Dear colleagues,
As I write this blog at the end of January 2025, we’ve just achieved a remarkable milestone: over 110,000 PAX tests have been conducted on sera from allergy-suspected dogs across the EU, US, and UK!
It is needless to say that we already began analyzing this gigantic trove of data. You will soon see reports on the clustering of sensitizations, seasonality of results, and variations of sensitization patterns between countries and species. Stay tuned!
For those eager to learn more about the PAX's intricacies, you can point your browser to this link (here) to download an extensive article detailing its design and validation for dogs. Never before will you have seen such a transparent publication about the inner workings of a commercial serological assay for allergen-specific IgE in companion animals!
In this newsletter, let me highlight some of the most common sensitizations detected in dogs in the last two years. I will then follow with a preview of what will be coming in the PAX2, the next version of the PAX.
Below is a comparison of the top 10 sensitizations detected in European and American dogs in 2023 and 2024.
On both sides of the Atlantic, the most detected sensitization in dogs is the well-known positivity to the Dermatophagoides farinae (Der f) house dust mite extract. This predominance of reactivity to Der f is somewhat surprising, as sensitization to the closely related species D. pteronyssinus (Der p) is more than ten times less common, even though the latter species is more prevalent in Europe and the coastal US. In 2024, we established that the previously believed most common Der f allergens for dogs (Der f 15 and Zen-1, and likely Der f 18) cross-react through their sugars with mucins from Toxocara canis larvae. Surprisingly, the Der p extract does not seem to cross-react with Toxocara canis, and this difference explains the far lower sensitization to Der p than that to Der f! The lower sensitization rate to Der f in the US may just reflect the decreased prevalence of Toxocara canis infestations in puppies in that country. Do note that Der f 15 and Der f 18 in the PAX do not carry the sugars that cross-react with Toxocara canis, so the test will rarely return positives to these allergens. For more details, click here.
Currently, we still recommend immunotherapy when an allergy-suspected dog (and cat!) has a positive PAX to Der f. Indeed, we suspect that normal dogs with positive tests to this extract are tolerant to its allergens, while atopic pets are not. We will research this topic further in 2025.
In both Europe and the US, the main storage mite that sensitizes dogs is Tyrophagus putrescentiae (Tyr p). This is not surprising in light of this mite being the main one proliferating in pet foods (here).
Another common sensitization pattern observed on both continents is sensitivity to Hymenoptera venoms. Like humans, a sensitization to venom allergens from bees and wasps in pets only indicates that the patient received one or more previous stings in the past. A positive sensitization does not necessarily mean that pets will experience a severe clinical reaction upon being stung again. Nevertheless, a previous publication did point out that the PAX is highly sensitive in detecting Hymenoptera venom sensitization in dogs experiencing anaphylaxis related to bee or wasp stings (here). Because similar sensitizations to these venoms are not found in laboratory beagles kept indoors, the PAX has both high sensitivity and specificity for detecting this type of sensitization.
Finally, the primary difference between Europe and the US in the “top ten” dog sensitivities is the apparent higher frequency of sensitization to weeds in the latter. This observation will not surprise European veterinarians, who have historically noted uncommon skin test positivity to pollens.
In 2024, we confirmed that IgE against CCDs (cross-reactive carbohydrate determinants), which are complex glycans shared by many plant-based and Hymenoptera venom allergens—but not by proteins from mammals—were not capable of inducing positive skin test reactions and mast cell degranulation in vitro (here). We also established that the PAX has fewer positive results to pollens than traditional ELISA because of its unique CCD-IgE blocking strategy and its use of detectors of the efficiency of such blocking. We will soon share data confirming that several commercial IgE serological tests in Europe and the US have insufficient CCD-IgE blocking and, thus, commonly report false-positive sensitizations to pollen extracts.
At the time we began developing the first version of the PAX, fewer than 30 molecular allergen components had been identified for dogs, and none had been identified for cats! We then postulated that the unique sequence, function, and structural characteristics that made certain proteins allergenic for humans would also make them allergenic for animals. After including some of the allergen components unique to dogs and removing those seemingly irrelevant to animals (like those of kiwi fruit), the first PAX for dogs had 247 allergen spots, with 69% of them coated with molecular components.
Two years later, I am happy to report that the chosen approach was indeed the right one! All PAX components and extracts have been found to sensitize at least some dogs! The lowest recognized allergens were those of the mold Penicillium chrysogenum (Pen ch) and the Ory c PKM (rabbit protein kinase M), which sensitized less than 0.05% of dogs worldwide.
A question that we frequently receive are variations of the following: Are you sure that you have the most relevant allergens for animals in the PAX?
This was indeed a question we had asked ourselves too! In 2023, we took advantage of the newly available AllerSIGHT from the U.S. biotech startup Infinity Bio (here) to begin our search for relevant allergens for pets. This library contains nearly 30,000 peptides that cover the complete sequence of about 4,500 different allergens for humans. In 2024, we tested close to 500 dogs and 500 cats (including healthy pets) using this library, which enabled us to identify nearly 1,900 and 600 different proteins targeted by IgE from allergy-suspected dogs and cats, respectively. The proteins bound by IgE from 10% or more of dogs and cats were a more modest 67 and 9, respectively. This technique proved particularly useful in helping us identify some highly prevalent mite allergens, such as Der f 4, Der p 33, and Tyr p 35 for dogs, and Der p 24 for cats! These allergens had not previously been determined to be relevant in animals!
The main limitation of AllerSIGHT is that it only includes protein sequences already recognized as allergens for humans. But what about those proteins that no one knows yet could be allergenic?
Our colleagues from the Nextmune UK labs set out to identify components targeted by IgE from dogs and cats that showed some positives to extracts (like Der f) without any positivity to corresponding components in the PAX. Through a combination of two-dimensional immunoblotting and mass spectrometry, new IgE-targeted proteins were identified in Der f (e.g., enolase) and Tyr p (e.g., superoxide dismutase). Recently available algorithms indicated that many of these proteins were predicted to be highly allergenic.
In our Madrid Nextmune laboratory, we similarly investigated food allergens. When we became aware that a dog had an immediate reaction to a specific food (for example, urticaria within 30 minutes of eating beef) but received a negative PAX for this same food, we suspected that the test was missing crucial components. Using the serum remaining after PAX testing from 10 dogs, we performed immunoblotting and mass spectrometry and identified new food allergens recognized multiple times across various species. Examples include the carbonic anhydrase found in several mammal meats and adenylate kinase present in different poultry species.
Overall, 2023 and 2024 have brought many discoveries! Following the investigations mentioned above, we created a “master list” of new allergen components that will be tested for inclusion in the upcoming PAX2. Work has begun at MADx, the manufacturer of PAX, on 25 of these proteins. They need to be produced recombinantly, and several stages of validation must occur before they are suitable for inclusion in the PAX2. This production and testing are expected to continue over the next 2-3 years, with each year bringing an additional list of a couple dozen of new components. We plan to roll out new PAX versions in several updates as new components are validated and included.
As you might have heard us say before, “The PAX is a living test!” We are continuously modifying some of its features and adding new quality control behind the scenes, with the goal of having a “masterpiece” with the highest possible accuracy for determining IgE sensitizations in companion animals. We are committed to continuously analyzing results and reporting our investigations to help advance molecular allergology in veterinary medicine.
We hope you will join us on this exciting journey!
Respectfully submitted,
Thierry Olivry, Head of R&D, Nextmune