Immunotherapy in Hay Fever with Two Major Allergens 19, 25 and Partially Purified Extract of Timothy Grass Pollen

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.     

Specific IgE and IgG antibody responses in children to timothy pollen components during immunotherapy

Fourteen children with timothy grass pollinosis were given immunotherapy (IT) for 3 years with a purified and characterized timothy grass pollen preparation or a crude aqueous timothy pollen extract. Crossed radioimmunoelectrophoresis (CRIE) showed that 75% of the children under 11 years of age developed new specificities of IgE antibodies against timothy antigens, in contrast to older children, where no development of IgE antibodies against new timothy antigens could be detected. IgE antibodies were only detected against antigens formerly known as allergens. Timothy-specific IgG antibodies increased in most children during hyposensitization against the major allergens Ag 19 and Ag 24/25 and several other IgE-binding timothy antigens.     

Anti-IgG Antibodies during Immunotherapy with Purified Grass Pollen Extracts

In a double blind study 40 patients were allocated specific immunotherapy (hyposensitization) with partially purified timothy extract or two timothy major allergens 19, 25. All patients had typical grass pollen hay fever, in 27% associated with grass pollen asthma and in 13% with birch pollen allergy. Serum IgG anti-IgG antibodies were determined after dithiothreitol treatment. Before hyposensitization, IgG anti-IgG titres greater than or equal to 9 were demonstrated in 45% of the patients. During hyposensitization IgG anti-IgG titres showed a slight initial increase followed by a decrease below pretreatment level. Neither increase nor decrease was statistically significant. Reactions to rabbit IgG F(ab')2 fractions were only obtained during hyposensitization. The occurrence of anti-IgG antibodies did no correlate with symptoms, side effects, or the level of allergen-specific IgG. In a previous study it was demonstrated that patients with multiallergy hyposensitized with combined allergen extracts showed a statistically significant increase in IgG anti-IgG titres during treatment. The increase failed to appear in the present patients allergic only to pollen and treated with purified allergen extracts. It is therefore suggested that a multiallergic condition and the combination and/or purification of allergen extracts administered during hyposensitization may influence the production of IgG anti- IgG antibodies.     

Immunotherapy with Grass Pollen Major Allergens

Perenial hyposensitization with a partially purified timothy extract resulted in a statistically significantly higher degree of clinical protection than treatment with the two timothy major allergens (Nos. 19 and 25) and protected better from the second—than during the first grass pollen season. The extracts were standardized biologically and adsorbed to aluminium hydroxide for administration. The therapy had a more beneficial influence on sneezing than on rhinorrhoea and blockage of nasal airways, and an excellent effect on grass pollen asthma was obtained with the partially purified timothy extract. Associated birch pollen allergy was not influenced by hyposensitization with grass pollen.     

Nasal and Skin Sensitivity during Immunotherapy with Two Major Allergens 19, 25 and Partially Purified Extract of Timothy Grass Pollen

In a double blind 3-year prospective study 40 grass pollen allergic patients were allocated to specific immunotherapy (hyposensitization) with two timothy major allergens, 19, 25, or partially purified timothy extract. The extracts were biologically standardized and coupled to aluminium hydroxide for treatment.
Efficacy of hyposensitization was evaluated by titrated nasal provocations and skin prick tests with the two treatment extracts and a five-grass mixture. The threshold dose for skin prick test (skin (threholds) produced reactions identical to histamine chloride 5.43 mmol/l, and the threshold dose for nasal provocations (nasal thresholds) produced two of the three reactions: at least 0.5 ml of nasal secretion, at least five sneezes, and/or at least a 20 % fall in nasal peak flow.
Nasal thresholds showed highest efficacy from partially purified timothy extract but equal protection against timothy and I he five -grass mixture. Nasal thresholds of 14 untreated patients corresponded to treatment with the two major allergens.
Changes in skin and nasal thresholds after 12 and after 64 weeks of treatment predicted the severity of hay fever. Increase in nasal thresholds coincided with a marked effect on asthma. Nasal thresholds below 1 HEP before treatment predicted major systemic side effects.     

Clinical efficacy of microencapsulated timothy grass pollen extract in grass-allergic individuals.

BACKGROUND: Conventional allergen immunotherapy is clinically effective in reducing the symptoms of allergic rhinitis and asthma. It differs from other pharmacotherapies in that it can induce long-term clinical remission of these diseases. However, it requires years of treatment and is associated with serious allergic reactions. OBJECTIVE: To evaluate the safety, clinical efficacy, and immunologic mechanisms of immunotherapy with an oral, microencapsulated form of timothy grass allergen. METHODS: In this double-blind, placebo-controlled study, 24 patients aged 19 to 55 years with grass pollen allergy were randomized to receive either microencapsulated timothy grass pollen extract or placebo once a day for 10 weeks. The dose of study drug was doubled weekly. Safety was evaluated through weekly visits, daily symptom diaries, and routine laboratory tests. Efficacy was evaluated by comparing medication use and symptoms scores during peak grass pollen season before and after treatment. Allergen-specific T-cell responses, cytokine production, and IgG, IgE, and skin reactivity were measured to evaluate immunologic mechanisms. RESULTS: Eleven of 12 patients in the active treatment group had a decrease in the combined medication and symptom score, but only 4 of 10 patients in the placebo group had a decrease in scores. The proliferative response to timothy grass was reduced by at least 30% in 9 of the 12 grass-treated patients, but only 3 of 11 placebo patients had a proliferative response reduction. Timothy grass-induced interleukin-5 messenger RNA was reduced in the active group, but not in the placebo group. There were no significant changes in either group in IgG, IgE, and skin reactivity. CONCLUSIONS: Oral immunotherapy with microencapsulated allergen induces a form of immunologic tolerance to the allergen and is a safe, efficient, and effective method of allergen immunotherapy.     

Clinical effects of hyposensitization using a purified allergen preparation from Timothy pollen as compared to crude aqueous extracts from Timothy pollen and a four-grass pollen mixture respectively

Most extracts used in hyposensitization are complex and ill-defined mixtures of a large number of antigenic components. A highly refined (purified) and well-characterized allergen preparation from Timothy pollen (Phleum pratense) is now available. This paper describes the results of hyposensitization for 3 years comparing the purified preparation Timothy N. the crude extract Timothy O and a four-grass mix in sixty patients with allergic rhinitis due to grass pollen. The sixty patients were randomized into three groups and compared with a control group not hyposensitized. All three groups showed a significant decrease in clinical symptoms compared with the control group. The Timothy N group had a significantly higher nasal tolerance shown by nasal challenge test after 3 years' treatment than the group treated with the crude extract (P= 0.05). In addition, the Timothy-N-treated patients needed significantly less antihistaminic medication than the patients having received the crude extract or the four-grass mix (P=0.02 and P= 0.01 respectively).     

Use of glutaraldehyde-modified timothy grass pollen extract in nasal hyposensitisation treatment of hay fever.

12 patients suffering from grass pollen hay fever were treated for 14 weeks pre- and co-seasonally by intranasal self-administration of an aqueous solution of a glutaraldehyde-treated timothy grass pollen allergen. These patients had a statistically significant decrease in nasal symptom scores during the grass pollen peak period and in nasal challenge end-point titre after the season compared to placebo-treated patients. No significant effect was seen on the eye symptoms. 1 patient withdrew from the trial as a consequence of too strong local nasal reactions during treatment. Most other patients treated with active material reported mild local reactions during the first minutes after administration of the nasal spray. In the actively treated group a significant increase in serum and nasal secretion of grass pollen specific IgE, IgG and IgA antibodies was obtained during the treatment. In contrast, in the placebo group a significant increase in IgE antibody levels in serum and secretion occurred during the pollen season. The reduction in symptoms and increase in antibody production together with the simplicity of the procedure makes this approach to immunotherapy attractive.     

Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis

Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen‐specific and disease‐modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross‐reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease‐relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross‐reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen‐based technologies.     

Basophil histamine release in patients with hay fever. Results compared with specific IgE and total IgE during immunotherapy.

Histamine release from leucocytes was demonstrated in grass pollen hay fever patients on in vitro challenge with extract of Pleum pratense (timothy). No release was found in persons without a history of grass pollen allergy. During preseasonal hyposensitization the following tendencies were found in cell sensitivity to allergen as well as in specific IgE antibody level of serum: an initial increase at the beginning of the therapy followed by a decrease during the pollen season. This is in contrast to untreated hay fever patients in whom an increase or no change at all of cell sensitivity and specific IgE was observed in the pollen season. Immunotherapy, therefore, can prevent such an increase in the pollen season. The mechanism might be due to a depression of the IgE production. In untreated as well as in treated patients the cell sensitivity was found to be significantly correlated to the grass specific IgE determined by RAST but not to the total serum level of IgE estimated by RIST. It seems likely that the sensitivity would be useful for evaluating the degree of allergy in grass pollen hay fever patients treated or not treated with immunotherapy.     

Skin test diagnosis of grass pollen allergy with a recombinant hybrid molecule

BACKGROUND: A recombinant hybrid molecule (HM) consisting of 4 major allergens from timothy grass (Phl p 1, 2, 5, and 6) was expressed in Escherichia coli, purified, and characterized regarding its immunologic properties. OBJECTIVE: We sought to determine whether the recombinant HM can be used for the diagnosis of grass pollen allergy by means of skin testing. METHODS: Skin prick testing was performed in 32 patients with grass pollen allergy and in 9 control individuals by using increasing concentrations (4, 12, 36, and 108 mug/mL) of the HM and using commercial grass pollen extract. Specific IgE reactivities against the HM, grass pollen extract, and a panel of purified grass pollen allergens (recombinant Phl p 1, 2, 5, 6, 7, 12, and 13 and natural Phl p 4) were measured by means of ELISA, and timothy grass pollen-specific IgE levels were determined by using ImmunoCAP. RESULTS: Grass pollen allergy was diagnosed in all patients by means of skin testing with the HM. No false-positive skin test responses were obtained in the control individuals. There was an excellent correlation between IgE levels obtained with the HM and natural grass pollen extract measured by means of ELISA (r = 0.98, P < .0001) and by means of ImmunoCAP (r = 0.98, P < .0001). CONCLUSIONS: The recombinant HM permitted accurate and specific in vivo diagnosis of grass pollen allergy in all tested patients. It can be considered a well-defined tool for the diagnosis and perhaps for immunotherapy of grass pollen allergy. CLINICAL IMPLICATIONS: A recombinant HM can replace traditional allergen extracts for skin test-based diagnosis of grass pollen allergy.     

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.     

Systemic immunological changes induced by administration of grass pollen allergens via the oral mucosa during sublingual immunotherapy

Twenty-four patients suffering from grass pollen allergy underwent sublingual immunotherapy (SLIT) with standardized grass pollen extract for 1 year. In order to investigate immunological changes induced by the administration of allergens via the oral mucosa, the SLIT-spit method was applied. The cumulative dose of approximately 80 microg of major allergen (grass group 5 allergen), was relatively low. During the time of treatment, we could observe a significant increase in the levels of specific IgG and IgG4 antibodies. However, the titers of allergen-specific IgE antibodies showed a significant increase in the course of SLIT as well. Analyzing lymphoproliferative responses, a significant decrease in reactivity in response to stimulation with complete grass pollen extract (p = 0. 001) and to recombinant Phl p 1 (a major allergen of timothy grass, p<0.001) could be observed, indicating the induction of immunological tolerance. Proliferative responses to a control antigen (tetanus toxoid) were not influenced by the treatment. At different time points during SLIT, allergen (Phl p 1)-specific T cell clones (TCC) were established from the peripheral blood of the patients. Cytokine production by allergen-stimulated T cells did not reveal any changes consistent with immune deviation, i.e. the ratio of Th1/Th2 TCC did not change during SLIT. In conclusion, we provide evidence that sublingual treatment leads to systemic changes in immunoreactivity to the administered allergen.     

Hyposensitization therapy with whole pollen extract or purified allergens monitored by immunoblotting.

Patients allergic to grass pollen were hyposensitized with two major allergenic components or whole extract of timothy grass pollen. Specific IgE, IgG1, and IgG4 formed during immunotherapy were analyzed by immunoblotting. Similar antibody-binding patterns were observed in both patient groups. Inhibition experiments using allergenic components isolated by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that all antigenic components of timothy grass pollen detected in immunoblot dispose of private and cross-reactive determinants for binding of human IgE. The worse clinical outcome of immunotherapy after hyposensitization with two cross-reactive components did, therefore, not correlate with a specific antibody formation pattern.     

Does the effect of immunotherapy last after termination of treatment?

In a previous study, 39 adults with grass pollen allergy were hyposensitized for approximately 2 1/2 years. Treatment was performed in a double-blind fashion with extract made from timothy grass--either Alutard SQ 20-component extract or a purified 2-component extract, including only the two major allergens Phl p V and VI. Standardized symptom + medicine scores and challenge tests demonstrated a clinical effect, most markedly in the group receiving 20-component extract. Six years after termination of treatment, 38 patients could be approached and 16 in each group were examined and repeated symptoms scoring during the subsequent season. When adjustment for variations in pollen counts were made, medicine + symptom scores stayed low during the follow-up period. Specific IgE-antibodies against timothy showed an increase to initial values during the same period, whereas total IgE antibodies remained low. Skin prick test reactions with timothy allergen tended to increase but were still smaller than before treatment. Retrospectively, the patients reported symptoms to have stabilized or even further decreased after termination of treatment, with no significant difference between groups. In conclusion, the clinical effect was still present more than 6 years after termination of treatment. Some in vitro parameters tended to return to pretreatment level. The spontaneous course of the disease in non-hyposensitized patients was not investigated.     

Orally Administered Grass Pollen

In 1900 it was claimed that oral administration of ragweed could be used for the hyposensitization of hay fever patients. Several uncontrolled trials have been published, all showing an effect of oral hyposensitization. Only one study was controlled and showed no effect of oral hyposensitization. It was decided to undertake controlled clinical trials to determine the safety and effectiveness of orally administered enteric-coated grass pollen tablets in patients with hay fever. The actual grass pollen dose in the first trial was 30 times the dose that is normally recommended for preseasonal oral pollen hyposensitization using pollen aqueous solution or pollen powder. The safety study will be described here. Twelve young adults with a history of grass pollen hay fever positive skin prick test and positive nasal provocation test with extracts of timothy grass pollen were randomly allocated to one of the treatment groups with four patients in each group taking enteric-coated Conjuvac Timothy tablets or enteric-coated Whole Timothy pollen tablets or enteric-coated placebo tablets. The study was double blind. Preseasonally, the patients received 342,500 PNU and in total they received 4,500,000 PNU during 6 months. The patients receiving active treatment did not have any side effects. No significant changes were shown in the skin and nasal reactivity to grass pollen during the study. Neither were there any changes in timothy-specific IgE, IgG, total IgE nor histamine liberation from basophils.     

An assessment of the role of intradermal skin testing in the diagnosis of clinically relevant allergy to timothy grass

BACKGROUND: Immediate skin testing is generally the preferred method for establishing the presence of allergy in clinical practice. There is no agreement, however, as to whether intradermal testing should be routinely performed if skin prick test results are negative. PURPOSE: The study was done to address the value of intradermal skin testing in the diagnosis of clinically significant sensitivity to grass pollen in patients exhibiting negative skin prick test responses to timothy extract. METHODS: Four groups were studied. Group I had a history of seasonal allergic rhinitis, negative skin prick test responses to timothy and Bermuda grass, but positive intradermal skin test responses to timothy grass. Group II had a history of seasonal allergic rhinitis and positive skin prick test responses to timothy grass. Group III had a history of seasonal allergic rhinitis but had negative responses to both prick and intradermal testing with timothy and Bermuda grass. Group IV had no history of rhinitis, had negative responses to skin testing with a panel of locally important allergens, as well as Bermuda and timothy grass, and had a serum IgE value of less than 20 IU/ml. Clinical sensitivity to grass was assessed by two methods: (1) nasal challenge with threefold increasing amounts of timothy pollen performed out of the pollen season and (2) correlation of subjects’ daily symptom and medication scores with daily grass pollen counts during the grass pollen season. RESULTS: On the basis of nasal challenge with timothy grass, pollen allergic reactions were present in 11% of group I, 68% of group II, 11% of group III, and 0% of group IV. As determined by correlation of symptoms during the grass pollen season with grass pollen counts, 22% of group I, 64% of group II, 21% of group III, and 0% of group IV were considered allergic. If both criteria were required for a diagnosis of clinical allergy to grass, the percent positive was 0 for group I, 46 for group II, 0 for group III, and 0 for group IV. CONCLUSION: Under the conditions of this study the presence of a positive intradermal skin test response to timothy grass (1000 AU/ml) in the presence of a negative skin prick test response to timothy grass (100,000 AU/ml) did not indicate the presence of clinically significant sensitivity to timothy grass, and by inference, to other cross-reacting grasses. (J Allergy Clin Immunol 1996;97:1193-201.)     

Multiallergen nasal challenges in nonallergic rhinitis

BACKGROUND: It has been proposed that some patients with nonallergic rhinitis may have "localized allergy" of the nasal mucosa. Nasal challenges with aeroallergens can help determine whether a patient is clinically allergic via an IgE-mediated pathway. OBJECTIVE: To determine the prevalence of localized allergy in patients with negative skin prick test results via nasal challenges with an array of allergens. METHODS: Twenty individuals with perennial rhinitis and negative epicutaneous test results to common perennial aeroallergens underwent nasal challenges to glycerin, Alternaria, cockroach, timothy grass, cat hair, and Dermatophagoides pteronyssinus. Total symptom scores, peak nasal inspiratory flow rates, and nasal eosinophil counts were determined. RESULTS: Of 20 patients with nonallergic rhinitis, 4 were hyperresponsive to glycerin and were not subsequently challenged. Eleven patients had negative nasal challenges. Five patients developed positive challenges (total symptom score > or = 5) to 7 allergens. These 5 patients returned for nasal provocation testing to their offending allergens, and these repeated challenges were negative. Three control subjects with allergic rhinitis developed positive challenges after nasal allergen challenge. CoNCLUSIONS: Although some individuals with nonallergic rhinitis can have positive nasal allergen challenges, these results were not reproducible in the present patient population.     

A double-blind, placebo-controlled trial by the sublingual route of immunotherapy with a standardized grass pollen extract

Fifty-eight patients with well-documented history of seasonal rhinoconjunctivitis caused by grass pollens were allocated randomly on a double-blind basis to receive either sublingual therapy with a solution of purified, standardized allergen preparation (Stallergenes) or a matched placebo for 17 weeks. The assessment of the effect of oral immunotherapy, done with drops of five-grass allergen extract, was on the clinical symptoms and on the medication score of the authorized rescue treatments. The actively treated patients had significantly (P<0.05 to P<0.01) fewer symptoms of rhinitis (sneezing and rhinorrhea) and of conjunctivitis (redness and tears) during the pollen season than the placebo group. Consumption of nasal solution of sodium cromoglycate and of betamethasone and dexchlorpheniramine was significantly less in the desensitized group (P<0.01). Side-effects were negligible. This study concludes that perlingual immunotherapy with grass pollen extract in grass-pollen-sensitive seasonal hay fever and conjunctivitis patients is effective, easy to perform, inexpensive, and safe.     

Key pollen allergens in North America.

OBJECTIVE: To discuss major pollen aeroallergens in North America that are essential for effective immunotherapy and to propose a list of pollen aeroallergens that could be prioritized for allergen standardization. DATA SOURCES: PubMed was used to search the existing medical literature. No date restrictions were used. Keywords included allergy, aeroallergen, taxonomy, cross-reactivity, pollen, and specific genus and species names. RESULTS: Tree species possess relatively unique allergens, and representative members should be chosen at the genus or family level. In the Composite family, there is significant cross-reactivity between ragweed species within the Ambrosia genus. Selection of one species should be sufficient for skin testing and immunotherapy. Extensive allergenic cross-reactivity exists among grasses. Selection of timothy grass alone or in combination with a single northern grass species provides adequate coverage in the northeastern regions of North America. CONCLUSIONS: One of the goals within the field of allergy should be to identify high-priority targets for future development of standardized commercial extracts. The standardization of increasing numbers of allergen extracts potentially benefits the discipline of allergy by facilitating transfer of care among physician practices, improving uniformity of patient care, and providing a template on which geographically specific extract choices can be built.