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Ann Allergy Asthma Immunol xxx (2014) 1e6

Objective approach for fending off the sublingual immunotherapy placebo effectin subjects with pollenosis: double-blinded, placebo-controlled trialTanya Z. Kralimarkova, MD; Todor A. Popov, MD, PhD; Maria Staevska, MD, PhD; Roxana Mincheva, MD;Cvetelina Lazarova, MD; Rumyana Racheva, MD; Tihomir B. Mustakov, MD; Violina Filipova, MD;Margarita Koleva, MD; Kalina Bacheva, BA; and Vasil D. Dimitrov, MD, PhDClinic of Allergy & Asthma, Medical University Sofia, Sofia, Bulgaria

A R T I C L E I N F O

Article history:Received for publication December 24, 2013.Received in revised form March 7, 2014.Accepted for publication March 25, 2014.

A

BsOm

Reprints: Todor A. Popov, MD, PhD, Clinic of AllerSofia, 1, Sv Georgi Sofiyski Street, BG-1431 Sofia, Bucom.Disclosure: Authors have nothing to disclose.Disclaimer: Laboratoire Stallergenes SA had no inimplementation, or interpretation.Funding: This study was funded by an unrestricteStallergenes SA (Antony, France).

1081-1206/14/$36.00 - see front matter � 2014 Ahttp://dx.doi.org/10.1016/j.anai.2014.03.019

B S T R A C T

ackground: Symptom scoring for the assessment of allergen immunotherapy is associated with a sub-tantial placebo effect.bjective: To assess the ability of exhaled breath temperature (EBT), a putative marker of airway inflam-ation, to evaluate objectively the efficacy of grass pollen sublingual immunotherapy in a proof-of-concept

study.Methods: This was a double-blinded, placebo-controlled clinical trial in 56 subjects (mean � SD 30 � 12years old, 33 men) sensitized to grass pollen. The objective measurements were EBT, spirometry, and per-iostin and high-sensitivity C-reactive protein in blood. Overall discomfort scored on a visual analog scale wasused as a proxy for subjective symptoms. Evaluations were performed before, during, and after the grasspollen season.Results: Fifty-one subjects (25 and 26 in the active treatment and placebo groups, respectively) wereassessed before and during the pollen season. The mean pre- vs in-season increase in EBT was significantlysmaller (by 59.1%) in the active treatment than in the placebo group (P ¼ .030). Of the other objectivemarkers, only the blood periostin level increased significantly during the pollen season (P ¼ .047), butwithout intergroup differences. Subjectively, the mean pre- vs in-season increase in the visual analog scalescore was 32.3% smaller in the active treatment than in the placebo group, although this difference did notreach statistical significance (P ¼ .116).Conclusion: These results suggest that the efficacy of grass pollen sublingual immunotherapy can beassessed by EBT, a putative quantitative measurement of airway inflammation, which is superior in its powerto discriminate between active and placebo treatment than a subjective assessment of symptoms assessedon a visual analog scale.Trial Registration: clinicaltrials.gov Identifier: NCT01785394.� 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Introduction total combined score taking into account symptom scores and

Allergen immunotherapy (AIT) for atopic airway diseases hasgone through a long and difficult period of doubt and skepticism.1

This is due largely to the fact that evidence of the efficacy of AIT isbased mainly on the subjective assessment of symptoms. The Eu-ropean Medicines Agency has recommended that the preferredprimary outcome measurement for AIT clinical trials should be a

gy & Asthma, Medical Universitylgaria; E-mail: ted.popov@gmail.

volvement in the study’s design,

d research grant from Laboratoire

merican College of Allergy, Asthma &

medication usage scores.2 In turn, symptom scoring is known to beassociatedwith a substantial placebo effect in blinded studies.3 Thisshortcoming also affects alternatives to conventional subcutaneousAIT routes for allergen administration, such as sublingual immu-notherapy (SLIT).4 This implies that to prove the efficacy of AIT,large placebo-controlled clinical trials need to be performedinvolving hundreds of patients and large amounts of data collectedby the patients in diaries for the entire duration of the treatmentperiod. Thus, the ever stricter standards for the quality of outcomereportingmean that traditional clinical research in the field of AIT isincreasingly challenging and expensive to perform.5

Grass polleneinduced allergic respiratory disease (also known aspollenosis) is a classic atopic condition in which the inhalation ofpollen grains by sensitized patients triggers a sequence of inflam-matory events in the airways. At least 1 clinical manifestation (such

Immunology. Published by Elsevier Inc. All rights reserved.

T.Z. Kralimarkova et al. / Ann Allergy Asthma Immunol xxx (2014) 1e62

as rhinitis, conjunctivitis, and asthma) subsequently affects varioustarget organs and is influenced more by immediate hypersensitivitythan by significant inflammatory events.6 As a potentially disease-modifying treatment, AIT is thought to modulate the aberrant im-mune response associated with the inflammatory cascade.

Grass polleneinduced allergic respiratory disease is common inmany parts of the world and is certainly the most prevalent pollen-induced allergy in Europe.7 From a research point of view, pollen-osis provides a convenient, natural model of atopic respiratorydisease for approximately 2 to 3 months during the late spring andearly summer, so that different pathogenetic aspects and treatmentmodalities can be studied before, during, and after the pollen sea-son. The authors took advantage of this particular feature of thedisease to assess a novel noninvasive method for evaluatinginflammation of the airways.

Measurement of exhaled breath temperature (EBT) has beensuggested as a new method for detecting and monitoring diseaseprocesses in the respiratory system.8e10 This approach is based onthe hypothesis that blood flow changes in the conducting airwaysare characteristic of different disease states and influence thetemperature of the exhaled gases. This influence is minimal andrequires high-precision gauging devices to assess it reliably so as toprovide ground for clinical inferences. Similarly to fractional nitricoxide in conjunction with which the first measurements of EBTwere made, EBT captures the signal related to eosinophil-driveninflammation. Different open and closed circuit systems using asingle breath or continuous breathing have been used by differentresearch teams. A handheld device has been developed, repre-senting an isolated thermal chamber with a valve system ensuringthe accumulation of heat frommultiple breaths until a temperatureplateau is reached.11,12 It is user friendly and allows accurate andreproducible EBT measurements within a very narrow temperaturerange. In a previous study with this instrument, the authorsdocumented a highly significant increase in EBT during the pollenseason in patients sensitized to grass pollen with upper airwaysymptoms (irrespective of whether they experienced signs ofasthma).13

The next logical step was to determine whether the initiation ofAIT before the pollen season could affect the in-season EBT values.The authors reasoned that if AIT created tolerance to the disease-inducing allergen, the subsequent inflammatory response wouldbe dampened.14 Thus, a study was designed to test this hypothesis.

Methods

Study Design

This was a double-blinded, placebo-controlled, parallel-group,proof-of-concept study of AIT-naive patients with grass polleneinduced allergic respiratory disease who were randomized 1:1 to

Figure 1. Flow diagram summarizing study protocol. EBT, exhaled breath te

SLIT with a 5-grass pollen extract or placebo (Fig 1). Objectivemeasurements and subjective assessment using visual analog scale(VAS) scores as a proxy for the classic approach for an evaluation ofthe efficacy of AIT were performed before the grass pollen season(when atmospheric grass pollen counts were well below criticallevels), during the season (when high atmospheric grass pollencounts triggered symptoms in the sensitized population), and afterthe season (when atmospheric grass pollen counts had decreased).The study’s objectives and protocols were approved by the localinvestigational review board (Medical University Sofia, Sofia,Bulgaria; reference number 86/01/06/2011) and the study wasconducted in accordance with good clinical practice and the tenetsof the Declaration of Helsinki (as amended in Edinburgh in 2000).All participants gave their prior, written, informed consent toparticipate in the study.

Subjects

Fifty-six AIT-naive patients referred to Clinic of Allergy &Asthma of Medical University Sofia were invited to participate inthe study. The study population’s demographic characteristics arepresented in Table 1. Participants were sensitized to grass pollen (asevidenced by a wheal diameter >3 mm larger than the negativecontrol in a skin prick test with a mix of 5 grass allergens:Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne,Phleum pratense, and Poa pratensis; Stallergenes, Antony, France).According to the study’s main inclusion criteria, participants had tobe 7 to 55 years old and have displayed documented symptomsrequiring symptomatic medication for at least the preceding 2 grasspollen seasons. The main exclusion criteria were moderate tosevere asthma (as defined by the Global Initiative for Asthmaguidelines15), symptoms related to or strong skin test positivity toother seasonal or perennial allergens, immunosuppression, malig-nancies, autoimmune diseases, intake of b blockers, pregnancy, andlactation at the time of initiation of immunotherapy. The investi-gating physicians also were asked not to include patients in whom(in their clinical judgment) comorbidities could affect the studyresults. At least 3 months before the expected start of the grasspollen season, 53 patients (3 patients revoked their informedconsent) underwent the preseason visit and were randomized toSLIT with a 5-grass pollen extract that contained the same com-ponents as the mixture used for skin prick testing (Staloral 688;Stallergenes) or to placebo. Once-daily treatment was continuedthroughout the pollen season. The AIT induction phase was carriedout with preparations with 10 and 300 indices of reactivity (Stal-lergenes) using the doses specified in the summary of productcharacteristics. The daily maintenance dose was 10 squirts underthe tongue before breakfast from the preparation with a 300 indexof reactivity, corresponding to approximately 25 mg/mL of the

mperature; SLIT, sublingual immunotherapy; VAS, visual analog scale.

Table 1Disposition of study population

Characteristics Treatment groups

5-Grass pollen SLIT Placebo

Before pollen seasonITT population, n 28 28Men/women 15/13 18/10Age (y), mean � SD 30.3 � 12.6 30.0 � 12.5Allergic rhinitis, n (%) 28 (100) 28 (100)Allergic conjunctivitis, n (%) 28 (100) 28 (100)Asthma, n (%) 10 (36) 10 (36)Atopic dermatitis, n (%) 10 (36) 7 (25)Oral allergy syndrome, n (%) 9 (16) 6 (10)Disease duration (y), mean � SD 14.2 � 11.4 10.8 � 5.2Smokers/nonsmokers/ex-smokers 2/17/9 4/18/6

During grass pollen seasonDropouts, n (men/women) 3 (1/2) 2 (2/0)PP population, n 25 26

After grass pollen seasonDropouts, n (men/women) 4 (2/2) 2 (2/0)PP population, n 21 24

Abbreviations: ITT, intention-to-treat; PP, per-protocol; SLIT, sublingual immuno-therapy.

T.Z. Kralimarkova et al. / Ann Allergy Asthma Immunol xxx (2014) 1e6 3

group with 5 major allergens. For the in-season visit, all partici-pants were instructed to report to the clinic if and when theyexperienced the first symptoms of pollenosis. The authors alsoenvisaged the ideal scenario when participants might not have anysymptoms by the time grass pollen counts had increased signifi-cantly according to the country’s aerobiology service and whenmost other grass pollenesensitized patients had developed clinicalsigns; such patients had to be invited by telephone to attend theclinic.

Assessments

Objective measurementsThe following objective measurements were performed: EBT,

spirometry, and blood levels of periostin and high-sensitivityC-reactive protein. Evaluations were performed before the grasspollen season, during the season, and after the pollen count haddecreased.

At each visit, experienced operators measured the EBT (at thesame time in the morning) with a portable breath thermometer (X-Halo, Delmedica Investments Ltd, Singapore) according to themanufacturer’s instructions. The description of the device andmeasurement procedure has been detailed in a specialized tech-nical article,12 so only a brief outline is provided here. The devicehas been certified to comply with the European CE markingdirective (Medical Device Directive 93/42/EEC) by the TechnischerÜberwachungs-Verein (Munich, Germany) and validated for pre-cision (up to the second digit after the decimal point for tempera-ture measurements in degrees Centigrade) and for safety use inhumans. Subjects seal their lips around the mouthpiece of thehandheld instrument and exhale repeatedly into it until the tem-perature of the heat sink within it reaches a plateau, indicating thata thermal equilibrium has been reached inside. The plateau isautomatically detected by an electronic processor connected to ahighly sensitive temperature, which produces audio and visualsignals to indicate the end of the measurement. The curve of therising temperature can be monitored on the screen of a computer,which ensures that the subject is performing the measurementproperly. To avoid confounding influences that are known to affectthe measurements,10 patients were asked to come early in themorning before breakfast and to avoid smoking and vigorous ex-ercise. They were seated comfortably to rest for 30 minutes in anair-conditioned room with the indoor temperature kept at 21�C to23�C.

A pneumotachograph-based spirometer (SP-10, Schiller AG,Baar, Switzerland) was used for the lung function measurements.The operators had been trained to comply with the AmericanThoracic Society’s spirometry guidelines.16 High-sensitivity C-reactive protein, a nonspecific indicator of inflammation, wasassayed using a latex immunoturbidimetric technique (Giesse Di-agnostics srl, Rome, Italy). Periostin, a matricellular protein that hasbeen recently proposed as a simple serum measurement to assessthe response to treatment directed at the T-helper type 2pathway,17 was measured with enzyme-linked immunosorbentassay kits from USCN Life Sciences Inc (Wuhan, China; range478e5,000).

Subjective symptom scoringOverall discomfort from allergic symptoms relevant for the time

of the visit and the last 72 hours before the visit was marked on a100-mm VAS ranging from “worst hay fever symptoms ever” at0 mm to “no hay fever symptoms” at 100 mm. The stretch from0 mm to the mark made by the patient was measured in millime-ters and used as a quantitative continuous variable in the analysis.

Statistical Analysis

The primary end point was the pre- vs in-season difference inEBT in each of the 2 study arms. Because the present pilot study isthe first to assess the value of EBT in differentiating between activeand placebo SLIT in patients with grass pollen allergy, 50 subjectswas considered a convenience sample as proof of this concept. Therelations and trends identified in the present studywill be of use forsample size calculations in future research.

The secondary end points were pre- vs in-season differences inVAS score, forced expiratory volume in 1 second (percentage pre-dicted), and blood levels of high-sensitivity C-reactive protein andperiostin. For each variable, the normality of distribution wasassessed with the Kolmogorov-Smirnov test and further analyzedwith the Student t test. All tests were 2-tailed and the threshold forstatistical significance was set to a P value less than .05. All statis-tical analyses were performed with SPSS 13.0 for Windows (SPSS,Inc, Chicago, Illinois).

Results

Assessment of the Study Population as a Whole

The objective data and subjective assessments by VAS scores ofall patients in the active treatment and placebo arms before andduring the pollen season are presented in Table 2. All patients(other than 3 in the active SLIT group) sensed the advent of thepollen season, as evidenced by a significant pre- vs in-seasondecrease in VAS scores from a mean � SEM of 92.2 � 1.5 to 66.7� 3.0 points (P < .001). This was paralleled by a significant increasein EBT from 34.32 � 0.08 to 34.71 � 0.05 (P < .001). There was asignificant, inverse correlation between the change in VAS scoreand the change in EBT (R ¼ �0.47, P ¼ .001). When considering theother objective measurements, only the blood periostin levelincreased significantly during the pollen season (from 1,127 � 72pg/mL before the season to 1,429 � 134 pg/mL during the season,P ¼ .048). None of the patients (and not even those with allegedlower airway involvement) experienced a severe asthma attack.

When considering only the 45 patients who returned for thethird (postseason) visit, the significant in-season vs postseasondifferences were similar to the pre- vs in-season differences (P <

.001 for VAS score and EBT; data for EBT only are shown in Fig 2).

Intergroup Comparisons

The pre- vs in-season changes in VAS score and EBT weresmaller in the active SLIT group than in the placebo group. This

Table 2Subjective and objective data for all patients and for active treatment and placebo arms before and during the pollen season

Variables All patients Patients on 5-grass pollen SLIT (active) Patients on placebo (placebo) P value, active vs placebo

Mean � SEM P value Mean � SEM P value Mean � SEM P value

ObjectiveEBT (�C) <.001 .05 <.001Before pollen season 34.32 � 0.08 34.40 � 0.13 34.25 � 0.10 .37During pollen season 34.71 � 0.05 34.61 � 0.07 34.80 � 0.07 .06

FEV1 (% predicted) .39 .10 .71Before pollen season 102.4 � 2.4 100.0 � 3.5 104.8 � 3.4 .33During pollen season 104.2 � 2.2 104.8 � 2.7 103.6 � 3.6 .80

Periostin (pg/mL) .05 .71 .41Before pollen season 1,127 � 72 1,112 � 111 1,101 � 100 .93During pollen season 1,429 � 134 1,135 � 186 1,688 � 178 .15

hsCRP .16 .07 .49Before pollen season 4.33 � 0.49 3.32 � 0.56 5.31 � 0.76 .11During pollen season 5.27 � 0.51 4.36 � 0.43 6.15 � 0.88 .08

SubjectiveVAS score (0e100 mm) <.001 <.001 .001Before pollen season 92.2 � 1.5 91.2 � 2.1 93.2 � 2.2 .52During pollen season 66.7 � 3.0 70.7 � 3.4 62.9 � 4.8 .19

Abbreviations: EBT, exhaled breath temperature; FEV1, forced expiratory volume in 1 second; hsCRP, high-sensitivity C-reactive protein; SLIT, sublingual immunotherapy;VAS, visual analog scale.

T.Z. Kralimarkova et al. / Ann Allergy Asthma Immunol xxx (2014) 1e64

intergroup difference was statistically significant for EBT but not forthe VAS score (Fig 3A, B). There were no significant intergroupdifferences for the other objective measurements.

Discussion

To the best of the authors’ knowledge, this study is the first tohave assessed the value of EBT measurement in subjects experi-encing symptoms of grass polleneinduced allergic respiratorydisease. These results suggest that measuring EBT during a courseof AIT may be an effective way of assessing an individual’s treat-ment response. However, several issues appear to be surprising atfirst sight and might have further implications.

Although most patients had seasonal rhinoconjunctivitis anddid not perceive symptoms emanating from the intrathoracicairways, all displayed an increase in EBT during the grass pollenseason. The authors believe that this constitutes further proof ofthe crosstalk between the upper and lower airways and, hence, ofthe “united airways” hypothesis.18,19 These findings also are inline with the long-known seasonal appearance of airway hyper-responsiveness in patients with allergic rhinitis20 and might beinterpreted as further proof that allergic rhinitis is a risk factor forasthma.21

SLIT with a 5-grass pollen extract did not completely abolish theseasonal increase in EBT. This raises questions about the effect of AIT

Figure 2. Exhaled breath temperature (EBT) before, during, and after the pollenseason. These data correspond to the 45 patients who completed all 3 study visits.

on airway inflammation. One study has suggested that the ability ofAIT to prevent the natural progression of allergic rhinitis to asthma isdisconnected from the therapy’s lack of effect on airway respon-siveness to methacholine or to changes in sputum eosinophilia.22

Similarly, negative findings have been reported for other relevantindices of airway inflammation (such as fractional exhaled nitricoxide and the pH of exhaled breath condensate23). However, a morerecent study has reported an association between bronchial hyper-responsiveness and these indices in patients with seasonal allergicrhinitis24; this fits better with the present results for EBT.

The lack of a more pronounced effect of AIT on airway inflam-mation (as judged by the presence of an increase in EBT during theseason) might be due to insufficient treatment duration. Additivebenefits have been demonstrated when AIT is maintained oversuccessive years.25 Accordingly, the authors plan to extend theirstudy in time.

At first sight, the lack of significance between the active treat-ment and placebo arms of the study in VAS scores is disappointing.However, it should be noted the tentative sample size calculationwas based on EBT variability. Some 10 patients in each arm wouldhave allowed reaching statistical significance based on VAS scores.Actually, the authors believe that statistical significance based onEBTachievedwith a smaller number of subjects is themajor asset ofthis study.

One might argue that VAS score is not the correct variable to usefor an evaluation of the effect of AIT. However, the aim was not toprove the efficacy of AIT; this has been demonstrated for theparticular sublingually applied 5-grass pollen vaccine used in thepresent study in 3 large multicenter placebo-controlled trialsinvolving hundreds of patients, which used daily symptom andmedication scores for the entire duration of the pollen season.26e28

The primary end point was the EBT and the purpose of the VASscore was to inform that the SLIT effectiveness trend was presenteven with the much smaller number of subjects in this study.Moreover, the “VAS approach” has been extensively investigatedand validated in allergic rhinitis and has been shown to correlatesignificantly with the severity of rhinitis graded by the AllergicRhinitis and Its Impact on Asthma criteria and quality of life.29,30 Areal-life prospective study of patients with symptomatic rhinitisfound VAS to be a sensitive and valid tool to monitor the burden ofallergic rhinitis in clinical practice and concluded that VAS changesof more than 23 mm should be considered of clinical importanceand reflects responsiveness to treatments.31

Figure 3. Pre- vs in-season differences in (A) visual analog scale (VAS) score and (B)exhaled breath temperature (EBT) in the placebo group and the active 5-grasssublingual immunotherapy (SLIT) group.

T.Z. Kralimarkova et al. / Ann Allergy Asthma Immunol xxx (2014) 1e6 5

The focus of the present study was the EBT, which has proveduseful in assessing disease control32 and small airway involve-ment in patients with asthma.33 Despite the narrow band withinwhich EBT changes, the pre- vs in-season differences assessedwith the paired-samples t test were highly significant, reachinghigher than 1�C in some cases. EBT appears to be an individualcharacteristic varying within the human population in health anddisease and exceeding the simplistic notion of an indicator ofinflammation.34 From a practical standpoint, it remains to be seenwhether EBT could be used for personalized monitoring andpredicting an individual’s response to AIT with specific allergens.This approach would require a specifically tailored time-seriesdesign.

The authors acknowledge that the present pilot study had somelimitations related to the scope of the measurements and thetreatment duration. However, they believe their findings set thestage for a robust evaluation of a novel, practical, objective way ofassessing patients on AIT.

In conclusion, the present results provide evidence for the ef-ficacy of SLIT with 5-grass pollen extracts based on EBT, an objec-tive measurement that reportedly reflects the level of airwayinflammation. It further confirms the difficulty in “sifting out” theplacebo effect in studies based on the subjective self-reporting ofsymptoms.

Acknowledgments

The authors thank Mrs Antonia Kapanova for technical assistance.

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