Gastropod Allergy: A Comprehensive Narrative Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Authors provided an interesting account of gastropod allergy, which is not that frequently addressed in original research or review literature.

Very clear and comprehensive description of taxonomy is provided which well introduces less familiar readers into the topic and facilitates further reading.

There are some concerns to be addressed, please find them below.

Major issues

Lines 265-266 section 7.1.1  - this part seems to be ;eft without further development – please clarify why only an abbreviation and reference is provided

Section 7.1.2 – please indicate here that allergic reactions to abalone have been addressed in section 5.2, otherwise this section looks unfinished

Lines 288-290: as regarding lines 265-266:  this part seems to be left without further development – please clarify why only  abbreviations and references are provided. The same applies to lines 301-302, section 7.3.2, Cra g 1 allergen

Minor issues

Line 66 „and” instead of „y”

Line 138 H. aspersa – when used for the first time provide full name Helix aspersa

Line 270 „previous” or „hitherto performed” rather than „former”

Author Response

 

Reviewer 1

 

 

Comments and Suggestions for Authors

The Authors provided an interesting account of gastropod allergy, which is not that frequently addressed in original research or review literature.

Very clear and comprehensive description of taxonomy is provided which well introduces less familiar readers into the topic and facilitates further reading.

There are some concerns to be addressed, please find them below.

Major issues

Lines 265-266 section 7.1.1  - this part seems to be ;eft without further development – please clarify why only an abbreviation and reference is provided

Answer: Thank you for the comment. The following text has been added:

7.1.1. Haliotis laevigata & Haliotis rubra

Hal l 1: A single reference is provided below concerning information related to its protein structure, along with some annotations regarding cases related to this sensitization. Specifically, 5 out of 9 individuals with case histories of allergy to consumption of crustacean shellfish and one after handling prawns were regarded as positive. All patients showed a positive (≥0.35 kU) ImmunoCAP result for oyster. Additionally, tests confirmed IgE binding to abalone tropomyosin in reducing immunoblots [41].

Section 7.1.2 – please indicate here that allergic reactions to abalone have been addressed in section 5.2, otherwise this section looks unfinished

Answer: Thank you for the accurate comment. The following text has been added: Please note that allergic reactions to abalone have been previously addressed in section 5.2. of the present manuscript.

Lines 288-290: as regarding lines 265-266:  this part seems to be left without further development – please clarify why only  abbreviations and references are provided. The same applies to lines 301-302, section 7.3.2, Cra g 1 allergen

Answer: Thank you for the precise remark, the following text have been added:

7.3.1. Crassostrea angulata

Cra a 1: Tropomyosin (TM), an important allergen of Crassostrea angulata, was purified and identified by mass spectrometry. Subsequently, TM was cloned and expressed, with a sequence of size 852 bp, encoding 284 amino acid residues. The results of circular dichroism, digestion assay, inhibition enzyme-linked immunosorbent assay, and basophil activation test showed that recombinant TM had similar physicochemical properties and immunological properties to native TM. Furthermore, two conformational mimotopes were obtained and 10 IgE linear epitopes were verified. Meanwhile, different degrees of cross-reactivity were observed between C. ngulate ™ and the other 8 shellfish (TMs) using antibodies and serological analysis, which may relate to the 3 conserved epitope regions. These findings are expected to provide a theoretical basis for the molecular diagnosis of oyster allergy and cross-reactivity among shellfish [43].

Cra a 2: Arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata. The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs [44].

Minor issues

Line 66 „and” instead of „y”

Answer: Corrected. Thank you.

Line 138 H. aspersa – when used for the first time provide full name Helix aspersa

Answer: Corrected. Thank you.

Line 270 „previous” or „hitherto performed” rather than „former”

Answer: Corrected. Thank you.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors should indicate the type of study (narrative review) and clearly state the main goals in the abstract. At the end of this section, it is expected to read what we learned with the carried-out investigation and what is still to be researched in the future.

The introduction is very short and poor. It must be in-depth, as it is essential to provide a strong background that justifies the need for the study. Section 2 (Epidemiology of shellfish allergy) is also too brief and superficial.

Lines 98-108: References are missing.

Lines 265-266: Please clarify this. I don’t understand section 7.1.1. The same for the beginning of section 7.3.1. and 7.3.2.

The background provided in section 8 should be moved to the Introduction.

Section 10 is too extensive, please revise it properly. Some parts of this section are Discussion.

 

Format the references according to the journal’s guidelines.

Comments on the Quality of English Language

 Minor editing of English language required.

Author Response

 

Reviewer 2

 

 

Comments and Suggestions for Authors

The authors should indicate the type of study (narrative review) and clearly state the main goals in the abstract. At the end of this section, it is expected to read what we learned with the carried-out investigation and what is still to be researched in the future.

Answer:  Thank you for the accurate remark, the following text have been added in the abstract: This narrative review highlights the limited research on gastropod allergy. It provides a comprehensive list of purified and recombinant allergens, and dicusses the applications of component-resolved diagnosis as well as current therapeutic developments.    

The introduction is very short and poor. It must be in-depth, as it is essential to provide a strong background that justifies the need for the study. Section 2 (Epidemiology of shellfish allergy) is also too brief and superficial.

Answer:  The introduction has been expanded as follows: As a result, its consumption has surged in recent years, paralleling an increase in allergic reactions to seafood [1,2]. While shellfish includes both crustaceans and mollusks, crustacean allergy stands out as the most prevalent and extensively studied. Consequently, much of the research in this field has been focused on crustaceans leaving studies on mollusk allergies, particularly gastropod allergies, notably sparse.

Allergens are proteins or glycoproteins capable of inducing IgE-mediated allergic reactions [6]. Typically, they are protein molecules, although carbohydrates have also been associated with some allergic capacity [7]. The molecular characteristics that determine if a molecule can be an allergen are unknown, but factors such as size, solubility, and structural stability influence their allergenic potency [6]. The part of the allergen recognized by IgE is called the epitope or antigenic determinant and consists of a series of amino acids, which can vary in size. IgE recognizes epitopes, which can be continuous or discontinuous [8].

Allergens are classified into two groups, major or minor, based on the frequency with which specific IgE is detected against them in patients sensitized to that allergenic source. An allergen is considered major if recognized by more than 50% of allergic patients and minor if recognized by less than 50%, but the frequency of allergen recognition among sensitized individuals varies geographic region and could also change over time [9].

Tropomyosin (TM) was the first allergen identified in Penaeus indicus (shrimp), and it has long been recognized as the primary allergen associated with shellfish allergy. Interestingly, this panallergen has also been found in various invertebrate species such as cockroaches, Anisakis simplex, and dust mites, suggesting potential cross-reactivity between shellfish and other invertebrates [10].

However, subsequent investigations have revealed the complexity and diversity of the allergenic composition of shellfish. Several proteins shared between mollusks and crustaceans have been identified, potentially contributing to cross-reactivity. These include arginine kinase (AK), myosin light chain, sarcoplasmic calcium-binding protein (SCBP), troponin C, hemocyanin, triose phosphate isomerase, and others [10].

Current diagnostic methods are inadequate for predicting cross-reactivity between crustaceans and mollusks. Detecting mollusk hypersensitivity should still rely on skin tests with fresh material. Exclusion of mollusks from the diets of shrimp-allergic patients should be based on clinical history, available diagnostic tools, and/or tolerance tests.

Answer: The epidemiology has been expanded as follows: In Europe, there is significant variation in the prevalence of shrimp allergy across different studies, ranging from 10,2% in Italy, 2,8% in Iceland, to 0,3% in Denmark [15-17].

Lines 98-108: References are missing.

Answer: Corrected. Thank you.

Lines 265-266: Please clarify this. I don’t understand section 7.1.1. The same for the beginning of section 7.3.1. and 7.3.2.

Answer: The sections have been modified as follows:

This section provides a brief overview of the biochemical properties and protein structure of the most relevant identified mollusk allergens.

7.1. Lepetellida

7.1.1. Haliotis laevigata & Haliotis rubra

Hal l 1: A single reference is provided below concerning information related to its protein structure, along with some annotations regarding cases related to this sensitization. Specifically, 5 out of 9 individuals with case histories of allergy to consumption of crustacean shellfish and one after handling prawns were regarded as positive. All patients showed a positive (≥0.35 kU) ImmunoCAP result for oyster. Additionally, tests confirmed IgE binding to abalone tropomyosin in reducing immunoblots [48].

 

7.3.1. Crassostrea angulata

Cra a 1: Tropomyosin (TM), an important allergen of Crassostrea angulata, was purified and identified by mass spectrometry. Subsequently, TM was cloned and expressed, with a sequence of size 852 bp, encoding 284 amino acid residues. The results of circular dichroism, digestion assay, inhibition enzyme-linked immunosorbent assay, and basophilactivation test showed that recombinant TM had similar physicochemical properties and immunological properties to native TM. Furthermore, two conformational mimotopes were obtained and 10 IgE linear epitopes were verified. Meanwhile, different degrees of cross-reactivity were observed between C. ngulate ™ and the other 8 shellfish (TMs) using antibodies and serological analysis, which may relate to the 3 conserved epitope regions. These findings are expected to provide a theoretical basis for the molecular diagnosis of oyster allergy and cross-reactivity among shellfish [50].

 

Cra a 2: Arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata. The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs [51].

Cra a 4 A 20 kDa protein was purified from oyster and confirmed 27 to be sarcoplasmic-calcium-binding protein (SCP) by LC-MS/MS. A 537 bp open 28 reading frame was obtained from oyster SCP total RNA, which encoded 179 amino 29 acids, and was expressed in Escherichia coli. According to the circular dichroism 30 results, digestion assay, and inhibition ELISA, the recombinant SCP (rSCP) exhibited 31 similar physicochemical properties and IgG-binding activity to native SCP. rSCP 32 displayed stronger IgE-binding activity by immunological method. Moreover, a 33 different intensity of cross-reactivity and sequence homology were demonstrated 34 between shellfish species. Collectively, these findings provide novel insight into 35 shellfish allergens, which can be used to aid in the in vitro diagnosis of 36 oyster-sensitized patients (GenBank: QIJ32297.1).

7.3.2. Crassostrea gigas

Cra g 1: A 20 kDa protein was purified from oyster and confirmed to be a sarcoplasmic-calcium-binding protein (SCP) by LC-MS/MS. A 537 base pairs open reading frame was obtained from oyster SCP total RNA, which encoded 179 amino acids, and was expressed in Escherichia coli. According to the circular dichroism results, digestion assay, and inhibition ELISA, the recombinant SCP (rSCP) exhibited similar physicochemical properties and IgG-binding activity to native SCP. rSCP displayed stronger IgE binding activity by immunological method. Moreover, a different intensity of cross-reactivity and sequence homology were demonstrated between shellfish species. Collectively, these findings provide novel insight into shellfish allergens, which can be used to aid in the in vitro diagnosis of oyster-sensitized patients [52].

 

 

The background provided in section 8 should be moved to the Introduction.

Answer: Corrected. Background from section 8 has been moved to the Introduction as follows:

Among the various subgroups within shellfish allergy, crustacean allergy stands out as the most prevalent and extensively studied. Consequently, much of the research in this field has been focused on crustaceans. Tropomyosin (TM) was the first allergen identified in Penaeus indicus (shrimp), and it has long been recognized as the primary allergen associated with shellfish allergy. Interestingly, this panallergen has also been found in various invertebrate species such as cockroaches, Anisakis simplex, and dust mites, suggesting potential cross-reactivity between shellfish and other invertebrates [7].

However, subsequent investigations have revealed the complexity and diversity of the allergenic composition of shellfish. Several proteins shared between mollusks and crustaceans have been identified, potentially contributing to cross-reactivity. These include arginine kinase (AK), myosin light chain, sarcoplasmic calcium-binding protein (SCBP), troponin C, hemocyanin, triose phosphate isomerase, and others [7].

Section 10 is too extensive, please revise it properly. Some parts of this section are Discussion.

 Answer: Section 10 has been shortened and the following text has been moved to section 8 (State of the Art):

Allergy to gastropods is inadequately documented in the scientific literature, with only a limited number of reported cases. This scarcity of documentation may be attributed to the localized consumption of this type of shellfish, primarily in regions such as Spain, France, Italy, and Portugal. Additionally, coastal regions of Asia are known for their significant consumption of mollusks (10), contributing to the prevalence of gastropod allergy in these areas [10].

Unlike other shellfish allergies, reactions to gastropods often manifest later and tend to be more severe, frequently involving severe respiratory symptoms. Due to the potential severity of these reactions, it is advisable for individuals experiencing suggestive symptoms to avoid not only ingesting gastropods but also inhaling cooking vapors or coming into contact with these shellfish.

At present, our diagnostic capabilities for gastropod allergy are limited. We rely on commercial snail extract available for conducting skin prick tests and specific IgE testing against snail allergens. Unfortunately, there are no commercial extracts or specific IgE avalaible for limpet and/or abalone, necessitating the use of fresh raw and cooked food for skin prick tests. Additionally, we have access to the ALEX technique, which includes a panel of five shellfish allergens (Pen m 1, Pen m 2, Pen m 3, Pen m 4 y Cra c 6). However, further studies are required to ascertain the reliability of these allergens for diagnosing gastropod allergy in our patients. It's worth noting that our group has presented preliminary findings at the EAACI 2023, indicating some degree of allergen recognition among a subset of patients using the ALEX technique [25].

While the gold standard for diagnosing food allergies remains the oral tolerance test, in many cases, a comprehensive medical history combined with positive results from a skin prick test or specific IgE testing may provide sufficient confirmation. This approach is particularly applicable given the often-severe reactions experienced by patients following ingestion of certain types of shellfish. Additionally, in mild cases, many patients may opt out of undergoing the oral tolerance test.

Format the references according to the journal’s guidelines.

Answer: References have beer formatted according to the journal’s guidelines.

 

 

 

Comments on the Quality of English Language

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing all my recommendations.