will be possible to construct hybrid molecules consisting of hypoallergenic allergen derivatives. In conclusion, we suggest vaccination with recombinant hybrid molecules as a generally applicable strategy whenever the induction of simultaneous immune responses against unrelated antigens with different immunogenicity is desired.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1012fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-1012fje 
This article has been cited by other articles:
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  S. Vrtala, M. Focke, J. Kopec, P. Verdino, A. Hartl, W. R. Sperr, A. A. Fedorov, T. Ball, S. Almo, P. Valent, et al. Genetic Engineering of the Major Timothy Grass Pollen Allergen, Phl p 6, to Reduce Allergenic Activity and Preserve Immunogenicity J. Immunol., August 1, 2007; 179(3): 1730 - 1739. [Abstract] [Full Text] [PDF]
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