Relevance of lipid transfer protein (LTP) to Cannabis sensitization in North America

Allergic reactions to cannabis, although sporadic, have been reported over the last 5 decades.¹ With increased access and decriminalization of cannabis, cases of symptomatic exposures have become increasingly frequent.² Clinical investigations into allergic reactions to cannabis are challenging owing to broad symptomology that makes it difficult to distinguish cannabis allergy from general physiological reaction to the distinct plant components. More recently, investigations from our laboratory and our collaborators have resulted in identification and subsequent validation of 4 putative allergens of cannabis,^2-5 thus paving the way for the development of novel diagnostic platforms.

One major cannabis allergen deemed critical for the development of allergic sensitization is the non-specific lipid transfer protein (nsLTP or Can s 3).⁵ In Europe, Can s 3 was identified as a critical driver of cross-reactivities to homologous allergens commonly found in other plants and foods.⁶ Further, Can s 3 was reported in cannabis pollen, thus extending the scope of its allergenic potential.⁷ However, in our previous study, while examining type I hypersensitivity to cannabis among symptomatic recreational users in North America, we did not identify IgE binding to nsLTP using protein extracts.³ Although, this observation raised questions on the relevance of Can s 3 in North America, concerns existed on the methodological limitations presented at the time. Specifically, drawbacks included the relatively crude nature of the cannabis protein extracts, intrinsic limitations of immunoproteomics approach and lack of complimentary analytical platforms required for validation.

In this report, we detail methodological advancements that have aided in establishing the relevance of Can s 3 to cannabis-linked allergic reactions in North America. First, we collected serum samples from fourteen symptomatic recreational users of cannabis from Canada. Patients with suspected allergic sensitization to cannabis demonstrated typically diverse symptoms including urticaria, angioedema, rhinitis, chest tightness, and exacerbation of asthma. Importantly, all patients tested positive on skin prick test (SPT) with cannabis leaf and bud extract when tested per methods described previously.⁸

To overcome the limitations presented by crude cannabis protein extracts employed in our prior study,³ we developed a purified Can s 3 expressed in E.coli using recombinant protein expression approaches (RE-CS3-1; InBio, Charlottesville, VA). Recombinant Can s 3 (rCan s 3) contains a 6xHis-tag on the C-terminus of the protein. Purity of the rCan s 3 was resolved using standard sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) approach and subsequent protein staining with Imperial^™ protein stain (Thermo Fisher), which revealed a protein band at ~ 12 kDa (Fig. 1; left panel). Western blot approach (15% SDS-PAGE) was used to probe rCan s 3 (5 μg) with serum (40 μl each; diluted 1:5 v/v in phosphate buffered saline (PBS) containing bovine serum albumin (BSA) from 14 cannabis allergy patients. Patient serum IgE binding to rCan s 3 was established using an anti-human IgE mouse monoclonal antibody (1:2000 v/v in PBS containing BSA) and subsequent incubation with an anti-mouse antibody coupled to infra-red dye (IRdye^®, LiCOR) as per methods described previously.³ Immunoreactive signals were detected using a LiCOR instrument. Sera from five patients (ID# 1,2,3,9 and 14) revealed IgE binding to rCan s 3 indicating positive reactivity (Fig. 1; right panel). We did not observe IgE binding to rCan s 3 in remaining serum samples.

Figure 1. Recombinant Can s 3 and IgE immunoreactivity in western blot.

Recombinant Can s 3 (500 ng) subjected to SDS-PAGE approach migrates as a ~12 kDa protein (left panel). Western blot analysis of serum IgE reactivity to rCan s 3 (right panel).

To further investigate Can s 3-specific IgE in symptomatic cannabis users, two (polyclonal and monoclonal antibody) serum samples were compared by chimeric ELISA using either polyclonal or monoclonal anti-IgE for detection. In addition to the samples indicating positive immunoreactivity (IgE binding to rCan s 3) in western blot analysis (Fig. 1; right panel), the polyclonal anti-IgE ELISA also detected positive reactivity in 3 additional serum samples (Fig. 2). In comparison, the monoclonal anti-IgE ELISA was mostly consistent with western blot analysis with only one additional sample demonstrating positive IgE immunoreactivity. These studies suggest that chimeric ELISA methods provided better resolution of serum IgE binding to Can s 3protein than immunoblotting.

Figure 2. Chimeric ELISA approaches for detecting Can s 3-specific IgE.

Can s 3 IgE (IU/ml) levels examined by chimeric ELISA using either polyclonal or monoclonal anti-IgE. Red circles indicate patient samples with positive result, while open circles indicate patient samples with negative result in western blot approach. Black line defines the threshold for ‘positive’ immunoreactivity. IU: International units.

nsLTPs from cannabis and other food-plant sources have been suggested as critical molecular drivers of cross-reactive allergies in Europe.⁵ However, reports on cannabis allergies in North America have been limited and the relevance of Can s 3 in North American cohorts has been inconclusive to date. Our studies demonstrate that cannabis nsLTP (Can s 3) is a relevant allergen for symptomatic exposure to cannabis. Further, patients with symptomatic exposure to cannabis in Europe and North America alike exhibit IgE antibodies to Can s 3 at comparable rates. As accessibility to cannabis increases for recreational and medicinal use, allergic reactions to cannabis may emerge as a public health issue. Further, a significant workforce is now engaged in cannabis industry with latest estimates identifying over 420,000 workers employed in growing and processing cannabis (www.leafly.com). Finally, our report is one of the few to demonstrate the relevance of nsLTP class of allergens in North American cohorts, underscoring the need to examine nsLTP sensitization, particularly in context of food allergy. This is a clarion call for the continued development of component resolved diagnostics (CRD) approaches and increased participation by allergists to address an emerging public health issue.

CLINICAL IMPLICATIONS.

Clinical determination of allergic sensitization to cannabis is difficult owing to lack of objective diagnostic measures. Our studies demonstrate that cannabis non-specific lipid transfer protein (nsLTP or Can s 3) is a clinically relevant allergen in North American cohorts.