Group-specific Multi-allergen Sublingual/Swallow Immunotherapy Improves the Quality of Life of Polysensitized Children and Adults with Allergic Rhinitis

To date, and since the 1970s, non-specific multi-allergen sublingual/ swallow immunotherapy (NS-SLIT) in Brazil has represented a cheap alternative approach for treating allergic diseases, and has been commonly prescribed by general practitioners [1]. This widespread use was encouraged by its low cost, easy administration, remarkable lack of collateral effects, prescription by brand name, extensive distribution (easily found in most drugstores) and broad allergen spectrum (composed of a comprehensive mixture of aerial, bacterial and food allergens). Usually, NS-SLIT was prescribed based entirely on clinical symptoms without a thorough etiological investigation and was associated with antihistamines/steroids. The lack of a graded concentration schedule and double-blinded placebo-controlled clinical trials, combined with the difficulty in recognizing the unequivocal presence/absence of benefits in this clinical context, explains why NS-SLIT was almost completely discredited by the Brazilian certified Allergy and Immunology specialists, despite the fact that some patients report amelioration of their symptoms with this treatment [1].


Introduction
To date, and since the 1970s, non-specific multi-allergen sublingual/ swallow immunotherapy (NS-SLIT) in Brazil has represented a cheap alternative approach for treating allergic diseases, and has been commonly prescribed by general practitioners [1]. This widespread use was encouraged by its low cost, easy administration, remarkable lack of collateral effects, prescription by brand name, extensive distribution (easily found in most drugstores) and broad allergen spectrum (composed of a comprehensive mixture of aerial, bacterial and food allergens). Usually, NS-SLIT was prescribed based entirely on clinical symptoms without a thorough etiological investigation and was associated with antihistamines/steroids. The lack of a graded concentration schedule and double-blinded placebo-controlled clinical trials, combined with the difficulty in recognizing the unequivocal presence/absence of benefits in this clinical context, explains why NS-SLIT was almost completely discredited by the Brazilian certified Allergy and Immunology specialists, despite the fact that some patients report amelioration of their symptoms with this treatment [1].
Subcutaneous immunotherapy (SIT), however, represents a longestablished treatment for respiratory allergies. Because the aggregation of many components into the composition of the SIT shots increases local cutaneous reactions [2], SIT is mandatorily allergen-specific and is given mainly to patients with monoallergy [3]. Therefore, most trials designed to compare sublingual/swallow immunotherapy (SLIT) with SIT use one or a limited number of allergens, despite the fact that the use of a large number of allergens does not increase the collateral effects of SLIT, as observed with the unique Brazilian NS-SLIT experience [1], and in the scientific literature that reports the safety profile of SLIT even in 3 years old children [4]. The treatment of polysensitized patients with the use of a single major allergen or multiple allergens is currently a matter of debate [5]. The clinical use of SLIT is based on the experimental observation that the most frequent outcome of an oral encounter with a soluble antigen is systemic tolerance [6,7]. The oral and intestinal mucosa are preferred sites for tolerogenic dendritic cells, which collect soluble antigens at the mucosal surface and migrate to the proximal draining submandibular/mesenteric lymph nodes and Peyer's patches. The tolerogenic dendritic cell will induce regulatory T-cells if this encounter occurs during a non-inflammatory response [8,9]. In addition to promoting allergen-specific desensitization, oral immunotherapy is able to prevent and reverse co-existent unrelated sensitizations by a cross-suppression mechanism [10]. The local and regional secretion of TGF-β and IL-10 by tolerogenic dendritic cells and T-reg cells inside lymph nodes affects neighborings naïve T-cells and T-helper cells with diverse antigen specificities and induces them into an unspecific tolerogenic immune deviation [10][11][12][13][14][15].
Despite being widely used in several countries [16][17][18][19], SLIT is not officially approved by the American Food and Drug Administration, and the few clinics in America that have experience with SLIT employ the allergens approved for injection immunotherapy in an off-label manner [20,21], therefore most researchers studying the benefits of SLIT are based in Europe [22][23][24]. Despite the difference in administration approach, allergen-specific SLIT and SIT may be indirectly compared [25]. However, due to the difficulty associated with direct or indirect comparison with SIT in clinical trials, NS-SLIT is still an orphan therapy without sufficient evidence-based studies to define its advantages and/ or disadvantages in relation to the subcutaneous reference treatment.
To rationalize the clinical use of multi-allergen SLIT, we designed a protocol for a graded concentration schedule of group-specific multiallergen SLIT (GSM-SLIT) treatment inspired by the pioneers of allergy desensitization. The first GSM-SLIT treatment reported in the literature was performed in 1900 by Holbrook Curtis who used "drops" of a mixture of ragweed, ambrosia and golden rod to successfully treat seasonal "hay fever" patients sensitized to pollens of these and other plants [26]. Using a different approach than Curtis' , we studied cutaneous sensitization using an extensive allergen panel of 46 extracts to establish 8 therapeutic groups consisting of combinations tailored to the subjects' sensitizations. The GSM-SLIT treatment was administered for 6 months, and the subjects who successfully completed the treatment without the additional use of any medication (steroids and/or antihistamines) were evaluated using a quality of life (QOL) questionnaire.

Study design and subjects
Two independent teams performed the diagnostic and treatment procedures (Instituto Alergoimuno de Americana) and QOL evaluations (Faculty of Nursing -Anhanguera University) in this open study. The study was approved by the ethical review board of the Anhanguera University (2442/2012), registered with the Brazilian clinical trial registry platform (CONEP 1038/2006) and was conducted according to the principles of the Declaration of Helsinki. The subjects' informed assent and consent were obtained before study enrolment, and their information was kept confidential. There were no financial or other potential conflicts of interest declared.
In total, 60 subjects (27 males) who completed 6 months of exclusive GSM-SLIT were interviewed. The mean age was 20.9 (SD ± 16.4) years, including 23 children from 3 to 12 years.
The polysensitized subjects with allergic rhinitis were treated exclusively with one or more specific groups of sublingual/swallow allergens, according with their cutaneous sensitizations. All of the patients had previous clinical diagnoses of persistent rhinitis according to the ARIA criteria [27] and evidence of IgE-mediated hypersensitivity by both skin prick test and skin scrape test performed as previously described [28].

Allergens extracts
The diagnosis of sensitization and immunotherapy were performed with extracts purchased from the FDA Allergenic/Immunotech, Rio

GSM-SLIT composition
The allergens for GSM-SLIT were divided into 8 groups: a) mites; b) animal tegument; c) fungi; d) pollens; e) insects; f) bacterial allergens and toxoids; g) latex/latex-related food; and h) food unrelated to latex. The individual prescription of GSM-SLIT was determined according to the cutaneous reactivity pattern of skin tests. If there was at least one allergen reagent from a given group, all the constituents of this group were incorporated into the prescription. The groups prescribed were mixed in equal parts. The original extract concentration of each immunotherapeutic group was adjusted to 1 mg of protein per mL as determined by colorimetric analysis, and 1:10 serial dilutions were derived from these adjusted solutions [29].
The food allergen immunotherapy was specifically composed of two main parts. The first part included a mixture of the antigens extracted from each food by Coca's solution. For this mixture, each extract was adjusted to 1 mg of protein per mL. The second part was composed of a digested mixture of each food extract in simulated gastric fluid with pepsin and HCl [30]. The objective was to provide the oral mucosa with peptides generated by gastric digestion which otherwise would not be able to reach the submandibular lymph nodes [31]. For simulated gastric digestion, the protein preparation was incubated with pepsin (Sigma-Aldrich, St Louis, Mo) in the proportion of 0.87 mg pepsin to 1 mg protein at 37°C with continuous agitation. The pH was titrated down to 2.0 with 0.1 N HCl in 1 minute; the solution was then refrigerated, and the pH was titrated up with 0.1 N NaOH to 7.0. The final concentration of the digested extract was adjusted to 1 mg/mL of food protein. The digestion of the individual extracts was examined by SDS-PAGE ( Figure 1) as previously described [32]. The non-digested food extract solution (1 mg/mL protein) and the digested food extract solution (1 mg/mL protein) were mixed at 1:1 volumes. The six-month treatment comprised 3 phases of two months each with treatment dilutions of 1:10.000, 1:1.000 and 1:100. The patient was oriented to put 3 drops under the tongue following teeth brushing, twice a day, to retain the solution in the mouth for approximately 5 minutes and then to swallow. The patient was also oriented to avoid eating or drinking anything for 20 minutes after the GM-SLIT administration.
In total, 49 subjects were treated with mite extract, 20 subjects with animal tegument extract, 33 subjects with fungi extract, 28 subjects with pollens extract, 15 subjects with insects extract, 12 subjects with bacterial and toxoids extracts, 24 subjects with latex/latex food related extract and 25 subjects with food extract not related to latex. The extracts were mixed in equal parts according to the subjects' individual sensitivities, as determined by skin tests, and were administered as a single therapy.

Quality of Life questionnaire
An independent team (Faculty of Nursing-Anhanguera University) analyzed the outcome after six months of treatment by means of a QOL questionnaire specific for allergic rhinitis, adapted and validated for the Brazilian population [33,34]. The patient or responsible parent answered the same questionnaire twice, with the first concerning the QOL before the GSM-SLIT treatment and the second concerning the QOL after completion of 6 months of GSM-SLIT treatment. The patient was also checked for the correct use of GSM-SLIT and the absence of use of any medication for allergy (topical and/or systemic corticosteroids and/or antihistamines) during the 6 months period of GSM-SLIT treatment.
The QOL questionnaire represents seven categories: sleep, systemic symptoms, practical problems, nasal symptoms, eye symptoms, activities and emotions. The subject was presented with a zero to six points Likert scale. This scale attributed points to the intensity of symptoms with the answers quantified from 0 to 6 as follows: 0=I did not feel discomfort, 1=disturbed me sometimes, 2=disturbed me few part of the time, 3=disturbed me moderately, 4=disturbed me many time, 5=disturbed me most of the time, and 6=disturbed me extremely all the time. The

Statistical analyses
The data were reported as the arithmetic mean with 95% confidence interval (CI). Comparisons between QOL index obtained before and after six months of GSM-SLIT were performed using a two-tailed paired t-test. The mean differences were considered significant at p < 0.05. Statistical analyses were performed using the GraphPad Prism for Windows (version 5.0; GraphPad Software, Inc., San Diego, CA, USA).

Results
The mean number of reagent cutaneous tests was 15.2 (CI 13.5 to 16.8).
The mean total QOL score of the entire group (Q1 to Q27) was 84.8 (CI 76.6 to 93.2) before GSM-SLIT and 24.1 (CI 19.2 to 28.9) after GSM-SLIT. The mean of the differences was 60.7 (CI 53.2 to 68.2) and this was significant for the paired t test (p < 0.0001) Figure 2.
The mean QOL score of the sleep symptoms group (Q1 to Q3) was 10.25 (CI 8.7 to 11.7) before GSM-SLIT and 2.3 (CI 1.6 to 3.0) after GSM-SLIT. The mean of the differences was 7.9 (CI 6.5-9.3) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the systemic symptoms group (Q4 to Q10) was 16.6 (CI 13.8 to 19.47) before GSM-SLIT and 5.6 (CI 3.9 to 7.3) after GSM-SLIT. The mean of differences was 11.0 (CI 8.7 to 13.2) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the practical problems group (Q11 to Q13) was 11.5 (CI 10.1 to 12.8) before GSM-SLIT and 3.3 (CI 2.5 to 4.1) after GSM-SLIT. The mean of the differences was 8.1 (CI 6.8 to 9.5) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the nasal symptoms group (Q14 to Q16) was 12.9 (CI 11.7 to 14.9) before GSM-SLIT and 3.7 (CI 2.8 to 4.6) after GSM-SLIT. The mean of the differences was 9.2 (CI 7.9 to 10.5) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the ocular symptoms group (Q17 to Q20) was 8.8 (CI 7.1 to 10.5) before GSM-SLIT and 2.7 (CI 1.8 to 3.6) after GSM-SLIT. The mean of the differences was 6.0 (CI 4.5 to 7.5) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the activities group (Q21 to Q23) was 10.4 (CI 8.9 to 11.8) before GSM-SLIT and 2.8 (CI 2.1 to 3.5) after GSM-SLIT. The mean of the differences was 7.5 (CI 6.3 to 8.7) and was significant for the paired t test (p<0.0001) (Figure 2).
The mean QOL score of the emotions group (Q24 to Q27) was 14.5 (CI 12.8 to 16.2) before GSM-SLIT and 3.6 (CI 2.6 to 4.6) after GSM-SLIT. The mean of the differences was 10.9 (CI 9.3 to 12.5) and was significant for the paired t test (p<0.0001) (Figure 2).
Of all individual questions, question 12 (need to rub nose/eyes) had a higher mean score before treatment (4.6) and after treatment (1.7). Question 19 (photophobia) had the lowest mean score before treatment (1.1) and after treatment (0.3). All individuals' questions had mean differences between pre-treatment and post-treatment answers that were significant for the paired t test (p<0.05).

Discussion
Allergic rhinitis, a multifactorial disease with immune and non- immune components, requires a multifactorial treatment. The etiologic diagnosis is itself therapeutic, if the knowledge of the causative allergens is a stimulus for the patient to avoid their particular triggers. However, environmental exposure is a variable factor that is difficult to control to avoid allergen inhalation. Equally difficult to control is the avoidance of oral antigen ingestion. Food allergies may produce respiratory symptoms [35]. Additionally, several airborne allergens also crossreact with food proteins as in the case of mite-shrimp-insects or fruitpollen-latex syndromes [36][37][38] and it is not uncommon to diagnose shrimp or fruit allergies in patients with mite-associated or polleninduced allergic rhinitis. In our study, the treatment of these conditions included the avoidance of those related food as well as group-specific immunotherapy.

MW CM PCM
Polysensitization is still a challenge to allergists who are used to dealing with allergen-specific immunotherapy [39]. Most physicians do not apply a comprehensive allergen panel for their diagnostic routine, so most sensitizations remain undiagnosed. The diagnosis of several sensitizations appears to be unnecessary if the previous intent is to treat the patient with an allergen-specific immunotherapy. However, even polysensitized patients treated with allergen-specific immunotherapy show improvements in their symptoms and this fact may be attributed to an unspecific action of allergen-specific immunotherapy. The offlabel use of allergen-specific immunotherapy to treat polysensitized patients seems to be driven by the lack of multi-allergen formulations approved for clinical use in the countries under the American Food and Drug Administration policies. This governmental restriction for multi-allergen immunotherapy may be justified when considering that the administration, by subcutaneous route, may be dangerous or hazardous to the patients, but the administration by sublingual/swallowing is relatively safe when prescribed with a progressive concentration schedule under specialized medical supervision. In addition to safety, there are other advantages in prescribing allergen immunotherapy orally, particularly if it is administered as part of a tolerance-induction strategy. The administration of an antigen by the oral route diminishes its further absorption [40]. This immune exclusion is mediated mainly by IgA which induces specific systemic tolerance. The oral mucosa is a privileged site for inducing tolerogenesis [41], but usually the oral mucosa is naturally provided only with large undigested proteins. Incomplete gastrointestinal digestion may generate immunogenic peptides devoid of their mediator-releasing activity, but not T cellactivating properties [42]. Tolerance-induction strategies may include homologous unmodified allergens associated with their modified forms [43]. The administration of intact proteins together with the peptides resulting from their peptic digestion diminishes the sensitizing capacity of oral allergens [44]. Tropomyosin, for instance, is the common allergen of cockroaches, mites and shrimps and is found in mammalian meat, therefore, it is reasonable to assume that their peptic digestion may produce similar peptides. If these peptides contain the same linear epitopes presented to T cell receptors by the antigen presenting cells, they can potentially induce cross-suppression when administered within a tolerance-induction strategy [45]. However, unless these peptides reach the oral mucosa by gastroesophageal regurgitation, they  will be targeted by gut immune cells only if not previously digested by pancreatic trypsin and microvilli peptidases. Thus, as part of the induction-tolerance strategy and to provide additional peptides directly to the oral mucosa (where they will not be digested by trypsin and peptidases), we pepsinized an aliquot of each food extract and added this mixture to food GSM-SLIT [46,47].
The treatment of allergic conditions is not based on specific interventions, but on global strategies involving educational guidelines, antihistamine/anti-inflammatory/symptomatic drugs and a desensitizing immunotherapy based on a thorough diagnosis (syndromic, anatomical and etiological). Despite the efficacy of these measures, which could be accessed by several biological markers, what really counts for the patient is to be free of the symptoms and released of the use of medications associated with collateral effects. Most subjects enrolled in this study had previously been treated with conventional therapies (nasal steroids and antihistamines) without satisfactory improvement of their symptoms, therefore, they were comfortable with the idea of the exclusive use of GSM-SLIT. The QOL questionnaire represents the resulting of the overall toleranceinduction strategy over these subjects and cannot differentiate between the influence of environmental control, dietary avoidance and GSM-SLIT. As the three pillars of this treatment act synergistically, they were prescribed together and resulted in a pronounced improvement in the QOL as demonstrated by the questionnaires scores. The conclusion of this study is that polysensitized patients with allergic rhinitis diagnosed using cutaneous tests by a specialized team may be safely and effectively treated with GSM-SLIT without the concomitant use of any medication.