Intralymphatic immunotherapy with tyrosine-adsorbed allergens: a double-blind, placebo-controlled trial

Background Most previous studies used aluminum hydroxide-absorbed allergen extracts in evaluating the potential therapeutic roles of intralymphatic allergen-specific immunotherapy (ILAIT). In this study, we evaluated the therapeutic efficacy and safety of ILAIT with L-tyrosine-adsorbed allergen extracts of Dermatophagoides farinae, D. pteronyssinus, cat, dog, or mixtures thereof, in patients with allergic rhinitis induced by these allergens. Methods In this randomized, double-blind, placebo-controlled trial, study subjects received three intralymphatic injections of L-tyrosine-adsorbed allergen extracts (active group) or saline (placebo group) at 4-week intervals. Results Although ILAIT reduced daily medication use and skin reactivity to HDM and cat allergens at 4 months after treatment, overall symptom score on a visual analog scale (VAS), sinonasal outcome test-20 (SNOT-20), rhinoconjunctivitis quality of life questionnaire (RQLQ), daily symptom score (dSS), daily medication score (dMS), daily symptom medication score (dSMS), nasal reactivity to HDM allergen, and basophil activity to HDM, cat, and dog allergens at 4 months and 1 year after treatment were similar between the treatment and control groups. Intralymphatic injection was more painful than a venous puncture, and pain at the injection site was the most frequent local adverse event (12.8%); dyspnea and wheezing were the most common systemic adverse events (5.3%). Conclusions ILAIT with L-tyrosine-adsorbed allergen extracts does not exhibit profound therapeutic efficacy in allergic rhinitis and can provoke moderate-to-severe systemic reactions and cause pain at the injection site. Trial registration: clinicaltrials.gov: NCT02665754; date of registration: 28 January 2016 Supplementary Information The online version contains supplementary material available at 10.1186/s12931-021-01766-0.

In this study, we evaluated the treatment outcomes and adverse effects of ILAIT using L-tyrosine-adsorbed allergen extracts of D. farinae, D. pteronyssinus, dog, cat, or mixtures thereof in patients with AR induced by one or more of these allergens.

Study subjects
The study was conducted at the outpatient clinics of five university hospitals in the Republic of Korea. From July 2016 to December 2018, the study included individuals with AR induced by individual or combined allergens from D. farinae, D. pteronyssinus, dogs, and cats. Patient selection was based on the following inclusion criteria [14,15]. (1) Sensitization confirmed by a skin prick test (SPT) or allergen-specific immunoglobulin E (IgE) serum levels. Subjects with an allergen-to-histamine ratio of ≥ 1 or an allergen-specific IgE serum level of ≥ 0.35 kU/L were regarded as sensitized to an allergen. (2) Complaints of AR symptoms upon exposure to house dust, dogs, or cats. Patients with mild asthma and those sensitized to pollen allergens were included in the study if there was no definite allergic symptom during the pollen season. Patients with severe or uncontrolled asthma (forced expiratory volume in 1 s ≤ 50%, predicted); tyrosinemia; alkaptonuria; severe underlying conditions, including hepatic, renal, hematologic, oncologic, immunologic, infectious, or cardiovascular diseases; acute (within 1 month) or chronic respiratory diseases other than asthma, such as the common cold, flu, bronchiectasis, or chronic obstructive pulmonary disease; or previous AIT, were excluded. Pregnant or lactating women were also excluded. Written informed consent was obtained from all subjects included in the study.

Randomization and blinding
Radiologists who performed intralymphatic injections randomly assigned subjects (1:1) into treatment and control groups using Microsoft Excel (Microsoft, Redmond, WA, USA) with a block size of 10. Other researchers prescribing medication and assessing treatment outcomes and adverse events were blinded to the treatment; blinding was maintained until all assessments and recordings were completed.

Intervention
Subjects in the active group were administered three injections (0.1 mL) of the respective allergen extract at 4-week intervals. Using ultrasound guidance and a 25-gauge needle, L-tyrosine-adsorbed allergen extracts (Tyrosine S ® ; Allergy Therapeutics, Worthing, UK) were aseptically injected into the superficial inguinal lymph node in the right side of the groin. Before the injections, aspiration was performed to avoid inadvertent intravascular administration. After each injection, subjects were closely monitored for 1 h, and vital signs were assessed at 5-min intervals; any adverse events were recorded. Adverse events due to previous injections were also evaluated before the next injection. Allergens were initially administered at a 1000fold dilution of the highest dose used for subcutaneous immunotherapy (SCIT) [14]. For the second and third injection, the administered dose was 3 and 10 times the initial allergen concentration, respectively [6,12,14]. Subjects in the control group received three injections (0.1 mL) of normal saline at 4-week intervals.

Outcome measures
Patients were asked to answer the questionnaires and complete diary that measured allergic symptoms and rescue medication, as well as underwent a SPT and intradermal test (IDT) before treatment, at 4 months after treatment, and at 1 year after treatment ( Fig. 1) [14]. For subjects who were enrolled at the central institution and whose target allergens were HDMs, a nasal allergen provocation test (NAPT) was also performed [14].

Overall treatment effects
Subjects were asked to score their overall AR symptoms on a visual analog scale (VAS) ranging from 0 (none) to 100 (extremely severe).

QOL
Quality of life (QOL) was evaluated using the Korean version of the sinonasal outcome test-20 (SNOT-20) and rhinoconjunctivitis QOL questionnaire (RQLQ) [14,17,18]. The RQLQ score was used as the primary outcome measure.

Symptom medication score
Subjects self-reported a daily symptom score (dSS) for runny nose, blocked nose, sneezing, and itchy nose the month before treatment, as well as at 4 months and 1 year after treatment. For the dSS, a four-point scale was used: 0, no symptoms; (1) mild symptoms (easily tolerated); (2) moderate symptoms (bothersome but tolerable); (3) severe symptoms (hard to tolerate and interfering with daily activities) [19]. The patients were provided antihistamines or nasal glucocorticosteroid spray in a stepwise fashion according to Allergic Rhinitis and its Impact on Asthma (ARIA) recommendations [14,[19][20][21]. Rhinitis medication use was scored to obtain the daily medication score (dMS), and the daily symptom medication score (dSMS) was calculated as the sum of the dSS and dMS [19].

SPT and IDT
The SPT and IDT were performed using serially diluted extracts of the respective allergen (D. farinae, D. pteronyssinus, dog, or cat; Tyrosine S ® ; Allergy Therapeutics). The skin test results were interpreted after 15 min by measuring the mean wheal diameter induced by each allergen, using calipers with resolution of 0.01 mm.

NAPT
Subjects sensitized to HDMs underwent NAPT at the central institution with saline and freshly reconstituted freeze-dried allergen solutions of Der f 1 (0.04, 0.4, 1, 2, and 4 μg/mL) at 15-min intervals [14,22]. At each step, two puffs (0.05 mL each) of the solution at room temperature were applied to each nasal passage using a metered pump spray; nasal obstruction, rhinorrhea, itching, sneezing, and ocular symptoms were recorded by placing a vertical mark on a horizontal 100 mm line or using a VAS, with the total VAS score calculated as the sum of five VAS scores (total range, 0-500) [14,22,23]. Measurements were taken before the NAPT (baseline VAS) and at 15 min after each challenge [14]. Acoustic rhinometry was performed using an SRE 2000 rhinometer (Rhinometrics, Lynge, Denmark) according to the guidelines of the Standardization Committee on Acoustic Rhinometry [24]. The mean volume (cm 3 ) in the anterior nasal segment (2-6-cm volume) was measured before the NAPT (baseline test) and at 15 min after each challenge [14,22]. Following the protocol in a previous report [4], the total VAS score and the 2-6 cm volume at different time points (15 min intervals) were plotted. The area under the curve (AUC) values for those two variables were calculated for every patient.

Basophil activation test
Blood was drawn (at the central institution) from 15 subjects (eight in the active group and seven in the control group) for a basophil activation test (BAT) before treatment, as well as at 4 months and 1 year after treatment. The cells were stimulated with the respective allergen within 2 h of blood sampling using a commercially available Flow-CAST kit (Bühlmann Laboratories AG, Schönenbuch, Switzerland) according to the manufacturer's and previously described instructions [25].

Statistical analyses
Statistical analyses were performed using PASW 20.0 (SPSS Inc. Chicago, IL, USA) and GraphPad Prism 6.01 software (GraphPad Software, La Jolla, CA, USA). Continuous variables were analyzed with the Man-Whitney U test for intergroup analysis, and the Wilcoxon signedrank test for intragroup analysis. Categorical variables were analyzed with the χ 2 test or Fisher's exact test.  Fig. 1 Study outline. At visit 1, rescue medications were prescribed, and diary cards were issued after informed consents were obtained; at visit 2, pre-ILAIT status was evaluated; at visits 3 to 5, subjects received intralymphatic immunotherapeutic allergen injection; at visits 6 to 7, post-ILAIT status was evaluated, respectively. ILAIT intralymphatic allergen-specific immunotherapy, HDM house dust mite P-values < 0.05 were considered to indicate statistical significance.

Minimum sample size calculation
A power calculation was performed with a two-sample t-test for the primary outcome (mean total score of RQLQ). We expected a clinically significant score difference of 0.50 in mean total score of RQLQ between the two groups at 4 months after treatment [26]. Assuming a standard deviation of 0.61 based on a previous pilot study [14], and aiming for a power of 0.80, type-1 error rate of 0.05, and loss to follow-up rate of 40%, the required total sample size was calculated to be 38.

Ethics
The study was conducted in accordance with good clinical practice guidelines [27].

Study subjects
A schematic representation of patient selection is shown in Fig. 2. Thirty-eight subjects were initially enrolled in the study; among them, five subjects rejected to be enrolled, and one developed an anaphylactic reaction during the IDT with a target allergen. Thirty-two subjects were randomly allocated into treatment (n = 19) and control (n = 13) groups, and each subject received three intralymphatic injections of target allergens or a placebo; one patient in the active group withdrew from the study after receiving only one injection but was still considered for the ILAIT safety evaluation. Another subject in the active group withdrew from the study after receiving three intralymphatic injections due to a lack of time. The therapeutic efficacy of ILAIT was evaluated (in 17 subjects in the active group and 13 in the control group) at 4 months after the first intralymphatic injection. Subsequently, the therapeutic efficacy of ILAIT was re-accessed at 1 year after the first intralymphatic injection; as eight subjects (four in the active group and four in the placebo group) withdrew due to lack of time, the 1-year follow-up was conducted for 22 subjects (13 in the active group and nine in the control group).
The baseline characteristics of the study subjects are summarized in Table 1 34.2 ± 9.5 years, and 19 subjects (59.4%) were female. The mean symptom duration of AR was 14.5 ± 9.5 years. Subjects in the active group were younger than those in the control group (32.4 ± 11.1 years vs. 36.9 ± 5.9 years, P = 0.037). By contrast, gender and symptom duration did not differ significantly between the two groups. The most common allergic comorbidity was asthma (59.4%), followed by urticaria (21.9%), food allergy (18.8%), drug allergy (15.6%), and atopic dermatitis (12.5%). Twentyone subjects (65.6%) had a family history of allergy. The frequencies of comorbid allergic diseases and a family history of allergy did not differ significantly between the two groups. The target allergens were D. farinae, D. pteronyssinus, cat, and dog extracts for 22 (68.8%), 21 (65.6%), 19 (59.4%), and 8 (25.0%) subjects, respectively; no significant differences in the frequencies of each target allergen were observed between the groups. Among the subjects followed-up at 4 months and 1 year, age, gender, symptom duration, allergic comorbidities, family history of allergy, and target allergens did not differ between the two groups.

Safety of ILAIT
On the day of the first intralymphatic injection, subjects reported that intralymphatic injection-caused pain was mild but more intense than venous punctures (VAS score: 3.5 ± 2.0 mm vs. 2.8 ± 1.7 mm, P = 0.019; Fig. 3).
The extent of pain caused by intralymphatic injection and venous puncture did not differ significantly between the two groups.
Local and systemic adverse events are summarized in Table 2. Pain was the most frequent local adverse event (12.8%), followed by itchiness (8.5%), paresthesia (6.4%), wheal (1.1%), and sensation of heat (1.1%) at the site of intralymphatic injection. The most frequent systemic adverse events were dyspnea (5.3%) and wheezing (5.3%), followed by chest discomfort (4.3%), headache (3.2%), chills (3.2%), palpitation (2.1%), urticaria (2.1%), itchy eyes (2.1%), itchy palms (2.1%), abdominal discomfort (1.1%), fever (1.1%), rhinorrhea (1.1%), postnasal drip (1.1%), and sneezing (1.1%). No significant differences in systemic and local adverse events were observed between the two groups. According to Müller's classification, four (one in the active group and three in the control group) and one (in the active group) subjects developed systemic hypersensitivity reactions of grade 1 and grade 3, respectively. The subject that developed a severe hypersensitivity reaction (grade 3) after the first intralymphatic injection complained of paresthesia at the injection site  Fig. 3 Pain of intralymphatic injection. On the day of the first intralymphatic injection, blood sampling was also performed. subjects were asked to compare the pain of intralymphatic injection to that of a venous puncture. Box plots show median (line), 24th and 75 percentiles (box), and ranges (whiskers). *P < 0.05 accompanied by dyspnea, chest and abdominal discomfort, palpitation, chills, and itchy palms. Although the allergen was further diluted 1,000-fold for the second intralymphatic injection, the subject experienced mild paresthesia at the injection site. The allergen's concentration was increased tenfold for the third intralymphatic injection, and the subject complained of pain and paresthesia at the injection site accompanied by fever and chills. For other subjects in the active group who had no reactions or a mild hypersensitivity reaction (grade 0-1), allergen concentrations were increased three-fold for the second intralymphatic injection and tenfold for the third intralymphatic injection.

Symptom relief and rescue medication use
The overall AR symptoms scored on a VAS did not change significantly in the treatment or control groups at 4 months or 1 year after treatment, and the scores did not differ between active and control groups at 4 months (VAS score: 37.9 ± 27.6 mm vs. 38.4 ± 21.7 mm, P = 0.805) and 1 year (VAS score: 47.7 ± 26.5 mm vs. 33.3 ± 22.9 mm, P = 0.186) after treatment (Fig. 4A). In the active group, the total mean scores of SNOT-20 and RQLQ and the mean domain score of the emotional function domain in RQLQ did not change significantly at 4 months or 1 year after treatment; however, these scores were significantly reduced at 4 months and/or 1 year after treatment in the control group (  Table 2 Adverse events associated with the intralymphatic injections in the cohort for testing procedural safety (n = 32) Data are shown in frequencies (%). *Hypersensitivity reactions were graded according to Müller's classification    Fig. S1E, F). Diaries of daily symptom and medication were returned by 27 subjects (16 in the active group and 11 in the control group); however, only 21 (13 in the active group and eight in the control group) and 10 (six in the active group and four in the control group) completed the reports for the 4-month and 1-year follow-up, respectively. According to these reports, the dSS did not change significantly in either group (Fig. 5A). The dSS did not differ between active and control groups at 4 months (mean daily score: 3.8 ± 3.4 vs. 2.4 ± 1.9, P = 0.750) and 1 year (mean daily score: 3.8 ± 3.2 vs. 3.0 ± 2.8, P = 0.610) after treatment. Meanwhile, the dMS decreased significantly at 4 months after treatment in the active group but not in the control group (Fig. 5B). However, the dMS was not different between active and control groups at 4 months (mean daily score: 2.7 ± 3.1 vs. 6.6 ± 5.7, P = 0.076) and 1 year (mean daily score: 3.4 ± 3.6 vs. 0.3 ± 0.6, P = 0.067) after treatment, which may be due to the small number of subjects who completed the diary. In both groups, the dSMS decreased significantly at 4 months and/or at 1 year after treatment (Fig. 5C). The dSMS was not different between active and control groups at 4 months (mean daily score: 6.7 ± 4.7 vs. 7.7 ± 7.4, P = 0.970) and 1 year (mean daily score: 11.0 ± 5.1 vs. 6.9 ± 7.8, P = 0.181) after treatment.

Skin reactivity
In the active group, skin reactivity to serial dilutions of HDM allergen decreased at 4 months after treatment (P < 0.05 for the 10 3 -fold dilution), then this reduction in skin reactivity diminished at 1 year after treatment, however the reduction in skin reactivity to 10 3 -and tenfold dilutions of HDM allergen remained significant (Additional file 2: Fig. S2A). On the contrary, in control subjects, skin reactivity after exposure to serial dilutions of HDM allergen increased at 4 months after treatment (P < 0.05 for the 10 7 -, 10 6 -, 10 5 -, 10 4 -, 10 3 -, and 10 2 -fold dilutions compared with baseline).
Skin reactivity to serial dilutions of cat allergens decreased at 4 months after treatment in the active group, and it was significantly lower than that in the control group (P < 0.05 for the 10 7 -, 10 6 -, 10 5 -, 10 4 -, and 10 3 -fold dilutions compared with the control group), then this reduction in skin reactivity diminished at 1 year after treatment (Additional file 2: Fig. S2B). In control subjects, skin reactivity to serial dilutions of cat allergens generally increased at 4 months, then decreased at 1 year after treatment, but the changes were not significant.
In the active group, skin reactivity after exposure to serial dilutions of dog allergens decreased at 4 months after treatment and increased at 1 year after treatment (Additional file 2: Fig. S2C). In control subjects, skin reactivity to serially diluted dog allergens increased at 4 months and 1 year after treatment; nevertheless, skin reactivity changes were not statistically significant in either group.
The IDT results revealed that skin reactivity to serially diluted HDM, cat, and dog allergens did not change significantly during the study period and that no differences existed between the two groups (Additional file 3: Fig.  S3).

Nasal reactivity
Among the 12 subjects who underwent the NAPT with the D. farinae allergen, nasal and ocular symptoms decreased at 4 months and 1 year after treatment in both groups; however, these changes were not statistically significant (Fig. 6A). Similarly, no significant alterations in nasal cavity volume were observed during the study period (Fig. 6B). Nasal or ocular symptoms and nasal cavity volume decrease during NAPT were not different between two groups before treatment and at 4 months and 1 year after treatment.

Basophil reactivity
Among the 15 subjects who provided blood samples, the percentages of activated CD63 + basophils after in vitro stimulation with D. farinae, D. pteronyssinus, and cat allergens, decreased at 4 months and 1 year after treatment in the active group, although not significantly. No changes in the percentages of activated CD63 + basophils were observed in control subjects (Additional file 4: Fig.  S4A-C). We could not evaluate the changes in the percentage of activated CD63 + basophils after in vitro stimulation with dog allergens due to the small number of subjects (two in the active group and one in the control group; Additional file 4: Fig. S4D.

Discussion
In 2008, Senti et al. first described ILAIT as a promising AIT that can alleviate nasal reactivity to pollen allergens as fast as within 4 months after treatment, which is significantly faster than with SCIT. In the open-label randomized study by Senti et al., ILAIT alleviated nasal reactivity for up to 3 years in patients with polleninduced AR [1]. Moreover, ILAIT has been shown to reduce allergic symptoms, serum pollen-specific IgE levels, and skin reactivity to the allergen for up to 3 years after treatment. Notably, compared with patients treated with SCIT, fewer ILAIT-treated patients required rescue medications. Subsequent open-label pilot studies confirmed that ILAIT alleviated nasal or ocular symptoms, improved the QOL, decreased nasal reactivity as observed in the NAPT and allergen-specific IgE serum levels, as well as increased allergen-specific IgG4 serum levels and the number of plasmablasts producing grass pollen-specific immunoglobulins other than IgE [5,7]. However, nasal reactivity, nasal and ocular symptoms, and QOL are largely self-reported and subject to confounding effects, thus introducing bias and uncertainty regarding the therapeutic efficacy of AIT. Therefore, double-blind placebo-controlled (DBPC) trials are required to elucidate the clinical efficacy of AIT objectively [28,29]. Thus far, six DBPC trials have evaluated the therapeutic efficacy and adverse effects of ILAIT in patients with pollen-induced AR [2-4, 6, 8-11]. In a DBPC trial with 15 subjects, Hylander et al. reported that ILAIT with birch or grass pollen improved seasonal allergic symptoms, decreased rescue medication use, and increased the activation levels of CD4 + T cells in the peripheral blood [2]. ILAIT also alleviated nasal reactivity to allergens and reduced the levels of the proinflammatory cytokine interleukin (IL)-8 in nasal fluids, although these effects were not statistically significant.
In a follow-up study with 20 additional subjects, they found that ILAIT modestly alleviated seasonal allergic symptoms. Moreover, ILAIT decreased nasal reactivity and IgG4 affinity to allergens in individuals who exhibited symptom improvement, although nasal reactivity did not differ significantly between treated and control subjects [3]. In a subsequent clinical trial with 60 patients sensitized to both birch and grass pollen allergens, ILAIT significantly improved the QOL during birch pollen but not grass pollen season [4]. They also found that ILAIT decreased rescue medication use during birch and grass pollen seasons, as well as reduced nasal reactivity to grass pollen and skin reactivity to birch and grass pollen; however, the overall improvement in symptoms did not differ significantly between the treatment and control groups. In a recent study, allergic patients received a booster dose at 1 year after three pre-seasonal intralymphatic injections of birch or timothy pollen; the booster dose further decreased the symptom and medication scores during the second pollen season compared with the first pollen season [8]. However, the study was unblinded after the end of the first pollen season, and no symptom or medication scores were recorded in the control group during the second pollen season.
Witten et al. assessed the usefulness of ILAIT for treating timothy grass (Phleum pretense) allergy and found that seasonal symptoms, QOL, skin or nasal reactivity to allergens, intracellular cytokine levels, regulatory T-cell marker expression, and histamine release levels did not differ significantly between the treatment and control groups [11]. By contrast, Patterson et al. reported that in subjects with a timothy grass allergy, ILAIT reduced the total combined score of symptoms and medications; however, Patterson et al. used different allergen concentrations and injection intervals from those used by Witten et al. [6]. Two recent DBPC trials reported that in individuals with Japanese cedar and mountain cedar allergies, ILAIT alleviated allergic symptoms and reduced rescue medication use [9,10]; nevertheless, further investigations are required to confirm these therapeutic outcomes. The usefulness of ILAIT for treating pollen-induced AR remains unclear [30,31].
The clinical efficacy of ILAIT against non-polleninduced allergies was first assessed by Senti et al. [12]. In a DBPC study, Senti et al. showed that ILAIT with recombinant cat allergens reduced nasal and skin reactivity, as well as increased cat-specific IgG4 serum levels and IL-10 production levels. In a recent open-label pilot study in China, cervical ILAIT improved nasal and ocular symptoms and QOL, as well as reduced rescue medication use in individuals with a HDM allergy [16]. However, the study did not include a control group, and the safety of cervical ILAIT was not comprehensively investigated.
Unlike the aforementioned studies, which used aluminum hydroxide-absorbed allergen extracts, in our previous open-label pilot studies, we evaluated the clinical efficacy and safety of ILAIT with aqueous allergen extracts of D. farinae, D. pteronyssinus, cat, dog, or mixtures thereof [14,15]. Although ILAIT improved allergic symptoms and the QOL, in some study subjects, ILAIT provoked severe local and systemic hypersensitivity reactions, especially in individuals treated with aqueous HDM allergens [14,15]. Hence, we substituted aqueous allergen extracts with tyrosine-adsorbed allergen extracts, which are considered safer [32]. However, one patient experienced a severe hypersensitivity reaction (grade 3) after ILAIT using tyrosine-adsorbed allergen extracts. With regard to local reactions, intralymphatic injection was proven to be more painful than venous puncture in this study. Previous studies also reported moderate-to-severe hypersensitivity reactions after ILAIT [5,7]. Thus, future studies are required to improve the safety of ILAIT. Additionally, a novel AIT such as epicutaneous immunotherapy using microneedles or adhesive-tape stripping can be an alternative modality that can minimize the risks of local and systemic adverse reactions [33,34].
The findings of this study suggest that ILAIT using tyrosine-adsorbed allergen extracts does not significantly alleviate AR induced by HDM, cat, or dog allergens. The relatively small number of subjects might have contributed to the insignificant efficacy of ILAIT for treating AR, although previous DBPC trials reported conclusive findings with a smaller number of subjects than that used in our study [2,6,12]. Alternatively, the ILAIT schedule (three intralymphatic injections at 4-week intervals) and doses used might have been insufficient to exert a therapeutic effect, given that most outcomes assessed did not differ significantly between the two groups. Additionally, only short-term reductions in rescue medication use, as well as partial improvements in QOL and skin reactivity response were observed after treatment.

Conclusion
ILAIT with tyrosine-adsorbed allergen extracts had no definite therapeutic effect on AR induced by HDM, cat, or dog allergens, although it may reduce rescue medication use, and partially improve QOL and skin reactivity responses. Additionally, ILAIT may provoke a severe hypersensitivity reaction; thus, future studies are required to elucidate the clinical efficacy and safety of ILAIT with various allergen extracts.