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.     

IgE, IgG and IgA antibodies in serum and nasal secretion during parenteral hyposensitization

Eighteen adult patients with allergic rhinitis due to Timothy pollen were observed for 36 weeks before, during and after the grass pollen season. Eight patients were treated by parenteral hyposensitization with grass pollen extract, and ten patients who were given no immunotherapy served as controls. Timothy-specific IgE, IgG and IgA antibodies in samples of serum and nasal secretion were quantified by radioimmunological technique. In comparison with the control group, the serum concentration of Timothy-specific IgE antibodies increased significantly (P <0.05) during the preseasonal hyposensitization treatment and then decreased significantly (P <0.05) during and after the pollen season while this therapy was being continued. In the hyposensitized patients the serum concentration of both IgG and IgA antibodies increased highly significantly (P <0.01 and P <0.001, respectively) during immunotherapy. In nasal secretion quantitative changes of the three types of antibodies were usually less pronounced or not detectable at all. The concentration of IgG antibodies, however, showed some increase in the nasal secretion during hyposensitization. These minor increases in allergen-specific IgG and IgA antibodies in nasal secretion might explain why parenteral hyposensitization in allergic rhinitis often does not give complete relief from symptoms.     

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

Forty grass pollen hay fever patients were randomly divided into two equal groups and treated from January 1978 with different timothy grass pollen extracts: whole pollen allergens (WPA) (Alutard® SQ), a partially purified extract and purified pollen allergens (PPA) consisting of the major allergens 19, 25. The effect was evaluated by symptom scores in grass pollen season 1, 1978, and titrated thresholds of skin prick test (SPT) and nasal provocation test (NFT) with extracts WPA, PPA, and FGM (five-grass mixture). Extracts were biologically standardized in the HEP (histamine equivalent prick) system (1 HEP = 1,000 biological units (BU)/ml) and used in concentrations of 0.01-0.1-1.0-10 HEP for tests and treatment. The extracts for treatment were attached to Al (OH)₃. Extracts from the same batch were used throughout the investigation. The two patient groups, WPA and PPA, were comparable us regards age, sex, total dosage (range 2,187–73,887 BU) and frequency of side effects. Fewer symptoms (P=0.0001) were observed with WPA than with PPA. Titrated threshold increase showed a treatment efficacy of 70–100% in SPT compared with 15–65% in NPT and tended to show higher protection after treatment with WPA titan PPA hut equal protection against WPA and FGM, in accordance with common allergens in various grass pollen. In SPT with WPA the threshold increase was correlated (P<0.05) to symptom scores. Frequency of major (urticaria, angioedema, asthma) general side effects was positively correlated (P0.05) to pretreatment NPT thresholds below 1 HEP and standard dosage at 1 HEP or more, but tended to a negative correlation with local side effects. All local and general side effects first appeared 1–5h after injection and a more careful dosage pattern than standard is recommendable at maximum concentration, 10 HEP, and also with extracts 1 HEP at pretreatment NPT thresholds below 1 HEP.     

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.     

Hyposensitization in Childhood Hay Fever

Thirty-six children with well-defined criteria for hay fever (mean age 8 and range 4-15 years) were allocated at random for hyposensitization (HS) with a refined (R) or whole (W) timothy pollen extract during 3-4 years. HS was performed as rush HS with the patients hospitalized for about 1 week and thereafter with monthly injections. Scores for symptoms and antihistamine use were recorded during the season before HS and all seasons during HS. Skin and conjunctival tests were made at the start of HS and postseasonally. Blood samples for IgE and IgG measurements were drawn before and during rush HS and pre- and postseasonally each year. The R-group patients tolerated a higher allergen dose at the end of the rush HS than the W-group ones. They also demonstrated a higher, significant increase in total and specific IgE levels within 7 days and specific IgG levels within 60 days after the start of HS. In both groups postseasonal increases in total and specific IgE levels were seen. The IgG levels increased successively during the treatment. The most remarkable difference between the groups was in scores for symptoms and antihistamine use, which in group R decreased significantly while they increased significantly in group W. Based on these data we recommended that HS, if indicated, should be performed with purified allergen extracts.     

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.     

Immunotherapy with Grass Pollen Major Allergens

Preseasonal hyposensitization stimulated an intercorrelated increase in both serum-specific IgE and allergen-specific IgG. Subsequent perennial treatment depressed the stimulated IgE response and the basophil cell sensitivity, whereas the allergen-specific IgE response showed further increase and persisted at a high level. Nasal IgE response was stimulated from the second pollen season and subsequently became depressed. One year after the end of hyposensitization the allergen-specific IgE response had fallen by 25–50%.     

Hyposensitization

Most extracts used in hyposensitization (immunotherapy) are complex and ill-denned mixtures of a large number of non-antigenic and antigenic components, only a few of the latter being of significance for allergy and allergen specific immunotherapy. A new purified and well-characterized allergen preparation from timothy pollen is now available, and it has been shown to be superior to the corresponding crude aqueous extract in the diagnosis of IgE-mediated human allergy to timothy pollen.
This paper describes the results of hyposensitization for 2 years, with the purified preparation and the crude extract compared. The changes in in vivo and in vitro tests Following this treatment in 40 patients with allergic rhinitis due to grass pollen are reported.
Both patient groups showed a significant decrease in clinical symptom scores when compared with a control group during the grass pollen season. For all groups the symptom scores correlated well with atmospheric pollen counts. Nasal challenge tests showed a significant increase in nasal tolerance to timothy pollen after 2 years of treatment, but nasal tolerance was unchanged in the control group. Nasal function as a criterion for evaluating the effect of specific hyposensitization is discussed.
Serum concentrations of timothy pollen-specific IgE antibodies showed a significant decrease for the group treated with the purified preparation and a slighter and non-significant reduction for the other treated group. There was no significant change in total serum IgE levels.
The results indicate that the purified preparation is preferable to the crude aqueous extract in hyposensitization.     

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.     

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.     

Allergen-antibody complexes can efficiently prevent seasonal rhinitis and asthma in grass pollen, hypersensitive patients

We have prepared antigen-antibody complexes from grass pollen allergens and autologous specific antibodies isolated by immunoadsorption from the serum of allergic patients. These complexes were inoculated into patients in a double-blind trial to evaluate their effect on grass pollen-related rhinitis and bronchial asthma. Thirty-eight grass pollen-hypersensitive patients were allocated to three groups; patients in the first two groups were treated with antigen-antibody complexes at different ratios and dosages and were compared with the third group who received the placebo carrier buffer alone. In addition, we treated a fourth group who had already received antigen-antibody complex inoculation during the previous pollen season. Injections were given every 2 weeks during the pollen season, starting 5 weeks prior to it. Tolerance was excellent with no signs of local or systemic side effects. The treatment prevented nasal symptoms while enabling the patients to reduce antihistamine intake. Bronchial asthma was virtually absent in the treated groups even though no bronchodilators or corticosteroids had to be taken. Specific IgE antibodies did not increase during the pollen season nor did IgG "blocking" antibodies. Inoculation of allergen-antibody complexes could provide a valuable alternative for the treatment of immediate hypersensitivity to airborne allergens as it appears to be safe and rapidly efficacious. This treatment offers several advantages compared to conventional hyposensitization and is characterized by the absence of an increase in specific IgG antibodies.     

Immunocytochemical mapping of IgE/IgG binding sites within the birch pollen grain using serum from a patient undergoing hyposensitization therapy. Comparison with biochemically determined data

Serum from an atopic patient undergoing a hyposensitization therapy to birch pollen allergens was used to carry out immunocytochemical mapping of specific IgE/IgG binding sites within ultrathin sections of birch pollen grains. There was a distinct rise in the density of specific IgG labelling in the course of therapy, whereas the density of IgE labelling remained fairly constant. However, the patterns of IgG and IgE binding in the pollen grain did not completely coincide since there was only IgE binding to certain pollen structures such as the apertural region. If the widely accepted concept of specific antibodies as 'blocking antibodies' is taken as a basis, the success of therapy must be questioned in this case because no IgG antibodies were formed to some of the allergens localized in the pollen grain and relevant to this patient. Very probably this result must be attributed to an incomplete pollen extract used in hyposensitization therapy. The results of the biochemical measurements (RAST, ELISA) agreed well with the immunocytochemical observations.     

The IgG subclasses of antibodies to grass pollen allergens produced in hay fever patients during hyposensitization

Total IgG, IgG subclass and IgE antibodies specific for grass pollen allergens were measured by the red cell linked antigen-antiglobulin reaction (RCLAAR) in serum samples from nineteen patients who had undergone a course of hyposensitization. Increases in both specific IgG and IgE antibodies were seen after treatment in most patients. In the IgG subclasses the predominant response was for IgG1 and IgG4 antibodies. Attempts were made to correlate the antibody responses with the clinical response and the results are discussed with reference to the possible mechanisms of hyposensitization.     

Evaluation of immunotherapy-induced changes in specific IgE, IgG and IgG subclasses in birch pollen allergic patients by means of immunoblotting

Sera from 27 birch pollen-allergic patients who had undergone hyposensitization treatment for 22-41 months were studied by immunoblotting before and after therapy, whereby the levels of IgE, IgG and IgG1-4 antibodies directed against the major allergen Bet v I and minor allergens of birch pollen were monitored. The clinical benefit of immunotherapy (IT) was evaluated using a symptom specific questionnaire. In patients with good clinical response (responders, n = 18), as defined by improvement of symptoms, anti-Bet v I IgE antibodies were found to decrease in 10/18 patients (55.5%), whereas in 6/18 (33.3%) no change and in two cases (11.2%) an increase of specific IgE was observed. In the group of patients with unsatisfactory clinical outcome (non-responders, n = 9), 3/9 patients (33.3%) showed a decrease, 3/9 (33.3%) no change and 3/9 (33.3%) an increase in levels of IgE antibodies directed against Bet v I. In the case of minor allergens, 5/18 responders (27.7%) and 8/9 non-responders (88.8%) showed specific IgE before IT. In the responder group, no increase of specific IgE could be observed after IT. In non-responders, however, an increase of IgE directed against minor allergens was seen in 3/9 patients (33.3%). In all patients, regardless of therapeutical success, IT-induced elevated levels of specific IgG, IgG1 and in particular IgG4 directed against Bet v I were found. Regarding minor allergens, a heterogeneous pattern of IgG responses without significant correlation to clinical benefit was observed. Our results indicate that changes in IgG reactivity patterns against Bet v I and minor allergens, as shown by the immunoblot technique, did not correlate with good or bad clinical outcome.     

Clinical and immunological studies of timothy antigen D immunotherapy.

The response to preseasonal immunotherapy with aqueous grass extract, timothy antigen D, or water-soluble timothy (WST) in alginate was compared in patients sensitive to grass pollen. Injections of antigen D in alginate produced little evidence of clinical or immunologic response. Treatment with aqueous grass extract or WST in alginate, on the other hand, significantly reduced the seasonal rise in grass-specific IgE. Aqueous extract therapy was also associated with a decline in leukocyte sensitivity during the pollen season, while WST treatment produced the greatest rise in hemagglutinating antibodies.     

Changes of serum cytokines after the long term immunotherapy with Japanese hop pollen extracts

Japanese hop (Hop J) pollen has been considered as one of the major causative pollen allergens in the autumn season. We developed a new Hop J immunotherapy extract in collaboration with Allergopharma (Reinbeck, Germany) and investigated immunologic mechanisms during 3 yr immunotherapy. Twenty patients (13 asthma with rhinitis and 7 hay fever) were enrolled from Ajou University Hospital. Sera were collected before, 1 yr, and 3 yr after the immunotherapy. Changes of serum specific IgE, IgG1, and IgG4 levels to Hop J pollen extracts and serum IL-10, IL-12, TGF-beta1 and soluble CD23 levels were monitored by ELISA. Skin reactivity and airway hyper-responsiveness to methacholine were improved during the study period. Specific IgG1 increased at 1 yr then decreased again at 3 yr, and specific IgG4 levels increased progressively (p<0.05, respectively), whereas total and specific IgE levels showed variable responses with no statistical significance. IL-10, TGF-beta1 and soluble CD23 level began to decrease during first year and then further decreased during next two years with statistical significances. (p<0.05, respectively). In conclusion, these findings suggested the favorable effect of long term immunotherapy with Hop J pollen extracts can be explained by lowered IgE affinity and generation of specific IgG4, which may be mediated by IL-10 and TGF-beta1.     

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.     

Childhood asthma: clinical and immunological changes over a decade

A group of 26 Australian asthmatic children with laboratory-proven bronchial hyper-reactivity to the allergens of rye grass pollen and/or the house dust mite has been studied over a 9-year period. Clinical symptoms and drug scores were used to evaluate the severity of the patients' asthma and, wherever possible, blood samples were obtained before, during and after the rye grass pollen seasons. The cumulative symptom and drug scores for the 20 patients with bronchial hyper-reactivity to rye grass pollen extract tended to increase during and fall after each pollen season but the peaks were of decreasing amplitude over the 9 years. Since a proportion of these patients underwent hyposensitization to rye grass during year 1, longitudinal comparisons were made between year 2 and year 9. Comparing the individuals at the same three seasonal time-points revealed significantly lower drug scores in year 9 compared with year 2, and in parallel with this, significantly lower total IgE, IgE anti-rye and IgG anti-rye antibodies at all three assessment points. In the 14 patients with bronchial hyper-reactivity to house dust mite the severity of the asthma and the median levels of IgE and IgG mite specific antibodies all decreased over the study period. Despite the progressive improvement in asthma and diminishing immune responses to both rye grass and house mite in the patients, no immunological feature could be identified that correlated significantly with clinical outcome.     

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.