The Immunology of Alpha‐Gal Syndrome: History, Tick Bites, IgE, and Delayed Anaphylaxis to Mammalian Meat

ABSTRACT

The primary features of the alpha‐gal syndrome (AGS) are (i) The IgE ab that are causally related to anaphylaxis with infusions of Cetuximab are specific for galactose alpha‐1,3‐galactose. (ii) In the USA, this IgE ab is induced by bites of the tick Amblyomma americanum . (iii) The anaphylactic reactions to food derived from non‐primate mammals are delayed in onset by three to five hours. A further important fact is that all humans make a “natural” response to alpha‐gal which includes IgM, IgG, and IgA, but not IgE. The clinical features of AGS are recognized in many parts of the world, but different species of ticks are involved. The immune response to tick bites includes T cells specific for tick protein, while IgE producing B cells appear to be derived from B cells specific for IgM or IgG. With repeated tick bites, the T cells develop a strong Th2 signal with IL‐4 and IL‐13 This obviously relates to IgE production, but may also be relevant to itching after tick bites which can last for weeks. The current hypothesis about the cause of the delayed reactions is based on the time that it takes to digest glycolipids from meat to LDL. The management of AGS symptoms is based on the avoidance of food derived from mammals; however, the only thing that can allow IgE to decrease is avoidance of tick bites.

1. How Allergic Reactions to Cetuximab, Delayed Reactions to Meat and Tick Bites Led to the Discovery of Alpha‐Gal Syndrome

1.1. Historical Introduction

In 2004 we knew of four observations that were interesting, but unexpected and each poorly understood. However, most important there was no reason to connect them: as the French would say “sans lien entre elle!!”

First: We had seen adult patients who reported allergic reactions occurring several hours after eating meat such as beef or pork, which they had eaten for many years without any problems.

Second: Roger Cohen, an oncologist at the University of Virginia, asked us to help in investigating acute reactions occurring during the first infusion of Cetuximab, a monoclonal antibody specific for epidermal growth factor receptor (EGFR) that was then in clinical trials to treat colon cancer.

Third: In a Kenyan village 100 miles north of Nairobi, 70% of the 11‐year‐old children that we studied had IgE antibodies to cat allergens in their serum, although there were no cats in the village and they did not appear to have any relevant symptoms [1].

Fourth: It had become difficult to grow azaleas in suburban Virginia because of the dramatic increase in the deer population since 1980.

Although each of these observations ultimately contributed to our current understanding of alpha‐gal syndrome (AGS), it was the second that led to our recognition of the specificity of the IgE antibodies, and to the availability of the assay for specific IgE (sIgE) to alpha‐gal. In 2005, Dr. Cohen arranged for us to receive sera from some of the patients who had reacted to Cetuximab, and we asked Phadia to make ImmunoCAPs coated with this monoclonal antibody. Those experiments were carried out by Dr. Tina Merrit, then a fellow, and provided preliminary evidence that some of the patients had sIgE to the drug. However, in 2005, an interesting sequence decreased interest in Cetuximab; firstly, Martha Stewart sold her stock in ImClone, and the next day the FDA declined to approve the drug and required ImClone to carry out a further controlled trial.

In 2006, Cetuximab went on the market approved for the treatment of both cancer of the head and neck and stage 4 colon cancer [2]. Within months, it was clear that this monoclonal Ab was causing severe reactions in several areas of the south, including North Carolina, Tennessee, Arkansas, and Missouri as well as Virginia. In particular, two severe reactions occurred during the first infusion in Bentonville, the first rapidly fatal, and the second severe. The senior nurse in the oncology clinic in Bentonville was married to the practice manager for Dr. Tina Merritt, who by then was in practice in Fayetteville, Arkansas. As a result of this connection, we got serum from the second case and obtained IRB approval to study the problem. In addition, we established connections with both ImClone, who made the molecule, and Bristol Myers Squibb (BMS), who carried out the clinical trials and marketed Cetuximab. Both companies were consistently helpful and provided critical reagents for the studies to define the relevant epitope on Cetuximab.

The high prevalence of the allergic reactions among cancer patients has been documented at both UNC and Vanderbilt, and the results were reported by Dr. Robert O'Neil at their national meeting. At that meeting, a senior oncologist from New York stated publicly that he was not sure whether Dr. O'Neil was “lying or crazy.” [3] That is, he thought the chances of an FDA approved cancer drug causing so much trouble among patients treated at major universities in the southeast, but no similar trouble in New York, Boston, or Chicago, was too unlikely to be worth considering [3]. None the less, the observation was correct and was published in the Journal of Clinical Oncology in 2007 [4]. The best documentation of those cases, including detailed scoring of the reactions and storage of pre‐treatment sera, came from the oncology group at Vanderbilt. Although there were several possible explanations for the rapid reactions, including complement activation, the most likely was pre‐existing IgE antibodies. Primarily because of our experience with measuring IgE antibodies to novel antigens, both Vanderbilt and BMS agreed that we should assay the sera from the treatment cases as well as sera from controls in the Nashville area. That study provided highly significant data that the patients who reacted severely and rapidly to Cetuximab infusion had pre‐existing IgE specific for the alpha‐gal epitope on Cetuximab [5].

ImClone provided reagent grade Cetuximab, and they provided the same molecule expressed in a different cell line. In our lab we had developed the technique of binding biotinylated proteins to ImmunoCAPs coated with Streptavidin which were by then provided by Phadia/Thermo Fisher [6]. We had also started to get positive results for IgE to Cetuximab in sera from cancer patients and also from clinic patients who had “idiopathic” anaphylaxis. Cetuximab was and continues to be made in the Sp2/0 mouse cell line, and the second line used in the experimental studies was Chinese Hamster Ovary (CHO) cells. The most striking feature was that the molecule expressed in CHO cells did not bind IgE antibodies, although it had the same amino acid sequence and the same binding activity for the epidermal growth factor receptor (EGFR). The fact that the “same protein molecule” expressed by a different cell line did not bind IgE antibodies in the serum of patients had to mean that the epitope for sIgE antibody was a post translational modification of the molecule. However, the great majority of post translational modifications relate to glycosylation [7]. Thus, we were primarily focused on the glycosylation of Cetuximab. ImClone again provided critical data. Dr. Quinwie Zhou was working at ImClone to define the full glycosylation of the drug and we discussed his data with him during a visit to the company. His results included evidence that the FAB part of the heavy chain of the molecule was consistently glycosylated with galactose alpha‐1,3‐galactose [8]. At that point, we digested Cetuximab with pepsin and found that the binding of IgE to Cetuximab was selectively to the F(ab')2 fragment of the molecule. Further detailed studies with absorption established that the epitope on Cetuximab was galactose alpha‐1,3 galactose (alpha‐gal) [5, 7, 8].

During the latter half of 2007 and early in 2008 we started to talk about the results in relation to Cetuximab, and also the preliminary data on delayed reactions to red meat. The first public talk was to a meeting in Kansas City, and the second was during the QuadAI meeting in March of 2008. In Kansas City Missouri, Dr. Barrett Lewis came up after the talk and said “I have seen a lot of those red meat cases in Springfield.” At that time, we arranged for him to send us brief histories and sera on ten of his patients. In fact, he sent us histories and sera on eleven patients and of those, ten had positive sIgE to alpha‐gal. Tragically, Dr. Lewis died suddenly before publication of the paper describing the initial cases of delayed anaphylaxis or urticaria in patients with IgE to alpha‐gal, but his cases were included in the paper and he had played an important role [9].

1.2. Background Information on the Oligosaccharide Galactose Alpha‐1,3 Galactose (Alpha‐Gal)

The first information about an oligosaccharide that is present on non‐primate mammalian cells, but not on the cells of higher primates, came not surprisingly from Karl Landsteiner [10, 11]. During his studies that defined the ABO group of human red cell antigens, he recognized that the serum of humans and other higher primates contained natural antibodies that were specific for a “B‐like” antigen on mammalian cells which was not present on the cells of primates, except for New World Monkeys [11]. Subsequently, he extended that statement to say that all immunocompetent humans had antibodies to this antigen [11]. In the 1930's the isotype of those antibodies was not well defined because the basic structure of immunoglobulin had not been resolved and specifically, IgM, IgA, IgD, and the IgG subclasses had not been defined [12, 13, 14].

At a later stage, that is, 1970 onwards, several groups with much better ability to investigate oligosaccharides provided extensive information about the “B‐like” antigen (Figure 1). This included major groups in the USA and Australia, including seminal work by Galili [15, 16]. Notably, alpha‐gal only differs from the B antigen by the absence of a fucose attached to the penultimate galactose. Subsequently, Sandrin and Mackenzie in Melbourne, as well as Macher and Galili in Chicago, provided the evidence that alpha‐gal was an important, or the most important, barrier to transplantation from animals, such as pigs, into humans [17]. In 2006, the transplantation group in Pittsburgh published a paper arguing that the ability to make alpha‐gal had been lost by humans because it was not relevant [18]. By contrast, Dr. Galili made a strong case that the antibodies to alpha‐gal must have provided a major survival benefit for those primates that did not make this antigen. The argument being that natural antibodies to alpha‐gal could be a major benefit in surviving against an epidemic or several epidemics caused by an organism or organisms that carried this oligosaccharide. The importance of this would be that ultimately none of the “Old World” primates that made alpha‐gal went on to survive. However, that process which Dr. Galili called “catastrophic evolution,” may well have taken a few thousand years or longer to become complete [19].

FIGURE 1.

The alpha 1–3 galactosyltransferase that establishes the alpha‐gal linkage is different from the alpha 1–3 galactosyltransferase that can make a very similar linkages to a substituted galactose on blood group B. The alpha‐gal hapten as well as the A and B blood group oligosaccharides can be linked either to asparagine or O‐linked to the sphingolipid ceramide.

The level of understanding of the significance of alpha‐gal led to the production of alpha‐gal knock‐out (KO) mice by several groups [20, 21]; and subsequently, it led to the first alpha‐gal KO pigs [22, 23]. The mice are being used for many experiments studying the role of alpha‐gal, while the alpha‐gal knock‐out pigs form the centerpiece of experiments to transplant pig kidneys into humans [23]. What matters here is that it is already clear that kidneys from alpha‐gal KO pigs do not undergo hyper‐acute rejection and will function in humans normally for at least a few weeks or months. At this point in 2025, with development in genetically modified pigs going on at Revivicor in Blacksburg, Virginia, USA, and at E‐genesis in Boston, Massachusetts, USA, and many other companies, it appears possible that the use of pigs for kidney transplantation could be established within as little as ten years [24].

1.3. The Route to Ticks in Virginia and the World

Once we had established the assay for IgE antibodies to Cetuximab, there was a short period when we did not know the nature of the epitope on the monoclonal antibody and neither did we understand the cause or causes of this form of sensitization. During that time, we assayed the positive sera for specific IgE antibodies to a range of common allergens. The early results showed that low levels of IgE specific for cow's milk were common, and also that the results for cat and dog extracts were remarkably similar. In hindsight, those results should have provided a strong clue that the antigen we were looking for was mammalian in origin, but we could not see that because “we were still blind.” We considered the possibility of fungal allergens, nematode allergens, a variety of pollens, as well as food allergens but nothing fit the distribution. However, from talking to other physicians and knowing where reactions to Cetuximab were happening, we had a preliminary map of those reactions. At that time, Jake Hosen, who was working on red meat allergy in our laboratory was asked to search Google for comparable maps. After two days he said the only similar map was the CDC map of Rocky Mountain Spotted Fever (RMSF) cases (http://medbox.iiab.me/modules/en‐cdc/www.cdc.gov/ticks/tickbornediseases/rmsf.html) [25]. In 2006, the diagnosis of RMSF was based on a febrile illness, and positive serology. Surprisingly, the only widely available assay for identifying IgG antibodies to RMSF was an assay for IgG antibodies specific for the Rickettsia genus. Because of this it identified a significant number of cases of a relatively mild infection caused by Rickettsia amblyommatis (previously known as R. amblyommii) rather than Rickettsia rickettsii both of these can be derived from bites of the tick Amblyomma americanum [26]. However, R. amblyomii is far more common in Virginia and for us “the cat (or the tick) was out of the bag” and in late 2006 and early 2007 we started, for the first time ever, asking patients in our clinic about tick bites. The responses were astonishing. Almost all the patients who had positive IgE to alpha‐gal and delayed reactions to red meat were aware of tick bites and in most cases multiple bites. The strength of the answers was sufficient to convince our group that the lone star tick, A. americanum , was the primary cause of this form of sensitization in Virginia. By that time, we had identified a group of cases that fit the known criteria of the new syndrome (See Table 1 for cardinal cases). In some cases, the species of the tick was identified from seeing an adult female with the typical white spot. However, we also became aware that both nymphs and larvae of this tick are known to bite humans. In fact, it is generally thought that the lone star tick is the only tick species present in the United States whose larvae commonly bite humans. On the other hand, there are major problems with identifying the bites of lone star larvae: firstly, the larvae look like “little black dots” and if further studied with a hand lens they only have six legs. (The typical eight legs of a tick only appear at the nymphal stage). Secondly, among the rural population of Virginia the normal name used for larval ticks is “chiggers” and occasionally “seed ticks” or “turkey ticks” (See Box 1).

TABLE 1.

Cases of AGS. (A) Four early cases with clearly defined tick bite history, the presence of serum specific IgE (sIgE) to alpha‐gal which is greater than 2% of total serum IgE and who had experienced reactions including generalized hives and/or anaphylaxis, which responded rapidly into antihistamines or adrenalin. (B) Three severe cases that had blood pressure fall sufficient to cause “shock” and who responded slowly to treatment such that they required a stay in hospital. In two cases serum tryptase was elevated at the time of admission.

Cases	Age	sIgE	Total IgE	Tick history	Reactions
(A) Four cardinal cases
April 2007. E022	73/F	13	65.6	Gardening	Hives. 4 h after eating pork
2006. E001	55/M	9.2	157	Hunting	Anaphylaxis. ED 5 h after eating beef a
2007. E031	62/F	135	500	Hunting on horseback	Anaphylaxis. ED 4½ h after eating beef a
2007. E036	64/M	130	425	Hiking	Hives. 5 h after eating lamb chops
(B) Three severe cases: requiring hospital admission
2018. A578	56/M	10.8	164	Rural Living	Ate beef. 3½ h later collapsed, BP 40/0. Hospital; tryptase of 90≠
Aug 2012. E783	73/F	28	111	Gardening	Ate pork. 3½ h later severe anaphylaxis. Hospital; tryptase 30≠
April 2017. A412	36/F	8.4	98	Hiking	Ate red meat. 4 h later severe abdominal pain collapsed confusion; hospital one night b

≠ admitted to UVA.

^a Treated Warrenton.

^b Admitted VCU.

BOX 1. Case report of an otherwise non allergic lady who was bitten by larval ticks.

A 43‐year‐old lady came to clinic in May of 2006 and reported several episodes of hives over the previous eight months. Each of those occurred 3–4 h after eating pork or beef. She and her husband were very specific about the timing and the lack of reactions to chicken or fish. When questioned about tick bites she answered “no.” By contrast when questioned about “chiggers” she gave a strong positive reply, and then she said that she had 300 “black chiggers” on her leg in 2005 which she had shown to her physician. Her husband was clear about the number of chiggers and the physician confirmed the visit.

The first part of her blood test was a total serum IgE of 11 international units (IU/mL), that is, very low and making an allergic cause less likely. However, the specific IgE to Cetuximab, by then known to be specific for alpha‐gal, was 4 IU/mL, that is, 35% of the total IgE.

She has had no further attacks while avoiding mammalian meat, and has had no trouble with dairy.

The case for a role of larval ticks became clear later in 2007, after the senior author of this review experienced approximately 200 profoundly pruritic “little black dots” on his legs after hiking for four hours mainly “off trail” in August. Those acarids were sent to Dr. Robert Lane at UC Berkeley who identified them definitively as larval lone star ticks. Interestingly, Dr. Lane had previously published a case of tick bite anaphylaxis related to Ixodes pacificus [27]. Following the larval bites in Virginia, that subject's blood was taken each week and the level of IgE specific for alpha‐gal increased over the next three months to 120 IU/mL. In November 2007 he ate three lamb chops and two glasses of wine without symptoms at the dinner table, but at 2:00 AM, he started itching and then became covered with hives without any symptoms of anaphylaxis. Thus, in Virginia, the syndrome was now formally confirmed as (i) bites from adult, nymphal, or larval Lone star ticks (ii) increased sIgE to alpha‐gal over several weeks to a few months, and (iii) allergic reactions to meat or organs, derived from a non‐primate mammal, which start three to five hours after eating [9].

At the QuadAI meeting in 2008, two separate people approached us after we gave a talk on the cases during the TIGERS meeting. Firstly, Dr. Ray Mullins from New South Wales told us that Dr. Sheryl Van Nunen had reported to the Sydney Allergy Society that she had seen patients who had received tick bites on the North Shore and had subsequently become allergic to red meat. That observation was published in abstract form in 2007 and as a paper in 2009 [28]. The second report came from a member of the Georgia Allergy Society who recalled that Dr. Antony Deutsch had reported five cases that were communicated to him by Mrs. Sandra Latimer in 1989. Those patients had received tick bites in a wooded area near Athens and had subsequently become allergic to red meat. In 2010, we contacted Mrs. Latimer and confirmed the details of the cases that she was aware of. Her understanding included onset in adults, tick bites, and delayed reactions to beef or pork but not to fish or chicken. Dr. Deutsch also reported the cases to the CDC in Atlanta. Overall, it is obvious that several different patients, as well as doctors, had observed the primary phenomena of tick bites and subsequent development of allergic reactions to red meat. However, it was the investigation of reactions to Cetuximab that made it possible to identify the specificity of the IgE antibodies induced by tick bites [5, 29, 30].

1.4. Progressive Awareness of AGS and the Relevance of Tick Bites in Different Parts of the World

Over the years after we established the assay for IgE to galactose alpha‐1,3‐galactose (alpha‐gal) several research groups identified the syndrome with its typical symptoms. In each case, they included both specific IgE to alpha‐gal and a history of tick bites. However, studies from many different parts of the world identified different species of ticks, and indeed, the lone star tick is only relevant in North America.

In Stockholm, Dr. Marianne Van Hage and her colleagues were well aware of tick bites because the locally dominant tick, that is, Ixodes ricinus , is a known vector of both Lyme disease and a European tick‐borne Encephalitis (TBE) [31, 32]. In addition, they had found that some patients with positive blood tests for cat had IgE specific for an oligosaccharide present on cat IgA molecules [32]. They had also recognized patients who had experienced delayed reactions to “red meat.” In 2008, we shared reagents with the group in Stockholm and confirmed that the epitope on cat IgA was also alpha‐gal [32, 33]. They then established that the patients they had seen with delayed anaphylaxis also had IgE to alpha‐gal. That group was also the first to identify the presence of the alpha‐gal epitope in the gut of a tick, in that case I. ricinus, which is the relevant tick in most parts of Europe [34].

Dr. Sheryl Van Nunen in New South Wales was one of the first allergists to recognize the connection between tick bites and the development of allergy to meat derived from non‐primate mammals [28]. In Australia, the breeding hosts of the tick Ixodes holocyclus include the bandicoot, which is a rodent, and the meats that cause reactions include kangaroo. Although the syndrome was similar in most of the patients, this tick is also recognized as a cause of tick anaphylaxis, which has not been recognized with A. americanum or I. ricinus. At present, it has not been fully established whether the subjects who have anaphylaxis can also have IgE to alpha‐gal (Table 2).

TABLE 2.

Selected conditions related to ticks in different parts of the world: alpha‐gal syndrome (AGS), Lyme disease, and tick anaphylaxis.

Region	Disease	Genus and species
Australia, New South Wales, and Victoria	Tick anaphylaxis**	Ixodes holocyclus
	AGS***	I. holocyclus
Sweden	Tick borne encephalitis (TBE)++	I. ricinus
	Lyme disease**	I. ricinus
From Upsalla South	AGS***	I. ricinus
Central Europe	Lyme disease***	I. ricinus
	AGS***	I. ricinus
	Tick anaphylaxis++	Argus reflexus
United States	Lyme disease***	I. scapularis
	AGS***	Amblyomma americanum
	Tick anaphylaxis+	I. pacificus
Japan	Encephalitis**	Haemaphysalis longicornis
	AGS*	H. longicornis, A. testudinarium
	Tick anaphylaxis+	H. longicornis

Note: For Lyme disease and AGS *only case reports, **case series, ***prevalent in areas where tick is common. Lyme disease is not reported in Australia. Tick anaphylaxis and TBE have only been published as case reports⁺ and/or case series⁺⁺.

2. Oral Challenges With Meat, Organs, or Other Material Derived From Mammals in Patients With AGS

In the diagnosis of peanut allergy, double‐blind placebo‐controlled challenges with peanut are recognized as the gold standard for diagnosis. Not surprisingly, several groups have used challenges with red meat to investigate AGS. However, with a delay of three or even five hours after exposure to meat, it is not practical to carry out sequential doses in a day. For this reason, we carried out single‐dose challenges and have avoided challenging patients with a history of anaphylactic reactions. Even so, in a series of 13 patients challenged with a pork or beef patty, we had two patients who required an injection of adrenaline and one subject who had a home visit from Scott Commins because of persistent abdominal pain [35]. Despite the logistical difficulties, there were a few challenges carried out with a traditional increasing dose regimen. In one of those challenges, the subject was covered in hives three hours after the challenge; the second challenge at National Jewish in Denver resulted in frank anaphylaxis. In three of the active challenges in Virginia, we observed a significant rise in tryptase at the same time as the increase in CD63 on basophils [35]. Interestingly, four of the controls who were negative for sIgE to alpha‐gal also had a significant rise in CD63 but did not have any symptoms, hives, or an increase in tryptase [35]. The important conclusion was that elevated CD63, increases in tryptase, and skin symptoms could all start at the same time. Evidence from challenge studies in Virginia and other areas suggested that in AGS, the patient's abdominal symptoms, which were comparable to irritable bowel syndrome, generally start at the same time as skin symptoms, that is, at three to five hours [35, 36] (Figure 2).

FIGURE 2.

Alpha‐gal syndrome involves a human with a pre‐existing natural antibody response to the oligosaccharide galactose alpha‐1‐3‐galactose (alpha‐gal), receiving tick bites which induce an IgE antibody response to the same epitope. Over the next few weeks or months, IgE antibodies may increase. After that time, eating meat or organs can induce an allergic reaction which does not start for 3–5 h.

Other challenge studies have been reported from Europe and also South Africa. Dr. Tilo Biedermann and his colleagues in Munich reported evidence that challenges with pork kidney could induce delayed reactions in patients who did not react to pork meat [36, 37]. Their decision to study pork kidney came from patient reports of severe, perhaps more rapid reactions after eating this organ: which is a delicacy in some parts of Germany. The result of those challenges would lead to a future collaboration in Europe on studies into the glyco‐proteins and glycolipids that are present in pork kidney, carried out in collaboration with Dr. Christiane Hilger [38]. However, as they were studying the substances present in pork kidney that could trigger a response, not surprisingly, alpha‐gal emerged as the target of IgE in those patients [39, 40, 41]. Dr. Biedermann also established that a challenge with gummy bears could induce anaphylaxis in a patient with positive IgE to alpha‐gal [42]. That study provided excellent evidence that gelatin could induce reactions in patients with AGS; however, it is important to remember that gelatin is a foreign protein often derived from cattle or pigs and there are patients who have IgE to protein epitopes on gelatin, who can have severe reactions unrelated to alpha‐gal [43, 44, 45].

The other group that has carried out a significant number of challenges with mammalian meat is led by Dr. Mike Levin in South Africa with subjects who live in the Eastern Cape [46]. In that study, there were a significant number of children, and in addition, they reported a large number of patients who experienced abdominal pain after the challenge. Dr. Sarah McGill, who is in gastroenterology at UNC, reviewed the published studies on challenges, focusing on the abdominal symptoms [47]. Occasionally, abdominal pain is experienced as a form of AGS without any other symptoms, but many of those patients come to the notice of allergists because they have had at least one episode that also included hives or anaphylaxis. It is increasingly clear that the major abdominal symptoms often start after a similar delay to that experienced in the skin and other organs (see Table 1, Figure 2) [35, 36, 47].

2.1. Possible Conclusions From Challenge Studies That Support a Role for Mast Cells

The results of the challenge tests in Virginia provide convincing evidence that systemically absorbed alpha‐gal becomes present in the circulation three to five hours after eating meat, at which time it has to be on particles that are capable of activating basophils resulting in increased surface CD63 [35, 36]. Subsequent work by Eller and colleagues confirmed the timing by studying serum collected after pork kidney challenges and assessing functional activity in a passive basophil histamine release assay [48]. Moreover, in our study, three of the allergic subjects had an elevation of tryptase at the same time that they experienced symptoms. The possible mechanistic explanation for these findings is that circulating alpha‐gal three to five hours after challenge can only activate basophils or mast cells in the skin or other tissues if those cells are sensitized with sIgE to alpha‐gal (Figure 3). Moreover, given the dominant role mast cells are thought to play in anaphylactic physiology, our primary hypothesis is that the delay between eating mammalian meat and allergic reactions reflects the time taken to digest, absorb, and process alpha‐gal that is present in the form of glycolipids. Given that the delay is often 3 h or longer, the particles carrying foreign glycolipids are most likely to be low density lipoproteins (LDL) [49]. The argument is that the glycolipids including alpha‐gal would, after transitioning from chylomicrons and VLDL, be on the surface of the LDL which could leave the circulation by passing between cells in the endothelium of the blood vessels [50]. At present, we need to concede that there is not sufficient evidence that foreign oligosaccharides such as alpha‐gal are present on lipid particles as small as 20 nm in diameter at the time that symptoms occur.

FIGURE 3.

Basophils are primarily present in the circulation and only accumulate in tissues at the site of an inflammatory reaction. Basophils can be triggered in the circulation to activate CD‐63 as they release histamine. Release of mediators from mast cells requires a protein with multiple epitopes, or a particle carrying multiple epitopes of the same antigen, which enters the tissues.

Interestingly, many of the studies and discussions about AGS are focused on explaining the delay of the reactions while the absence of “immediate reactions” has generally been ignored. Even allowing 30 min or 1 h for the term “immediate,” very few of the AGS patients report reactions of that kind [51]. The question is, why do proteins carrying alpha‐gal such as beef thyroglobulin or porcine aminopeptidase not cause clinical reactions in patients with high levels of sIgE to alpha‐gal ? The likely explanation is that the relevant sugars are digested rapidly by glycosidases in the mouth or esophagus. Alternatively, it is possible that these glycoproteins do not transfer through the mucosal membranes. Dr. Swoboda and her colleagues in Vienna have published evidence from in vitro models that alpha‐gal present on glycolipids can pass through gut membranes in vitro while alpha‐gal on glycoproteins does not [52]. Interestingly, the findings in that paper acted as a stimulus to include an addendum on carbohydrate epitopes in the website on allergen nomenclature that was uploaded to the website in 2020 and published in 2021. See www.allergen.org [53].

2.2. The Relevance of Ticks in Different Parts of the World

Any attempt to understand the role of ticks in sensitization to alpha‐gal will have to include:

1. Understanding that all immunocompetent humans and primates other than new world monkeys have pre‐existing antibodies to the same epitope: that is, alpha‐gal.

2. That the “natural” responses to alpha‐gal include IgG, IgA, and IgM antibodies, but that in developed countries they only include IgE antibodies in individuals who have received tick bites.

3. That there are at least a dozen species of ticks that are known to cause sensitization to alpha‐gal.

Over the period that we have been doing research in the USA, there has been increasing awareness of the importance of ticks as vectors of a variety of infections, including Lyme, Ehrlichiosis, Anaplasmosis, and RMSF. In addition, there has been increasing understanding, particularly by the CDC, that the increase in deer populations on the East Coast has played a major role in increasing exposure to ticks, but specifically to A. americanum [54]. When we published our data on the relevance of tick bites to AGS, we also realized that prolonged pruritus at the site of a tick bite was an important characteristic of the response in patients with AGS [29]. The itching can be both prolonged and severe, and in occasional cases, persists for months. Interestingly, the response to bites from Ixodes scapularis, which is the primary tick carrying Borrelia burgdorferi in the USA, in most cases does not include itching. Indeed, a major infectious disease study about Lyme disease on Block Island, which is a part of Rhode Island, found that individuals who reported three or more episodes of pruritic tick bites were 90% less likely to develop positive Lyme serology over a ten‐year period [55]. Furthermore, it has been observed in Germany that the bites of I. ricinus that are related to Lyme disease do not itch (Tilo Biederman personal communication).

Comparing different countries, it is obvious that not only are different ticks relevant but that the biology of the ticks and/or the immune responses is different. In Australia, there is evidence that the same tick, that is, I. holocyclus, can induce tick anaphylaxis and sensitization to alpha‐gal. In the USA, the primary tick that induces IgE responses to alpha‐gal is A. americanum , but we are unaware of cases of tick anaphylaxis related to this tick. I. scapularis on the east coast and I. pacificus on the west coast are major carriers of the Lyme disease pathogen. Both have anecdotal reports linking them to AGS, though these cases seem to be exceptionally rare, and only I. pacificus has been linked to tick anaphylaxis (Table 2).

The intense focus on ticks raises the question about why these organisms are special and why other biting arthropods are not known to induce sIgE to alpha‐gal. The arthropods we could consider are bed bugs, mosquitos, fire ants, scabies, and also larval harvest mites or chiggers. Of these, the only published data relates to the red larval mites or chiggers, which may induce sensitization [56]. However, bites from larval mites are rare compared to tick bites. It is important to remember that studies on the sialome of ticks identify a large variety of agents that can induce activities as varied as controlling coagulation and fixing the mouth parts in the skin with “cement” [57]. In addition, Dr. Ribeiro at the NIH argues that the evolution of the constituents of tick saliva is more rapid than the evolution of any other feature of those animals [58]. There are thousands of species of ticks, and the complexity of their mouth parts is very elegant (Figure 4); after biting, there is a rapid activation of the salivary glands, such that any study of saliva will be different when the glands are active [59].

FIGURE 4.

Confocal microscopy of ventral side of an adult female Lone Star tick: The barbs on the hypostome, stained here in blue, make ticks difficult to remove. Pigment bleached with peroxide, cuticle stained with Congo Red and Calcofluor White imaged on Zeiss LSM at 100× magnification. Image courtesy of Igor Siwanowicz Janelia Research Campus of the Howard Hughes Medical Institute, Ashburn, Virginia, USA.

3. Epidemiology of the Alpha‐Gal Syndrome

Understanding the prevalence of AGS in the USA has involved: maps of the different ticks; maps of the prevalence of Lyme, Ehrlichiosis, Anaplasmosis, and RMSF; as well as maps of AGS; including maps of cases identified by allergists in practice; as well as random population studies of adults in some cases based on occupation; a map based on sensitization of recruits to the US armed forces; and finally, the CDC map of state and county of origin of subjects who had positive assays for IgE to alpha‐gal, which was published in 2024 [60]. The obvious thing is that all of these except the maps of ticks or zoonotic diseases transmitted by ticks are dependent on the assay for specific IgE to alpha‐gal.

3.1. Details About the Assays for sIgE to Alpha‐Gal

At the time of the early work on AGS, the assay for IgE to cat extract (e1) used an epithelium extract made from cats, which included a wide range of serum proteins such as cat IgA. Because of this, that assay acted as a reliable screening assay for IgE antibodies that bind to alpha‐gal. Thus, we knew that subjects in Norbotten who were negative for IgE to cat did not have IgE to alpha‐gal. Equally, the sIgE to cat we had observed in the village in Kenya turned out to be almost entirely due to sIgE to alpha‐gal [61, 62]. In addition, the positive assays for IgE to cat and dog that we reported in the original paper on Cetuximab were primarily detecting IgE to alpha‐gal [5, 9]. However, in 2010, the European Union passed regulations to control the use of any material derived from feline tissue. Because of this, the primary assay for cat changed to being made from dander only and effectively became an assay for sIgE to Fel d 1 [63]. The initial assay for sIgE to alpha‐gal used Cetuximab on the solid phase, which we developed using Streptavidin caps provided by Phadia. That assay used a level of detection (LOD) of 0.35 IU/mL [5, 9]. In 2010, the company recommended changing the LOD of the assay to 0.1 IU/mL. When data on a larger number of cases being treated with Cetuximab were reported with the new cut‐off, the association between hypersensitivity reactions and sIgE was less convincing [64]. However, this was largely because among the 17 subjects with low‐level alpha‐gal IgE, that is, ≥ 0.1 IU/mL and < 0.35 IU/mL, only one had a significant hypersensitivity reaction (HSR). What matters here is that lower levels of sIgE to alpha‐gal, for example, < 0.35 IU/mL, may not predict reactions during infusion of the monoclonal or after eating red meat [46, 65, 66, 67]. As with other forms of IgE mediated food allergy, alpha‐gal specific IgE results need to be interpreted in the context of clinical history.

A recent detailed study from Aarhus in Denmark has provided excellent evidence that IgE (and IgG) antibodies to MUXF3 can have distinct specificity to different parts of this oligosaccharide epitope: MUXF3 is a primary plant‐derived oligosaccharide with xylose (X) and fucose (F) substitutions (see reference [53]). The results suggest that sIgE antibodies may have diverse biological effects that are not appreciable from an ImmunoCAP binding assay [68]. This kind of phenomenon has also recently been investigated by Scott Smith and his colleagues at Vanderbilt using monoclonal human IgE antibodies to alpha‐gal.

3.2. Maps of Ticks and Diseases

In the maps of ticks and disease, there was an early and obvious distinction between the distribution of Lyme Disease and the increasingly clear distribution of alpha‐gal cases. This fit well with the strikingly different distribution of I. scapularis and that of A americanum [25]. However, there was a discrepancy appearing which was that from our data derived from interviews with allergists, there were very few cases of AGS on the gulf coast, including the whole of Texas, while the maps indicated the presence of the lone star tick as late as 2015 [25]. At a tick summit in 2018, Colonel Ting from the Air Force gave us an important piece of information about ticks in Texas. He said that the fire ants had killed or otherwise depleted A. americanum . At first, we had trouble finding that literature, but then found an excellent book by Stephen Taber on fire ants, which had a collection of old references making it clear that fire ants were capable of killing ticks [69]. In addition, we found the detailed maps of the spread of fire ants between 1932 and 2020 [70].

There are three maps that together make it clear that sensitization to alpha‐gal and the cases of AGS diagnosed by allergists in practice have a distribution that is very similar to the established distribution of cases of Ehrlichiosis and the most recent map of A. americanum [71]. Our favorite is the map of cases derived by questioning practitioners initially about AGS and secondly about cases of fire ant anaphylaxis (Figure 5) [72, 73]. That map shows a range of clinics where ≥ 50 cases had been seen that includes Southern Missouri, Oklahoma, Arkansas, Tennessee, Kentucky, North Carolina, and Virginia. We are also aware of areas in northern Georgia and Alabama, as well as the recent extension of cases north into Indiana, New Jersey, and Maryland. That map also showed a highly significant inverse correlation between cases of AGS and cases of fire ant anaphylaxis (Figure 5) [72]. The recent CDC map of positive assays for sIgE to alpha‐gal fits well with our map of cases and also with the recent map of sensitization among army recruits. That map came from assays of sera from 3000 recruits, which was part of a collaboration between Dr. Jeff Wilson and Dr. Cade Nylund at The Uniformed Services University of Health Science. That map was particularly clear about the hot spot in what we call “three corners”; that is Eastern Oklahoma, Southern Missouri, and Northwest Arkansas, as well as central Virginia (Figure 6) [73].

FIGURE 5.

South Eastern USA, showing the area where A. americanum was established prior to 2015, and also the expansion of fire ants following their introduction in Mobile, AL (71, 72). The red bars represent the number of AGS cases reported by allergy clinics while the dashed bars indicate responses related to fire ant anaphylaxis (FAA), in the same clinics. The data for fire ant quarantine dates is curtesy of the USDA (www.aphis.usda.gov).

FIGURE 6.

Alpha‐gal IgE prevalence by state. States in light green without annotation did not have any subjects with positive alpha‐gal specific IgE. In 2024 we published a study on serum assays for IgE antibodies to alpha‐gal among 3000 recruits to the armed services whose county and state of origin was known. The results for the states are presented as a % of individuals with positive assays for IgE antibodies to alpha‐gal and that varied from 39% in Arkansas and 35% in Oklahoma, to 2% in Arizona and 0% in New Mexico.

Several random surveys have been reported on subjects whose occupation included an extensive time spent outdoors. The first of these came from Dr. Fischer and his colleagues in Germany [74]. That survey was influential in confirming the correlation between sensitization to alpha‐gal and tick bites, and also in showing that only a small proportion of the sensitized subjects had experienced symptomatic reactions following eating red meat [74]. A more recent survey was carried out by the infectious disease group in Bialystok, Northwest of Warsaw. That study was initially designed to study serological and other evidence of zoonotic diseases, primarily Lyme and TBE; however, Dr. Rutkowski, who was working at St. Thomas' Hospital, suggested that they should also assay the sera for sIgE to alpha‐gal. That collaboration confirmed the low prevalence of symptoms among sensitized individuals, a strong correlation between sensitization to alpha‐gal and tick bites, and provided the best evidence yet that there was no association between those zoonotic diseases and sensitization to alpha‐gal [75, 76].

Recently we have become aware of a new pharmaceutical product that appears to have a regional distribution of hypersensitivity reactions (HSR). This antivenom is in the form of F(ab') 2 fragments digested from a polyvalent antiserum made in horses (ANAVIP). That antivenom product gave very little trouble in Arizona or New Mexico but has been associated with a high frequency of HSR in Oklahoma and Arkansas, which fits with our map of sensitization to alpha‐gal among young men in those states [71, 73, 77]. Recent evidence from our group in collaboration with Dr. Joshua Kennedy in Little Rock shows that ANAVIP has far greater activity than CroFab in the basophil activation test (BAT) using blood taken from patients with AGS [77] (Figure 7).

FIGURE 7.

Basophil activation test results from 2 subjects with α‐gal syndrome and one non‐allergic control. Basophils were exposed, ex vivo, to bovine thyroglobulin (BTG; serially diluted 1:10 from 100 to 0.01 μg/mL), or to the antivenom biologics Anavip (diluted 1:5 from 1000 to 0.32 μg/mL) or CroFab (diluted 1:5 from 10,000 to 1.6 μg/mL). Flow cytometry was used to detect the percentage of CD63‐positive basophils (a measure of basophil activation). The % CD63 positive data were normalized based on the anti‐FcεRI positive control (100%) and baseline negative control (stimulation buffer; 0%).

4. The Effects of Alpha‐Gal Syndrome (AGS) on Diseases That Are Not Generally Recognized as Having an Allergic Connection

As we have already emphasized, there were many reasons why it was initially difficult to recognize the features of AGS. For patients with symptoms that are not obviously allergic presenting to a general physician or an emergency department (ED) it was much worse. The patients presenting to an ED with abdominal pain and answering negatively to all the normal questions including “what have you eaten in the last two hours?” had no chance of being diagnosed correctly. Indeed, this was also true for some cases presenting to an allergist because of episodes of abdominal symptoms and hives starting three to five hours after eating a food that they had eaten without trouble for several decades.

4.1. Irritable Bowel Syndrome

During our challenge studies, two of the subjects developed abdominal symptoms three to four hours after eating a pork patty [35]. Thus, we were already aware that participants could develop abdominal symptoms including pain, at the same time as the obviously allergic symptoms such as urticaria or angioedema. Indeed, we considered that this could be taken as evidence that the mechanism by which mast cells in the gut were activated after several hours delay was the same as the mechanism in the skin and other tissues.

As far as we know, the first gastroenterologist who started using an assay for IgE to alpha gal routinely in a clinic which was focused on irritable bowel syndrome (IBS) was Dr. Robert Richards in Lynchburg [78]. He published in 2021 that as many as 30% of his IBS patients had sIgE to alpha‐gal, and that as many as 75% of those cases who adopted a diet responded to a diet avoiding mammalian products [78]. Subsequently, Dr. Sarah McGill at the University of North Carolina (UNC) recognized cases of IBS who had sIgE to alpha‐gal and responded to the diet [79]. Most recently, in a collaboration between UNC and UVA, a clinical practice update on alpha‐gal syndrome (AGS) for the G.I. physician was published [80].

We have recently become aware of two more cases where repeated or single severe abdominal pain episodes were followed by an episode with severe features of anaphylaxis. These cases draw attention to the need for investigation of unexpected severe episodes of GI distress occurring 3–5 h after eating mammalian meat.

4.2. The Possible Relevance of Alpha‐Gal to Coronary Artery Disease (CAD)

The relevance of AGS to coronary artery disease (CAD) was first considered because of an unexpectedly high number of cases of myocardial infarction among these patients being followed in the allergy clinics in Charlottesville. In part, of course, this may reflect a modest male dominance and the age of the patients presenting with AGS. Initially, we took advantage of a study using intravascular ultrasound (IVUS) being carried out at the University of Virginia (UVA) on subjects enrolled for coronary artery catheterization. The cardiologists, headed by Dr. Coleen McNamara and Dr. Angela Taylor, encouraged us to assay sera from their cases. The results published by Wilson et al. showed a significant association between positive specific IgE to alpha‐gal and overall atheroma burden and also a highly significant correlation with atheromatous lesions that were considered necrotic in the left main artery [81]. One of the striking features of that study was that it appeared from subsequent questionnaires that only a few of the subjects with significant atheroma burden and positive sIgE to alpha‐gal were aware of symptoms related to eating red meat.

About the same time, the cardiac surgeons at UVA reported three cases whose biovalves deteriorated rapidly and who were found to have sIgE to alpha‐gal [82]. The obvious implication was that the valves carried the alpha‐gal epitope, although the manufacturers denied that. We also became aware that Dr. Jan Ankersmit in Vienna had reported in 2005 that patients who received biovalves had an increase in sIgG to alpha‐gal over the following year [83]. The same group in Vienna subsequently reported that the IgG antibodies induced by biovalves were predominantly IgG3 [84]. In addition, they were involved in an evaluation of the long‐term consequences of aortic valve transplantation in Austria and supported the emerging evidence that implanting biovalves in recipients less than 65 years old is associated with increased mortality, compared to mechanical valves [85, 86].

In 2015, Vernon et al. in Sydney were carrying out a prospective study on adults with cardiac risk, which was in part designed to identify novel risk factors for CAD. In collaboration with two of the local allergists, they assayed the sera for sIgE to alpha‐gal and found a significant positive association with CAD as assessed by cardiac CT [87]. The results showed a significant association between positive sIgE and both atheroma burden and ST elevated myocardial infarction (STEMI). Interestingly, in a discussion with Dr. Gemma Figtree in 2022, we recognized that, in both our studies and the study in Sydney, a large proportion of the subjects with positive results for sIgE to alpha‐gal had low levels of sIgE and had not been aware of clinical reactions in relation to eating mammalian derived foods [81, 87].

While the question about the delay between allergic responses and eating red meat applies to all forms of this IgE dependent allergic response, it is specifically relevant to evaluating the possible relationship to cardiac disease [81, 87]. The best current hypothesis is that the delay relates to the digestion of glycolipids: firstly, the chylomicrons which pass through the thoracic duct, and after approximately one hour enter the circulation: secondly, the processing of chylomicrons that are approximately 400 nm in diameter to LDL approximately 25 nm (Figure 8). The important feature of these particles is that it is now well established that both the proteins and the lipids on these particles are glycosylated [88]. Equally, there is now extensive evidence that the glycosylation of both proteins and lipids on LDL particles can be altered either by diet or medicines [89]. The possible relevance to clinical reactions assumes that these particles gain access to mast cells in the tissue when they pass out of the circulation using LDL receptor or other receptors on the endothelial surface. Our hypothesis includes the possibility that LDL can be glycosylated with galactose‐alpha‐1,3‐galactose (alpha‐gal) coming from their diet and can cross‐link sIgE on mast cells, or in some way accelerate entry of LDL and contribute to the overload of the vessel walls with cholesterol [49, 89]. Overall, it is clear that more research is needed about the ways in which lipid particles change during the period after eating a meal derived from a mammalian source.

FIGURE 8.

Time course of the digestion of glycolipids after eating mammalian meat or organs. Over the first hour, glycolipids but not glycoproteins can pass through the intestinal wall and be formed into the chylomicrons in the lacteals, which lead to the thoracic duct. Chylomicrons are large (i.e., 300–1000 nm) and are largely made of fat, so they float on the top of serum. Over the next 2–6 h they are transformed into VLDL and finally to LDL, which are 12–25 nm in diameter, and can pass through endothelial walls. In the tissues of an allergic subject, the LDL, with alpha‐gal on them, could cause release of mediators such as histamine, leukotrienes, and tryptase from mast cells.

5. The Immunology of the Alpha‐Gal Syndrome

The realization that the target of the IgE antibody response to Cetuximab was an oligosaccharide epitope opened up many questions about the control of glycosylation of protein molecules produced in non‐mammalian cell lines [5, 7, 90]. However, the fact that the same specificity of IgE antibodies was present in almost all the patients who presented with reports of urticaria or anaphylaxis starting three to five hours after eating meat or other products derived from non‐primate mammals dramatically extended the clinical significance of these antibodies [30, 53]. Traditionally, antibody responses to oligosaccharides are predominantly of the IgM or IgG2 isotypes and are thought to be relatively T independent. By contrast, IgE responses, which are largely specific for proteins, are known to be T cell dependent. Thus, there was no obvious established view of the role that T cells were expected to play in promoting IgE responses to alpha‐gal. The second problem, or question, is why this oligosaccharide specific IgE response should be associated with food‐related clinical symptoms while the plant‐based cross‐reactive carbohydrate determinants (CCD) such as MUXF3 are generally not considered to be related to symptoms [53, 91, 92, 93].

The central fact is that galactose–alpha‐1,3‐galactose is an oligosaccharide and is also a hapten; this, of course, is a common feature of the CCDs. One of the cardinal features of the natural immune response to alpha‐gal is that, despite producing significant or large quantities of IgG, IgM, and IgA antibodies, it almost never includes sIgE antibodies to alpha‐gal [93]. Thus, people who live in Boston or Northern Sweden, where ticks are extremely rare, do not have sIgE to alpha‐gal, and cases of AGS are absent or rare [5, 61]. The natural antibody response to alpha‐gal that does not include IgE antibodies is assumed to be stimulated by the microbiota of the gut. If so, it is logical to believe that the response is controlled by the kind of homeostasis that Belkaid and Harrison describe occurring between regulatory T cells and effector T cells that promotes oral tolerance [94]. While there are many different factors that influence the balance in that response, there is good evidence that unmethylated DNA derived from organisms in the gut can make an important contribution [95]. That observation is potentially very interesting in considering why the response in the gut does not include sIgE. Early experimental work on the control of sIgE production in animal models by Moneret‐Vautrin, Provost‐Danon, and others in mice as well as by Kishimoto and Ishizaka in rabbits provided strong evidence that Complete Freund's Adjuvant (CFA), which includes bacterial cell wall and unmethylated DNA, was the most effective way to inhibit IgE production [96, 97].

What is now certain is that the primary way that this form of tolerance to mammalian foods can be broken is by the effect of tick saliva injected into the skin. In Virginia, this is almost exclusively by Lone Star Ticks; however, as we have outlined, there are multiple species that can induce the effect, and the clinical syndrome is similar in each county where individuals with an IgE response to alpha‐gal have been documented. Much of the primary research on the saliva of lone star ticks, including the sialome, came from the laboratory of Dr. Ribeiro at the NIH [57, 58]. Work by Crispell and colleagues revealed strong upregulation of alpha‐gal production in lone star tick salivary glands [98]. Importantly, this upregulation occurred even in ticks fed on an artificial feeding system that relied on human blood, which argues against an important role for tick acquisition of alpha‐gal from a prior feed on a mammalian host (e.g., deer). In addition, Dr. Park in Kansas collaborated with Dr. Azadi and her colleagues at the Complex Carbohydrate Center in Athens, Georgia, to publish a detailed study on cross‐reactive carbohydrates in saliva [99]. That study also defined the complexity of the pathways for the synthesis of oligosaccharides in the Rough Endoplasmic Reticulum (RER) and the Golgi apparatus. The first experiment to use tick saliva to immunize alpha‐gal KO mice was reported by Araujo et al. in Brazil. In 2016, they reported that intradermal injection of saliva from Amblyomma sculptum could induce both IgE and IgG antibodies specific for alpha‐gal [100]; subsequently, Choudhary et al., using salivary gland extracts obtained from feeding ticks by their colleague Shahid Karim, established a model in mice that had many of the features of AGS, including sIgE and IgG antibodies to alpha‐gal and anaphylactic responses after oral exposure to meat [59]. By contrast, using extracts of whole body adult or larval ticks, the specific IgE response was modest but could be boosted effectively by adding alpha‐gal BSA [21]. Importantly, that study provided evidence that the IgE response required CD4+ T cells and that the antibody production was dependent on the MyD88 pathway. That the T‐cells, which are involved in this response, would have characteristics of Th2 cells seems obvious and may well be relevant to both the production of sIgE and the fact that bites from these ticks can start itching in the skin within a few hours [101]. Our initial observation about itching was that reports that a tick bite or bites had itched for two weeks or more were strongly associated with the presence of sIgE to alpha‐gal [29]. As we have already considered, repeated episodes of itching tick bites on Block Island were negatively associated with positive serology to Lyme [55]. Logically, a considerable amount of research has focused on understanding both (i) the effect of tick saliva in the skin and (ii) which elements of the saliva are most important [59, 102]. However, there is other evidence that fully activated salivary glands contain alpha‐gal [21, 98, 103]. The recent review by Raj Sharma and Karim highlights many important points on this topic, including evidence that tick‐derived factors such as prostaglandins, sphingomyelinase, and cysteine protease inhibitors favor Th2 induction. What is worth re‐emphasizing here is that there is no evidence that zoonotic organisms transmitted by tick bites contribute to the alpha‐gal sensitization [72].

Patients with the syndrome have provided strong evidence that they have T cells which respond in vitro to tick proteins, but such T cells were not observed that recognized alpha‐gal [101]. On the other hand, multiple studies have pointed to an important role for T cells in promoting the alpha‐gal antibody response [21, 104, 105]. Moreover, Hils et al. recently reported a critical role for IL‐4 in alpha‐gal IgE sensitization, suggesting a role for Th2 cells. Many questions about T cell involvement in AGS remain but it seems likely that they are acting as carrier specific T cells which are important for the IgE response. A role for NKT cells, which can be a major early source of IL‐4 and can directly bind certain glycolipid structures, has also been proposed, but to date there is limited direct evidence for this [106]. It is also in keeping with the fact that sIgE responses to alpha‐gal tend to decline in the absence of recurrent tick bites [107]. On the other hand, there is a large quantity of observational evidence that continued consumption of mammalian food does not increase the production of specific IgE to alpha‐gal. Thus, we would conclude that the development of an IgE response to alpha‐gal induced through the skin does not extend to the “natural” response in the gut associated lymphoid tissue (GALT).

In natural exposure or challenge studies with mammalian meat or organs, we believe that a large part of the delay in symptoms is best explained by the time taken to absorb glycolipids and form chylomicrons, as well as the time taken to process these lipid‐laden particles down to the size that can escape from the circulation and reach areas where sensitized mast cells are present [49]. This model implies that a clinical response to a CCD such as alpha‐gal requires a particle with multiple epitopes of this hapten on it (Figure 8). Interestingly, no such model has been described for MUXF3 or any other plant or venom‐derived CCD [91, 92, 93]. Equally, it implies that the glycoproteins derived from meat or other tissue of a mammal do not reach the circulation in an appropriate form that could reach mast cells and trigger allergic symptoms [88]. To date, no human challenges have been described where the challenge material is selectively present or primarily a glycoprotein. At present, it seems most likely that the striking absence of immediate symptoms following oral exposure is best explained by the difference in absorption of glycoproteins compared to glycolipids [38, 52]. Although there is good evidence that carrier‐specific T cells are involved in the antibody response to alpha‐gal induced by tick bites, there is no reason to think that T cells play a role in the delayed response that follows consumption of meat or organs of mammals by subjects with AGS.

6. Conclusions

Studying the immune response that underlies alpha‐gal syndrome (AGS) only became possible once the primary features of cases were clear. That included (i) the specificity of IgE antibodies to cetuximab; (ii) the relevance of tick bites as the cause of sensitization; (iii) the fact that in most cases the reactions to eating mammalian meat start 3–5 h later. The immunology then starts with the IgG, IgM, and IgA “natural” response to alpha‐gal which is thought to be driven by proteins or glycolipids from the gut microbiome. However, that could explain the fact that IgE antibodies to alpha‐gal among people who only eat mammalian meat but do not get tick bites are very rare or non‐existent. The driving force of a tick bite is the injection of the complex mixture present in tick saliva. The subsequent IgE response to alpha‐gal involves T cells that are specific for tick proteins and have a strong Th2 signal; those T cells are almost certainly responsible for the switch to IgE of the existing IgG or IgM committed B cells in the circulation, and may well be the primary cause of the intense itching that occurs after repeated bites from lone star ticks. A major question remains about exactly why the allergic response that follows eating mammalian meat takes 3–5 h before it starts. However, the most convincing hypothesis at present is that the delay represents the time taken to process chylomicrons to LDL. In keeping with our current understanding of the syndrome, the primary treatment for the symptoms is to avoid eating meat or other foods derived from non‐primate mammals. On the other hand, the only way to reduce or eliminate IgE to alpha‐gal is to avoid tick bites [108]. Finally, we are studying an IgE response to a mammalian oligosaccharide that is induced by tick saliva in the skin, while the symptoms can be induced by eating mammalian meat or organs or by injection of medicine carrying this epitope, such as cetuximab.

7. Major Ongoing Research Questions

Having largely resolved the causes and clinical details of the syndrome, there are several major questions that are the focus of current research.

1. What is the nature of the antibody response to alpha‐gal that is present in all non‐primate mammals? Is this an important model of tolerance to food?

2. How do tick bites induce isotype switch, and is this restricted to switching of pre‐existing IgG or IgM B cells in the circulation? What is the nature of the relevant T cell?

3. Why is the clinical response to meat or organs of non‐primate mammals always or nearly always delayed by two to six hours? The primary hypothesis is that the delay relates to the time taken for glycolipids, including alpha‐gal, to be processed to the size of LDL, that is, 20 nm.

4. What is the difference between ticks that induce IgE to alpha gal, A. americanum , I. ricinus, I. holocyclus , and so forth, and those that can cause anaphylaxis, for example, Argus reflexus and I. pacificus , but are not known to induce IgE to alpha‐gal ?

Why has the tick A. americanum and bites of adults, nymphs, and larvae increased so dramatically in a large swath of the southeastern United States as well as “Three Corners”?

The primary breeding host of this tick is deer, so the primary question is, why have the deer increased so dramatically since 1980? (See Box  2 ).

BOX 2. The relevance of dogs to the rise in the number of white‐tailed deer in Virginia as consequent increased tick bites.

Over the last 40 years the primary concern of our research group has been to understand why allergic diseases develop and, in particular, have increased [Ref A]. Given that our group including Dr. Heymann, Dr. Borish, and Dr. Platts‐Mills have been practicing in central Virginia for a long time we are convinced that classical cases of the alpha‐gal syndrome have increased dramatically. In addition, we are certain that the Lone Star tick is the primary cause of sensitization. The white‐tailed deer in Virginia are the primary breeding host for this tick. Thus, the increase in the deer population has to be relevant. In 1950 deer were not present in Albemarle County, VA or the county around Chapel Hill, NC, USA and were essentially extinct in the state of Missouri. Since 1950 the deer have increased in Albemarle County from from <10 to >5,000. This involved reintroduction between 1948 and 1950 by the Virginia State Game Commission, followed by the invention of invisible fences, and then by leash laws for dogs in the early 1980's. Understanding the effects of controlling dogs requires appreciation of the role of dogs in the development of villages and larger units of human civilization. The first animal species to show signs of synanthropic behavior were ravens and wolves [Ref B]. The first signs of wolves living close to humans occurred approximately 25,000 years ago, and by 15,000 years ago there was evidence that dogs which were genetically different from wolves were present. The relevance of dogs to the domestication of other species is obvious in protecting sheep, and cattle from wolves and chickens from foxes. The relevant fact here is that deer are now common close to suburban houses. In keeping with that, approximately half the cases of AGS that we diagnose, report getting tick bites on their own land. The obvious point is that it may be foolish to restrict our dogs so completely that we are now suffering from a disease caused by ticks from infested wild animals living next to our houses.

[Ref A] Platts‐Mills TA. The allergy epidemics: 1870–2010. J Allergy Clin Immunol 2015; 136: 3–13.

[Ref B] Bauman C. The paleo‐synanthropic niche: a first attept to define animal's adaptation to a human‐made environment. Arch Anthropol Sci. 2023; 15: 63.

Conflicts of Interest

Dr. Platts‐Mills and Dr. Wilson report grant support from the NIH and material support for assays from ThermoFisher Phadia.

Platts‐Mills T. A. E., Gangwar R. S., Workman L., and Wilson J. M., “The Immunology of Alpha‐Gal Syndrome: History, Tick Bites, IgE, and Delayed Anaphylaxis to Mammalian Meat,” Immunological Reviews 332, no. 1 (2025): e70035, 10.1111/imr..

Data Availability Statement

For Figures 2, 3, 5, 6, and 7 data availability rests with the authors of the published references and all come from the University of Virginia.