Dimerisation increases the immunogenicity of recombinant Parj1/Parj2 allergens

Abstract

Purified recombinant Parj1 and Parj2 allergens bind an IgE repertoire common to the Parietaria species, allowing their use as marker molecules for diagnosis and therapy of allergic disease induced by the Urticaceae family. Preclinical studies on the in vivo immunogenicity of recombinant Parj1, Parj2 and their isoforms indicated differential capacity to induce IgG1 antibody responses, as indication of potential clinical use. A recombinant hetero-dimeric hybrid derivative (PjED), encompassing the shorter Parj1 isoform (Parj1.0201) and Parj2 allergen, was characterised. In vivo immunisation with PjED induces IgG1 antibodies capable of binding all the isoforms of Parietaria major allergens, overcoming the poor immunogenicity of single monomeric allergens. This feature makes PjED a promising candidate molecule to be further characterised for clinical applications in the treatment of Parietaria allergy.

Parietaria pollen is one of the major causes of pollinosis and asthma. Purified recombinant Parj1 and Parj2 allergens have been used as marker molecules for diagnosis and therapy of allergic disease induced by the Urticaceae family. ¹ Allergen-specific immunotherapy (SIT) is the only known curative approach to treat Parietaria allergic diseases. However, classical SIT performed with natural allergenic extracts can induce side effects, ranging from mild and local to severe and life-threatening symptoms, such as anaphylactic shock.

Several clinical studies indicate that recombinant allergen derivatives hold promise to improve SIT safety and efficacy.^2,3 Immunogenicity is improved when low molecular weight allergens are produced as oligomeric derivatives.^4,5 In vivo immunisation in mouse models has been applied to test reagents for immunotherapeutic treatment ⁶ as well as to predict the potential immunogenicity and allergenicity of novel proteins. ⁷

Recently, we demonstrated that Parj1 allergen displays LPS-binding activity due to the presence of a 37 amino acid COOH terminal region and that this region could be associated with differential cell and antibody responses in vitro and in vivo. ⁸ In fact, the recombinant ‘short’ Parj1 isoform (Parj1.0201, characterised by the absence of LPS binding region) did not give rise to detectable levels of IgG antibodies capable of recognising both rParj1 isoforms after in vivo immunisation, as well as not inducing spleen cell proliferation in response to in vitro re-stimulation with the same antigens. ⁸ A recombinant hetero-dimeric hybrid derivative (PjED) generated by the fusion of this shorter Parj1 isoform (Parj1.0201) with Parj2 allergen has been recently described by our group. ⁹

We report the study of the in vivo immunogenicity of PjED molecule in comparison with its monomeric components.

First, we tested the human IgE-binding capacity of PjED in comparison to the other recombinant monomeric allergens (‘long’ Parj1.0101, ‘short’ Parj1.0201 and Parj2.0101), and to the whole Pj extract (PjE). Sera used in ELISA experiments were collected from eight patients with a clear history of allergic disease and who had positive skin prick test (SPT) to P. judaica commercial extract. None of them had received specific immunotherapy against allergens or were using steroids or antihistamines. Each subject signed an informed consent before the sampling. This study was approved by the Ethics Committee of Policlinico Universitario, Palermo. IgE from all the human sera, individually tested, were able to recognise the three recombinant monomeric allergens, the recombinant heterodimer PjED and the natural extract, without statistically significant differences as indicated by the Kruskall-Wallis non-parametric test (no differences among the medians of all groups) and the Wilcoxon non-parametric test (P >0.05 for all the combinations) (Figure 1a). Recombinant allergens showed a higher trend in IgE recognition when compared to the natural PjE, probably associated with their higher purity and bioavailability as antigens on the ELISA plates. It is worth mentioning that the PjED heterodimer is recognised equally or better than the three monomeric allergens.

Figure 1.

(a) Human IgE reactivity to the two recombinant isoforms of Parj1, the recombinant Parj2, the heterodimer PjED and the Parietaria pollen extract (PjE). Sera from eight patients clinically allergic to Parietaria pollen were individually tested against the different antigens in ELISA. Mean ± SEM are reported. No statistically significant differences have been evidentiated by the Kruskall-Wallis non-parametric test (no differences among the medians of all groups) and the Wilcoxon non-parametric test (P >0.05 for all the combinations). (b) Allergen-specific IgG1 response induced in mice immunised with the three monomeric allergens Parj1.0101, Parj1.0201, Parj2.0101 and tested in ELISA against the corresponding antigens. (c) Allergen-specific IgG1 response induced in mice immunised with PjED and tested in ELISA against the same three monomeric allergens and the PjED itself. n = 6–10 mice/group. Mean ± SEM are reported. Statistically significant differences reported in the panels were evaluated by Wilcoxon (panels a, c) and Mann-Whitney (panel b) tests.

Physico-chemical characterisation of PjED was then performed by photon correlation spectrometry (PCS), in comparison to the monomeric allergens. By this analysis, the PjED showed a size of about 12.19 nm with a polydispersity index (PDI) value of 0.449 in PBS. On the other hand, the monomeric allergens displayed PDI values (Parj1.0201 [0.6951] and Parj2.0101 [0.991]) higher than the heterodimer, demonstrating that the PjED has a more homogeneous size distribution than the monomeric allergens in solution. Moreover, the measurements of the zeta potential by laser Doppler velocimetry and phase analysis light scattering (M3-PALS technique, in PBS), gave a value of −2.05 mV at 25°C similar to that one displayed by the Parj2.0101 allergen (−2.95 mV) and lower than that one measured for the Parj1.0201 (−5.47 mV). This value remains constant at 37°C, demonstrating that the PjED is self-stabilising in this buffer inasmuch as its surface charge inhibits coalescence and enhances stability. After several freeze-and thaw cycles, the measurements showed similar values demonstrating that the hybrid presents high stability in PBS.

We then moved to study in parallel the in vivo immunogenicity of the three recombinant allergen molecules. Three groups of six female Balb/c mice were immunised intraperitoneally (I.P.) with equimolar (0.150 nM) amounts of either rParj1.0101 or rParj1.0201 isoforms or with rParj2.0101, in Alum as adjuvant according to a previously published protocol. ¹⁰ Levels of specific IgG1 antibodies in the serum of sensitised mice were evaluated against homologous allergens in ELISA assays. Immunisation with rParj1.0101 induced a strong IgG1 response in mice. On the other hand, when mice were immunised with the shorter rParj1 isoform (Parj1.0201) and with rParj2.0101, barely detectable IgG1 antibodies were induced against the homologous allergens, in all cases at significantly lower levels than those obtained after rParj1.0101 immunisation (Figure 1b). These results confirm our previously reported results on Parj1.0201 ⁸ and suggest that the in vivo immunogenicity of monomeric rParj1.0201 and rParj2.0101 is significantly reduced compared with rParj1.0101 in mice.

To assess whether the poor immunogenicity of these monomeric allergens could be improved by dimerisation, mice were immunised I.P. with an equimolar amount of PjED at the same conditions reported above, and IgG antibody response was tested by ELISA against the two monomeric allergens included in PjED, the ‘long’ Parj1.0101 and the PjED itself as control.

PjED immunisation induced high levels of IgG antibodies able to recognise both the major allergens of the Parietaria pollen as well as the shorter isoform of Parj1 (Figure 1c). This finding suggests that immunisation with this single hybrid molecule can induce antibodies capable of binding all the clinically relevant isoforms of Parietaria allergens.

In summary, PjED displays attractive advantages, in terms of physico-chemical characteristics and immunological properties. First, it collects in a unique molecule all the relevant determinants of different isoforms of the two major allergens of Parietaria pollen, and shows a more homogeneous size distribution than the monomeric allergens, as well as high solubility and stability in aqueous solutions. These properties make the standardisation procedure required for clinical applications easier, taking also into account that the shorter isoform of Parj1 (Parj1.0201) contained in PjED lacks the LPS-binding region at the COOH terminal, thus reducing the possibility of endotoxin contamination in the final recombinant product. Indeed, endotoxin contamination may not only affect the outcome of the immune response to such allergenic molecules, but also affect both safety and efficacy of these products as immunotherapeutic reagents. For these reasons, standardised allergen vaccines should be controlled for the endotoxin content for the application to clinical purposes. ¹¹ The preliminary in vivo characterisation of PjED supports its high immunogenicity and ability to induce sustained IgG antibody responses overcoming the poor immunogenicity of single monomeric allergens.