Cracking the cashew nut : Strategies to identify novel allergens

Bastiaan-Net, Shanna


Cashew nut allergy has been recognized as a severe tree nut allergy amongst (Dutch) children and young adults and its prevalence seems to be increasing. For clinical diagnosis of an allergy, it is essential to know the causative agents in the food product causing the allergic symptoms.

In this thesis entitled ‘Cracking the cashew nut: strategies to identify and characterize novel allergens’, we aimed to apply innovative strategies and technologies to identify and characterize putative allergenic proteins in cashew nut, to broaden the current knowledge on cashew nut allergens beyond those already known (Ana o 1, Ana o 2 and Ana o 3). Our knowledge of cashew nut proteins that can trigger an allergic reaction is currently very limited, especially compared to other nuts or seeds in which the allergen repertoire has been researched much more widely. Using several different strategies, we evidenced that additional allergens must be present in cashew nuts, which presumably contribute to the elicitation of allergic symptoms in cashew nut allergic patients. Knowledge of newly identified cashew nut proteins provides a basis for further research to extend clinical diagnostic tests and treatments currently available for cashew nut allergy.

Chapter 2 includes an opinion on the use of current in vivo and ex vivo endpoints in murine food allergy models and their suitability for evaluating the sensitizing capacity of protein concentrates and/or food products. An overview is given of the best predictive risk assessment methods and endpoint parameters currently relied on in in vivo food allergy models with a focus on milk, egg and peanut allergens, addressing their strengths and limitations for assessing sensitization risks. Findings indicated that, although the current available models are suitable for studying the pathophysiology of food allergy, they still couldn’t predict the magnitude of the allergic potential of a particular allergen. Thus, there is still a strong need to better define the allergic reaction to predict the clinical outcomes of sensitization to novel food proteins. In addition, there is an urgent need for a consensus on key food allergy parameters to be applied in future food allergy research, to guarantee optimal lab- to-lab reproducibility and reliable use of predictive tests for protein risk assessment.

Cashew nut allergic individuals may develop cross-reactive responses to foods that are phylogenetically related to cashew nut. In Chapter 3, we therefore aimed to determine the IgE cross-sensitisation and cross-reactivity profiles in cashew nut sensitised subjects. Profiling was specifically aimed at botanically related proteins of common tree nut species and other Anacardiaceae family members like pistachio, mango, pink peppercorn or sumac. Half of cashew nut positive sera on dot blot were co- sensitised; 19% to solely Anacardiaceae species and 31% to tree nuts, which indicated that cross-sensitisation/cross-reactivity is widespread among cashew nut allergic individuals. Interestingly, subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. Putative underlying novel allergens were identified in cashew nut, pistachio and pink peppercorn, which demonstrated that indeed additional allergens might exist in cashew nut that may pose factors underlying cashew nut allergic symptoms.

In line with these findings, we applied a novel IMMULITE®-based inhibition methodology in Chapter 4, to investigate the IgE cross-reactivity between cashew nut-, hazelnut- and peanut proteins in children that are multi-allergic to these foods. Observations indicated that hazelnut extract was a strong inhibitor of cashew nut sIgE while cashew nut extract was less able to inhibit hazelnut extract. In contrast, peanut extract showed the least inhibition potency. Importantly, there were strong indications that a birch pollen sensitisation to Bet v 1 might play a role in the observed symptoms provoked upon ingestion of cashew nut and hazelnut, suggesting the existence of putative Bet v 1-like protein homologs in cashew nut.

Based on the strong indications that additional allergenic proteins may exist in cashew nut, cashew nut transcript profiling was conducted resulting in a RNA-seq database that can be used to screen for protein homologs of allergens identified in phylogenic related species. In Chapter 5, we applied this method to identify and characterize three PR10 proteins in cashew nut. The identification and partial characterization of two additional 2S albumin proteins, next to the major cashew nut 2S albumin Ana o 3.0101, are described in Chapter 6.

Finally, Chapter 7 discusses the major findings of the different research chapters and pros and cons of the applied strategies. Additional putative cashew nut allergens are presented, identified using the RNAseq screening approach mentioned in chapter 5 and 6 which, although not yet characterized, likely contribute to the allergen repertoire of cashew nut. To conclude, future research opportunities are presented that could take our current knowledge of cashew nut allergy to a higher level.