A randomised placebo-controlled trial investigating efficacy and mechanisms of low-dose intradermal allergen immunotherapy in treatment of seasonal allergic rhinitis

Article history

The research reported in this issue of the journal was funded by the EME programme as project number 11/20/05. The contractual start date was in September 2012. The final report began editorial review in September 2015 and was accepted for publication in March 2016. The authors have been wholly responsible for all data collection, analysis and interpretation, and for writing up their work. The EME editors and production house have tried to ensure the accuracy of the authors’ report and would like to thank the reviewers for their constructive comments on the final report document. However, they do not accept liability for damages or losses arising from material published in this report.

Declared competing interests of authors

Stephen J Till reports personal fees and grants from ALK Abelló, and personal fees from Thermofisher Scientific, outside the submitted work. Mohamed H Shamji reports grants from BioTech Tools and Regeneron USA, outside the submitted work. David J Cousins reports grants from GlaxoSmithKline, Asthma UK, and the Medical Research Council, outside the submitted work. Stephen R Durham reports grants from ALK Abelló, grants and personal fees from Merck, grants from Regeneron USA, personal fees from Biomay Austria and personal fees from Circassia UK, outside the submitted work; in addition, Stephen R Durham has a patent pending. Emily Lam is a Health Technology Assessment Primary Care, Community and Preventive Interventions panel member.

Copyright statement

© Queen’s Printer and Controller of HMSO 2016. This work was produced by Slovick et al. under the terms of a commissioning contract issued by the Secretary of State for Health. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK.

Setting

This single-centre RCT was conducted in the Clinical Research Facility of the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy’s Hospital from September 2012. The final study visit was on 27 August 2014. The study was conducted in accordance with the principles of Good Medical Practice (GMP) for clinical trials, and approved by the National Research Ethics Service Committee (London–Harrow; 12/LO/0941), with oversight by King’s Health Partners Clinical Trial Office, together with an independent Trial Steering Committee and Data Monitoring and Ethics Committee. The clinical trial protocol was published20 and the statistical analysis plan finalised prior to randomisation. All participants provided written informed consent prior to participation.

Patient and public involvement

The recruitment campaign for this trial involved development of a dedicated advertising campaign and website (developed by Media with Impact Ltd, London, UK) (see Appendices 1 and 2). The website contained a number of online prescreening questions. With the assistance of Asthma UK, patient representatives reviewed the design and helped to ensure appropriate engagement with the target audience. Patient representatives also reviewed all advertisement materials, participant information sheets and consent forms. In response to this feedback, substantial changes were made to the branding of the trial website and advertising materials in particular, to ensure appropriate engagement with the target population. Patient representatives also reviewed materials prior to disseminating the results to study participants.

Primary objective

The primary objective was to determine if preseasonal low-dose intradermal grass pollen allergen immunotherapy [seven 2-weekly injections of 10 BU (33.3 SQ-U)] reduces symptoms and requirements for antiallergic drugs in seasonal allergic rhinitis during the 2013 grass pollen season compared with the control intervention (histamine only).

Secondary objectives

The secondary objectives were to:

1. determine if this intervention is associated with improvement in quality of life compared with the control intervention, as assessed during the 2013 grass pollen season

2. evaluate if this intervention is safe and well tolerated

3. investigate immunological changes in response to repeated intradermal allergen injections by examining humoral and cellular responses, both in peripheral blood and in tissue

4. explore if the intradermal late-phase response desensitisation effect is long-lived, that is, persists following cessation of intradermal injections.

Participants

Participants were identified via a recruitment campaign including advertisements in press, online and on public transport. Potential participants were invited to visit the trial website (www.pollenlite.co.uk) to answer seven prescreening questions before registering. Participants passing the prescreening on the trial website were contacted for further telephone screening, and, if considered potentially eligible, they were invited to attend the Clinical Research Facility at Guy’s Hospital for a formal screening visit. Full eligibility criteria were as follows.

Randomisation

Randomisation was performed by King’s Clinical Trials Unit (KCTU; UK Clinical Research Collaboration registered) at King’s College London (KCL) using a 24-hour, web-based randomisation system. Participants were randomised 1 : 1 to active intradermal immunotherapy or the control arm by the method of block randomisation with randomly varying block sizes, stratified by the size of skin test response to grass pollen at screening visit (the cut-off SPT size being the median value of all subjects to be randomised, ≥ 11 mm) and presence/absence of rhinitis symptoms outside the grass pollen season. Study medication was blinded. To minimise bias through accidental unblinding, as a result of common injection site reactions in the active trial arm, the control intervention consisted of a reducing dose of histamine to produce similar clinical effects as the active medication. All physicians, researchers, research nurses, outcome assessors and patients remained blinded to treatment allocation until the primary analysis was completed. The trial statistician was subgroup unblind only. Only the KCTU randomisation service provider and the manufacturing pharmacy had access to the blinding information for the study.

In August 2013, the KCTU also randomly selected participants to be approached to undergo skin biopsies. The first 40 participants who gave agreement then underwent biopsy after giving additional procedure-specific informed consent. Furthermore, in August 2013, the KCTU randomised all participants for a second time to one of three groups. These three groups then underwent repeat intradermal allergen injections, at 7, 10 or 13 months after the final intradermal immunotherapy or control injection, to assess if low-dose intradermal allergen immunotherapy was associated with prolonged suppression skin responses.

Trial medication

Each active intradermal allergen injection contained 10 BU (33.3 SQ-U) of P. pratense soluble grass pollen extract (Aquagen SQ^TM Timothy Grass Pollen extract, ALK Abelló, Reading, UK) contained in a 20-µl volume [i.e. 500 BU/ml (1666.7 SQ-U/ml)]. Individual vials for each participant and each visit were preprepared and prelabelled by Guy’s Hospital Pharmacy under GMP conditions. In brief, Aquagen SQ Timothy Grass Pollen extract was reconstituted in manufacturer-supplied diluent to the maximum recommended concentration [30,000 BU/ml (100,000 SQ-U/ml), i.e. 60 times the final working strength; shelf-life 6 months at 2–8 °C after reconstitution] and 0.15 ml was aliquoted into glass study vials. At each visit for intradermal injection the investigator added 8.85 ml of clinical grade 0.9% normal saline at ambient temperature to the vial corresponding with that participant’s visit, to achieve a 60-fold dilution. Then 20 µl was aspirated from this vial and administered directly. The allergen required dilution on the day of administration, as the recommended shelf-life of Aquagen SQ Timothy Grass Pollen extract at 500 BU/ml (1666.7 SQ-U/ml) is 14 days. The control drug was histamine only, administered at a concentration of 100 µg/ml for the first and second injections. To help preserve blinding, histamine concentrations were reduced to 30 µg/ml for the third and fourth injections, and 10 µg/ml for fifth, sixth and seventh injections. To match the grass pollen extract dilution and preserve blinding, histamine was also aliquoted into study vials at 60 times the final working strength in 0.15-ml volumes, for further dilution with 8.85 ml of clinical grade 0.9% normal saline immediately prior to injection. Active and control study medications appeared to be identical.

Following manufacture, vials were packed into individual dispensing packs and dispensed by Guy’s Hospital Pharmacy against a single study prescription for each study participant, covering all visits. At randomisation, an e-mail was sent from the randomisation system to the dispensing pharmacy. The blinded dispensed packs were thereafter stored in the Clinical Research Facility in temperature-monitored fridges, in a secure environment. Study drug accountability was assessed and documented by Guy’s Hospital Pharmacy. Study vials that had been reconstituted in saline for injection were stored separately at room temperature after use for return to pharmacy for drug accountability to be assessed.

Intervention

A series of seven intradermal active or control histamine injections was administered 2-weekly into the forearm before the 2013 grass pollen season (Figure 1). The first injection for each participant was administered between 18 February and 1 March 2013, with the seventh and final injection given between 13 May and 24 May 2013. The injection site was alternated between left and right arms at each visit. Intradermal injections were administered in a 20-µl volume using a 29 gauge insulin syringe (BD Micro-Fine^TM, Becton Dickinson, Oxford, UK). In the event of an injection being administered too deeply (i.e. into subcutaneous tissue) to elicit an immediate injection ‘bleb’ and subsequent characteristic wheal, the injection was repeated 1 cm from the original site. Most participants were not taking antihistamines at the time of intradermal injections, as these were performed before the grass pollen season. Nevertheless, all of the participants were asked to avoid taking antihistamines for 5 days before receiving an intradermal injection, so that the presence of a wheal could be confirmed. Following an intradermal injection, participants were able to take an antihistamine to reduce the local itching and swelling if they so wished.

FIGURE 1.

Study design.

Assessment of efficacy

The primary end point was the area under curve (AUC) of the combined symptom and medication score (CSMS) during the grass pollen season period spanning 13 May to 31 August 2013 (111 days), the clinical end point recommended by World Allergy Organization (WAO) guidelines for clinical trials of immunotherapy for allergic rhinitis. 21 Participants were provided with daily diary cards (see Appendix 3) to record symptoms in the nose (sneezing, blockage and running), eyes (itching, redness, tears and swelling), mouth and throat (itching and dryness) and chest (breathlessness, cough, wheezing and tightness), on a scale of 0–3 (with a score of ‘0’ indicating no symptoms and ‘1’, ‘2’ and ‘3’ indicating mild, moderate and severe symptoms, respectively). Daily rescue medication was scored as follows: desloratadine (Merck Sharp and Dohme Ltd., Moddesdon, UK), 5 mg, up to one tablet daily (6 points per day); olopatadine eye drops, 1 mg/ml, up to one drop per eye twice daily (1.5 points per drop, up to 6 points per day); fluticasone proprionate nasal spray, 50 µg per spray, up to two sprays per nostril once daily (2 points per spray, up to 8 points per day); and prednisolone, 5 mg per tablet, up to six tablets per day (2 points per tablet, up to 12 points per day). Symptom and medication scores were expressed as AUC for the entire grass pollen season. As maximum scores for symptoms (39) and medications (32) were different in magnitude, these parameters were normalised as per WAO guidelines. 21

Secondary clinical end points were:

1. symptom scores (AUC) over entire pollen season

2. medication scores (AUC) over entire pollen season.

3. Mini Rhinitis Quality of Life Questionnaire (Mini-RQLQ) scores22 (overall score and domain scores), recorded three times during the pollen season (12 June, 26 June and 10 July) and once after the season in September 2013.

4. Health-related quality of life: evaluated using the European Quality of Life-5 Dimensions, 5-levels (EQ-5D-5L) questionnaire23 three times during the pollen season (12 June, 26 June and 10 July) and once after the season in September 2013.

5. Visual analogue scales (VASs) for nasal and eye symptoms (see Appendix 4). These were recorded every 2 weeks during the pollen season and AUC values calculated.

6. Global evaluation scores (see Appendix 5).

7. The number of primary care [i.e. general practitioner (GP)] visits for hay fever during summer 2013.

8. CSMSs during the peak of the 2013 grass pollen season.

9. Number of medication-free days covering the grass pollen season period of 13 May to 31 August 2013.

10. Number of symptom-free days covering the grass pollen season period of 13 May to 31 August 2013.

11. Individual symptoms scores (AUC) for each organ: nose, mouth, eyes and lungs.

12. Total number of days during which prednisolone was used between 13 May and 31 August 2013.

13. Frequency of adverse events (AEs).

The peak of grass pollen season was prospectively defined as starting on the first three consecutive days between 13 May and 31 August 2013, when grass pollen counts in central London were ≥ 30 grains/cm³, using counts supplied by the UK Meteorological Office. The end of the peak season was defined as the first of 3 consecutive days when grass pollen counts were < 30 grains/cm³.

Data management

Data were managed using the regulatory compliant [GCP (Good Clinical Practice), 21CRF11, EC Clinical Trial Directive] InferMed MACRO database system (MACRO 4, Elsevier, Amsterdam, the Netherlands). An electronic case report form (eCRF) was created in collaboration with the trial statisticians and the chief investigator, and maintained by the KCTU. Data were hosted on a dedicated secure server within KCL, and all source data were entered into the eCRF by authorised staff with a full audit trail. Trial data may be obtained from the corresponding author on request.

Safety

Adverse events and side effects were recorded in the eCRF after randomisation and then throughout the study, regardless of their severity or relation to study participation. As a precaution against systemic allergic reactions, all participants were observed after the first intradermal injection for 1 hour and, if there was no systemic reaction, for 30 minutes after subsequent injections. In the event of a participant experiencing a grade 1 reaction, the clinical observation period for that individual was maintained at 1 hour after subsequent injections.

The following AEs were anticipated and not reported:

1. symptoms attributable to aeroallergen exposure: that is, nasal blockage, rhinorrhoea, itching or sneezing; itching, watering, redness or swelling of eyes; itching or dryness of mouth/throat; breathless, cough, wheeze and chest tightness

2. transient discomfort from intradermal injections

3. appearance of an itchy oedematous wheal, with surrounding erythema, after intradermal injection

4. appearance of swelling (oedema) within hours of intradermal injection

5. temporary discomfort, bleeding, bruising, swelling at the needle site following venesection

6. mild localised itching arising from skin prick testing during screening.

Withdrawal criteria and stopping rules

The prespecified criteria for discontinuation of the study therapy (active or control) were as follows.

1. Inability or failure to attend for intervention within 3 weeks of previous administration.

2. Inability or failure to receive seven or eight injections within the dates specified.

3. Two grade 2 systemic reactions, or a single systemic reaction of grade 3 or above after administration of study therapy. Systemic reactions were graded according to the WAO criteria:

   • Grade 1 Symptoms of 1 organ system (cutaneous, upper respiratory tract, conjunctival, gastrointestinal, other).

   • Grade 2 Symptoms of more than one organ system present or asthma symptoms/signs [cough, wheezing, shortness of breath but, < 40% drop in peak expiratory flow (PEF) or FEV₁].

   • Grade 3 Asthma symptoms/signs (with ≥ 40% drop in PEF or FEV₁), upper respiratory tract (laryngeal, uvula, tongue) oedema with or without stridor.

   • Grade 4 Respiratory failure or hypotension with or without loss of consciousness.

4. An AE that, in the judgement of the principal investigator or the medical monitor, presented an unacceptable consequence or risk to the participant.

5. An illness or infection not associated with the condition under study and which required treatment that was not consistent with protocol requirements or if a participant developed an intercurrent illness that, in the judgement of the principal investigator, in any way justified discontinuation.

6. An inability or unwillingness to comply with the study protocol, with the protocol deviations being sufficient to jeopardise the participant’s well-being or the integrity of the study.

7. Pregnancy occurring during study participation.

Predefined study-stopping rules included the occurrence of five grade 3 reactions or a single grade 4 reaction.

Concomitant medications

Rescue medications were provided to participants before and throughout the pollen season. These included: desloratadine (5 mg, up to one tablet daily), olopatadine eye drops (1.0 mg/ml, up to one drop per eye twice daily), fluticasone propionate nasal spray (50 µg per spray, up to two sprays per nostril once daily) and prednisolone (for use at 30 mg per day for up to 5 days). Participants were asked to use only these medications to treat their hay fever symptoms on an ‘as required’ basis. However, participants who were not experiencing hay fever symptoms were encouraged to try not to use these medications. Participants were asked to use only these medications. A short course of prednisolone was made available for severe symptoms, although participants were instructed to contact a trial doctor prior to starting this treatment. Concurrent treatment with beta-blockers, calcium channel blockers, tricyclic antidepressant drugs, monoamine oxidase inhibitors or anti-IgE monoclonal antibody (mAb) were not permitted.

Measurement of skin early- and late-phase responses

All participants underwent intradermal skin challenge testing 4 months after the final intradermal allergen immunotherapy or control injection (September 2013). Participants were then randomised to undergo a repeat follow-up test at either 7, 10 or 13 months later to assess persistence of late-response suppression by comparing late-phase response sizes in those who had received active intradermal immunotherapy with those who had received the control intervention. The procedure for the intradermal skin challenge testing and the dose of allergen used were identical to that for an active intradermal allergen immunotherapy injection. In brief, grass pollen extract (10 BU, equivalent to 33.3 SQ-U, of P. pratense Aquagen ALK Abelló) in a 20-µl volume of allergen diluent was injected intradermally into the extensor aspect of each forearm. A negative control injection of 20 µl of diluent was injected into the contralateral forearm. Although the trial was not unblinded at this stage, these intradermal injections were performed open label. Participants were asked to refrain from taking antihistamines or oral steroids for a minimum of 5 days and 2 weeks beforehand, respectively. Early phase responses were measured 15 minutes after the intradermal injection. The wheal outline was traced and transferred into the patient record. Late-phase responses were measured after 24 hours by palpating the outline of oedema. The areas of the late response was also traced and transferred to the patient record. A single clinician performed all measurements under double-blind conditions. The early- and late-phase response areas were calculated from scaled scanned images of the tracings with NIS Elements v4.2 software (Nikon Instruments, Surrey, UK). Early- and late-phase response areas were then compared in the intradermal immunotherapy and control arms at each time point.

Skin biopsy

Forty participants (20 in each trial arm) were randomised to undergo 3-mm skin punch biopsies immediately after measurement of late-phase responses (i.e. 24 hours after challenge), 4 months after the final treatment injection, in September 2013. Biopsies were collected from both allergen-challenged and diluent control sites. Local anaesthesia was achieved with 10 mg/ml of lidocaine hydrochloride with 5 µg/ml of adrenaline (1 in 200,000). In the first 20 subjects, biopsies were divided with a scalpel into two pieces and one half piece was fixed in 4% paraformaldehyde (Sigma-Aldrich, Poole, UK) for 2 hours. In the rest of the subjects, entire biopsies were processed for immunohistochemistry by fixation in 4% paraformaldehyde at room temperature for 4 hours. After washing twice in 15% sucrose, biopsies were mounted in Optimal Cutting Temperature compound (OCT) embedding medium (Bayer UK Ltd, Basingstoke, UK) and stored at –80 °C pending analysis. The remaining unfixed half-biopsy pieces were cultured directly for T-cell analysis.

Analysis of T cells cultured from skin biopsies

Skin biopsy tissue was finely dissected and suspended in complete medium [Roswell Park Memorial Institute Medium, Sigma-Aldrich^® (RPMI) supplemented with 10% foetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and l-glutamine (2 mM); all Life Technologies, Warrington, UK]. Tissue was then cultured at 37 °C in a humidified atmosphere containing 5% carbon dioxide in the presence of interleukin 2 (IL-2; 50 U/ml). After 3–4 days, cells were passed through a 0.2-µm cell strainer to obtain single cell suspensions, and restimulated with immobilised anti-cluster of differentiation 3 (CD3)/cluster of differentiation 28 antibodies for a further 3 days, followed by expansion for 4 days in the presence of IL-2. Expanded T cells were stained with the viability dye eFluor^®780 (eBioscience, Vienna, Austria) prior to surface staining with anti-cluster of differentiation 4 (CD4) PerCP-Cy5.5 (BioLegend, London, UK), anti-cluster of differentiation 8 BV510 (BD Biosciences, Oxford, UK), anti-CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) PE (BioLegend), anti-CXCR3 [chemokine (C-X-C Motif) receptor 3] BV421 (BioLegend), anti-chemokine receptor 6 (CCR6) PE-Cy7 (BD Biosciences) and anti-interleukin 25 receptor AF647 (kind gift of Dr Andrew McKenzie). Samples were resuspended for flow cytometric analysis (FACSCalibur™, BD Biosciences). Data were analysed using FlowJo^TM v7.6 software (Tree Star Inc., Ashland, OR, USA). For microarray studies, cells were activated for 4 hours with ionomycin (0.5 µg/ml) and phorbol 12-myristate 13-acetate (5 ng/ml) (both Sigma-Aldrich). Ribonucleic acid (RNA) was isolated from cell pellets using the miRNeasy Mini Kit and RNeasy MinElute Cleanup Kit (both Qiagen, Manchester, UK) in accordance with the manufacturer’s instructions. Complementary deoxyribonucleic acid (cDNA) synthesis and amplification were performed with the Ovation PicoSL WTA Systems V2 kit (NuGEN, Leek, the Netherlands) as per the manufacturer’s instructions. Purity and yield was then analysed using the Bioanalyzer Platform (Agilent, Stockport, UK) and NanoDrop 2000 spectrophotometer (Thermo Scientific, Loughborough, UK), respectively, before amplified cDNA was biotin-labelled with the NuGEN Encore BiotinIL Module according to the manufacturer’s instructions. Biotin-labelled cDNA was hybridised to an Illumina HumanHT-12 v4 Expression BeadChip (Illumina, Saffron Walden, UK) before scanning with the iScan System (Illumina) utilising GenomeStudio software. Data analysis was performed with the Partek Genomics Suite™ software (Partek Inc., Chesterfield, MO, USA).

Immunohistochemistry

Immunohistochemical staining of skin biopsies was performed using the modified alkaline phosphatase anti-alkaline phosphatase (APAAP) method to stain for eosinophils, neutrophils, CD4⁺ T cells and CD3-positive (CD3⁺) T cells. 24,25 In brief, 8- to 10-µm thickness tissue sections were air dried overnight on poly-l-lysine-coated slides. For immunostaining, slides were incubated at room temperature in a humidified chamber with the primary mouse mAb [neutrophil elastase (Dako, Ely, UK); eosinophil major basic protein (Abcam, Cambridge, UK); CD3 and CD4, both Dako] suspended in 5% human serum/phosphate-buffered saline (PBS) for predetermined optimised incubation times. Sections were then washed in PBS and incubated with rabbit anti-mouse immunoglobulin (Dako) for 30 minutes then washed again. Slides were then incubated with a third layer of soluble complexes of alkaline phosphatase (AP) and mouse anti-APAAP (Serotec, Kidlington, UK) for 30 minutes, washed and developed with Fast Red (Sigma-Aldrich) for a further 20 minutes. Sections were washed extensively in PBS before counterstaining with Harris haematoxylin (BDH, Poole, UK) and mounting in glycerol gel. For negative controls, each primary antibody was substituted with the appropriate isotype-matched irrelevant mAb. Slides were counted blind in random order by two observers. Allergen and diluent biopsy sections were evaluated from each subject. The total number of positive cells was expressed as the number of cells per square millimetre of biopsy. Interobserver variability was 7%, assessed on repeat counts of 19 slides. The difference between the two counts was plotted against the mean of the two counts; all but one of the differences fell within two standard deviations (SDs) of the mean difference, indicating satisfactory agreement between observers.

Serum antibody measurements

Sera were analysed for concentrations of pre- and post-treatment P. pratense-specific IgG, IgG4 and IgE, and IgE specific to the major allergens Phl p 5 and Phl p 1 using a commercial assay system (ImmunoCAP™, ThermoFisher Scientific, Horsham, UK) in accordance with the manufacturer’s instructions.

Basophil activation tests

Basophil activation tests were performed in 92 participants following administration of the final intradermal allergen immunotherapy or control injection (May 2013). Whole blood was collected and tested within 2 hours of sampling under blinded conditions by a single investigator. Heparinised whole blood was immunostained with anti-human CD3 PE-Cy7 (BD Biosciences), CD294 PE (Miltenyi Biotec, Woking, UK), CD203c PerCP-Cy5.5 (BioLegend), CD303 APC (Miltenyi Biotec), CD107a Brilliant Violet 421 (BioLegend), CD63 FITC (BioLegend) and isotype controls. Basophils were then stimulated with anti-human IgE (1000 ng/ml, positive control; Abcam) or P. Pratense extract (ALK Abelló) at 10 ng/ml and 100 ng/ml for 15 minutes at 37 °C. Samples were then lysed (BD FACS Lysing Solution, BD Biosciences), washed and resuspended (CellFIX™, BD Biosciences) for flow cytometric analysis (FACSCalibur™, BD Biosciences). Data were analysed using FlowJo^TM v7.6 software (Tree Star), gating on CD3^–CD303^–CD294⁺ basophils. Basophil activation was expression as the percentage of CD63⁺, CD203c⁺ or CD107a⁺ basophils of the entire basophil population, and compared between the two groups.

Statistical analysis

Sample size calculations for the primary outcome (CSMS) were performed, based on raw data from a previous clinical trial of subcutaneous grass pollen immunotherapy. 26 The power calculation was conservatively based on the detection of a clinical effect size of 80% of that reported in that trial. Using this method and a two-sided non-parametric test based on a Monte Carlo approach, group sample sizes of 35 and 35 achieved 90% power to detect such a difference in AUC of the CSMSs at a significance level of 0.05. To make allowance for the unknown distribution of the primary outcome, and based on the lower bound for the asymptotic relative efficiency of the Mann–Whitney U-test, the sample size was increased by a further 15% to 40 in each arm. Further accounting for a post-randomisation dropout rate of up to 10%, consistent with previous trials of grass pollen immunotherapy, a total sample size of 90 (45 each arm) was estimated as required.

The statistical analysis plan was finalised and agreed before any analysis was undertaken (see Appendix 6). Statistical analyses were performed on an intention-to-treat (ITT) basis, with data from all participants who could be assessed for the primary outcome. Summary measures for the baseline characteristics of each group were calculated as mean and SD for continuous (approximate) normally distributed variables, medians and interquartile ranges (IQRs) for non-normally distributed variables, and frequencies and percentages for categorical variables. The AUC of the CSMSs was plotted against time as a summary measure of the primary outcome. The primary efficacy analysis, that is, the difference between the two arms in AUC of the CSMSs, was analysed on randomised patients using a stratified Mann–Whitney U-test (van Elteren test), adjusted for the baseline stratification factors of size of the skin test to grass pollen, and presence or absence of rhinitis symptoms outside the grass pollen season. Median differences between the groups were calculated using the stratified Hodges–Lehmann method. Similar analyses were conducted for symptom scores, medication scores, symptoms in different organs and VAS scores. Linear mixed models were used to evaluate Mini-RQLQ and EQ-5D-5L scores in order to isolate the effect of the intervention on each arm after adjusting for stratification factors. Differences between the groups were reported with their 95% confidence intervals (CIs). All mechanistic between-group comparisons were performed by Mann–Whitney U-test, with the exception of serology and immunohistochemistry comparisons, which were analysed by analysis of covariance (ANCOVA). Comparisons of serology between pre and post treatment, and skin biopsy immunohistochemistry between diluent control and allergen challenge were made by Wilcoxon signed-rank test.

The primary outcome and secondary outcomes are reported in the ITT population without imputation of missing data. However, a sensitivity analysis was also performed, with missing data imputed for the primary outcome and secondary outcomes in the ITT population. A multiple imputation technique was applied, whereby missing data on a particular date were substituted with the mean CSMS on that date in the corresponding trial arm. Further sensitivity analyses were undertaken for the primary outcome and secondary outcomes in the predefined per-protocol population. Participants who were on holiday outside continental Europe during the daily collection period were considered as ‘missing data’ for the days concerned, in accordance with the Trial Steering Committee and statistical analysis plan. When > 50% of the data were missing, participants were excluded from the per-protocol analysis.

The principal software package was SAS/STAT^® version 9.2 (SAS Institute Inc., Cary, NC, USA), with verification of results from syntax for selected analyses analysed in Stata^® version 12.1 (StataCorp LP, College Station, TX, USA).

Study population

From 1660 people who completed initial online prescreening, 150 potential participants attended the Clinical Research Facility for full screening. Of these, 93 were enrolled and randomised to receive intradermal allergen or histamine control injections between 18 February and 1 March 2013 (Figure 2). Study arms were well balanced for baseline characteristics (Table 1). All 46 participants assigned to intradermal allergen immunotherapy completed the seven-injection treatment course, although one participant deviated from the administration schedule by 1 day for one injection. Of the 47 participants who were assigned to control injections, one did not complete the treatment course, withdrawing after the second injection because of work commitments, and another participant deviated from the administration schedule by 4 days because of an unrelated upper respiratory tract infection that necessitated postponement of the injection. There was a high rate of diary card data collection for the primary outcome: 99% of participants supplied > 50% of data for all days; 96% of participants supplied > 75% of daily data; and 94% of participants supplied > 90% of daily data. One patient holidayed outside continental Europe for 52% of the data collection period, so was excluded from the per-protocol analysis, in accordance with the predefined statistical analysis plan. Five participants, all in the control arm, significantly deviated from use of the rescue medications that were specified in the trial protocol according to criteria specified prior to unblinding. Participants were unable to identify if they had received active intradermal allergen treatment or histamine control (Table 2).

FIGURE 2.

Consolidated Standards of Reporting Trials diagram. All randomised participants were included in the ITT analysis. Only participants who adequately adhered to treatment and rescue medications were included in the per-protocol analysis.

Clinical outcomes

All 93 randomised participants were evaluated for the primary outcome and were included in the ITT analysis. The CSMS showed a clear correlation with daily pollen counts in London (Figure 3), which peaked at levels in the above-average range. However, intradermal immunotherapy did not significantly affect the primary end point, that is, the CSMS over the whole grass pollen season (difference in median AUC = 14; 95% CI –172.5 to 215.1; p = 0.80) (see Figure 3 and Table 3). Furthermore, significant differences were not seen between the trial arms in the secondary end points of overall symptom scores or rescue medication use (p = 0.44) during the whole season, or the CSMSs during peak season (12 June to 26 July 2013) (p = 0.99; see Table 3).

FIGURE 3.

Primary outcome and daily symptom and daily medication scores in the primary intention-to-treat analysis. (a) Mean daily combined symptom and medication; and (b) median daily symptom scores (sum of scores for nose, eyes, lungs, mouth according to treatment arm. The p-values are based on (c) mean medication scores; and (d) daily grass pollen counts in central London over the 2013 grass pollen season. AUC values for each participant were compared Mann–Whitney U-tests. Broken vertical lines indicate the beginning and end of the peak pollen season (12 June to 26 July 2013).

Among other prespecified secondary end points, allergic rhinitis symptoms, measured by daily nasal symptom scores, were significantly higher in the intradermal allergen immunotherapy group than in the histamine control group, with a difference in median AUC of 35 (95% CI 4.0 to 67.5; p = 0.03) (see Table 3 and Figure 4). Furthermore, there was a trend for rhinitis symptoms measured by VAS to be higher in the arm that received intradermal immunotherapy group, with a difference in median AUC of 53 (95% CI –11.6 to 125.2; p = 0.05) (see Table 3 and Figure 4). No significant differences were seen between groups in daily eye or lung symptoms (see Table 3), although there was a trend for mouth symptoms to be higher in the intradermal allergen group (difference in median AUC 10.0; 95% CI –3.8 to 24; p = 0.05). No significant group differences were observed in eye symptoms measured by VASs, Mini-RQLQ scores, EQ-5D-5L scores, global evaluation of symptoms scores, numbers of symptom-free or medication-free days or number of days during which prednisolone was used as rescue medication (see Table 3). Analysis of the ITT population after imputation of missing data values gave results that were consistent with the main ITT analysis (see Appendix 7). The per-protocol analysis included 45 participants who received intradermal allergen immunotherapy and 39 who received the histamine control treatment (see Appendix 8). In this population, daily individual nasal (p = 0.05) and mouth symptoms (p = 0.02) were also higher in the actively treated group, with a trend for worse lung symptoms (p = 0.05). Participants in the intradermal allergen group in this population also had significantly worse nasal symptoms measured by VAS (p = 0.01) and recorded fewer symptom-free days than subjects in the control group (p = 0.04).

FIGURE 4.

Nasal symptoms. (a) Mean daily nasal symptom scores (sum of scores for sneezing, blockage and running); and (b) mean nasal symptoms measured by VAS (total of blockage, running, itching and sneezing). AUC values for each participant were compared according to treatment arm. The p-values are based on Mann–Whitney U-tests.

Given the unexpected observation that allergic rhinitis nasal symptom scores were higher in participants who had received intradermal allergen immunotherapy, a post hoc analysis was performed to compare the daily data for each individual allergic symptom in the two trial arms (see Appendix 9). Sneezing (p = 0.01) and cough scores (p = 0.03) were both significantly higher in the intradermal immunotherapy group, with non-significant trends for greater chest tightness (p = 0.08) and mouth itching (p = 0.06). In contrast, eye swelling was lower in the intradermal immunotherapy group (p = 0.03). Further post hoc analysis of individual nasal symptoms measured by VAS also revealed higher scores after intradermal immunotherapy for running (p = 0.006), sneezing (p = 0.006) and itching (p = 0.003) (see Appendix 10).

Safety

There was a low rate of AEs that were related to treatment (Table 4). There were three serious AEs, although all were unrelated to treatment. One participant in the intradermal allergen immunotherapy group was hospitalised for severe tonsillitis. One control arm participant was admitted for overnight polysomnography, and another control participant required treatment to remove an infected dental plate. There were no deaths during the study. Three participants in the intradermal immunotherapy group and six in the control group were recorded with treatment-related AEs – all mild grade 1 systematic reactions. These reactions manifested as generalised pruritus without wheals, except for one intradermal allergen participant who developed erythema, which tracked from the injection site in a lymphatic distribution (IgE-mediated lymphangitis) approximately 20 minutes after every intradermal injection.

Immunological end points

Serum immunoglobulins specific for whole P. pratense (Timothy grass) and major Timothy grass allergens Phl p 1 and Phl p 5 were compared before (between October 2012 and January 2013) and after (May 2013) intradermal allergen or control injection therapy. In the histamine control arm, there was a typical small seasonal decline in allergen-specific IgE antibodies (all p < 0.001; Figure 5). This seasonal decline in IgE was, however, significantly less in the intradermal allergen immunotherapy group than the control group (all p = 0.001), indicating that intradermal allergen treatment stimulated allergen-specific IgE production. A treatment effect was also seen on P. pratense-specific IgG titres, which fell in the control but not the intradermal allergen group over the same period (p = 0.03; Figure 6), although no effect was seen on IgG4 responses.

FIGURE 5.

Immunological outcomes. Levels of (a) P. pratense-specific IgE; (b) Phl p5-specific IgE; and (c) Phl p1-specific IgE before and after completion of seven intradermal allergen or histamine control injections. Solid bars represent median values. The p-values for pre- and post-treatment serology comparisons are based on the Wilcoxon signed-rank test. The p-values for between-group IgE comparisons are based on ANCOVA.

FIGURE 6.

Immunological outcomes. Levels of (a) P. pratense-specific IgG; and (b) P. pratense-specific IgG4 before and after completion of seven intradermal allergen or histamine control injections. The p-values for pre- and post-treatment serology comparisons are based on the Wilcoxon signed-rank test. The p-values for between-group IgG comparisons are based on ANCOVA.

For surface phenotype analysis, CD4⁺ T cells were successfully expanded from 19 of 20 skin biopsies (10 from the intradermal immunotherapy group and nine from the control group) collected 24 hours after an intradermal grass pollen challenge, at the end of the 2013 grass pollen season. Cutaneous CD4⁺ T cells derived from grass pollen challenged sites showed higher expression of Th2 surface marker CRTH2 in the intradermal allergen immunotherapy group (median 13.4%, IQR 6.3–25.4) than the control group (median 6.3%, IQR 1.9–7.6; p = 0.04), whereas expression of the T helper type 1 cell (Th1) marker CXCR3 was lower in the intradermal allergen immunotherapy group [median 33.5 (IQR 24.7–47.3) vs. median 56 (IQR 45.8–63.8); p = 0.01] (Figure 7). No differences were seen in the expression of T helper type 17 cell marker CCR6 or the interleukin 25 receptor (not shown). Insufficient T cells could be expanded from diluent-challenged skin biopsies for analysis. Microarray transcriptional profiling was performed on cultured T cells that were derived from 15 allergen-challenged skin biopsies (seven intradermal allergen treatment and eight control arm subjects). Only 14 genes were significantly overexpressed by skin T cells in the intradermal allergen immunotherapy group [defined as > 1.5-fold higher expression than in control group and p < 0.05 using a three-way analysis of variance (ANOVA) model] including the Th2 cytokine interleukin 5 (IL-5) (p = 0.03) (Table 5; Microarray Gene Expression Omnibus accession number GSE72324).

FIGURE 7.

Expression of (a) CRTH2 (Th2 marker); (b) CXCR3 (Th1 marker); and (c) ratio of CRTH2 to CXCR3 expression on CD4⁺ cells expanded from skin biopsies (24 hours post-skin challenge). The p-values are based on Mann–Whitney U-tests.

Immunohistochemistry performed on the entire 40 diluent- and 40 allergen-challenged skin biopsies (20 intradermal allergen treatment and 20 control arm subjects) showed grass pollen-induced recruitment of eosinophils, neutrophils, CD3⁺ T cells and CD4⁺ T cells, but no significant treatment effect (Figure 8). Furthermore, no significant treatment effect was seen on surface expression of peripheral blood basophil activation markers (Figure 9).

FIGURE 8.

Immunohistochemistry analysis of skin biopsies. Comparison of allergen-induced inflammatory cell numbers in skin biopsies from intradermal immunotherapy and control arm participants. Data shown indicate numbers of (a) neutrophils; (b) eosinophils; (c) CD3 ⁺ T cells; and (d) CD4⁺ T cells in skin biopsies taken after diluent and P. pratense intradermal skin challenges in September 2013. Cells were stained using the APAAP method. Solid bars represent median values. The p-values comparing diluent- and allergen-challenged biopsies are based on the Wilcoxon signed-rank test. The p-values for between-group comparisons are based on ANCOVA.

FIGURE 9.

Basophil activation tests. Percentage of basophils staining positive for activation markers. (a) CD63; (b) CD107a; and (c) CD203c. Whole blood was stimulated under the conditions described. The p-values are based on Mann–Whitney U-tests.

Intradermal skin challenge responses

Early- (15 minutes) and late-phase (24 hours) skin responses could be measured in 86 participants 4 months after the final treatment injection in September 2013, and the measurements were repeated at either 7, 10 or 13 months (Figure 10a). The size of late-phase responses in the control group was consistent with that previously reported under the same conditions9 (shown for comparison in Figure 10). Late-phase responses remained significantly suppressed in the group that had received intradermal immunotherapy at both 4 and 7 months (both p = 0.03), although the degree of suppression at these time points was clearly less than that which we previously reported immediately after completion of six injections. Late responses were not suppressed at 10 or 13 months. These data suggest that the suppressive effect of intradermal immunotherapy on late-phase responses was wearing off within 4 months. In contrast with the late-phase response, no significant differences between treatment arms were seen in early-phase responses at 4-, 7-, 10- or 13-month time points (see Figure 10b).

FIGURE 10.

Late-phase skin responses. Areas of cutaneous (a) early-; and (b) late-phase responses (15 minutes and 24 hours after intradermal skin challenge, respectively), performed 4 months and either 7, 10 or 13 months post treatment (September 2013). Late response suppression is shown from our previous study (Rotiroti et al. 6) immediately after six 2-weekly intradermal injections. Solid bars represent median values. The p-values are based on Mann–Whitney U-tests.

Conclusions

The results of this study provide evidence that low-dose intradermal allergen injection immunotherapy is not clinically effective, even if it is able to suppress late-phase skin responses. Furthermore, we found evidence that this intervention resulted in immunological priming in certain assays in parallel with worsening of certain symptoms during the grass pollen season. These findings support the concept that dermal allergen exposure has the potential to exacerbate, rather than ameliorate, allergic responses. We conclude that novel immunotherapy strategies that promote dermal allergen exposure have the potential to be deleterious, even if local macroscopic responses appear to be suppressed by this approach.

Contributions of authors

Ms Anna Slovick (Clinical Fellow, KCL, and Ear, Nose and Throat trainee) was overall trial co-ordinator and participated in the set-up of the trial, recruitment, administration of intradermal injections and collection of clinical outcome data; performed mechanistic assays; and participated in preparation of the first draft of this manuscript.

Dr Abdel Douiri (Senior Lecturer in Medical Statistics, KCL) participated in the design of the trial, preparation of the manuscript and was the trial statistician.

Dr Rachel Muir (Research Matron) participated in the set-up of the trial, recruitment, administration of intradermal injections and collection of clinical outcome data.

Dr Andrea Guerra (Clinical Fellow, KCL) participated in the set-up of the trial, recruitment, administration of intradermal injections and collection of clinical outcome data, and performed mechanistic assays.

Mr Konstantinos Tsioulos (Clinical Fellow, KCL) participated in the set-up of the trial, recruitment, and administration of intradermal injections.

Ms Evie Haye (Research technician) performed mechanistic assays.

Dr Emily PS Lam (PhD student, degree now awarded) performed mechanistic assays.

Dr Ms Joanna Kelly (Strategic Data Management Lead, KCTU) participated in the design and set-up of the trial.

Professor Janet L Peacock (Professor of Medical Statistics, KCL) participated in the statistical analysis and data interpretation.

Dr Sun Ying (Reader in Allergy, KCL) participated in design, supervision and co-ordination of mechanistic assays.

Dr Mohamed H Shamji (Research Fellow, Imperial College London) participated in design, supervision and coordination of mechanistic assays.

Professor David J Cousins (Professor Respiratory Science, University of Leicester) supervised mechanistic assays.

Professor Stephen R Durham (Professor of Allergy & Respiratory Medicine, Imperial College London; Consultant Allergist, Royal Brompton & Harefield NHS Trust) participated in the study conception, design, interpretation of the results and manuscript preparation.

Dr Stephen J Till (Reader in Allergy, KCL; Consultant Allergist, Guy’s and St Thomas’ NHS Foundation Trust) was chief investigator of the PollenLITE trial and conceived of the study, participated in its design and co-ordination, and prepared the first draft of this manuscript with the assistance of AS.

Publications

Slovick A, Douiri A, Muir R, Guerra A, Tsioulos K, Hay E, et al. Intradermal grass pollen immunotherapy increases T_H2 and IgE responses and worsens respiratory allergic symptoms [published online ahead of print October 20 2016]. J Allergy Clin Immunol 2016.

Data sharing statement

All available data can be obtained by contacting the corresponding author.

Disclaimers

This report presents independent research. The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect those of the NHS, the NIHR, the MRC, NETSCC, the EME programme or the Department of Health. If there are verbatim quotations included in this publication the views and opinions expressed by the interviewees are those of the interviewees and do not necessarily reflect those of the authors, those of the NHS, the NIHR, NETSCC, the EME programme or the Department of Health.