University of Veterinary Medicine Vienna

Bos d 13, A Novel Heat-Stable Beef Allergen.

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown. IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco-2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. MYLs are identified as novel heat-stable bovine meat allergens.

Editorial: Characterization, Biosynthesis and Biological Functions of Novel Glyco-Epitopes.

Assessment of the Safety and Efficacy of an Oral Probiotic-Based Vaccine Against Aspergillus Infection in Captive-Bred Humboldt Penguins ().

Aspergillosis is a fungal infection caused mainly by that often results in respiratory disease in birds. Aspergillosis is a major cause of morbidity and mortality in captive-bred penguin species. Currently, there is no registered vaccine to prevent aspergillosis. Recent research demonstrated that oral administration of gram-negative bacteria expressing high levels of galactose-α-1,3-galactose (α-Gal) modulates anti-α-Gal immunity and protects turkeys from clinical aspergillosis caused by experimental infection. The role of anti-α-Gal immunity in penguins has not been studied. Here, we tested the distribution of α-1,3-galactosyltransferase (α1,3GT) genes in the fecal microbiome of Humboldt penguins (). The occurrence of natural anti-α-Gal antibodies (Abs) in sera and eggs of healthy Humboldt penguins was also assessed. A trial was then conducted to test whether oral administration of Nissle, expressing high α-Gal levels, modulates anti-α-Gal immunity in a colony of Humboldt penguins. Animals in the vaccination and placebo groups were evaluated before the trial and followed for one year for aspergillosis detection using a diagnostic panel including computed tomography scans, capillary zone electrophoresis, 3-hydroxybutyrate levels, and anti- Abs. Anti-α-Gal Abs were detected in sera (IgM and IgY) and eggs (IgY) of healthy penguins. Microbiota analysis and functional predictions revealed the presence of α1,3GT genes in the microbiota of Humboldt penguins and other penguin species. A strong decrease in anti-α-Gal IgM levels was observed in all animals in the placebo group three months after vaccination protocol. This decrease was not observed in Nissle-treated penguins. After the vaccination protocol, we found a positive correlation between anti- IgY and anti-α-Gal IgY in the Nissle group, suggesting a correlation between the presence of the bacteria and these Abs. During the study period, three penguins exhibited respiratory signs consistent with aspergillosis. Two were from the placebo group whose symptoms resolved with specific treatments, while a single vaccinated individual developed fatal respiratory aspergillosis eight months after the trial. We conclude that Nissle represents a safe potential probiotic with a protective effect against aspergillosis in Humboldt penguins that deserves to be further explored for therapeutic uses in these animals.

Tick-human interactions: from allergic klendusity to the α-Gal syndrome.

Ticks and the pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. The ability of some animal species to acquire resistance to blood-feeding by ticks after a single or repeated infestation is known as acquired tick resistance (ATR). This resistance has been associated to tick-specific IgE response, the generation of skin-resident memory CD4+ T cells, basophil recruitment, histamine release, and epidermal hyperplasia. ATR has also been associated with protection to tick-borne tularemia through allergic klendusity, a disease-escaping ability produced by the development of hypersensitivity to an allergen. In addition to pathogen transmission, tick infestation in humans is associated with the α-Gal syndrome (AGS), a type of allergy characterized by an IgE response against the carbohydrate Galα1-3Gal (α-Gal). This glycan is present in tick salivary proteins and on the surface of tick-borne pathogens such as Borrelia burgdorferi and Anaplasma phagocytophilum, the causative agents of Lyme disease and granulocytic anaplasmosis. Most α-Gal-sensitized individuals develop IgE specific against this glycan, but only a small fraction develop the AGS. This review summarizes our current understanding of ATR and its impact on the continuum α-Gal sensitization, allergy, and the AGS. We propose that the α-Gal-specific IgE response in humans is an evolutionary adaptation associated with ATR and allergic klendusity with the trade-off of developing AGS.

The α-Gal Syndrome and Potential Mechanisms.

The α-Gal syndrome is a complex allergic disease characterized by the development of specific IgE antibodies against the carbohydrate galactose-α-1,3-galactose (α-Gal), an oligosaccharide present in cells and tissues of non-primate mammals. Individuals with IgE antibodies to α-Gal suffer from a delayed form of anaphylaxis following red meat consumption. There are several features that make the α-Gal syndrome such a unique allergic disease and distinguish it from other food allergies: (1) symptoms causing IgE antibodies are directed against a carbohydrate moiety, (2) the unusual delay between the consumption of the food and the onset of the symptoms, and (3) the fact that primary sensitization to α-Gal occurs tick bites. This review takes a closer look at the immune response against α-Gal, in healthy and in α-Gal allergic individuals. Furthermore, the similarities and differences between immune response against α-Gal and against the other important glycan moieties associated with allergies, namely cross-reactive carbohydrate determinants (CCDs), are discussed. Then different mechanisms are discussed that could contribute to the delayed onset of symptoms after consumption of mammalian meat. Moreover, our current knowledge on the role of tick bites in the sensitization process is summarized. The tick saliva has been shown to contain proteins carrying α-Gal, but also bioactive molecules, such as prostaglandin E2, which is capable of stimulating an increased expression of anti-inflammatory cytokines while promoting a decrease in the production of proinflammatory mediators. Together these components might promote Th2-related immunity and trigger a class switch to IgE antibodies directed against the oligosaccharide α-Gal. The review also points to open research questions that remain to be answered and proposes future research directions, which will help to get a better understanding and lead to a better management of the disease.

Anti-Tick Microbiota Vaccine Impacts Performance during Feeding.

The tick microbiota is a highly complex ensemble of interacting microorganisms. Keystone taxa, with a central role in the microbial networks, support the stability and fitness of the microbial communities. The keystoneness of taxa in the tick microbiota can be inferred from microbial co-occurrence networks. Microbes with high centrality indexes are highly connected with other taxa of the microbiota and are expected to provide important resources to the microbial community and/or the tick. We reasoned that disturbance of vector microbiota by removal of ubiquitous and abundant keystone bacteria may disrupt the tick-microbiota homeostasis causing harm to the tick host. These observations and reasoning prompted us to test the hypothesis that antibodies targeting keystone bacteria may harm the ticks during feeding on immunized hosts. To this aim, in silico analyses were conducted to identify keystone bacteria in the microbiota of nymphs. The family Enterobacteriaceae was among the top keystone taxa identified in microbiota. Immunization of α-1,3-galactosyltransferase-deficient-C57BL/6 (α1,3GT KO) mice with a live vaccine containing the Enterobacteriaceae bacterium strain BL21 revealed that the production of anti- and anti-α-Gal IgM and IgG was associated with high mortality of nymphs during feeding. However, this effect was absent in two different strains of wild type mice, BALB/c and C57BL/6. This result concurred with a wide distribution of α-1,3-galactosyltransferase genes, and possibly α-Gal, in Enterobacteriaceae and other bacteria of tick microbiota. Interestingly, the weight of nymphs that fed on -immunized C57BL/6 was significantly higher than the weight of ticks that fed on C57BL/6 immunized with a mock vaccine. Our results suggest that anti-tick microbiota vaccines are a promising tool for the experimental manipulation of vector microbiota, and potentially the control of ticks and tick-borne pathogens.

Gut Microbiota Abrogates Anti-α-Gal IgA Response in Lungs and Protects against Experimental Infection in Poultry.

Naturally occurring human antibodies (Abs) of the isotypes IgM and IgG and reactive to the galactose-α-1,3-galactose (α-Gal) epitope are associated with protection against infectious diseases, caused by pathogens expressing the glycan. Gut microbiota bacteria expressing α-Gal regulate the immune response to this glycan in animals lacking endogenous α-Gal. Here, we asked whether the production of anti-α-Gal Abs in response to microbiota stimulation in birds, confers protection against infection by , a major fungal pathogen that expresses α-Gal in its surface. We demonstrated that the oral administration of O86:B7 strain, a bacterium with high α-Gal content, reduces the occurrence of granulomas in lungs and protects turkeys from developing acute aspergillosis. Surprisingly, the protective effect of O86:B7 was not associated with an increase in circulating anti-α-Gal IgY levels, but with a striking reduction of anti-α-Gal IgA in the lungs of infected turkeys. Subcutaneous immunization against α-Gal did not induce a significant reduction of lung anti-α-Gal IgA and failed to protect against an infectious challenge with . Oral administration of O86:B7 was not associated with the upregulation of lung cytokines upon infection. We concluded that the oral administration of bacteria expressing high levels of α-Gal decreases the levels of lung anti-α-Gal IgA, which are mediators of inflammation and lung damage during acute aspergillosis.

COVID-19 in the Developing World: Is the Immune Response to α-Gal an Overlooked Factor Mitigating the Severity of Infection?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which has affected millions of people worldwide. Considerably lower prevalence and fatality rates resulting from COVID-19 are reported in Africa and Asia than in the industrialized world. In this Viewpoint, we discuss the possibility that this intriguing phenomenon could be, among other factors, due to protective immunity of the oligosaccharide galactose-α-1,3-galactose (α-Gal). The α-Gal immunity induced by gut microbiota that express the same glycan modification may prevent COVID-19 through the activation of different mechanisms involved in SARS-CoV-2 neutralization and the downregulation of the inflammatory response in the lungs of infected patients.

Infection with Inhibits the Production of IgE Antibodies to α-Gal in Humans: Towards a Conceptual Framework of the Hygiene Hypothesis?

α-Gal syndrome (AGS) is a type of anaphylactic reaction to mammalian meat characterized by an immunoglobulin (Ig)E immune response to the oligosaccharide α-Gal (Galα1-3Galβ1-4GlcNAc-R). Tick bites seems to be a prerequisite for the onset of the allergic disease in humans, but the implication of non-tick parasites in α-Gal sensitization has also been deliberated. In the present study, we therefore evaluated the capacity of helminths (, , ), protozoa (), and parasitic fungi () to induce an immune response to α-Gal. For this, different developmental stages of the infectious agents were tested for the presence of α-Gal. Next, the potential correlation between immune responses to α-Gal and the parasite infections was investigated by testing sera collected from patients with AGS and those infected with the parasites. Our results showed that and produce the terminal α-Gal moieties, but they were not able to induce the production of specific antibodies. By contrast, , and lack the α-Gal epitope. Furthermore, the patients with infection had significantly decreased anti-α-Gal IgE levels when compared to the healthy controls, suggesting the potential role of this nematode parasite in suppressing the allergic response to the glycan molecule. This rather intriguing observation is discussed in the context of the 'hygiene hypothesis'. Taken together, our study provides new insights into the relationships between immune responses to α-Gal and parasitic infections. However, further investigations should be undertaken to identify components with potent immunomodulatory properties and to assess their potential to be used in immunotherapy and control of AGS.

Tick Bites Induce Anti-α-Gal Antibodies in Dogs.

Due to the functional inactivation of the gene encoding for the enzyme that is involved in the oligosaccharide galactose-α-1,3-galactose (α-Gal) synthesis, humans and Old-World primates are able to produce a large amount of antibodies against the glycan epitope. Apart from being involved in the hyperacute organ rejection in humans, anti-α-Gal antibodies have shown a protective effect against some pathogenic agents and an implication in the recently recognized tick-induced mammalian meat allergy. Conversely, non-primate mammals, including dogs, have the ability to synthetize α-Gal and, thus, their immune system is not expected to naturally generate the antibodies toward this self-antigen molecule. However, in the current study, we detected specific IgG, IgM, and IgE antibodies to α-Gal in sera of clinically healthy dogs by an indirect enzyme-linked immunosorbent assay (ELISA) for the first time. Furthermore, in a tick infestation experiment, we showed that bites of induce the immune response to α-Gal in dogs and that the resulting antibodies (IgM) might be protective against . These findings may help lead to a better understanding of the underlying mechanisms involved in mammalian meat allergy and tick-host-pathogen interactions, but they also open up the question about the possibility that dogs could develop an allergy to mammalian meat after tick bites, similar to that in humans.

Delayed hypersensitivity reaction to mammalian galactose-α-1,3-galactose (α-Gal) after repeated tick bites in a patient from France.

The α-Gal syndrome is a tick-associated and emerging IgE-mediated hypersensitivity reaction directed against the carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) epitope after red meat intake. Herein, we describe a clinical case of a 44-year-old French patient who suffered from recurrent anaphylactic reactions after mammalian meat consumption for five years before the final diagnosis of the α-Gal syndrome was established in 2018. The patient also reported multiple tick bites prior to symptom onset. This unique type of allergy has increasingly been reported across the world, but it is still unknown in many European countries. Therefore, the present clinical case should increase awareness among primary care practitioners and further improve the early diagnosis of the α-Gal syndrome in affected individuals.

Alpha-gal allergy after a tick bite in Austria.

Tick bites can cause the alpha-gal syndrome, which is characterized by delayed anaphylactic reactions mainly to red meat and offal due to IgE antibodies against mammalian galactose-alpha-1.3-galactose carbohydrate (alpha-gal). Ixodes ricinus bites are considered the primary cause of IgE antibody responses specific for alpha-gal in Europe. This article reports on a 51-year-old Austrian male who acquired a tick bite in Austria in spring 2017, which, within 48 h, resulted in prolonged inflammation of the skin area around the bite. The patient experienced an allergic reaction 3 months later approximately 8 h after eating a medium rare steak for dinner. The symptoms included an itchy rash on both sides of the torso and on both arms which persisted for several hours. In spring 2018, the patient suffered another tick bite. The patient's skin reaction was similar to that of the previous year. In the following months, the patient experienced five episodes of severe allergic reactions, each during the night after having eaten beef for dinner. The symptoms included pruritic urticarial rash involving the entire body along with swollen hands, diarrhea, vomiting and in some episodes even shortness of breath. At the request of the patient, specific IgE antibodies against alpha-gal were determined, revealing a highly positive result (>100 kU/l). This brief report aims to raise awareness that recurrent delayed anaphylactic reactions to food can develop after tick bites.

Environmental and Molecular Drivers of the α-Gal Syndrome.

The α-Gal syndrome (AGS) is a type of allergy characterized by an IgE antibody (Ab) response against the carbohydrate Galα1-3Galβ1-4GlcNAc-R (α-Gal), which is present in glycoproteins from tick saliva and tissues of non-catarrhine mammals. Recurrent tick bites induce high levels of anti-α-Gal IgE Abs that mediate delayed hypersensitivity to consumed red meat products in humans. This was the first evidence that tick glycoproteins play a major role in allergy development with the potential to cause fatal delayed anaphylaxis to α-Gal-containing foods and drugs and immediate anaphylaxis to tick bites. Initially, it was thought that the origin of tick-derived α-Gal was either residual blood meal mammalian glycoproteins containing α-Gal or tick gut bacteria producing this glycan. However, recently tick galactosyltransferases were shown to be involved in α-Gal synthesis with a role in tick and tick-borne pathogen life cycles. The tick-borne pathogen increases the level of tick α-Gal, which potentially increases the risk of developing AGS after a bite by a pathogen-infected tick. Two mechanisms might explain the production of anti-α-Gal IgE Abs after tick bites. The first mechanism proposes that the α-Gal antigen on tick salivary proteins is presented to antigen-presenting cells and B-lymphocytes in the context of Th cell-mediated immunity induced by tick saliva. The second mechanism is based on the possibility that tick salivary prostaglandin E2 triggers Immunoglobulin class switching to anti-α-Gal IgE-producing B cells from preexisting mature B cells clones producing anti-α-Gal IgM and/or IgG. Importantly, blood group antigens influence the capacity of the immune system to produce anti-α-Gal Abs which in turn impacts individual susceptibility to AGS. The presence of blood type B reduces the capacity of the immune system to produce anti-α-Gal Abs, presumably due to tolerance to α-Gal, which is very similar in structure to blood group B antigen. Therefore, individuals with blood group B and reduced levels of anti-α-Gal Abs have lower risk to develop AGS. Specific immunity to tick α-Gal is linked to host immunity to tick bites. Basophil activation and release of histamine have been implicated in IgE-mediated acquired protective immunity to tick infestations and chronic itch. Basophil reactivity was also found to be higher in patients with AGS when compared to asymptomatic α-Gal sensitized individuals. In addition, host resistance to tick infestation is associated with resistance to tick-borne pathogen infection. Anti-α-Gal IgM and IgG Abs protect humans against vector-borne pathogens and blood group B individuals seem to be more susceptible to vector-borne diseases. The link between blood groups and anti-α-Gal immunity which in turn affects resistance to vector-borne pathogens and susceptibility to AGS, suggests a trade-off between susceptibility to AGS and protection to some infectious diseases. The understanding of the environmental and molecular drivers of the immune mechanisms involved in AGS is essential to developing tools for the diagnosis, control, and prevention of this growing health problem.