ORIGINAL RESEARCH published: 31 March 2022 doi: 10.3389/fimmu.2022.873019 Poly-L-Lysine-Based aGal- Glycoconjugates for Treating Anti- aGal IgE-Mediated Diseases Sara Olivera-Ardid 1, Daniel Bello-Gil 1*, Alexander Tuzikov 2, Ricardo N. Araujo 3, Yara Ferrero-Alves 1, Blanca Esther Garcı́a Figueroa 4,5,6, Moisés Labrador-Horrillo 6,7,8,9, Ana L. Garcı́a-Pérez 10, Nicolai Bovin 2 and Rafael Mañez 1,11,12 1 RemAb Therapeutics, Mòdul de Recerca B, UAB Bellaterra, Barcelona, Spain, 2 Department of Chemical Biology of Glycans and Lipids, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences (RAS), Moscow, Russia, 3 Laboratório de Artrópodes Hematófagos, Departamento de Parasitologia, ICB/UFMG, Belo Horizonte, Brazil, 4 MEGA: Asthma Inception and Progression Mechanisms, Complejo Hospitalario de Navarra (CHN), Pamplona, Spain, 5 Instituto de investigación sanitaria de Navarra (IdiSNA), Pamplona, Spain, 6 ARADyAL Research Network, Instituto de Salud Carlos III (ISCIII), Madrid, Spain, 7 Department of Medicine, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 8 Allergy Section, Internal Medicine Department, Hospital Universitari Vall d’Hebron (HUVH), Barcelona, Spain, 9 Immunomediated Diseases and Innovative Therapies, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain, 10 Departamento de Sanidad Animal, Instituto Vasco de Investigación de Desarrollo Agrario (NEIKER), Derio, Spain, 11 Hospital Universitari de Bellvitge, Servicio de Medicina Intensiva, Hospitalet de Llobregat, Barcelona, Spain, 12 Instituto de Investigación Biomédica de Bellvitge (IDIBELL), Grupo Edited by: Inmunidad Innata y Patologı´a del Paciente Crı´tico, Hospitalet de Llobregat, Barcelona, Spain Pedro A Reche, Complutense University of Madrid, Spain Anti-aGal IgE antibodies mediate a spreading allergic condition known as aGal-syndrome Reviewed by: (AGS). People exposed to hard tick bites are sensitized to aGal, producing elevated levels Lourdes Mateos Hernández, of anti-aGal IgE, which are responsible for AGS. This work presents an immunotherapy Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement based on polymeric aGal-glycoconjugates for potentially treating allergic disorders by et du Travail (ANSES), France selectively inhibiting anti-aGal IgE antibodies. We synthesized a set of aGal- Said Rabbani, University of Basel, Switzerland glycoconjugates, based on poly-L-lysine of different degrees of polymerization *Correspondence: (DP1000, DP600, and DP100), to specifically inhibit in vitro the anti-aGal IgE antibodies Daniel Bello-Gil in the serum of aGal-sensitized patients (n=13). Moreover, an animal model for aGal daniel.bello@remabtx.com sensitization in GalT-KO mice was developed by intradermal administration of hard tick’ Specialty section: salivary gland extract, mimicking the sensitization mechanism postulated in humans. The This article was submitted to in vitro exposure to all polymeric glycoconjugates (5-10-20-50-100 µg/mL) mainly Vaccines and Molecular Therapeutics, inhibited anti-aGal IgE and IgM isotypes, with a lower inhibition effect on the IgA and a section of the journal Frontiers in Immunology IgG, respectively. We demonstrated a differential anti-aGal isotype inhibition as a function Received: 10 February 2022 of the length of the poly-L-lysine and the number of aGal residues exposed in the Accepted: 02 March 2022 glycoconjugates. These results defined a minimum of 27 aGal residues to inhibit most of Published: 31 March 2022 the induced anti-aGal IgE in vitro. Furthermore, the aGal-glycoconjugate DP1000- Citation: Olivera-Ardid S, Bello-Gil D, Tuzikov A, RA0118 (10 mg/kg sc.) showed a high capacity to remove the anti-aGal IgE antibodies Araujo RN, Ferrero-Alves Y, (≥75% on average) induced in GalT-KO mice, together with similar inhibition for circulating Garcı´a Figueroa BE, Labrador-Horrillo M, anti-aGal IgG and IgM. Our study suggests the potential clinical use of poly-L-lysine- Garcı´a-Pérez AL, Bovin N and Mañez R (2022) Poly-L-Lysine-Based based aGal-glycoconjugates for treating allergic disorders mediated by anti-aGal aGal-Glycoconjugates for Treating IgE antibodies. Anti-aGal IgE-Mediated Diseases. Front. Immunol. 13:873019. Keywords: aGal-syndrome, poly-L-lysine-based aGal-glycoconjugates, anti-aGal IgE inhibition, GalT-KO doi: 10.3389/fimmu.2022.873019 mice, immunotherapy Frontiers in Immunology | www.frontiersin.org 1 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS INTRODUCTION Type-I allergic conditions are disorders mediated by IgE, eliciting hypersensitivity to various allergens (1). IgE antibodies orchestrate an abnormal adaptive response against non- infectious, harmless, exogenous, and environmental substances, including glycoproteins from grass, pollen, dust mites, insect venom, and food (1). The number of people affected by such disorders is continuously growing globally (2, 3). Plasma exchange, immunosuppressive drugs, and monoclonal antibodies are potential immunotherapies focused on reducing IgE levels (4). Omalizumab (Xolair®), an unspecific treatment directed to total IgE, is the only anti-IgE therapy approved to treat moderate to severe asthma and chronic idiopathic urticaria (5). However, natural IgE has been described to participate in the FIGURE 1 | Hard ticks as the cause for aGal syndrome. Worldwide map reflecting (in green) the 17 countries in which hard ticks from different species physiological host resistance against certain parasites such as have been detected to be causative of aGal syndrome (AGS). The list of arthropods and helminths (6, 7). Thus, the total removal of IgE is countries includes Australia, United States, Germany, France, Spain, Belgium, a concern. Switzerland, Sweden, United Kingdom, Italy, Norway, Korea, Japan, Panama, Increasing evidence describes the functional involvement of Brazil, Ivory Coast, and South Africa. anti-aGal antibodies in different human disorders (8), including aGal-syndrome (AGS) (9, 10). These antibodies bind to Gala1,3Gal and Gala1,3Galb1,4GlcNAc oligosaccharides condition is higher in countries where individuals are in contact (aGal) (11), although with higher affinity to the free with ticks (27). The natural habitat of this type of tick is being trisaccharide (12). Primates, including apes, and Old-World significantly impacted by climate change, occupying ever larger monkeys, do not express the aGal epitopes due to an evolutive regions worldwide (28). Therefore, a notable increase in patients inactivation of the gene coding for the a1,3-galactosyltransferase with AGS is expected in the coming years. Currently, the enzyme (13, 14); consequently, they naturally produce these seventeen countries where there are registries of hard ticks as antibodies. Some evidence links their origin to the gut the causative agent of the aGal syndrome include Australia microbiota (15, 16). aGal residue is expressed in glycolipids [Ixodes holocyclus (29)] and the United States [Amblyomma and glycoproteins of the cell membrane of different Americanum (30)]. In Europe, the endemic tick is Ixodes ricinus, microorganisms, including viruses, bacteria, and protozoans found in Germany (31), France (32), Spain (33), Belgium (34), (8). Hence, it has been associated with a possible protective Switzerland (35), Sweden (36), United Kingdom (37), Italy (38) role of anti-aGal antibodies (17). However, the existing and Norway (39). The list of affected countries also includes Korea epidemiological evidence is controversial. High serological [Ixodes nipponensis (40)], Japan [Haemaphysalis longicornis (41)], levels of anti-aGal IgM at the start of dialysis therapy have Panama [Ixodes cajennense (42)], Brazil [Amblyomma sculptum been described as a predictor of later risk for mortality and (43)], Ivory Coast [Amblyomma variegatum (44)] and South enteric peritonitis in peritoneal dialysis patients (18). Africa (45). Hard ticks’ saliva, salivary glands, and midgut Furthermore, anti-aGal IgM was associated with protection contain proteins decorated with aGal residues (42, 45). against malaria in infants (19) and children >4 years old (20). Sensitization is postulated to start with the allergen uptake by On the contrary, anti-aGal IgG has been associated with a higher APCs in the epidermis (46). Then, the sensitization mechanism risk of malaria infection in children (19, 20). Additionally, anti- promotes the class switch recombination in B cells and the aGal IgM and IgG, but not IgE antibodies, were significantly subsequent secretion of IgE antibodies in the skin-draining higher in uninfected than Plasmodium falciparum- and lymph nodes (46, 47). Anti-aGal IgE-switched B cell precursor Mycobacterium tuberculosis-infected individuals (21). Similarly, seems to be a naive non-switched B cell (48). Re-exposure to the experimental models have also demonstrated protection against allergen leads to the activation and degranulation of mast cells and lethal Trypanosoma cruzi challenge (22) and malaria by basophils. Moreover, the allergic reaction is triggered 3-6 h after prophylactic vaccination with an aGal-based compound and mammalian meat ingestion due to the binding of anti-aGal IgE oral administration of Escherichia coli O86:B7 (20), respectively. antibodies to aGal epitopes expressed in the meat (24, 49). Interestingly, oral administration of the same bacterium protects Regarding Cetuximab allergy, anti-aGal IgE binding to aGal turkeys from developing acute aspergillosis. Nevertheless, this residues in the monoclonal antibody induces an immediate effect was not associated with augmented anti-aGal IgY levels systemic allergic reaction that can be severe enough to trigger a but with an apparent reduction of anti-aGal IgA in the lungs of life-threatening anaphylactic shock (50, 51). infected animals (23). Patients with AGS exhibit various clinical symptoms, AGS symptoms occur after red meat intake (24) or exposition including urticaria, pruritus, angioedema, and systematic to other products containing aGal like Cetuximab (25). Hard anaphylaxis. In addition, some patients have reported specific ticks are associated with the anti-aGal IgE sensitization and AGS symptoms, such as nausea, indigestion, diarrhea, and abdominal spreading (26, 27) (Figure 1). The prevalence of this pathological discomfort (51, 52). Frontiers in Immunology | www.frontiersin.org 2 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS Anti-aGal antibodies (IgM, IgG) have previously been removed in rodents and primates using a poly-L-lysine-based aGal-glycoconjugate (GAS914), without side effects, minimal complement activation, and no sensitization (53). GAS914 was developed to overcome the hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation primarily caused by anti-aGal antibodies (53). However, GAS914 never reached the clinic due to the participation of anti-non-aGal antibodies in the mentioned rejection mechanism (54). Additionally, the development of a1,3-galactosyltransferase gene-knockout transgenic pigs made unnecessary the clinical development of GAS914 for this indication (55, 56). The lack of sensitization, together with the high capacity of GAS914 to inhibit anti-aGal antibodies (IgM and IgG), prompted us to study the in vitro and in vivo removal of anti-aGal IgE antibodies with a set of poly-L-lysine-based aGal-glycoconjugates as a potential treatment for diseases mediated by such antibodies. MATERIALS AND METHODS FIGURE 2 | General structure of polymeric aGal-glycoconjugates. Poly-L- Polymeric aGal-Glycoconjugates lysine backbone (degree of polymerization, DP: 100, 600, and 1000) were GAS914 (Novartis Pharma AG, Basel, Switzerland) is a poly-L- derivatized with the trisaccharide Gala1-3Galb1-4GlcNAcb-sp, (sp = -O(CH2) lysine backbone with an average degree of polymerization (DP) 3NH2). Percent of modification with aGal = a (9, 12, 18, 27 and 34%). of 1,000 L-lysines (DP1000) and with 23-28% of lysines Residual amino groups of poly-L-lysine were acylated with glycolic acid. derivatized with the oligosaccharide Gala1,3Galb1,4GlcNAc- (aGal) (53). GAS914 was used as anti-aGal inhibitor control. controls (CN, n=8). Serum samples from aGal-sensitized Analysis of 1H-NMR allowed determining the load of aGal in subjects (PT, n=13) were selected from previous epidemiological GAS914 (Figure S1). studies (59), where anti-aGal IgE prevalence in individuals with The RA01-compounds were specifically synthesized for this acute urticaria or anaphylaxis from different geographical areas work from a linear poly-L-lysine (57) of a final DP of 100, 600, of Spain was studied. 1000 (DP100, DP600, and DP1000, respectively) and increasing loads of Gala1,3Galb1,4GlcNAc- in the final structure. DP1000 In Vitro Inhibition of Anti-aGal With glycoconjugates were produced following the synthetic route Polymeric aGal-Glycoconjugates described by Duthaler et al., 2010 (58). For DP100 and DP600 To test the inhibition of anti-aGal antibodies, human serum glycoconjugates, poly-L-lysine hydrobromide was acylated with a samples (PT, n=13) were incubated for 15-17 h under mild calculated amount of Gala1,3Galb1,4GlcNAc (aGal)-sp-Ad-ONSu orbital shaking (225 rpm at 4°C) with each glycopolymer at active ester in DMSO in the presence of Et3N. The residual amino growing concentrations (5-10-20-50-100 µg/mL). The final groups were acylated (in the same reaction mixture) with an excess volume of the reaction was set at 100 µL. Vehicle (PBS)-treated of glycolic acid acetate succinimide ester AcOCH2(CO)ONSu in the serum was incubated under the same conditions as a control presence of Et3N. To remove acetyl protecting groups by hydrolysis, (baseline). After incubation, the serum samples containing the the reaction mixture was diluted with a twofold volume of water, different glycopolymers or PBS (control for each serum) were and Et3N was added (2% of the volume of the solution). conveniently diluted to determine the unbound fraction of the Glycopolymers were isolated by gel-permeating chromatography different anti-aGal isotypes, by ELISA, following the general on Sephadex LH-20 in MeCN-water 30:70 by volume. Fractions protocol previously described (15). Briefly, Nunc MaxiSorpTM contained pure conjugate were evaporated to ~2 mL volume and 96-well flat-bottom plates (Thermo Fisher Scientific, Waltham, freeze-dried. The purity and composition of the synthesized MA, USA) were coated with 2.5 mg/mL of Gala1,3Galb1,4GlcNAc glycoconjugates (the percentage of modification of poly-L-lysine glycan conjugated to human serum albumin (HSA). After with Gala1,3Galb1,4GlcNAc) were determined by the 1H NMR washing and blocking steps, serum samples diluted in PBS spectroscopy (Figure S2). The complete list of resulting polymeric (1:100 for IgM and IgG, 1:25 for IgA, and 1:10 for IgE) were glycoconjugates is shown in Table S1. Figure 2 provides the added to the wells and incubated for 1 h at 25°C. After washing, general structure for this set of glycopolymers. the incubated for 1 h at 25°C with the corresponding horseradish peroxidase (HRP)-labeled anti-human or anti-mouse secondary Human Serum Samples antibodies diluted in PBS. o-Phenylenediamine dihydrochloride Normal Human Sera (NHS) from donors of the Blood Bank of (OPD) was used as HRP substrate, and incubated at 25°C in the the Hospital Universitari de Bellvitge were used as healthy dark. The reaction was stopped with 3N hydrochloric acid (HCl). Frontiers in Immunology | www.frontiersin.org 3 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS The resulting absorbance was registered at 492 nm using a PowerWave™ XS Microplate Reader (Biotek, Winooski, VT, USA). The resulting data were graphed as optical density units. Moreover, the in vitro data was the result of three independent experiments. The inhibition rate for each glycopolymer (expressed as a percentage of anti-aGal inhibition) was calculated according to the levels of anti-aGal antibodies determined in baseline (PBS) and treated (glycopolymer) conditions for each serum. a1,3-Galactosyltransferase Knocked Out (GalT-KO) Mice This study was performed in 48 mice of 24-32 weeks-old (sex parity), in which the gene coding for the a1,3- galactosyltransferase enzyme had been knocked out (GalT-KO mice) and was derived from a highly inbred colony with a hybrid FIGURE 3 | Scheme of GalT-KO mice sensitization for anti-aGal IgE production. Group 1: double negative control (PBS) (n=16). Group 2 (n=16) genetic background (B6xCBAx129sv) (60). Animals were and Group 3 (n=16) (aGal-sensitized mice) were treated with PBS (control) handled and housed as previously described (15). Procedures and DP1000-RA0118, respectively. Black arrows: bleeding (for immunological concerning all animals were supervised and approved by the determinations), blue arrows: PBS id. (control of sensitization) or sc. (control ethics committee for animal experimentation of Bellvitge of treatment), yellow arrows: salivary gland extract (20 or 5 µg id.), dark-red arrows: DP1000-RA0118 10 mg/kg sc. Biomedical Research Institute (IDIBELL) and the Catalonia Government (Record FUE-2018-00931758). The care, as well as the handling of the animals, were following the Guide for the Group 2 was a negative control for the treatment (vehicle: PBS Care and Use of Laboratory Animals that the US National sc.). In Group 3, animals were treated with three consecutive Institutes of Health published (NIH Publication n° 85–23 DP1000-RA0118 doses (10 mg/kg, sc.) on days 16 (4 h after last revised 1996) as well as the European Agreement of Vertebrate challenge), 17, and 18. The challenge was repeated in two Animal Protection for Experimental Use (86/609). The animals of Group 3 one week after the last treatment with procedure for euthanasia was established following the DP1000-RA0118 (day 26). Although each experimental group European Directive on protecting animals used for scientific was composed of sixteen animals, not all the parameters were purposes (2010/63/EU). Death was never considered a determined in the totality of mice due to welfare reasons. In the human endpoint. case of multiple blood extractions, volume never exceeded 7.5% of the total blood volume (124-158 µL of fresh blood in ~30 g Amblyomma sculptum Salivary mice) weekly (65). Animal blood was collected by controlled Gland Extract submandibular bleeding on days -3 (baseline), 16 (3 h after As previously described (61), salivary gland extract (SGE) was challenge), 18 (3 h after treatment), and 28 (after rechallenging, produced from 200 females of unfed A. sculptum. To obtain the two animals of Group 3) as previously described (66). SGE, females were washed with sterile water, and their salivary glands were individually dissected in saline (0.9% NaCl). Each Total Circulating Mouse IgM, IgG1, IgG2a, pair of glands were transferred to 1.5 mL tubes containing saline IgG2b, IgG3, and IgE by ELISA solution, placed in an ultrasonic bath for 40 seconds, centrifuged Total mouse serum immunoglobulins (n=6) were determined on at 14,000g for 5 min. Next, the supernatant was transferred to a days -3, 16, and 18 using the commercial RayBio® mouse ELISA new tube, dried under vacuum at 56°C to yield an amount of kit following manufacturer instructions (RayBiotech, GA, USA). 3.11 mg, and kept at -20°C until use. The amount of protein in the sample was measured by Bradford et al., 1976 (62) using White Blood Cells by Flow Cytometry bovine serum albumin as standard. Sterile PBS was conveniently Flow cytometry analysis was performed on a Gallios analyzer used as a vehicle to prepare the final aqueous solution injected as (Beckman Coulter, IN, USA) equipped with violet (405 nm), blue an allergen to the GalT-KO mice. (488 nm), and red (633 nm) solid-state lasers as an excitation source. Purified rat anti-mouse CD16/CD32 was added to the GalT-KO Mice Sensitization fresh blood samples to block non-antigen-specific binding GalT-KO mice were randomly separated into three different (Beckton Dickinson, CA, USA). After 5 min incubation at 25°C, groups (sex parity). Group 1 (n=16) was a double negative fluorochrome-conjugated antibodies (Table S2, Beckton control (PBS id. or sc. for sensitization and treatment, Dickinson, CA, USA) were added to the samples. BD FACS™ respectively). Group 2 (n=16) and 3 (n=16) were sensitized lysing solution was then added to lyse red blood cells. Samples with two doses of 20 mg id. of the salivary gland extract (days were homogenized with vortex and incubated for 5 min at 25°C 0 and 7, Figure 3). Animals were then challenged with three in the dark. After centrifugation (5 min, 3,220g, 25°C), the consecutive doses of 5 mg id. of the extract on days 14, 15, and 16 supernatant was discarded, and the pellet was resuspended in to induce the production of anti-aGal IgE antibodies (63, 64). 400 µL of PBS for immunophenotyping of different White Blood Frontiers in Immunology | www.frontiersin.org 4 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS Cells (WBC) for every experimental group (n=6) on days -3, 16, and 18. Events collected from fresh blood mouse samples were displayed in a CD45 vs. side scatter intensity (SS INT) plot to discard debris and define a total WBC population. Every single FACS determination recorded about 150,000 total events, of which 50,000 were CD45 positive. Fluorescence was collected through the corresponding bandpass filters for each indicated surface cell marker. Data were analyzed using KALUZA software (Beckman Coulter, CA, USA). Statistics GraphPad Prism statics software was used for analysis and data graphing. The Gaussian distribution of data was checked by the D’Agostino-Pearson omnibus normality test (alpha = 0.05), and homogeneity of variances was determined by the F test (alpha = 0.05). Most of the statistical analyses were performed using paired or unpaired parametric t-tests. The Wilcoxon matched- pairs signed-rank and Mann-Whitney tests (unpaired data analysis) were used as non-parametric tests when data did not follow a Gaussian distribution. Tukey and Sidak were used as multiple comparison tests. Differences were considered statistically significant when p<0.05 (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001), ns: non-significant. RESULTS FIGURE 4 | Serological levels of anti-aGal antibodies by ELISA. aGal- sensitized patients (PT, n=13, blue dots) showed significantly higher Prevalence of Anti-aGal IgE in aGal- serological anti-aGal antibodies levels (expressed as optical density units, 492 Sensitized Subjects nm) compared to healthy subjects (CN, n=8, grey dots). To detect each The prevalence of anti-aGal IgE in aGal-sensitized subjects has immunoglobulin isotype, serum samples were accordingly diluted: 1:100 for IgM and IgG, 1:25 for IgA, and 1:10 for IgE. Unpaired t-test analysis was been extensively reviewed (67–69). We confirmed by ELISA the performed (**: p < 0.01; ****: p < 0.0001). significantly elevated circulating levels of anti-aGal IgE antibodies in patients compared to controls. PT also showed higher serological levels for the rest of the anti-aGal isotypes studies human serum from aGal-sensitized patients instead of (IgM, IgG, and IgA) than CN (Figure 4). affinity-purified fractions of anti-aGal antibodies. Overall, the exposure to all polymeric glycoconjugates mainly Influence of the Multivalent aGal Exposure inhibited anti-aGal IgE and IgM isotypes, with a lower inhibition on the Inhibition of Anti-aGal IgE Antibodies effect on the IgA and IgG isotypes, respectively (Figure 5). We Anti-aGal antibodies inhibition with monovalent compounds found very similar inhibition rates for all the anti-aGal isotypes has been demonstrated as inefficient due to their low affinity for when comparing DP1000-RA0118 vs. GAS914 (positive single oligosaccharides (53). Previously, GAS914 has shown a control). Indeed, for IgE, DP1000-RA0118 showed slightly maximal increase in avidity (relative to the monomer) by anti- higher inhibition rates than GAS914. Additionally, reducing aGal IgM and IgG antibodies (53, 70). Nevertheless, there is no the aGal load in the same poly-L-lysine backbone to 9% data on whether the anti-aGal IgE inhibition could be affected (DP1000-RA0109) significantly reduced the inhibitory capacity using multivalent aGal compounds. For that, we synthesized two of the glycopolymer (Figure 5). Hence, DP1000-RA0118 was glycopolymers composed of a backbone of 1,000 L-lysines selected for the in vivo proof of concept. (DP1000), with 9 and 18% of lysine residues derivatized with Gala1,3Galb1,4GlcNAc- (DP1000-RA0109 and DP1000- In Vivo Inhibition of Anti-aGal IgE in RA0118, respectively). GAS914 (DP1000) with 27% aGal load Sensitized Mice (Figure 5) was used as a positive control for anti-aGal inhibition. The preliminary data obtained in vitro prompted us to It was previously demonstrated that the chromatographic investigate, as proof of concept, the anti-aGal IgE inhibitory affinity purification process could favor the preferential binding capacity of DP1000-RA0118 in a small animal model of aGal and elution of IgM over the rest of anti-aGal isotypes masking sensitization. Therefore, the main objective of this study was to the real inhibitory capacity of polyacrylamide-based aGal- induce anti-aGal IgE in a relevant animal model and to study its conjugates (71). Therefore, we used in the in vitro inhibitory intracorporeal removal with DP1000-RA0118. Frontiers in Immunology | www.frontiersin.org 5 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS FIGURE 5 | Influence of aGal load on anti-aGal antibodies inhibition. Serum samples from aGal-sensitized patients (n=13) were incubated with DP1000-RA0109, DP1000-RA0118 or GAS914 (positive control of inhibition) at growing concentrations (5-10-20-50-100 µg/mL). PBS-treated serum was similarly incubated as a baseline condition. After incubation, samples were conveniently diluted to determine by ELISA the unbound fraction of the different anti-aGal isotypes (1:100 for IgM and IgG, 1:25 for IgA, and 1:10 for IgE). The inhibition rate for each glycopolymer (expressed as a percentage of anti-aGal inhibition) was calculated according to the quantity of anti-aGal antibodies determined in baseline (PBS) and treated (glycopolymer) conditions for each serum. IgE green line, IgG orange line, IgM pink line, and IgA purple line. GalT-KO mice are considered an adequate model for aGal sensitization and production of anti-aGal IgE antibodies (15, 61, 72, 73). The sensitization (Figure 3) was conducted in immunologically mature GalT-KO mice (15). Significant induction of anti-aGal IgE antibodies was achieved in all the animals of Groups 2 and 3 on day 16 (Figure 6). This induction was also accompanied by significantly augmented anti-aGal IgG and IgM. Elevated levels of anti-aGal IgE antibodies were again induced on day 28 in two mice of Group 3 rechallenged with the SGE on day 26 (Figure 6). The treatment with DP1000-RA018 (day 18) produced a significant decrease in the levels of anti- aGal IgE in Group 3 (≥75% on average, Figure 6) as well as in anti-aGal IgG and IgM compared to the induced condition (Group 3, day 16). This decrease in the levels of anti-aGal can only be attributed to the in vivo inhibitory capacity of DP1000- RA0118 because in Group 2 (treated with PBS) the levels of these antibodies remained constant from day 16 (end of challenge) to day 18 (end of PBS treatment). Impact of Sensitization and Treatment With DP1000-RA0118 on Humoral and Cellular Immune Mediators Anti-aGal antibodies (IgM, IgG) have previously been removed in rodents and primates using GAS914 (53). Since these studies FIGURE 6 | A. sculptum salivary gland extract significantly induced anti-aGal were conducted in healthy animals (no sensitized), we antibodies in GalT-KO mice. Animals from Group 1 (in blue, n=6) are a double investigated whether some of the humoral and cellular negative control (PBS for sensitization and treatment, respectively). Animals of mediators of the immune system were affected by the Group 2 (in green, n=6) and 3 (in dark-red n=6) were sensitized to aGal according to Figure 2. In Group 3, after sensitization, animals were treated sensitization protocol and the treatment with DP1000-RA0118. with three consecutive DP1000-RA0118 doses (10 mg/kg, sc.) on days 16, Regarding immunoglobulins, despite the increase obtained 17, and 18. The challenge was repeated in two animals of Group 3 on day for anti-aGal antibodies after sensitization for Groups 2 and 3, 26, one week after the last treatment with DP1000-RA0118. Red columns the total IgM, IgG1, IgG2a, IgG3 remained constant during the represent the anti-aGal inhibition on day 18 (% referred to sensitization on evaluated days for all the experimental groups (Figure 7). day 16) in Group 3 (treated with DP1000-RA0118). Wilcoxon matched-pairs signed-rank was used as a non-parametric test (*: p < 0.05). However, total IgM slightly decreased from day 16 to 18 only Frontiers in Immunology | www.frontiersin.org 6 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS FIGURE 7 | The pattern of total immunoglobulin levels (IgE, IgG1, IgG2a, IgG2b, IgG3, and IgM) and their association with the different isotypes assessed for anti- aGal antibodies (IgE, IgG, and IgM). Relative serological levels of total immunoglobulins (expressed as OD units, 450 nm) and anti-aGal antibodies (expressed as OD units, 492 nm) were determined by ELISA on days -3 (baseline), 16 (after sensitization), and 18 (after treatment). GalT-KO mice from Group 1 are a double negative control (PBS for sensitization and treatment, respectively) (n=6). Animals of Groups 2 and 3 were sensitized to aGal, according to Figure 2. In Group 3, after sensitization, animals were treated with three consecutive DP1000-RA0118 doses (10 mg/kg, sc.) on days 16, 17, and 18. in Group 3. The treatment with DP1000-RA0118 (day 18) on basophils. On day 16, Groups 2 and 3 showed a significant removed preexisting and induced anti-aGal IgM, which increase in fluorescence compared to day -3 (baseline) (Figures impacted the total IgM levels measured. Interestingly, a slight S3B, C). Since the gated basophil population between increase in total IgE and IgG2b subtype was observed for Groups experimental groups was similar, the augmented fluorescence 2 and 3 after mice sensitization (day 16, Figure 7). These trends obtained for Groups 2 and 3 seems to be associated with the in both experimental groups seem to be partially associated with increased serological levels of anti-aGal IgE antibodies after the the induction on day 16 of augmented levels of anti-aGal sensitization procedure. On day 18, the basophil fluorescence antibodies for the IgE and IgG isotype, respectively. returned to baseline levels (Figure S3C). During aGal sensitization, the major change in the WBC population was registered for basophils (Figure S3). The rest of Influence of Degree of Polymerization on the the evaluated WBC remained unchanged between days and Differential Inhibition of Anti-aGal Isotypes experimental groups. Basophils were gated using rat anti- Along with specific inhibition of anti-aGal IgE, DP1000-RA0118 mouse Ly6G and IgE (Figure S3A). The later monoclonal showed a high inhibitory capacity to the rest of circulating anti- antibody allowed quantifying the IgE bound to Fcϵ receptors aGal isotypes. Although removing most of the circulating anti- Frontiers in Immunology | www.frontiersin.org 7 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS aGal isotypes might not represent an obstacle to the potential DP600-Glycopolymers clinical development of DP1000-RA0118 (74), we investigated From DP600-RA0112 to DP600-RA0134, glycoconjugates whether the DP can impact the differential inhibition of anti- showed a similar inhibitory capacity for all isotypes. From 10 aGal isotypes. For that, we synthesized a complementary set of µg/mL, the inhibition was >70% for IgE and IgM, 33-60% for aGal-glycoconjugates using polymeric backbones containing IgG, and 40-72% for IgA. Surprisingly, IgM inhibition rates 600 (DP600) and 100 (DP100) L-lysine residues. In addition, slightly decreased from 50 µg/mL in the case of RA0118, DP1000 aGal-glycoconjugates with 12% and 27% RA0127, and RA0134. DP600-RA0109 showed a similar trend Gala1,3Galb1,4GlcNAc load were also synthesized. All but with lower inhibition rates. Regardless of the glycan load, all glycopolymers were assessed in the same experimental setting the glycopolymers inhibited >85% of circulating anti-aGal IgE at to avoid biases. Like the pilot study, the exposure to all polymeric 100 µg/mL (Figure 8). glycoconjugates mainly inhibited anti-aGal IgE and IgM isotypes, with a lower inhibition effect on the IgA and IgG DP100-Glycopolymers isotypes, respectively (Figure 8). From DP100-RA0112, all DP100 glycoconjugates showed similar inhibitory capacity regardless of the Gala1,3Galb1,4GlcNAc DP1000-Glycopolymers percentage. Starting at 5 µg/mL, the inhibition was 25-75% for From DP1000-RA0112 to DP1000-RA0127, glycoconjugates IgE, 40-56% for IgM, 12-25% for IgG, and 20-45% for IgA. showed a similar inhibitory capacity for all isotypes at the Similar to DP600, IgM inhibition rates decrease from 20 µg/mL assessed concentrations. Starting at 10 µg/mL, the inhibition in the case of RA0118, RA0127, and RA0134. To note, DP100- was >85% for IgE and IgM, ~60% for IgG, and 50-80% for IgA. In RA0127 and DP100-RA0134 (100 µg/mL) showed a high IgE- contrast, the inhibition for DP1000-RA0109 was significantly inhibition rate (>75%), with minimal IgG inhibition lower than the other glycopolymers for all Ig-isotypes (Figure 8). (<25%, Figure 8). FIGURE 8 | Differential antibody inhibitory capacity of aGal glycoconjugates with different DP and aGal loads in serum from aGal-sensitized patients. Serum samples (n=13) were incubated with the glycopolymers at growing concentrations (5-10-20-50-100 µg/mL). PBS-treated serum was similarly incubated as a baseline condition. After incubation, samples were conveniently diluted to determine by ELISA the unbound fraction of the different anti-aGal isotypes (1:100 for IgM and IgG, 1:25 for IgA, and 1:10 for IgE). The inhibition rate for each glycopolymer (expressed as a percentage of anti-aGal inhibition) was calculated according to the quantity of anti-aGal antibodies determined in baseline (PBS) and treated (glycopolymer) conditions for each serum. IgE green line, IgG orange line, IgM pink line, and IgA purple line. Frontiers in Immunology | www.frontiersin.org 8 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS Interestingly, RA0112, RA0118, and RA0127 (DP600) explained by the fact that anti-aGal IgE are strictly induced showed similar IgE and IgA inhibitory capacities compared to antibodies, whereas the circulating anti-aGal IgG are mainly DP1000 glycopolymers with the same aGal load, except for composed of natural antibodies. Thus, despite the relatively RA0112 at 10 µg/mL for IgE. Additionally, RA0118 and RA0127 lower levels of anti-aGal IgE, their affinity seems to be higher (DP600) showed similar IgM and IgG inhibitory capacity than anti-aGal IgG. Consequently, we hypothesize that anti- compared to DP1000-homologues. aGal IgE might come from IgG-switched B cells, unlike the classic atopic sensitization to pollen and mite allergens that come from naive B cells (48). In contrast, previous studies described DISCUSSION that anti-aGal IgE might be predominantly formed by class switch from non-switched (IgM) B cells (80). This work shows that immunotherapy based on the We also developed a model of aGal-sensitization that intracorporeal inhibition of anti-aGal IgE antibodies with reproduced the findings obtained for patients. We achieved a poly-L-lysine-based aGal-glycoconjugates may be a potential clear induction of anti-aGal IgE, together with IgM and IgG after solution to treat AGS. The rationale for using poly-L-lysine- intradermal administration of hard ticks’ SGE. Unlike natural based aGal-glycoconjugates on removing anti-aGal antibodies anti-aGal antibodies (15), anti-aGal IgE are induced after lies in: i) anti-aGal antibodies have naturally high avidity for processing the aGal residues in tick saliva by APCs (Langerhans multivalent antigens, ii) absence of immune response against and dermal Dendritic Cells). APCs migrate then to skin draining either the carbohydrate or the poly-L-lysine in different murine lymph-node where aGal-specific B cells undergo clonal selection and primate species that spontaneously produce the anti-aGal (46). The increased levels of anti-aGal antibodies in sensitized antibodies (53), and iii) anti-AB0 group antibodies (similar in animals confirmed the implication of components from A. origin and structure to anti-aGal antibodies) have been safely sculptum SGE in the induction of anti-aGal antibodies (43, 81). maintained at low concentrations (plasmapheresis plus Furthermore, DP1000-RA0118 showed high efficacy for the immunosuppression) for a prolonged time in blood type- intracorporeal removal of anti-aGal in GalT-KO mice. At the incompatible kidney transplantation without side effects (74). cellular level, the WBC population remained unaltered except for Firstly, elevated levels of anti-aGal IgE antibodies were basophils that showed a higher fluorescence after the sensitization detected in patients compared to healthy volunteers (67–69). process in Groups 2 and 3 because of an augmented IgE binding to The same trend was observed for the rest of the anti-aGal Fcϵ receptors. In humans, basophil activation can determine the isotypes. In the initial pilot study, we demonstrated the severity of the clinical picture in patients with delayed anaphylaxis feasibility of improving the inhibitory capacity of aGal- due to the consumption of red meat (82, 83). glycopolymers by reducing the aGal load. This finding was The immune stimulation with the SGE was not restricted to unexpected according to the differences in aGal density in IgE. Elevated titers of anti-aGal IgG antibodies have been DP1000-RA0118 (180 residues) and GAS914 (270 residues), previously described in IgE-positive subjects (67, 80). respectively. An unfavorable spatial conformation due to a Specifically, the authors found more anti-aGal IgG1 antibodies higher aGal density in GAS914 compared to DP1000-RA0118 in IgE-positive subjects, contrasting the expected more IgG2 could explain these results (sterical hindrances) (71, 75). production specific for natural anti-aGal antibodies (67). These Conversely, DP1000-RA0109 (90 residues) showed a drastically results suggest that IgE directed to a carbohydrate antigen results reduced capacity to inhibit anti-aGal antibodies. The limited from the stimulation of a glycoprotein or glycolipid, even a exposure of the antigenic determinant (antigen/antibody ratio) bacterial immune stimulation with essentially the same antigen could explain this finding. already exists (80). In our study, the increase of anti-aGal IgG Additionally, anti-aGal IgE was the most inhibited isotype in and IgE directly impacted the rise of total IgG2b and IgE vitro with similar behavior for IgM and a lower inhibition for IgA immunoglobulins, respectively. and IgG, respectively. The later isotypes typically have higher Additionally, high levels of anti-aGal IgE antibodies in aGal- affinities for protein antigens (single binding site) than IgM. sensitized patients have been directly correlated to a total IgE However, IgM is a pentamer with ten Fab capable of a antibody increase (9). However, the significant contribution of multivalent binding (76). In addition, 20% of serological IgA anti-aGal IgG to the total IgG2b was surprising, indicating a exists as oligomers with multi-binding sites. On the other hand, preferential induction of this IgG subclass. GalT-KO mice IgG only exists as a monomer with two binding sites (71). These naturally produce anti-aGal antibodies (15, 70), with IgG3 as properties of antibodies were confirmed in our in vitro study, the predominant IgG subclass (manuscript submitted to where the IgM avidity was increased, exceeding the affinity of publication). In mice, the IgG3 subclass is functionally IgA and IgG. Similar results have been reported for equivalent to human IgG2, which predominantly recognizes polyacrylamide-based aGal-glycoconjugates (71). However, the carbohydrate epitopes (84, 85). Accordingly, two immune behavior of anti-aGal IgE, similar to IgM, was unexpected since responses could be distinguished against the aGal epitope in it is a monomer like IgG (77). Despite some structural differences the animal model: (I) the typical T-independent response (78), IgE and IgG share the highest homology compared to other promoted by the continuous antigenic stimulation of the isotypes (78, 79). However, the inhibition of anti-aGal IgG in intestinal microbiota (15), with IgG3 as the predominant patients was lower compared to IgE. This finding could be subclass and (II) an “atypical” Th2-response (hypersensitivity Frontiers in Immunology | www.frontiersin.org 9 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS type-I) which leads to the production of IgG2b and IgE in GalT- infections must be considered in case our therapy is KO mice. This immunological response is similar to that implemented to treat any disease mediated by anti-aGal IgE. described in humans sensitized after tick bites (9, 80) and in Nevertheless, it is essential to clarify that our therapy produced an GalT-KO models (46, 86) confirming the robustness of our immediate but transient inhibition of anti-aGal antibodies. More animal model for aGal sensitization. importantly, our data suggest that it is possible to get a differential DP1000-RA0118 inhibited anti-aGal IgE and IgG, with a anti-aGal isotype inhibition as a function of the length of the poly- discrete impact on the total level of mouse IgE and IgG2b, L-lysine backbone and total residues of aGal exposure in respectively. However, since the sensitization process was the polymers. conducted with SGE, we hypothesize that, together with the The present work confirmed that hard ticks’ SGE elements anti-aGal, other IgE and IgG2b specificities may be induced are responsible for the induction of anti-aGal IgE antibodies. too. That is why the induction process impacted the total levels We postulate that the aGal sensitization mechanism may go of IgE and IgG2b antibodies. Conversely, the treatment with through an “atypical” Th2-response (hypersensitivity type-I), DP1000-RA0118 was specific and only removed the anti-aGal which primarily led to IgG2b and IgE production in GalT-KO antibodies. Interestingly, total circulating IgM was reduced mice. Due to the high affinity showed by anti-aGal IgE after DP1000-RA0118 treatment, likely due to the significant antibodies for the assessed polymeric aGal-glycoconjugates, contribution of anti-aGal to the circulating IgM repertoire of we hypothesize that the anti-aGal IgE in sensitized patients GalT-KO mice (15). might come from IgG-switched B cells, unlike the classic atopic Finally, glycopolymers with DP<1000 have shown a high sensitization to pollen and mite allergens where IgE come from capacity to inhibit anti-aGal IgM and IgG in vitro but have naive B cells. We demonstrated the potentiality of poly-L- been entirely ineffective in vivo (53). However, there is no data lysine-based aGal-glycoconjugates for treating allergic regarding the impact of DP on anti-aGal IgE inhibition. In our disorders mediated by anti-aGal IgE antibodies. As AGS is research, we found that DP100-RA0127 and DP100-RA0134 spreading due to the expansion and changes of hard ticks’ (100 µg/mL) showed high IgE-inhibition activity (>75%), with habitats, the immunotherapy concept presented here, based on reduced IgG inhibition (<25%). This IgE isotype-dependent the selective removal of induced anti-aGal IgE antibodies with inhibition might be helpful for IgE-mediated diseases. Despite poly-L-lysine aGal-glycoconjugates, may provide a clinical the in vivo administration of DP1000 aGal-conjugates has solution to this disorder. shown to be safe in primates and rodents (53), the impact of a lower DP on safety has not been evaluated so far. The polysaccharide chain length affects the immunogenicity of glycan-conjugated vaccines (87). In addition, the DATA AVAILABILITY STATEMENT polysaccharide hapten size is critical in the immune response The raw data supporting the conclusions of this article will be to carbohydrate vaccines (88). Indeed, reducing polysaccharide made available by the authors, without undue reservation. chain lengths improved vaccine immunogenicity (89, 90). Our experience developing monoclonal antibodies using Poly-L- lysine-OVA conjugate as the immunogen shows the poor immunogenicity of DP1000-Poly-L-lysine. Currently, we are ETHICS STATEMENT improving the immunogenicity of Poly-L-lysine–OVA conjugates by reducing the length of the poly-L-lysine The studies involving human participants were reviewed and backbone (data not shown). With all these findings, we might approved by Clinical Research Committee of the Hospital expect higher toxicities associated with DP100-glycopolymers Universitari de Bellvitge (PR212/17). The patients/participants than DP1000. Therefore, before studying the efficacy of DP100- provided their written informed consent to participate in this aGal-glycopolymers in the selective in vivo removal of IgE over study. The animal study was reviewed and approved by ethics the other anti-aGal isotypes, we consider conducting committee for animal experimentation of Bellvitge Biomedical immunogenicity studies with DP100 crucial to rule out Research Institute (IDIBELL). Serum samples from aGal- possible safety concerns. sensitized subjects (PT, n=13) were selected from previous Although the results presented here are promising, they have epidemiological studies (59), where anti-aGal IgE prevalence of some limitations. First, the primary objective of this work was to anti-aGal IgE in individuals with acute urticaria or anaphylaxis develop a model of aGal-sensitization in mice; however, a more from different geographical areas of Spain was studied. robust model of clinical AGS needs to be addressed to study the clinical impact of DP1000-RA0118 administration. Second, the most promising compounds (including DP100-RA0127 and AUTHOR CONTRIBUTIONS DP100-RA0134) will need to be tested for their ability to elicit B cell hyporesponsiveness in GalT-KO mice sensitized to aGal. SO-A contributed to the experimental design, performed the Finally, according to some epidemiological studies, anti-aGal, experimental work related to animal samples collection, ELISA, mainly IgM, may play a protective role against protozoan and white blood cell profiling, and participated in the manuscript infections (19–21). Therefore, the potential benefit of having drafting. DB-G designed all the experimental work, coordinated these induced antibodies in regions endemic for protozoan the study, made a substantial contribution to data management Frontiers in Immunology | www.frontiersin.org 10 March 2022 | Volume 13 | Article 873019 Olivera-Ardid et al. Potential Immunotherapy for AGS and analysis, and wrote the body of the manuscript. RA ACKNOWLEDGMENTS performed the tick saliva extraction and contributed to the manuscript drafting. YF-A performed the experimental work The authors want to acknowledge and express their gratitude to related to ELISA and white blood cell profiling. BG and ML-H Magdiel Pé rez-Cruz for initial technical insights related to the provided the clinical samples and significantly contributed to animal sensitization model. manuscript drafting. AG-P provided technical insights regarding the preliminary preparation of salivary gland extract and contributed during the manuscript drafting. NB and AT SUPPLEMENTARY MATERIAL participated in synthetic conjugation strategy and contributed during manuscript drafting. RM significantly contributed to the The Supplementary Material for this article can be found online at: manuscript conception and drafting. All authors contributed to https://www.frontiersin.org/articles/10.3389/fimmu.2022.873019/ the article and approved the submitted version. full#supplementary-material Repertoire in GalT-KO Mice Is Determined by Gut Microbiota. Front REFERENCES Immunol (2019) 10:342/full(March). doi: 10.3389/fimmu.2019.00342/full 16. Boussamet L, Montassier E, Soulillou JP, Berthelot L. Anti a1-3Gal Antibodies 1. Hu J, Chen J, Ye L, Cai Z, Sun J, Ji K. Anti-IgE Therapy for IgE-Mediated and Gal Content in Gut Microbiota in Immune Disorders and Multiple Allergic Diseases: From Neutralizing IgE Antibodies to Eliminating IgE+B Sclerosis. Clin Immunol (2021) :108693. doi: 10.1016/j.clim.2021.108693 Cells. 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Affinity Enhancement of Antibodies: How Low-Affinity Antibodies Produced Early in Immune Responses Are Followed by High-Affinity Conflict of Interest: SO-A, DB-G, and YF-A are employees of RemAb Antibodies Later and in Memory B-Cell Responses. Cancer Immunol Res Therapeutics SL. DB-G and RM are shareholders of RemAb Therapeutics SL. (2014) 2(5):381–92. doi: 10.1158/2326-6066.CIR-14-0029 SO-A and DB-G hold a patent on new glycoconjugates and medical uses thereof. 78. Sutton B, Davies A, Bax H, Karagiannis S. IgE Antibodies: From Structure to The remaining authors declare that the research was conducted in the absence of Function and Clinical Translation. Antibodies (2019) 8(1):19. doi: 10.3390/ any commercial or financial relationships that could be construed as a potential antib8010019 conflict of interest. 79. Wurzburg BA, Garman SC, Jardetzky TS. Structure of the Human IgE-Fc Cϵ3-Cϵ4 Reveals Conformational Flexibility in the Antibody Effector The authors declare that this study received funding from RemAb Therapeutics Domains. Immunity (2000) 13(3):375–85. doi: 10.1016/S1074-7613(00) SL. The funder had the following involvement in the study: design and complete 00037-6 execution, data generation, interpretation and graphing, article writing, edition, 80. Rispens T, Derksen NIL, Commins SP, Platts-Mills TA, Aalberse RC. IgE and submission. Production to a-Gal Is Accompanied by Elevated Levels of Specific IgG1 Antibodies and Low Amounts of IgE to Blood Group B. PloS One (2013) 8 Publisher’s Note: All claims expressed in this article are solely those of the authors (2):1–7. doi: 10.1371/journal.pone.0055566 and do not necessarily represent those of their affiliated organizations, or those of 81. Commins SP, Karim S. Development of a Novel Murine Model of Alpha-Gal the publisher, the editors and the reviewers. Any product that may be evaluated in Meat Allergy. J Allergy Clin Immunol (2017) 139(2):AB193. doi: 10.1016/ this article, or claim that may be made by its manufacturer, is not guaranteed or j.jaci.2016.12.628 endorsed by the publisher. 82. Choudhary SK, Commins SP. Association of Alpha-Gal Red Meat Allergy Severity With Concentration of Antigen in Basophil Activation. J Allergy Clin Copyright © 2022 Olivera-Ardid, Bello-Gil, Tuzikov, Araujo, Ferrero-Alves, Immunol (2017) 139(2):AB125. doi: 10.1016/j.jaci.2016.12.406 Garcıá Figueroa, Labrador-Horrillo, Garcıa-Pé ŕ ez, Bovin and Mañez. This is an 83. Commins SP, James HR, Stevens W, Pochan SL, Land MH, King C, et al. open-access article distributed under the terms of the Creative Commons Attribution Delayed Clinical and Ex Vivo Response to Mammalian Meat in Patients With License (CC BY). The use, distribution or reproduction in other forums is permitted, IgE to Galactose-Alpha-1,3-Galactose. J Allergy Clin Immunol (2014) 134 provided the original author(s) and the copyright owner(s) are credited and that the (1):108–15. doi: 10.1016/j.jaci.2014.01.024 original publication in this journal is cited, in accordance with accepted academic 84. Hussain R, Dawood G, Abrar N, Toossi Z, Minai A, Dojki M, et al. Selective practice. No use, distribution or reproduction is permitted which does not comply with Increases in Antibody Isotypes and Immunoglobulin G Subclass Responses to these terms. Frontiers in Immunology | www.frontiersin.org 13 March 2022 | Volume 13 | Article 873019