Allergen immunotherapy (AIT) has gained a permanent place in the therapeutic arsenal for the patient with allergy. Particularly, substantial evidence has been established for the efficacy of AIT in allergic rhinitis. A hallmark of AIT is it disease modifying effect resulting in persistent benefit after the treatment has been terminated. Both the subcutaneous and sublingual mode of administration appear to be safe. It is, however, a matter of debate whether AIT can be implemented for patients with asthma. EAACI and GINA guidelines recommend sublingual AIT in house dust mite driven asthma. The question however remains whether the different available forms of AIT should be used for allergic asthma in general.
Background: Nonimmediate (delayed) allergic reactions to penicillins are common and some of them can be life-threatening. The genetic factors influencing these reactions are unknown/poorly known/poorly understood. We assessed the genetic predictors of a delayed penicillin allergy that cover the HLA loci. Methods: Using next-generation sequencing (NGS), we genotyped the MHC region in 24 patients with delayed hypersensitivity compared with 20 patients with documented immediate hypersensitivity to penicillins recruited in Italy. Subsequently, we analyzed in silico Illumina Immunochip genotyping data that covered the HLA loci in 98 Spanish patients with delayed hypersensitivity and 315 with immediate hypersensitivity compared to 1,308 controls. Results: The two alleles DRB3*02:02:01:02 and DRB3*02:02:01:01 were reported in twenty cases with delayed reactions (83%) and ten cases with immediate reactions (50%), but not in the Allele Frequency Net Database. Bearing at least one of the two alleles increased the risk of delayed reactions compared to immediate reactions, with an OR of 8.88 (95% CI, 3.37–23.32; P <0.0001). The haplotype (ACAA) from rs9268835, rs6923504, rs6903608, and rs9268838 genetic variants of the HLA-DRB3 genomic region was significantly associated with an increased risk of delayed hypersensitivity to penicillins (OR, 1.7; 95% CI: 1.06–1.92; P=0.001), but not immediate hypersensitivity. Conclusion: We showed that the HLA-DRB3 locus is strongly associated with an increased risk of delayed penicillin hypersensitivity, at least in Southwestern Europe. The determination of HLA-DRB3*02:02 alleles in the risk management of severe delayed hypersensitivity to penicillins should be evaluated further in larger population samples of different origins.
SARS-CoV-2 caused one of the most devastating pandemics in the recent history of mankind. Due to various countermeasures, including lock-downs, wearing masks and increased hygiene, the virus has been controlled in some parts of the world. More recently, the availability of vaccines, based on RNA or Adenoviruses, have greatly added to our ability to keep the virus at bay, again in some parts of the world only. While available vaccines are effective, it would be desirable to also have more classical vaccines at hand for the future. Key feature of vaccines for long-term control of SARS-CoV-2 would be inexpensive production at large scale, ability to make multiple booster injections and long-term stability at +4 oC. Here we describe such a vaccine candidate, consisting of the SARS-CoV-2 receptor binding motif grafted genetically onto the surface of the immunologically optimized cucumber mosaic virus, called CuMV TT-RBM. Using bacterial fermenter production and continuous flow centrifugation, the productivity of the production process is estimated to be >2.5 million doses per 1000 liter fermenter run and the vaccine candidate is stable for at least 14 months at 4°C. We further demonstrate that the candidate vaccine is highly immunogenic in mice and rabbits and induces more high avidity antibodies compared to convalescent human sera and antibodies induced are more cross-reactive to mutant RBDs for variants of concern (VoC). Furthermore, antibody responses are neutralizing and long-lived. This, the here presented VLP-based vaccine may be a good candidate for use as conventional vaccine in the long-term.
Increased circulating CRTH2+Tregs are associated with asthma control and exacerbation Short running title:Increased circulating CRTH2+Tregs among patients with asthmaTeerapol Chantveerawonga, Sasipa Sangkangjanavanicha,b, Chirawat Chiewchalermsria,c, Panitan Pradubpongsaa, Wat Mitthamsiria, Sarawut Jindaratd, Ming Wange, Mübeccel Akdisf, Milena Sokolowskaf, Cezmi A. Akdisf, Atik Sangasapaviliyaa, Tadech Boonpiyathada,faDivision of Allergy and Clinical Immunology, Department of Medicine, Phramongkutklao Hospital, Bangkok, Thailandb Division of Allergy, Immunology and Rheumatology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, ThailandcDepartment of Medicine, Panyananthaphikkhu Chonprathan Medical Center, Srinakharinwirot University, Nonthaburi, ThailanddDepartment of Pharmacology, Phramongkutklao College of Medicine, Bangkok, ThailandeDepartment of Otolaryngology, Head and Neck Surgery, Beijing TongRen Hospital, Capital Medical University, and the Beijing Key Laboratory of Nasal Diseases, Beijing Institute of Otolaryngology, Beijing, ChinafSwiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, and the Christine Kühne-Center for Allergy Research and Education (CK-CARE), Davos, Switzerland
COVID-19 vaccination with BNT162b2 and ChAdOx1 vaccines induces nasal neutralizing antibodiesDeclercq Jozefien(1,2,5)*, Tobback Els(3)*, Vanhee Stijn(2,5), Natalie De Ruyck(1), Gerlo Sarah(4), Gevaert Philippe(1)**, Vandekerckhove Linos(3,4)***shared co-first authorship** shared last authorshipUpper Airways Research Lab URL, Department of Otorhinolaryngology, Ghent University, Ghent, BelgiumLaboratory of immunoregulation, VIB Center for Inflammation Research, Ghent, BelgiumDepartment of General Internal Medicine, Ghent University Hospital, Ghent, BelgiumHIV Cure Research Centre, Department of Internal Medicine and Pediatrics, Ghent University, Ghent, BelgiumDepartment of Internal Medicine and Pediatrics, Ghent University, Ghent, BelgiumCorresponding author:Prof. Dr. Philippe GevaertUpper Airways Research Lab URL, Department of Otorhinolaryngology, Ghent University,C Heymanslaan 10, 1P1Ghent, Belgium+3293324922Philippe.firstname.lastname@example.orgFinancial support: no fundingWord count: 591
Background Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the 4 structural proteins of SARS-CoV-2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography and characterized by tunable resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1 biased T cell responses, reduced virus load and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.
Serum pregnancy-associated plasma protein A (PAPPA) as a predictor of eosinophilic Type-2 high asthmaTo the Editor,Pregnancy-associated plasma protein A (PAPPA), a metalloproteinase that cleaves insulin-like growth factor (IGF)-binding proteins (IGFBPs) to increase IGF availability, is expressed systemically in pregnant women but also in other tissues (1). Higher serum PAPPA levels are reported in patients with newly-diagnosed asthma (1) and allergic rhinitis compared to healthy controls and are decreased following omalizumab treatment (2). We determined whether PAPPA could represent a novel biomarker for Type-2 (T2) asthma by exploring the relationship between asthma severity and phenotypes of severe asthma and PAPPA gene and protein expression (3).We recruited 288 severe non-smoking asthma (NSA), 102 smokers and ex-smokers with severe asthma (SSA), 86 mild/moderate non-smoking asthmatics (MMA) and 95 healthy non-smoking controls (HC) from the U-BIOPRED cohort (NCT01976767) (4) (Table S1 ). Transcriptomic and proteomic profiling of blood and sputum samples and specific serum periostin ELISA were performed (3). Gene set variation analysis (GSVA) was used to calculate the enrichment score (ES) of 34 genes that were upregulated following in vitro stimulation of primary human bronchial epithelial cells with IL-13 (T2_IL-13_IVS) (3). Eosinophilic inflammation was defined by sputum eosinophilia >1.49% (3). Local Ethics Committees of the recruiting centres approved the study and all participants gave written informed consent.Sputum cell PAPPA mRNA was elevated in NSA compared to SSA, MMA and HC subjects particularly in granulocytic asthmatics and in the transcriptomic-associated cluster (TAC)1; an eosinophilic cluster (5) (Figure 1A-C ). This was more pronounced with sputum PAPPA protein analysis according to asthma severity, in eosinophilic and mixed granulocytic asthmatics and in T2-high asthmatics identified by the T2_IL-13_IVS signature (Figure 1D-F ).PAPPA mRNA expression in blood cells was similar across asthma severities, blood granulocytes and molecular phenotypes (Supplementary Figure 1A-C ). However, serum PAPPA protein levels supported the discrimination seen in sputum with significant elevation seen in SA compared to HC, in eosinophilic and mixed granulocytic asthma and in T2-high asthma (SupplementaryFigure 1D-F ).Sputum eosinophil percentages were significantly correlated with sputum (r=0.88, p=10-6) and serum (r=0.41, p=10-6) PAPPA protein levels. Overall, sputum PAPPA protein gave a greater distinction between asthma severity, granulocyte composition and T2-high asthma than with serum although fewer samples were available.These results were validated in sputum from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study (6) (Supplementary Figure S2 ). Elevated PAPPA protein in the serum and sputum of severe asthmatics and in eosinophilic compared to non- eosinophilic subjects was seen (SupplementaryFigure S2A-D ). In addition, sputum PAPPA mRNA levels were also elevated in eosinophilic versus non-eosinophilic asthma in the ADEPT cohort (Supplementary Figure S2E ).The ES score of the T2_IL-13_IVS gene signature in bronchial brushings was significantly, but weakly, correlated with blood eosinophil counts (r=0.329, p=10-6), serum PAPPA (r=0.356, p=10-6), but not with serum periostin levels (r=0.07, p-value=0.48). In contrast, the T2 IL-13 IVS ES score was strongly correlated with sputum PAPPA levels (r=0.72, p=10-3). Sputum PAPPA protein levels also significantly correlated with markers of remodelling such as MMP10 (r=0.646, p<10-6) and MET (r=0.429, p<10-6).Receiver-operating characteristics (ROC) curve analysis was performed for sputum eosinophilia (Supplementary Table S2 ). The area under the ROC curve (AUC) for serum indicated that there was no good predictor although blood eosinophilia was the best (0.79) being marginally better than serum PAPPA and exhaled NO (Figure 2A ). In contrast, sputum PAPPA was an excellent predictor of sputum eosinophilia (0.98), better than blood eosinophilia and exhaled nitric oxide levels (Figure 2B ).Therefore, sputum PAPPA is an excellent biomarker for sputum eosinophilia and for T2-high asthma whilst serum PAPPA is as effective as blood eosinophilia in predicting high sputum eosinophil levels and with T2-high asthma.
Background: Early exposure to allergens through a defect skin barrier has been proposed as a mechanism for inducing sensitization and development of allergic diseases. We hypothesized that early-onset, severe atopic dermatitis (AD) is associated with development of aeroallergen sensitization and allergic rhinitis. Methods: We included 368 children from the Copenhagen Prospective Studies on Asthma in Childhood 2000 (COPSAC 2000) at-risk mother-child cohort. AD was diagnosed prospectively based on Hanifin&Rajka’s criteria and severity assessed using the Scoring Atopic Dermatitis (SCORAD) index. Early-onset AD was defined as debut ≤1 year, late-onset as debut from 1-6 years. Aeroallergen sensitization and allergic rhinitis were diagnosed at ages 6-7 and 12 years. Associations between early-onset and late-onset AD and allergy endpoints were calculated using general estimating equations (GEE) models to compute the overall odds ratios (OR) for both time points. Results: Early-onset AD (yes/no) and severity (SCORAD) were associated with development of aeroallergen sensitization during childhood; GEE OR=1.68 [1.08; 2.62], p=0.02 and 1.08 [1.03; 1.12], p<0.001, whereas late-onset was not; GEE OR=1.65 [0.92; 2.94], p=0.08 and 1.01 [0.97; 1.06], p=0.55. The same trend was seen for allergic rhinitis with significant association between early-onset AD and allergic rhinitis; GEE OR=1.56 [1.01; 2.41], p=0.04 and severity; GEE OR=1.09 [1.05; 1.13], p<0.001, whereas late-onset AD showed no association. The effects on sensitization and rhinitis of early-onset vs. late-onset AD severity were significantly different: p-interaction sensitization=0.03 and p-interaction rhinitis<0.01. Conclusion: Increasing severity of early-onset AD, but not late-onset AD, associates with aeroallergen sensitization and allergic rhinitis later in childhood.
This review presents state-of-the-art knowledge and identifies knowledge gaps for future research in the area of exercise-associated modifications of infection susceptibility. Regular moderate-intensity exercise is believed to have beneficial effects on immune health through lowering inflammation intensity and reducing susceptibility to respiratory infections. Infection-promoting consequences are attributed to strenuous exercise as performed by professional athletes. In about half of the athletes presenting respiratory symptoms, no causative pathogen can be identified. Acute bouts of exercise enhance release of proinflammatory mediators thus probably leading to appearance of infection-like respiratory symptoms. Studies assessing influence of regularly repeated exercise on immune response and systemic inflammation are far less numerous than those regarding acute exercise effects. This identifies another knowledge gap requiring further assessment both in recreational and in professional athletes Additionally, ambient and environmental conditions modify systemic inflammatory response and infection susceptibility in particular in outdoor athletes. Both acute and chronic regular exercise influence humoral and cellular immune response mechanisms resulting in decreased specific and non-specific response in competitive athletes. Most promising areas of further research in exercise immunology include: detailed immunological characterization of infection-prone and infection-resistant athletes; efficacy of nutritional and pharmaceutical interventions as countermeasures to infections’ symptoms; and influence of various exercise loads on susceptibility to infections with respiratory viruses, including SARS-CoV-2. Establishing uniform definition of “elite athlete’ shall hopefully allow for comparable and straightforward interpretation of data coming from different studies and settings.
Immune modulation is a key therapeutic tool for allergic diseases and asthma. It can be achieved in an antigen-specific way via allergen immunotherapy (AIT) or in endotype-driven approach using biologicals that target the major pathways of the type 2 (T2) immune response: IgE, IL-5 and IL-4/IL-13. COVID-19 vaccine provides an excellent opportunity to tackle the global pandemics and is currently being applied in an accelerated rhythm worldwide. It works as well through immune modulation. Thus, as there is an obvious interference between these treatment modalities recommendations on how they should be applied in sequence are expected. The European Academy of Allergy and Clinical Immunology (EAACI) gathered an outstanding expert panel under its Research and Outreach Committee (ROC). This expert panel was called to evaluate the evidence and formulate recommendation on the administration of COVID-19 vaccine in patients with allergic diseases and asthma receiving AIT or biologicals. The panel also formulated recommendations for COVID-19 vaccine in association with biologicals targeting the type 1 or type 3 immune response. In formulating recommendations, the panel evaluated the mechanisms of COVID-19 infection, of COVID-19 vaccine, of AIT and of biologicals and considered the data published for other anti-infectious vaccines administered concurrently with AIT or biologicals.
Needle-free Epicutaneous For t 2 DNA Vaccine is Effective for Preventing and Treating Biting Midge (Forcipomyia taiwana) allergy in a murine modelTo the Editor,Allergen-specific immunotherapy (ASIT) remains the only treatment capable of inducing immune tolerance to the corresponding allergen and potentially treating the root cause of the allergic disease.1 As the treatment course of protein-based vaccines for ASIT is time-consuming, an easily administered epicutaneous anti-allergic DNA-based vaccine is an attractive method, especially in light of the COVID-19 pandemic.2 We established a mouse model of biting midge allergy to test the concept of the epicutaneous DNA vaccine.The biting midge, Forcipomyia taiwana , is the most prevalent cause of biting insect allergy in Taiwan. It is a tiny hematophagous midge that attacks en masse. As many as 60% of exposed individuals develop allergic reactions to the bites.3 The midge is widely distributed throughout Taiwan and southern China. Among the identified allergens, For t 2 is the most predominant, with 75% of midge-allergic patients showing specific IgE to For t 2.4E.coli -expressed For t 2 recombinant protein (rFor t 2) was used as an allergen to sensitize and challenge the mice.5For t 2-encoding fragment (GenBank accession EU678971) was amplified by PCR. The PCR products were subcloned into pVAX1 (Life Technologies, Carlsbad, CA) . The experiments were designed using two approaches: therapeutic and prophylactic (Fig 1). Twenty-five μg For t 2 DNA was determined as the optimal dose after several dose-finding experiments (Fig S1, data not shown). For each treatment, the hair of the abdominal area of the mice was removed using a depilatory, tape-stripped, then patched with 25 μg For t 2 DNA vaccine for one hour and removed. A total of three treatments were given spaced one week apart (Fig 1 and Fig S2). For t 2 proteins were detected in the patched skin and the immune organ spleen at 24 hours and had significantly increased at 48 hours (Fig S3). Scratch bouts after rFor t 2 challenge were used as a clinical surrogate of itch. We measured total IgE and For t 2-specific IgG2a in the sera as well as mRNA and proteins of IL-13, interferon-gamma, IL-10, and FOXP3 in the culture supernatants of splenocytes after stimulation with various doses of rFor t 2 at 37℃ for 3-5 days by ELISA and real-time quantitative PCR. Histopathology of the challenged skins was examined.We found that after epicutaneous DNA vaccination, the allergen-induced itch of the mice significantly improved, and For t 2-specific IgG2a increased (Fig 2). Both mRNA and protein of IL-13, and eosinophils infiltration in the targeted skin, significantly decreased. Expression of FOXP3 mRNA increased (Fig S4-S6).This is the first study to demonstrate an epicutaneous anti-allergic DNA vaccine that is effective at both treating an established allergic condition and preventing the development of an allergic disease using biting midge allergy as a model. After epicutaneous DNA vaccination, in addition to the significant improvement in allergen-induced itch, the changes of biomarkers for allergic inflammation, including IgE, allergen-specific IgG2a, allergen-challenge-induced eosinophil infiltration in the skin, Th2 cytokines from the splenocytes, and regulatory T cell-related transcription factors, suggest that immune tolerance was induced after three patches of the epicutaneous DNA vaccine.Our data show that though the molecular weight of the For t 2 DNA vaccine is as high as 4000 base pairs, it is able to penetrate the dermal barrier and translates the corresponding protein in the targeted skin as well as the spleen of the vaccinated mice. It is possible that the DNA vaccine passes the epidermis via the hair follicles as the skin is tape-stripped before epicutaneous vaccination.6The mode of this anti-allergic epicutaneous DNA vaccine may have potential for use in other specific immunotherapies for other allergens.Mey Fann Lee1Chi Sheng Wu2 Shyh Jye Lin3Yi Hsing Chen2,4*1Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan2Division of Allergy, Immunology and Rheumatology, Taichung Veterans General Hospital, Taichung, Taiwan3School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan4School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
Background: The impact of physical activity (PA) on immune response is a hot topic in exercise immunology, but studies involving asthmatic children are scarce. We examine the level of PA and TV attendance (TVA) in asthmatic children to assess the role on asthma control and immune response to various stimulants. Methods: Weekly PA and daily TVA were obtained from questionnaires at inclusion of the PreDicta study. PBMC cultures were stimulated with phytohemagglutinin (PHA), R848, poly I:C and zymosan. Cytokines were measured and quantified in cell culture supernatants using luminometric multiplex immunofluorescence beads-based assay. Results: Asthmatic preschoolers showed significantly more TVA than their healthy peers (58.6% vs. 41.5% 1-3h daily and only 25.7% vs. 47.2% ≤ 1h daily). Poor asthma control was associated with less frequent PA (75% no or occasional activity in uncontrolled vs. 20% in controlled asthma; 25% ≥ 3x weekly vs. 62%). Asthmatics with increased PA exhibited elevated cytokine levels in response to stimulants, suggesting a readiness of circulating immune cells for type-1, -2 and -17 cytokine release compared to low-PA and high-TVA subjects. Low PA and high TVA were associated with increased proinflammatory cytokines. Proinflammatory cytokines were correlating with each other in in-vitro immune responses of asthmatic children, but not healthy controls. Conclusion: Asthmatic children show more sedentary behavior than healthy subjects, while poor asthma control leads to a decrease in PA. Asthmatic children profit from exercise, as elevated cytokine levels in stimulated conditions indicate an immune system prepared for a strong response in case of infection.
ABBREVIATIONSCA cannabis allergyCCDs cross-reactive carbohydrate determinantsCI confidence intervalCSA cannabis sativa allergic patientsnsLTP s nonspecific lipid transfer proteinsP+LTP- controls sensitized to pollen without nsLTP sensitizationP+LTP+ controls sensitized to pollen and nsLTPs(r) recombinantsIgE specific immunoglobulin EST skin teststIgE total immunoglobulin EKEYWORDSCannabis allergy, sIgE, total IgE, allergy diagnosis, sIgE-to-total IgE ratioTo the Editor,The most important “diagnostic test” for CA is a detailed history. However, a positive history is no absolute proof of CA, mainly because of physiological effects of cannabis i.e. (rhino)conjunctivitis presence and possibly because of incorrect interpretation or recollection of symptoms by the patients under the drug’s influence. Consequently, clinical suspicion of CA requires confirmatory testing. A cannabis challenge, being excluded for obvious ethical/legal reasons, documentation of CA generally starts with skin testing (ST) or specific IgE-quantification. However, in the absence of a standardized extract for ST, many will use prick-prick tests (e.g., with buds, leaves, seeds)1 2. Crude plant parts or extracts thereof, can contain both genuine and cross-reactive allergenic components. Consequently, positive results of crude extract ST and sIgE should always be interpreted cautiously, as it might merely reflect (cross-) sensitization instead of allergy.Here, we sought to investigate whether serological diagnosis of CA could benefit from an adjustment for tIgE. For this purpose, sIgE-to-tIgE ratios were calculated for sIgE hemp (FEIA, ImmunoCAP ThermoFisher Scientific), sIgE to recombinant (r) Can s 3 and rCan s 5 (Cytometric Bead Assay) as detailed elsewhere 3. A sIgE rCan s 3-to-rPru p 3 and sIgE rCan s 5-to-rBet v 1 ratio was also calculated. Negative sIgE values were excluded, as these cannot benefit from such an adjustment. Patients were selected from our previously published data3 4; cannabis sativa allergic patients (CSA), controls with a pollen but no nsLTP sensitization (P+LTP-) and controls with both pollen and nsLTP sensitizations (P+LTP+) were enrolled.Detailed demographic information is shown in table 1 of our previously published article3. As shown in figure 1, the distribution of the sIgE hemp-to-tIgE ratio and sIgE rCan s 5-to-tIgE differed significantly between controls and CSA. Using a cut-off of 0.02 for sIgE hemp-to-tIgE, it was shown that a specificity of 93% (95% confidence interval (CI), 85-98%) could be reached (table 1). A second, lower cut-off (0.005) can benefit most of the CSA population (sensitivity 77% (95% CI 67-85%)) and retains a relatively good specificity 79% (95% CI 68-87%).For sIgE rCan s 5-to-tIgE, a cut-off of 0.01 could possibly be beneficial. However, small group numbers make it difficult to estimate its true value. The ratio of sIgE rCan s 3-to-tIgE did not show added value (p=0.86).Comparing CSA patients with and without anaphylaxis; no added value was found to identify (a risk of) cannabis related anaphylaxis for any of the explored sIgE-to-tIgE ratios (sIgE hemp-to-tIgE->p=0.104; sIgE rCan s 3-to-tIgE->p=0.416; sIgE rCan s 5-to-tIgE->p=0.84, data not shown).Finally, ratios of similar protein families i.e. sIgE rCan s 3-to-sIgE rPru p 3 and sIgE rCan s 5-to-sIgE rBet v 1 were explored, showing no additional diagnostic value (see figure E1 in the online repository).These results indicate that sIgE-to-tIgE ratios might have a place in the diagnostic approach of CA. In the case of a definite history of cannabis related symptoms we recommend (figure E2) a sIgE hemp assay (sensitivity 82% (95% CI 74-89%) and specificity 32% (95% CI 20-45%)) 3. A negative result significantly reduces the chance of IgE-mediated CA. A positive result should be followed by a sIgE hemp-to-tIgE ratio as it notably increases test specificity. Where available, it is worthwhile using cannabis component resolved diagnostics as it was shown that over two-thirds of CSA who experienced anaphylaxis are Can s 3 sensitized 3. This study is bound by certain limitations; group numbers vary between the different analyses because of insufficient patients’ sera. Additionally, we could not explore sIgE rCan s 2-to-tIgE, sIgE rCan s 4-to-tIgE and sIgE rCan s 4-to-sIgE rBet v 2, because of insufficient group numbers. Finally, there is no guarantee that these results can be extrapolated to other CSA populations or different geographical regions. The results of these study are of a preliminary nature. Repetition of our methods in other, ideally larger populations are feasible to see whether these results can be confirmed and remain intact in different CA populations.FIGURES & TABLESFIGURE 1