Detection of pan-allergens in commercial pollen extracts for allergen immunotherapy

Multiple-allergen and single-allergen immunotherapy strategies in polysensitized patients: Looking at the published evidence

Although many patients with allergic rhinoconjunctivitis have symptoms due to sensitization with more than one pollen allergen, and mixed pollen extracts are widely used for allergen immunotherapy in practice, there are few published trials. We performed a 2-year multicentre, double-blind, placebo-controlled trial of subcutaneous immunotherapy with mixed depigmented-polymerized birch and grass pollen extract in 285 patients with allergic rhinoconjunctivitis symptomatic during both birch and grass pollen seasons. Primary outcome was combined symptom and medication score (SMS) assessed by daily visual analogue scales (VAS). Analysis included a placebo-based analysis examining the effect of treatment only on days when placebo patients were symptomatic. There was a significant reduction in median SMS for actively treated patients (median 5.70 (interquartile range 2.62-10.02) compared with 7.07 (3.47-10.71) for placebo, P=0.0385). Rhinitis quality-of-life scores were significantly better for active compared with placebo, and other secondary endpoints were not significantly different. Placebo-based analysis showed a 33.7% reduced SMS at year 2 for active treatment compared with placebo on days when placebo patients were symptomatic. Both birch pollen- and grass pollen-specific IgG4 increased with active treatment. This study shows efficacy of mixed pollen extracts for immunotherapy for patients symptomatic to both birch and grass pollen allergens. The relatively modest effect may reflect 50% dose reduction for each allergen in the mixture. It supports VAS for symptom assessment and placebo-based analysis as useful for the analysis of immunotherapy trials. The safety of modified extracts may allow study of mixed extracts without dose reduction to improve efficacy.

Similarities between antigens derived from ragweed pollen and from other botanical sources

Sublingual immunotherapy (SLIT) has been used to reduce symptoms in allergic rhinitis and to prevent asthma onset. Many studies lack level of adherence and standardized endpoints based on international guidelines. We aimed to study the long-term outcomes of allergic rhinitis severity and asthma onset by adherence of SLIT to grass and birch allergens in children and adolescents with allergic rhinitis. In a population-based register study, we included all children 5-17 years, with initiation of SLIT between 1 July 2006 and 30 June 2022. Allergic rhinitis severity and asthma onset were based on diagnosis and treatment. Adherence was measured for one, two, and 3 years and divided into low, moderate, and high and compared to only one prescription (reference). 7222 children were treated with grass pollen extract and 1184 with birch pollen extract. A significantly lower allergic rhinitis severity was seen in adjusted analyses with odds ratios (OR) of 0.85 (95% confidence interval: 0.72-0.99) and OR 0.81 (0.67-0.96) for the high adherence group (i.e., only one prescription) compared to the reference group in the second and third year respectively after SLIT grass initiation. No statistically significant difference was seen for asthma onset. Estimates for birch SLIT were less conclusive. We found reduced allergic rhinitis severity with higher adherence and longer treatment duration, particularly for grass SLIT. Shorter follow-up for birch SLIT and smaller sample size for asthma onset probably influenced our results. The results should encourage clinicians to prescribe SLIT for eligible children and motivate patients to 3-years full adherence.

IntroductionThere is a need to evaluate the safety and efficacy of intralymphatic immunotherapy (ILIT) for inducing tolerance in patients with allergic rhinitis.MethodsThirty‐seven patients with seasonal allergic symptoms to birch and grass pollen and skin prick test >3 mm and/or IgE to birch and timothy >0.35 kU/L were randomized to either ILIT, with three doses of 0.1 mL of birch pollen and 5‐grass pollen allergen extracts on aluminium hydroxide (10,000 SQ‐U/ml; ALK‐Abelló) or placebo using ultrasound‐guided intralymphatic injections at monthly intervals. Daily combined symptom medical score and rhinoconjunctivitis total symptom score were recorded during the peak pollen seasons the year before and after treatment. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were recorded annually starting 2 years after treatment. Circulating proportions of T helper cell subsets and allergen‐induced cytokine and chemokine production were analysed using flow cytometry and ELISA.ResultsThere were no differences between the groups related to daily combined symptom medical score the year before and after treatment. Two years after ILIT (after unblinding), the actively treated group reported significantly fewer symptoms, lower medication use and improved quality of life than did the placebo group. After the pollen seasons the year after ILIT, T regulatory cell frequencies and grass‐induced IFN‐γ levels increased only in the actively treated group.ConclusionIn this randomized controlled trial, ILIT with birch and grass pollen extract was safe and accompanied by immunological changes. Further studies are required to confirm or refute the efficacy of the treatment.

Associations between HLA type and IgE or IgG antibody (Ab) responses to two well-characterized, antigenetically non-crossreactive components of Lolium perenne (rye grass) pollen extract, Lol p I (Rye I) and Lol p II (Rye II) were studied in two groups of skin-test positive (ST+) Caucasoid adults. By both nonparametric and parametric statistical methods, significant associations were found between Ab responses to both Lol I and Lol II and the possession of HLA-DR3. In view of the well-known associations of both DR3 and B8 (which are in linkage disequilibrium) with many autoimmune diseases, differences in anti-Lol I and anti-Lol II mean log[Ab] levels between B8+, DR3- vs B8-, DR3- subjects and B8+, DR3+ vs B8-, DR3+ subjects were investigated. No differences were found. Our data, along with recent RFLP and DNA sequence studies, suggest that an Ia molecule involved in immune recognition of a similar major Ia recognition site of both the Lol molecules may consist of a DR3 alpha-beta I pair. Abbreviations used: Ab: Antibody. HLA: Human leukocyte antigen. Lol p I, Lol I: Group I allergen from Lolium perenne pollen (Rye I). Lol p II, Lol II: Group II allergen from Lolium perenne pollen (Rye II). Mr: Relative molecular mass. Rx: Immunotherapy with grass pollen extracts. ST: Skin test.

Background: Pollen of Bahia grass (Paspalum notatum) represents a major cause of type I allergy in diverse geographical areas, particularly in the southeastern coastal plain area of the United States. The aqueous protein extract of Bahia grass pollen contains the allergenically active components that produce skin-test-positive reactions in sensitive patients. Objective: The emphasis of this study included the identification and characterization of the allergenic proteins present in the crude protein aqueous extract of Bahia grass pollen. Methods: The crude extract of Bahia grass pollen, partially purified by isoelectric focusing and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was electroblotted onto nitrocellulose membranes, probed with sera from patients skin test positive to Bahia grass and detected using anti-human IgE conjugated peroxidase. Results: Four allergenic proteins of Bahia grass pollen with estimated molecular weights of 45, 33, 31 and 28 kD were identified and characterized. Following treatments with deglycosylation enzymes, the 4 allergens retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. Conclusion: The crude extract of Bahia grass pollen contains many proteins but only 4 have allergenic reactivity. Following deglycosylation treatment, Bahia grass allergenic proteins have retained their antigenic reactivity with Bahia-grass-allergic patient sera containing polyclonal IgE antibodies. Four proteins reactive with IgE were detected, but the 33-kD protein (pI of 6.59) was the most reactive.

Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

Rhinitis with negative skin tests and absent serum allergen-specific IgE: More evidence for local IgE?

Grass pollen hay fever patients were hyposensitized in a prospective 3‐year double blind study with two timothy extracts. Group WPA (20 patients) was treated with partially purified extract = whole pollen allergens (WPA) (Alutard®SQ) and group PPA (20 patients) was treated with a purified pollen allergen (PPA) = two isolated major allergens, Ag19 and Ag25. Both aluminiumhydroxide adsorbed extracts were biologically standardized. Clinical results from the first season have been recently published and WPA showed significantly fewer symptoms (P= 0.001) than PPA. Corresponding preseasonal and seasonal in vitro results are presented here.Serum total IgE, specific IgE versus total and individual allergens of timothy pollen and allergen‐specific IgG showed a rapid increase in both groups until the season but showed no further increase or decrease during the season. Specific IgE was shown to correlate to specific IgG during hyposensitization.Group WPA, with fewer symptoms in grass pollen season than group PPA showed a significantly higher increase of specific IgG and IgE than group PPA, but individual symptom scores were not correlated to specific IgG or IgE, or their ratio.Specific IgE and IgG increases were not correlated to dosage. Surprisingly, almost all females were low‐responders to specific IgG and IgE though they had equal symptom scores, dosage, and side effects as males, while the characteristics of high‐responders to antibody were: youngest individuals and shortest duration of symptoms prior to treatment.Crossed radioimmunoelectrophoresis (CRIE) showed specific reaction to stimuli but no development of allergy against new timothy antigens.High response of IgE to Ag 19 in CRIE during initial hyposensitization seems suitable as marker for prospective evaluation of clinical effect in grass pollen hyposensitization.Nasal secretion was collected after methacholine provocation, and total IgE and specific IgE detected. There was no response to treatment, only a slight increase during the season. No decrease in nasal reactivity to methacholine was noted during one preseasonal hyposensitization.

Allergic conjunctivitis is a common symptom amongst Type I (IgE-mediated) allergic diseases; and most frequently seen as rhinoconjunctivitis. However, the site of production and the significance of allergen specific IgE needs further elucidation. We investigated whether the presence of IgE in tears of grass pollen allergic patients correlated with disease and clinical symptoms, whether the IgE binding pattern to the different grass pollen antigens was different in sera and tears, and whether IgA antibodies to grass pollen allergens were present in tears. Finally, we looked whether specific IgE was produced locally or was exudated from serum. Sera from 44 grass pollen allergic patients suffering from either allergic rhinitis (n = 11) or allergic rhinoconjunctivitis (n = 33) and from healthy controls (n = 7) were used for the experiments. Binding of specific IgE and IgA antibodies to the different groups of grass pollen allergens (Phleum pratense) was evaluated by means of immunoblotting. Specific IgE was detected in sera as well as in tears of allergic patients, whereby tear-derived allergen-specific IgE exerted similar specificities to the corresponding IgE from serum. The correlation between symptoms of ocular allergy and the presence of allergen-specific IgE in tears was highly significant (P < 0.0001). In contrast, only a poor correlation was found between specific and/or total IgE in sera and the manifestation of ocular allergy (P = 0.73). Allergen-specific IgE antibodies in tears seem to be produced locally rather than exudated from serum. IgE in tears seems to be responsible for allergic conjunctivitis. IgA in tears cannot exert a protective function since the IgA antibodies recognize different antigens in a grass pollen (Phleum pratense) extract than IgE antibodies. The highly significant correlation between allergic conjunctivitis and the presence of specific tear IgE emphasizes the diagnostic value of immunoblots with tear IgE, especially in cases in which serum provides inconclusive results.

In spite of our recent insight into nasobronchial interaction mechanisms in allergic airway disease, the association between allergic rhinitis and voice complaints remains obscure. To evaluate the effects of nasal allergen provocation and seasonal grass pollen exposure on subjective and objective laryngeal parameters in singers with and without allergic rhinitis, an observational case control study was conducted. Prior to the pollen season, six grass pollen allergic and six non-allergic semiprofessional singers were exposed to nebulized sham solution and grass pollen extract (HAL°) in rising concentrations. After 3 min, 60 min and 24 h, nasal and laryngeal complaints were evaluated by the use of a visual analog scale (VAS). Laryngeal parameters like voice appearance (video stroboscopic images), voice range profile and subjective (GRBAS) and objective (jitter, shimmer, H/N, DSI) voice quality were evaluated before provocation, after 60 min and 24 h. During the pollen season, the allergic singers were re-evaluated. Results showed that in allergic singers both nasal (TNS of 4.0 ± 2.4 vs. 0.0 ± 0.0, p < 0.05) and laryngeal complaints (TLS of 1.4 ± 1.1 vs. 0.0 ± 0.2, p < 0.05) were induced at 3 min after the provocation. The induced laryngeal complaints were the feeling of laryngeal irritation, secretions and globus. No change in voice quality or stroboscopy score was measured. During the pollen season, laryngeal complaints were present (TLS of 2.4 ± 2.4) in allergic singers, without evidence for objective voice and laryngeal changes. In conclusion, we here demonstrate the rapid induction of laryngeal complaints in allergic singers by nasal allergen provocation and during the pollen season. There was no subject reported or investigator measured change in voice quality. No change in stroboscopy score was measured.

Intralymphatic allergen-specific immunotherapy: An effective and safe alternative treatment route for pollen-induced allergic rhinitis

Background: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. Objectives: To characterize the IgE–binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. Methods: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme–linked immunosorbent assay (ELISA), ELISA–inhibition, thin–layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well–known group 1, 4, and 5 allergen homologues in Ic pollen extract. Results: Out of 303 respiratory allergies patients skin–tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver–stained bands (pI 3.5–7.0) while SDS–PAGE resolved it into 24 Coomassie–Brilliant–Blue–stained bands (MW 100–10 kD). Immunoblotting with individual patient sera recognized 7 major IgE–binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. Conclusion: Ic pollen extract was characterized for the protein profile by TLIEF and SDS–PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

Hundred years ago, Leonhard Noon and John Freeman published their pioneering works on allergen-specific immunotherapy (ASIT) using grass pollen extracts. To honor their contribution to the development of ASIT as the only causal treatment of IgE-mediated allergies, we review the history of ASIT that started with the anecdotal descriptions of ASIT performed by the ancient king Mithridates (132-63 B.C.) and Jenner's development of a cowpox vaccine. Following Noon's and Freeman's first controlled human trials, ASIT was performed by a large number of modalities and with a myriad of pharmacologic preparations. These developments range from early aqueous pollen extracts and whole bee extracts to chemically modified allergens (allergoids) and various recombinant allergens. In addition to allergen-specific immunotherapy, non-specific immune response modifiers have been used in the past or are in the developmental stage. Also, currently many innovative experimental approaches of ASIT are studied in animal models and human in vitro systems and will hopefully further broaden the range of allergies that can be treated by ASIT, with enhanced efficacy and further reduced side-effects.