Weed Pollen Allergies
Recombinant Weed Pollen Allergens for IT
In contrast to trees or grasses, the term, 'weed' does not describe a distinct botanical group of plants, however, it is rather used to define plants without economic or aesthetic value. Worldwide, pollen from weed represents an important allergen source and sensitized patients, with some exceptions, typically develop allergic symptoms during late summer until autumn. Clinically, the most relevant weed species are ragweed (Ambrosia), mugwort (Artemisia), pellitory (Parietaria), chenopod (Chenopodium), Russian thistle (Salsola), plantain (Plantago) and annual mercury (Mercurialis). However, the major weed pollen allergens are mainly restricted to four distinct protein families, which are defensin-like proteins, Ole e 1-like proteins and pectate lyases, as well as nonspecific lipid transfer proteins (nsLTPs).[64] Moreover, the panallergens profilin and polcalcin, which represent allergenic molecules with a ubiquitous distribution, have been recognized in weed pollen.[65] To date, 36 weed pollen allergens have officially been acknowledged by the International Allergen Nomenclature Sub-Committee International Union of Immunological Societies,[101] and many of them were produced as recombinant proteins either in E. coli or the yeast P. pastoris.[64] Of note, the major mugwort pollen allergen, Art v 1, was successfully produced as a recombinant protein in N. tabacum, however the yields of recombinant Art v 1 obtained in either the leafs of tobacco plants or in cell cultures were insufficient to allow upscaled production;[66] therefore, unicellular organisms still represent the best choice for the production of recombinant weed pollen allergens.
Recombinant Asteraceae Allergens & Their Derivatives
Besides the production of recombinant wild-type allergens, many approaches for the production of hypoallergenic weed pollen allergen derivatives applicable for future therapy have been developed. The major ragweed pollen allergen Amb a 1, which belongs to the family of pectate lyases, causes sensitization rates of up to 95% in ragweed pollen allergic individuals.[64] The protein was produced as a wild-type recombinant allergen in E. coli and purified from bacterial inclusion bodies. Recombinant Amb a 1 behaved quite different with regard to IgE reactivity in various experimental setups (i.e., immunoblot, ELISA and mediator-release assays), thus suggesting that a high-quality preparation of the protein would be needed for reproducible immunologic evaluation.[67] Natural Amb a 1 is proteolytically processed resulting in two fragments termed α- and β-chain. Immunoblot analyses of Amb a 1 and its cleavage products revealed that the IgE reactivity of the allergen was mainly triggered by the β-chain representing the N-terminal part of the protein, whereas the C-terminally located α-chain harbored most of the relevant T-cell epitopes.[67,68] Therefore, hypoallergenic constructs as vaccine candidates for specific IT were developed based on the Amb a 1 α-chain. The pectate lyase Art v 6 from mugwort shares 65% sequence identity with Amb a 1 and both allergens show IgE cross-reactivity; however, Amb a 1 harbors more B-cell epitopes compared with its mugwort homolog and, also, the T-cell response to Art v 6 is more restricted.[69] Similar to Amb a 1, the recombinant production of Art v 6 in E. coli seems difficult, probably due to the intrinsic fold of pectate lyases, which is characterized by a parallel β-helix and stabilized by multiple disulfide bridges.[70,71] Unlike Art v 6, the major mugwort pollen allergen Art v 1 has successfully been produced in E. coli, even though the allergen has a complex head and tail structure, with a N-terminal defensin domain that contains four disulfide bonds and a C-terminal O-glycosylated hydroxyproline-rich domain.[72,73] Targeting the cysteine residues of Art v 1 resulted in the generation of low-IgE-binding fold variants, which still had the ability to activate Art v 1-specific T cells.[74]
Weed Pollen nsLTPs
A similar approach to Art v 1 was performed to modify the IgE-binding capacity of pellitory allergens. Parietaria represents the only clinically relevant allergy-eliciting weed of the Urticaceae. The plant is endemic in the Mediterranean area and can cause sensitization rates of 60–90% in some coastal regions in southern Europe.[75] The two major allergens of Parietaria, Par j 1 and 2, both belong to the family of nsLTPs; however, they share only 48–50% sequence identity and the cross-reactivity with other allergenic nsLTPs is limited.[76,77] Therefore, it has been suggested to use both Parietaria allergens as candidates for IT. The fold of nsLTPs is stabilized by disulfide bonds, thus cysteine mutants of both proteins were generated and produced as recombinant proteins in E. coli. For Par j 1, the destruction of three (PjC) or four (PjD) of the four disulfide bridges was most effective in reducing IgE binding, while the T-cell reactivity remained intact.[78] Moreover, PjC was evaluated in a mouse model demonstrating that immunization with PjC resulted in the induction of a robust IgG, but weak IgE immune response. Along this line, immunization with Par j 1 led to highly cross-reactive IgG antibodies against PjC, while IgE cross-reactivity was low.[79] To determine IgE-binding regions of Par j 2, the protein was first dissected into fragments and IgE binding was analyzed by immunoblot and mediator-release assays. Except for peptide 48–76, IgE binding to peptides could be detected, which was dependent on the serum used for the assay.[80] For further evaluation, two fragments, which cover the full sequence of Par j 2, were selected and produced as recombinants in E. coli. In addition, disulfide bonds within the peptides were destroyed by a mutation to reduce IgE-binding properties of the molecules. Interestingly, the N-terminal fragment of Par j 2 triggered the expression of Toll-like receptors and the production of IFN-γ upon stimulation of PBMCs from atopic children. Thus, such recombinant vaccines could help to redirect the allergic immune response.[81] Since Parietaria allergies are triggered by both nsLTPs, a head-to-tail fusion of disulfide variants of Par j 1 and 2 was generated. The hybrid demonstrated a drastically reduced IgE interaction by retained T-cell activation, as analyzed using PBMCs from Parietaria allergic donors. Moreover, in a preclinical model, the hybrid protein could induce IgG1 antibodies, which demonstrated cross-reactivity with both parental allergens.[82] Building on this concept, a head-to-tail fusion of Par j 1 and 2 was designed, which had mutations in a loop region previously identified as important for IgE binding within both nsLTPs. The mutant demonstrated reduced IgE binding, but again the T-cell reactivity remained unaltered. Furthermore, the loop-mutated hybrid could induce cross-reactive IgG antibodies, as well as T-cells, in a mouse model of sensitization.[83] Moreover, fusions of full-length Par j 1 and 2, and derivatives thereof showing deletions in both fusion partners were constructed. By analyzing the secondary structure elements all fusion proteins showed increased content of random coil, which reduced the IgE-binding capacity of the molecules. T-cell reactivity was only reduced in one of the hybrids indicating that fusing Par j 1 and 2 followed by deletions within the allergens could be a suitable method for designing safe vaccine candidates.[84]
Immunotherapy. 2013;5(12):1323-1338. © 2013 Future Medicine Ltd.
