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Ascaris lumbricoides cystatin induces specific IgE but not allergic response

Sandra Coronado1*, Maria Manotas1, Josefina Zakzuk1 and Luis Caraballo1
  • 1 Universidad de Cartagena, Instituto de Investigaciones Inmunologicas, Colombia

Introduction: Helminth infections, including those caused by gastrointestinal helminths are characterized by mast cell activation; eosinophilia and high levels of circulating IgE. There is evidence that these antibodies are involved in immunity against parasites. Ascaris lumbricoides has somatic antigens and excretory/secretory products that induce a strong Th2 and specific IgE response; being important examples the protein ABA-1, glutathione transferase (GST) and tropomyosin. Tropomyosinis the major allergenic component of Ascaris extract but other IgE binding components have been detected by one two dimensional SDS-PAGE immunoblotting; some of them homologous to other allergen sources. The identification and characterization of new allergenic molecules from A. lumbricoides is essential for understanding immunity to this helminth and its influence on the evolution of allergic diseases in humans. During the screening of a cDNA library with human IgE antibodies we found a clone coding for cystatin (Al-CPI), a molecule with potential allergenic properties. Objective: To evaluate the ability of recombinant Al-CPI to bind human IgE and induce an allergic response in vitro and in vivo. Methods: We amplify the Ascaris lumbricoides cystatin sequence by PCR strategies. The PCR product was sequenced to confirm correspondence with that reported in GeneBank. A codon-optimized version of the Al-CPI sequence (481 nucleotides) was inserted into the expression vector pQE30 and produced as recombinant protein in E coli; in addition, its inhibitory activity was determined by protease-activity assays using specific colorimetric substrates. The prevalence of specific IgE and IgG4 antibodies against Al-CPI was determined by indirect ELISA, testing 237 sera from a cohort of asthmatic patients naturally exposed to the parasite (range of age: 7 to 70 years old). The ability to activate an allergic response was evaluated ex vivo by basophile activation test (BAT) and histamine release assays (HRA) and in vivo by skin prick test (SPT) in 40 subjects with positive IgE antibody levels to Al-CPI (OD>0.141), ten of them with positive SPT to Ascaris extract. Furthermore, the allergenicity was analyzed in vivo using a passive cutaneous anaphylaxis (PCA) model in BALB/c mice. Results: A recombinant cystatin of 14.2 kDa with potent inhibitory activity of cysteine protease was produced and purified. This protein has 42% identity with the highly allergenic Anisakis simplex cystatin, Ani s 4. The IgE binding frequency to Al-CPI was 64.5% in the group of asthmatic patients sensitized to Ascaris and 56.6% in non-sensitized individuals. When comparing the levels of IgG4, IgE and the ratio IgG4/IgE in Ascaris extract sensitized (n=154) and non-sensitized individuals (n=83), only IgG4 levels were significantly higher in those sensitized (median OD: 0.190 vs 0.177; Mann Whitney test: p= 0.021). In vivo (SPT) or ex vivo (BAT or HRA) functional assays to test the allergenicity of Al-CPI in humans were negative in contrast to the house dust mite allergen Der p 2, which was used as a positive control. In mice, PCA test was also negative. Conclusions: Our findings suggest that Al-CPI induces a strong and frequent IgE response in human exposed to Ascaris lumbricoides but does not induce an allergic reaction, probably, because of the blocking effect of IgG4 antibodies. However, this and other factors underlying the lack of allergenicity of this molecule should be evaluated because it could be a general phenomenon of protective IgE mediated response without adverse allergic effects on the host. The impact of our findings on the relationship of helminthiasis and allergy should be further investigated. Also, it remains to be defined whether this occurs with molecules from other sources.

Acknowledgements

This work was performed under the funding of the research project "Identification and characterization of proteins Ascaris lumbricoides with immunomodulatory properties useful in immunotherapy of allergic diseases", code 1107540531403 contract 406-2011, University of Cartagena-Colciencias.

References

Acevedo N., Erler A., Briza P., Puccio F., Ferreira F., Caraballo L. (2011). Allergenicity of Ascaris lumbricoides Tropomyosin and IgE Sensitization among Asthmatic Patients in a Tropical Environment. Int Arch Allergy Immunol 154: 195-206.

Acevedo N., Mohr J., Zakzuk J., Samonig M., Briza P., Erler A., Pomés A., Huber C.G., Ferreira F., Caraballo L. (2013). Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides. PLoS One 8(11): e78353.

Acevedo N., Sanchez J., Erler A., Mercado D., Briza P., Kennedy M., Fernandez A., Gutierrez M., Chua K.Y., Cheong N., Jimenez S., Puerta L., Caraballo L. (2009). IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1. Allergy 64: 1635-1643.

Fitzsimmons C.M., Falcone F.H., Dunne D.W. (2014). Helminth allergens, parasite-specific IgE, and its protective role in human immunity. Frontiers in Immunology 5(61): 1-12.

Liu S., Dong J., Mei G., Liu G., Xu W., Su Z., Liu J. (2011). Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides. Acta Crystallographica Section, Structural Biology and Crystallization Communications 67: 228–230.

Mei G., Dong J., Li Z., Liu S., Liu Y., Sun M., Liu G., Su Z., Liu J. (2014). Structural Basis for the Immunomodulatory Function of Cysteine Protease Inhibitor from Human Roundworm Ascaris lumbricoides. PLOS ONE 9 (4 e96069): 1-9.

Rodriguez-Mahillo A., Gonzales-Muñoz M., Gomez-Aguado F., Rodriguez-Perez R., Corcuera M.T., Caballero M.L., Moneo I. (2007). Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor. International Journal for Parasitology 37: 907-917.

Keywords: Ascaris, Allergenicity Assessment, Cystatins, IgE, IgG4

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Oral Presentation

Topic: Allergy

Citation: Coronado S, Manotas M, Zakzuk J and Caraballo L (2015). Ascaris lumbricoides cystatin induces specific IgE but not allergic response. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00295

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Received: 13 Apr 2015; Published Online: 15 Sep 2015.

* Correspondence: Dr. Sandra Coronado, Universidad de Cartagena, Instituto de Investigaciones Inmunologicas, Cartagena, Colombia, 130010, Colombia, sandracoronado360@yahoo.com