A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders

Humans evolved by losing the capacity to synthesize the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal), which resulted in the development of a protective response mediated by anti-α-Gal IgM/IgG/IgA antibodies against pathogens containing this modification on membrane proteins. As an evolutionary trade-off, humans can develop the alpha-Gal syndrome (AGS), a recently diagnosed disease mediated by anti-α-Gal IgE antibodies and associated with allergic reactions to mammalian meat consumption and tick bites. However, the anti-α-Gal antibody response may be associated with other immune-mediated disorders such as those occurring in patients with COVID-19 and Guillain-Barré syndrome (GBS). Here, we provide a dataset (209 entries) on the IgE/IgM/IgG/IgA anti-α-Gal antibody response in healthy individuals and patients diagnosed with AGS, tick-borne allergies, GBS and COVID-19. The data allows correlative analyses of the anti-α-Gal antibody response with factors such as patient and clinical characteristics, record of tick bites, blood group, age and sex. These analyses could provide insights into the role of anti-α-Gal antibody response in disease symptomatology and possible protective mechanisms.

Factors that may affect the antibody response to α-Gal include but are not limited to age, repeat consumption of certain food and meats of different origin or innards with higher α-Gal content, exposure to tick bites, ABO blood group, co-occurring disorders and exposure to cats and other pets (Cabezas-Cruz et al., 2017;Cabezas-Cruz et al., 2019;Commins, 2016;Commins et al., 2014;de la Fuente et al., 2020a;Fischer et al., 2014;Fischer et al., 2016;Morisset et al., 2012;Platts-Mills et al., 2020;Wölbing et al., 2013).Additionally, the anti-α-Gal-specific IgE response has been associated with other diseases such as atopy, coronary artery disease and atherosclerosis (Gonzalez-Quintela et al., 2014;Wilson et al., 2017;Wilson et al., 2019).Furthermore, α-Gal-mediated innate and adaptive immune response mechanisms have been associated with protection against pathogen infection in various animal models (Hodžić et al., 2020a).However, little is known about the influence of anti-α-Gal immune response on immune-mediated disorders such as those occurring in patients with COVID-19 and Guillain-Barré syndrome (GBS).
These results raise questions and hypothesis regarding the role of α-Gal-mediated immune responses in disease symptomatology and possible protective mechanisms (de la Fuente et al., 2019b;de la Fuente et al., 2020b;Pacheco et al., 2021;Urra et al., 2021).Consequently, to advance in addressing these questions and hypothesis, here we provide data on the IgE/IgM/IgG/IgA anti-α-Gal antibody response in healthy individuals and patients diagnosed with AGS, tick-borne allergies, GBS and COVID-19.These data contribute to correlative analyses of the anti-α-Gal antibody response with factors such as patient and clinical characteristics, record of tick bites, blood group, age and sex.These analyses could provide insights into the role of anti-α-Gal antibody response in disease symptomatology and protection against immune-mediated disorders.

Patients and healthy individuals
A retrospective case-control study was conducted in patients suffering from COVID-19 admitted to the University General Hospital of Ciudad Real (HGUCR), Spain from March 1 to April 15, 2020.The infection by SARS-CoV-2 was confirmed in all patients included in the study by the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay from Abbott Laboratories (Abbott RealTime SARS-COV-2 assay, Abbott Park, Illinois, USA) from upper respiratory tract samples after hospital admission.Clinical features, as well as laboratory determinations were obtained from patient's medical records.The patients were grouped as hospital discharge, hospitalized and intensive care unit (Urra et al., 2021).Patients were hospitalized for developing a moderate-severe clinical condition with radiologically demonstrated pneumonia and failure in blood oxygen saturation.Patients with acute respiratory failure who needed mechanical ventilation support were admitted to a hospital ICU.The patients were discharged from the hospital due to the clinical and radiological improvement of pneumonia caused by the SARS-CoV-2, along with the normalization of analytical parameters indicative of inflammation, such as C-reactive protein (CRP), D-Dimer and blood cell count (Urra et al., 2021).Samples from asymptomatic COVID-19 cases with positive anti-SARS-CoV-2 IgG antibody titers but negative by RT-PCR were collected in May 22-29, 2020 and included in the dataset (Urra et al., 2021).Samples from healthy individuals (individuals without record of tick bites and allergic reactions) and patients diagnosed with tick-borne allergic reactions (AGS, anaphylaxis or urticaria) were collected prior to COVID-19 pandemic in April 2019(Pacheco et al., 2021).The use of human peripheral blood serum samples from healthy individuals and patients diagnosed with tick-borne allergic reactions was done with their written informed consent in compliance with the Helsinki Declaration.Nursing personnel at the General University Hospital of Ciudad Real, Spain, extracted blood samples.Samples and data from patients with GBS included in this dataset were provided by the BioB-HVS, integrated into the Spanish National Biobanks Network.All samples were processed following standard operating procedures with the appropriate approval of the Ethical and Scientific Committees (Toledo Hospitable Complex 29012014-No17, University Hospital of Ciudad Real C-352 and SESCAM C-73).

Preparation of serum samples
For the preparation of serum samples, a sterile tube without anticoagulant was used to collect blood samples.The blood from each patient and the healthy individual was maintained in standing position at room temperature (RT) for clotting (20-30 min) and centrifuged at 1,500 × g for 20 min at RT. Serum was collected and conserved at -20°C until used for analysis.
Human serum samples were diluted 1:100 in PBST with 1% HAS and 100 µl/well were added into the wells of the antigen-coated plates and incubated for 1 h at 37°C.Plates were washed four times with PBST and 100 µl/well of goat antihuman immunoglobulins-peroxidase IgG (FC specific) (Cat.No. I2136), IgM (µ-chain specific) (Cat.No. I1636), and IgE (ɛ-chain specific) (Cat.No. I6284) secondary antibodies (Sigma-Aldrich) diluted 1:1000, v/v in blocking solution were added and incubated for 1 h at RT. Plates were washed four times with 100 µl/well of PBST and 100 µl/well of 3,3,´5,5-tetramethylbenzidine TMB (Promega, Madison, WI, USA) were added and incubated for 20 min at RT. Finally, the reaction was stopped with 50 µl/well of 2 N H 2 SO 4 and the O.D. was measured in a spectrophotometer at 450 nm.The average of two technical replicates per sample was used for analysis after background (coated wells incubated with PBS and secondary antibodies) subtraction.(p < 0.01; https://www.socscistatistics.com/tests/spearman/default2.aspx)was conducted between anti-α-Gal IgE, IgM and IgG antibody titers and age (Figure 1B).

Dataset validation
The dataset (de la Fuente et al., 2020) was validated in studies reported by Urra et al. (2021), Pacheco et al. (2020) and Doncel-Pérez et al. (2020).A recent study correlated blood group with anti-α-Gal antibody response and SARS-CoV-2 infection (Hodžić et al., 2020b).Despite the presence of relatively high anti-α-Gal IgE levels in healthy individuals, factors such as tick bites or allergy correlate with higher IgE antibody titers against α-Gal (Pacheco et al., 2021).Additionally, a comparative analysis was conducted between the IgE+IgM+IgG antibody response to α-Gal and blood groups (Figure 1A), age (Figure 1B) and sex (Figure 1C) in healthy individuals (n = 75) to illustrate lower antibody titers in blood group B/AB individuals as previously reported (Cabezas-Cruz et al., 2017) but no differences regarding age and sex, which have been reported before as factors affecting the antibody response to α-Gal, infection and vaccination (Buonomano et al., 1999;Giefing-Kröll et al., 2015;Wang et al., 1995).
The main limitation of the dataset is sample size for some factors (i.e.age, sex or blood group), which were not disclosed by all individuals, and anti-α-Gal IgA antibody titers that could be considered in the analysis (Mateos-Hernández et al., 2020;Urra et al., 2021).

Mona Mahmoud
Parasitology and Animal Diseases Department, National Research Center, Giza, Egypt The article is scientifically sound.The data includes a dataset of anti-alphaGal IgM, IgG, and IgE levels in the serum of healthy people and patients, including COVID-19 patients, tick bite patients, and patients with Guillain-Barré syndrome.The rationale and methods are stated clearly, and the dataset is provided in a way that is both accessible and convenient.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 20 July 2021 https://doi.org/10.5256/f1000research.57019.r86098The authors validate their dataset using comparative analysis of antibody titers of healthy individuals with blood group, age and sex.What is the rational to use a summation of α-Gal IgG, IgM and IgE antibody levels for these analyses?Since different antibody isotypes are associated with certain immunological functions (effector functions mediated by Fc part of the antibody) and certain diseases are associated with certain antibody isotypes (e.g.AGS and α-Gal IgE), a summation of different antibody isotype levels is in my opinion not correct and does not provide meaningful scientific insights.Additionally, since entries for the key characteristics are incomplete for a large proportion of the individuals included, the used comparative analysis is not generally applicable for the presented dataset and thus not appropriate for its validation. 1.
The OD 450 values and thus the titers of α-Gal specific IgE are unexpectedly high in almost half of the healthy individuals.Is there an explanation for α-Gal IgE titers in serum of healthy individuals which are as high as in AGS patients or individuals with tick-bite associated allergic reactions?Can these individuals for certain be classified as "healthy" or how can sensitization to α-Gal be explained (atopy)?In my opinion, the ELISA data presented in this manuscript requires validation due to the unexpected and controversial high titers of α-Gal specific IgE antibodies in "healthy" individuals e.g. by using α-Gal ImmunoCAP or the commercially available α-Gal IgE ELISA Kit.

2.
As also stated by the authors as main limitation of their dataset, key characteristics such as age, sex and blood group are missing for a large proportion of the entries.This incompleteness significantly limits the benefit of the dataset for other research questions.Reviewer Expertise: Glycobiology, Host-pathogens interactions, histo-blood group antigens I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com methods used for the generation of the dataset (de la Fuente et al., 2020) were described in Urra et al. (2021) with additional information in Pacheco et al. (2021) and Doncel-Pérez et al. (2020).
Figure 1.An example of the effect of certain factors such as (A) blood group, (B) age and (C) sex on the antibody response to α-Gal in healthy individuals.Anti-α-Gal IgE, IgM and IgG antibody titers were determined by ELISA.(A, C) The ELISA O.D. at 450 nm values were compared for each Ig by one-way ANOVA test (p < 0.05).(B) A Spearman Rho correlation analysis (p < 0.01) was conducted between antiα-Gal IgE, IgM and IgG antibody titers and age.Correlation coefficient (R 2 ) is shown.Please refer to Pacheco et al. (2021) for validation of the ELISA with ImmunoCAP Phadia 250 automated platform (Thermo Fisher Scientific, Uppsala, Sweden) with the commercial ImmunoCap α-Gal bovine Thyroglobulin kit according to the manufacturer's instructions.

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73 (7): 1525-1531 PubMed Abstract | Publisher Full Text 3. Cabezas-Cruz A, Mateos-Hernández L, Alberdi P, Villar M, et al.: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases.Experimental & Molecular Medicine.2017; 49 (3).Publisher Full Text Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.CRCINA, Université de Nantes, Inserm, Nantes, France No further comments to make.Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.Reviewer Expertise: Glycobiology, Host-pathogens interactions, histo-blood group antigens I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Version 1 Reviewer Report 18 May 2021 https://doi.org/10.5256/f1000research.30388.r84326© 2021 Biedermann T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Tilo Biedermann 1 Department of Dermatology and Allergology, Technical University of Munich, Munich, Germany 2 Clinical Unit Allergology, Hemholtz Zentrum Munchen, German Research Centre for Environmental Health, Neuherberg, Germany The data note presented by José de la Fuente and colleagues contains α-Gal specific antibody titers of IgE, IgM, IgG isotypes in the serum and, for some individuals, IgA isotype in saliva of healthy individuals as well as patients with tick bite-associated symptoms, Guillain-Barré syndrome and COVID-19 infection, respectively.The dataset was already used in a published study by the authors correlating α-Gal specific antibodies and COVID-19 disease symptoms (Urra et al. 2020, 1 ).Although the dataset is given in an easily accessible format (Excel sheet) and methods are adequately described, I have some major concerns in regard to the benefit of the data for other research applications which are listed below.
JM, Ferreras-Colino E, Contreras M, Cabrera CM, et al.: The antibody response to the glycan α-Gal correlates with COVID-19 disease symptoms.J Med Virol.93 (4): 2065-2075 PubMed Abstract | Publisher Full Text Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?No Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Partly Competing Interests: No competing interests were disclosed.Reviewer Expertise: Immunology, Allergy, Dermatology.I would only like to see a clarification concerning the exact alphaGal-BSA antigen that was used for coating.The structure of the oligosaccharide should be given.Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.