Simplex Allergens Research Articles

TheAnisakis simplexAni s 7 major allergen as an indicator of trueAnisakisinfections

Ani s 7 is currently the most important excretory/secretory (ES) Anisakis simplex allergen, as it is the only one recognized by 100% of infected patients. The allergenicity of this molecule is due mainly to the presence of a novel CX(17-25)CX(9-22)CX(8)CX(6) tandem repeat motif not seen in any previously reported protein. In this study we used this allergen as a model to investigate how ES allergens are recognized during Anisakis infections, and the usefulness of a recombinant fragment of Ani s 7 allergen (t-Ani s 7) as a marker of true Anisakis infections. The possible antigenic relationship between native Ani s 7 (nAni s 7) from Anisakis and Pseudoterranova decipens antigens was also investigated. Our results demonstrate that nAni s 7 is secreted and recognized by the immune system of rats only when the larvae are alive (i.e. during the acute phase of infection), and that this molecule is not present in, or is antigenically different from, Pseudoterranova allergens. The t-Ani s 7 polypeptide is a useful target for differentiating immunoglobulin E antibodies induced by true Anisakis infections from those induced by other antigens that may cross-react with Anisakis allergens, including P. decipiens. The results also support the hypothesis that the Ani s 7 major allergen does not participate in maintaining the antigenic stimulus during chronic infections.

Anisakis simplex as a risk factor for relapsing acute urticaria: a case–control study

Background:IgE-mediated hypersensitivity reactions to Anisakis simplex have been described after ingestion of fish, suggesting that sensitisation to this parasite may induce acute urticaria and anaphylaxis. Anisakis simplex allergens are highly...

Cloning and expression of Ani s 9, a new Anisakis simplex allergen

The larvae of the nematode Anisakis simplex parasitize seafood. When people eat raw or undercooked parasitized fish, they can suffer anisakiasis, an important immune human response to parasitic infection of the gastrointestinal tract. Even more, allergic manifestations like angioedema, urticaria or anaphylaxis can occur in sensitized patients. The aim of this work was to clone Ani s 9-cDNA and overproduce this recombinant allergen in Escherichia coli. The finding of this allergen was an unexpected result of a PCR using degenerate primers designed to amplify Ani s 5. The complete cDNA for Ani s 9 was obtained by RACE-PCR, cloned and sequenced. Expression of recombinant allergen was performed in E. coli. Immunodetection and immunoblot inhibition assays tests were carried out with sera from Anisakis allergic patients. The recombinant Ani s 9 (rAni s 9) is a protein of 147 amino acids. By immunoblot inhibition assay, it was located as a 14 kDa band present in a crude extract of the parasite. This new allergen is heat stable and is present in excretory/secretory products. Ani s 9 belongs to the SXP/RAL-2 family and shares amino acid sequence identity of 60% with As-14, an Ascaris suum allergen. Five of thirty-six Anisakis allergic patients (13.8%) were positive to rAni s 9 and natural Ani s 9 by immunodetection. In conclusion, Ani s 9 is a new allergen in Anisakis allergy and it has been cloned and successfully expressed in E. coli.

Ani s 1, the major allergen of Anisakis simplex: Purification by affinity chromatography and functional expression in Escherichia coli

Third stage larvae of the nematode Anisakis simplex often infect marine fish and invertebrates. When the larvae are ingested orally via seafood, they can cause IgE-mediated allergic reactions as well as anisakiasis. Of the known A. simplex allergens, Ani s 1 (Kunitz/bovine pancreatic trypsin inhibitor family protein) has been demonstrated to be a major allergen, being expected to be a useful tool for diagnosis of A. simplex allergy. For a diagnostic purpose, sufficient amounts of either natural Ani s 1 (nAni s 1) or recombinant Ani s 1 (rAni s 1) with an IgE-binding capacity should be stably supplied whenever needed. In this study, therefore, we first developed a simple and rapid purification method for Ani s 1 that is based on affinity chromatography using anti-Ani s 1 antibodies as ligands. The method was shown to produce nAni s 1 with a higher yield than the previously reported methods. Then, an attempt was made to express rAni s 1 in Escherichia coli as a His-tagged protein. rAni s 1 obtained as an inclusion body was solubilized in a solvent containing denaturing and reducing reagents and purified by nickel-chelate chromatography. Refolding of rAni s 1 was accomplished by dialysis in the presence of arginine, followed by that in the absence of arginine. Fluorescence ELISA and inhibition ELISA data revealed that rAni s 1 is IgE reactive enough to be used as a diagnostic tool.

Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen*

Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O-deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3-ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis. An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096-amino acid fragment of Ani s 7 (GenBank: EF158010) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained. The rAni s 7 fragment comprised 19 repeats of a novel CX(17-25)CX(9-22)CX(8)CX(6) tandem repeat motif not seen in any previously reported protein sequence. An internal (435)Met-(713)Arg fragment of the rAni s 7 (t-Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme-linked immunosorbent assay (ELISA) with t-Ani s 7 identified as positive the same 60 sera as UA3-ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O-deglycosylated nAni s 7. In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis-induced allergy.

Purification and cDNA cloning of a new heat-stable allergen from Anisakis simplex

The nematode Anisakis simplex is a representative parasite for marine animals and occasionally causes not only anisakiasis but also allergic reactions in sensitized subjects. Besides the known allergens, a number of unidentified allergens have been suggested to still exist in A. simplex. In this study, a new heat-stable allergen of 15kDa (named Ani s 8) was purified from the third stage larvae of A. simplex by gel filtration on Sephacryl S-300, anion-exchange HPLC on Mono Q and reverse-phase HPLC on TSKgel Phenyl-5PW RP. Analysis by fluorescence ELISA showed that 7 of 28 Anisakis-allergic patients had elevated serum levels of IgE to Ani s 8. On the basis of the determined partial amino acid sequence, the complete sequence of Ani s 8 (composed of 150 amino acid residues) was elucidated by cDNA cloning, in which as many as 32 homologs of the cDNA encoding 10 isoforms of Ani s 8 were detected. Ani s 8 shares amino acid sequence homology (up to 36%) with several members of the SXP/RAL-2 protein family, including Ani s 5 (15kDa) previously identified as an A. simplex allergen. Inhibition ELISA data demonstrated the IgE cross-reactivity between Ani s 8 and Ani s 5.

Cloning and expression of a biologically active Anisakis simplex allergen Ani s 1 in the yeast Pichia pastoris

Cloning and expression of a biologically active Anisakis simplex allergen Ani s 1 in the yeast Pichia pastoris

Cloning and characterisation of the Anisakis simplex allergen Ani s 4 as a cysteine-protease inhibitor

Anisakis simplex is a nematode that can parasitise humans who eat raw or undercooked fish containing live L3s. Larvae invading the gastrointestinal mucosa excrete/secrete proteins implicated in the pathogenesis of anisakiasis that can induce IgE mediated symptoms. Misdiagnosis of anisakiasis, due to cross-reactivity, makes it necessary to develop new diagnostic tools. Recombinant allergens have proved to be useful for diagnosis of other parasitoses. Among the Anisakis allergens, Ani s 4 was considered to be a good potential diagnostic protein because of its heat resistance and its importance in the clinical history of sensitised patients. Therefore, the objective of this study was to clone and characterise the cDNA encoding this allergen. The Ani s 4 mRNA sequence was obtained using a PCR-based strategy. The Ani s 4 amino acid sequence contained the characteristic domains of cystatins. Mature recombinant Ani s 4 was expressed in a bacterial system as a His-tagged soluble protein. The recombinant Ani s 4 inhibited the cleavage of a peptide substrate by papain with a Ki value of 20.6nM. Immunobloting, ELISA, a commercial fluorescence-enzyme-immunoassay and a basophil activation test were used to study the allergenic properties of rAni s 4, demonstrating that the recombinant allergen contained the same IgE epitopes as the native Ani s 4, and that it was a biologically active allergen since it activated basophils from patients with allergy to A. simplex in a specific concentration-dependent manner. Ani s 4 was localised by immunohistochemical methods, using a polyclonal anti-Ani s 4 anti-serum, in both the secretory gland and the basal layer of the cuticle of A. simplex L3. In conclusion, we believe that Ani s 4 is the first nematode cystatin that is a human allergen. The resulting rAni s 4 retains all allergenic properties of the natural allergen, and can therefore be used in immunodiagnosis of human anisakiasis.

Données nouvelles sur l’anisakidose

Acute anisakiasis is generally due to the third-stage larvae of Anisakis simplex, and occasionally to other anisakidae. Human infection occurs through consumption of raw seafish, and especially herring, hake, black plaice and cod. Patients sensitized by prior consumption of parasitized fish develop, within a few hours, violent abdominal pain and an allergic reaction. Anisakis-induced urticaria is seen in about one in five cases. Preventive measures have reduced the number of cases. In France for example, the number of cases fell four-fold between 1977-1991 and 1992-2005. In 1990 Kasuya [1] reported chronic anisakiasis related to consumption of cooked parasitized fish. Seafish-induced urticaria might be an allergic response to Anisakis larval antigens rather than to the fish itself. Indeed, 11 patients with mackerel-related urticaria all had a positive reaction to Anisakis simplex larval antigen, while none reacted to mackerel antigen. A low-molecular-weight thermostable A. simplex allergen causes chronic urticaria, angioedema and even anaphylactoid reactions. Victims have a genetic predisposition (HLA class II alleles) that is uncommon in France and Germany but frequent in Japan. A number of cases have been observed in Spain, where fish is particularly popular. Immunoblotting shows cross-reactions between antigens of A. simplex and Toxocara canis, nematodes belonging to the same superfamily. At present, the only way to avoid contracting anisakiasis is not to eat raw or even cooked parasitized seafish.

Anisakis simplexallergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern

The study of the singular hypersensitivity reactions to Anisakis simplex (A.s) proteins, may help us to undestand many of the unknown immune interactions between helmiths infections and allergy. We have developed a murine model of allergy to A. simplex, that mimics human A. simplex allergy to study the specific aspects of anaphylaxis induced by parasites. Male C3H/HeJ mice were intraperitoneally sensitized to A. simplex. Mice were then intravenous or orally challenged with A. simplex. Antigen-specific immunoglobulins, polyclonal IgE, anaphylactic symptoms, plasma histamine levels and cytokine profiles were determined. Comparative IgE immunoblot analyses were also performed. Specific IgE, IgG(1) and IgG(2a) were detected in sensitized mice since week 3. Polyclonal IgE raised and peaked with different kinetics. Intravenous A. simplex challenge produced anaphylaxis in mice, accompanied by plasma histamine release. Oral A. simplex challenge in similarly sensitized mice did not caused symptoms nor histamine release. Numerous A. simplex allergens were recognized by sensitized mouse sera, some of them similar to human serum. The A. simplex stimulated splenocytes released IL-10, IFN-gamma, IL-4, IL-13 and IL-5. We describe a new animal model of anaphylaxis. It exhibits characteristics of type I hypersensitivity reactions to Anisakis simplex similar to those observed in allergic humans. Different responses to i.v. or oral A. simplex challenges emerged, which did not reflect a window tolerization period. The cytokine profile developed (mixed Th(1)/Th(2) pattern) differed from the observed in classical models of anaphylaxis or allergy to food antigens. This model may permit to investigate the peculiar allergic reactions to parasitic proteins.

Ultrastructural localization of Ani s 1, a major allergen from the fish parasite Anisakis simplex.

No data about the nature or function of the first major Anisakis simplex allergen, named Ani s 1, is available. The aim of this study was to investigate the ultrastructural localization of this protein, to obtain further information about it. Ani s 1 was detected in secretory granules of the excretory gland and occasionally lining the main excretory canal. Our results suggest that Ani s 1 could be a secretory product and could have an enzymatic function related to mechanisms of infection.

Association of hypersensitivity to the nematode Anisakis simplex with HLA class II DRB1∗1502-DQB1∗0601 haplotype

Anisakiasis as well as allergic and anaphylactoid reactions to Anisakis simplex antigens are recently identified clinical entities. They are relatively frequent in countries with habitual raw food consumption, often in the form of large amounts of fish and sea food products. In this communication the relationship between HLA class II alleles and the IgE-specific immune response to A. simplex allergen was studied in a defined population in Northern Spain. Individuals with immediate-type Anisakis hypersensitivity and healthy controls were examined for HLA-DRB1, DQB1 and DQA1 alleles by sequence-specific oligonucleotide probe typing. Analysis of the HLA data among patients revealed increased phenotypic frequencies for DRB1∗1502 and DRB1∗0404 compared to healthy controls ( p < 1 × 10 −7 and < 0.01, respectively). Analysis of haplotypic frequencies showed that the DRB1∗1502-DQB1∗0601 haplotype is significantly higher in patients with Anisakis hypersensitivity in comparison with the control population from the same region ( p < 4 × 10 −8). The data suggest that this haplotype can be considered to be a susceptibility factor for hypersensitivity to A. simplex antigens.

Immune response to Anisakis simplex and other ascarid nematodes

Anisakis simplex is a nematode parasite belonging to the Ascaroidea group, which includes several species of medical importance, and the immune response to A. simplex is discussed with reference to some of these. The strong Th2 reaction usually provoked by nematodes seems to occur only in the context of infection, and exposure to nonviable antigens fails to do so. This would imply either that allergic reactions to A. simplex require prior infection, or that a classical atopic response is responsible, and the cause possibly varies among patients. The antibody response to secreted and exposed surface antigens of A. simplex is strong, and should be explored as potentially a better antigen source for serodiagnosis than somatic materials, and it might also be useful in addressing the question of the role of infection in predisposing to allergic reactions. As in toxocariasis, both secreted and shed surface antigens of A. simplex could provide a focus for inflammatory lesions in tissue‐invasive larvae. Humans respond heterogeneously to A. simplex allergens, and experience with other nematodes would indicate that this is due to genetic control of the immune repertoire by the major histocompatibility complex, a fact which, in turn, might have clinical implications. A. simplex is known to produce allergens similar to the ABA‐1 of Ascaris, which represents a structurally novel class of fatty acid and vitamin A‐binding protein. This and other recently described nematode proteins are known to be highly stable to thermal denaturation and can recover their structures, biochemical activities, and allergenicity upon cooling.