Journal Pre-proof

Sputum microbiome profiles identify severe asthma phenotypes of relative stability at

12-18 months

Mahmoud I. Abdel-Aziz, MSc, Paul Brinkman, MSc, Susanne J.H. Vijverberg, PhD,
Anne H. Neerincx, PhD, John H. Riley, PhD, Stewart Bates, PhD, Simone Hashimoto,
MD, PhD, Nazanin Zounemat Kermani, MSc, Kian Fan Chung, MD, DSc, Ratko
Djukanovic, MD, PhD, Sven-Erik Dahlén, MD, PhD, Ian M. Adcock, PhD, Peter H.
Howarth, MD, PhD, Peter J. Sterk, MD, PhD, Aletta D. Kraneveld, PhD, Anke H.
Maitland-van der Zee, PharmD, PhD, on behalf of U-BIOPRED Study Group

PII: S0091-6749(20)30565-0
DOI: https://doi.org/10.1016/j.jaci.2020.04.018
Reference: YMAI 14518

To appear in: Journal of Allergy and Clinical Immunology

Received Date: 10 November 2019

Revised Date: 6 April 2020
Accepted Date: 9 April 2020

Please cite this article as: Abdel-Aziz MI, Brinkman P, Vijverberg SJH, Neerincx AH, Riley JH, Bates
S, Hashimoto S, Kermani NZ, Chung KF, Djukanovic R, Dahlén S-E, Adcock IM, Howarth PH, Sterk
PJ, Kraneveld AD, Maitland-van der Zee AH, on behalf of the U-BIOPRED Study Group, Sputum
microbiome profiles identify severe asthma phenotypes of relative stability at 12-18 months, Journal of
Allergy and Clinical Immunology (2020), doi: https://doi.org/10.1016/j.jaci.2020.04.018.

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Immunology.
 Sputum microbiome profiles identify severe asthma
phenotypes of relative stability at 12-18 months

Airway obstruction

Pathogenic bacteria
More severe
Severe adult asthma patients asthma

Neutrophils

Unsupervised Cluster 2
Macrophages
microbiome-driven
clustering
Baseline 12-18 month Cluster 1
follow-up

Less severe
asthma

Commensal bacteria

Sputum microbial 2 robust microbiome

signature assessment clusters with relative
stability after 12-18 months
 1 Sputum microbiome profiles identify severe asthma phenotypes of relative stability at 12-18
2 months

3 Mahmoud I. Abdel-Aziz MSc1,2, Paul Brinkman MSc1, Susanne J. H. Vijverberg PhD1, Anne H.
4 Neerincx PhD1, John H. Riley PhD3, Stewart Bates PhD3, Simone Hashimoto MD, PhD1,4, Nazanin
5 Zounemat Kermani MSc5, Kian Fan Chung MD, DSc6, Ratko Djukanovic MD, PhD7, Sven-Erik
6 Dahlén MD, PhD8, Ian M. Adcock PhD6, Peter H. Howarth MD, PhD7, Peter J. Sterk MD, PhD1,
7 Aletta D. Kraneveld PhD9,10, Anke H. Maitland-van der Zee PharmD, PhD1,4, on behalf of the U-
8 BIOPRED Study Group

9 1 Department of Respiratory Medicine, Amsterdam UMC, University of Amsterdam,

10 Amsterdam, The Netherlands.
11 2 Department of Clinical Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt.
12 3 Respiratory Therapeutic Unit, GlaxoSmithKline, Stockley Park, United Kingdom.
13 4 Department of Pediatric Respiratory Medicine, Emma Children's Hospital, Amsterdam UMC,
14 Amsterdam, The Netherlands.
15 5 Data Science Institute, Imperial College London, London, United Kingdom.
16 6 National Heart and Lung Institute, Imperial College London, and Royal Brompton and Harefield
17 NHS Trust, London, United Kingdom.
18 7 NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental Sciences
19 and Human Development and Health, University of Southampton, Southampton, United
20 Kingdom.
21 8 Centre for Allergy Research, Institute of Environmental Medicine, Karolinska Institutet,
22 Stockholm, Sweden.
23 9 Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science,
24 Utrecht University, Utrecht, the Netherlands.
25 10 Institute for Risk Assessment Sciences, Faculty of Veterinary Medicine, Utrecht University,
26 Utrecht, the Netherlands.
27
28
29 Corresponding author details:
30 Prof. Dr. A.H. Maitland-van der Zee
31 Department of Respiratory Medicine, Amsterdam UMC, University of Amsterdam, Meibergdreef
32 9, Amsterdam, the Netherlands.
33 e-mail: a.h.maitland@amsterdamumc.nl, Telephone: +31 (0) 20 5668137
34
35 This article has an online data supplement.
36
37 Word count of the abstract: 250
38 Word count of the manuscript: approximately 3,800 without abstract, tables, figures, discloses
39 and references.
40 Number of Figures: 23 (1 as a graphical abstract, 5 in the main body of the manuscript; 4 in color
41 and 17 as a supplementary information)
42 Number of Tables: 5 (3 in the main body of the manuscript and 2 as a supplementary
43 information)
44

45 Key messages:

46 • Using unbiased clustering based on sputum microbiome profiles we show that, severe
47 asthma can be stratified into two phenotypic clusters.
48 • These clusters significantly differed by age of asthma onset, patient residential locations,
49 smoking history, percentage of blood and/or sputum neutrophils and macrophages,
50 spirometry and used asthma medications.
51
52
53 Capsule summary

54 Two distinct sputum-microbiome driven clusters of severe asthmatics were revealed, exhibiting
55 relative stability after 12-18 months. An interplay between bacteria and innate immunity was
56 suggested, which may help to design urgently needed precision medicine approaches.
57
58 Keywords:

59 Sputum Microbiome; Metagenomics; Asthma Phenotypes; Unbiased Clusters; Follow-up;

60 Neutrophils; Macrophages; Lung Function;
61 Abbreviations used:

62 ACQ5: 5-item asthma control questionnaire

63 AQLQ: Asthma quality of life questionnaire
64 BMI: Body mass index
65 COPD: Chronic obstructive pulmonary disease
66 FDR: False discovery rate
67 FENO: Fraction of exhaled nitric oxide
68 FEV1: Forced expiratory volume in 1 second
69 Figure E: Electronic figure (supplementary figure)
70 FVC: Forced vital capacity
71 GINA: Global Initiative for Asthma
72 ICS: Inhaled corticosteroids
73 IMI: Innovative Medicines Initiative
74 IQR: Interquartile range
75 LABA: Long acting beta agonist
76 OCS: Oral corticosteroids.
77 PAM: Partition around the medoids
78 PCoA: Principal co-ordinate analysis
79 RI: Rand index
80 rRNA: Ribosomal ribonucleic acid
81 SABA: Short acting beta agonist
82 TDA: Topological data analysis
83 U-BIOPRED: Unbiased BIOmarkers in PREDiction of respiratory disease outcomes
84 WCSS: Total within-cluster sum of square
85
86
87 List of author contributions:
88
89 MIA has performed the analysis and drafted the manuscript. MIA, AHM, PB, ADK, IMA, FC and
90 PJS have contributed to the design of the analysis plan. All co-authors have contributed to the
91 acquisition of data, interpretation of the analysis, revision, critical appraisal and ensuring
92 accuracy and integrity of the analysis. All co-authors have provided final approval of the version
93 to be published. AHM is the corresponding author. In addition, the U-BIOPRED is a consortium
94 effort and we wish to acknowledge the help and expertise of the individuals and groups whose
95 names are mentioned in the attached U-BIOPRED study group list.
96
97
98
99
100 Funding:

101 U-BIOPRED has received funding from the Innovative Medicines Initiative (IMI) Joint
102 Undertaking under grant agreement no. 115010, resources of which are composed of financial
103 contributions from the European Union’s Seventh Framework Programme (FP7/2007–2013) and
104 European Federation of Pharmaceutical Industries and Associations (EFPIA) companies’ in-kind
105 contributions (www.imi.europa.eu). The salary of MIA was sponsored by the Egyptian
106 Government PhD Research Scholarships.

107 Conflict of interests:

108 MIA, PB, SJHV, AHN, SH, NZK, and AK have no conflicts of interest to disclose. JHR, SB and PH are
109 employees by and share-holders of GlaxoSmithKline. RD has received fees for lectures at
110 symposia organized by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as
111 member of advisory boards, and participation in a scientific discussion about asthma organized
112 by GlaxoSmithKline. RD is a co-founder and current consultant, and has shares in Synairgen, a
113 University of Southampton spin out company. SED reports personal fees from AstraZeneca,
114 GlaxoSmithKline, Merck & Co, Novartis, Regeneron, Sanofi & Teva , outside the submitted work.
115 IMA, FC and PJS have received grants from Innovative Medicines Initiative (IMI), during the
116 conduct of the study. AHM has been reimbursed for visiting the ATS by Chiesi, received a fee for
117 participating in advisory boards for Boehringer lngelheim and Astra Zeneca, and received
118 unrestricted research grants from GSK, Chiesi and Boehringer Ingelheim.
119 Abstract

120 Background

121 Asthma is a heterogeneous disease characterized by distinct phenotypes with associated

122 microbial dysbiosis.

123 Objectives

124 To identify severe asthma phenotypes based on sputum microbiome profiles and assess their

125 stability after 12-18 months. Furthermore, to evaluate clusters’ robustness after inclusion of an

126 independent mild-to-moderate asthmatics.

127 Methods

128 In this longitudinal multicenter cohort study, sputum samples were collected for microbiome

129 profiling from a subset of the U-BIOPRED adult patient cohort at baseline and after 12-18

130 months of follow-up. Unsupervised hierarchical clustering was performed using the Bray-Curtis

131 β-diversity measure of microbial profiles. For internal validation, partitioning around medoids,

132 consensus cluster distribution, bootstrapping and topological data analysis were applied.

133 Follow-up samples were studied to evaluate within-patient clustering stability in severe

134 asthmatics. Cluster robustness was evaluated by an independent mild-moderate asthma cohort.

135 Results

136 Data were available for 100 severe asthma subjects (median age: 55 yrs, 42% males). Two

137 microbiome-driven clusters were identified, characterized by differences in asthma onset,

1
138 smoking status, residential locations, percentage of blood and/or sputum neutrophils and

139 macrophages, lung spirometry, and concurrent asthma medications (all p-values <.05). Cluster 2

140 patients displayed a commensal-deficient bacterial profile which was associated with worse

141 asthma outcomes compared to cluster 1. Longitudinal clusters revealed high relative stability

142 after 12-18 months in the severe asthmatics. Further inclusion of 24 independent mild-to-

143 moderate asthmatics was consistent with the clustering assignments.

144 Conclusion

145 Unbiased microbiome-driven clustering revealed two distinct robust severe asthma phenotypes,

146 which exhibited relative overtime stability. This suggests that the sputum microbiome may

147 serve as a biomarker for better characterizing asthma phenotypes.

2
148 Introduction

149 Severe asthma patients represent approximately 5% of the total asthma population.1 Severe

150 asthma places substantial health and cost burdens on patients and healthcare communities.2 It

151 is a heterogeneous disease consisting of multiple phenotypes that show differences in clinical

152 characteristics, inflammatory biomarkers, pathophysiological processes and therapeutic

153 requirements. Better characterization of the severe asthma patient population should

154 eventually lead to more effective tailoring of therapeutic decisions to meet patients’ needs and

155 thus improve outcomes and reduce burdens (precision medicine).

156 Asthma phenotyping aims to classify the asthmatic population into subgroups based on clinical

157 characteristics and/or biological parameters,3 underpinned by different pathophysiological

158 mechanisms that drive these phenotypes. Omics technologies utilize high throughput advanced

159 analytical and computational tools to elucidate biological pathways and/or highlighting novel

160 biomarkers that can improve diagnosis and therapeutic decisions.4 Classifying cohorts of

161 asthmatics by omics methods can be done in a supervised or an unsupervised approach. The

162 latter can be considered as “unbiased” since it does not involve any priori assumptions and is,

163 therefore, the preferred option. Applying this principle, the Unbiased BIOmarkers in PREDiction

164 of respiratory disease outcomes (U-BIOPRED) project has published asthma phenotypes driven

165 by sputum transcriptomics,5 proteomics,6 or breathomics.7

166 Over the past decade several studies have investigated the airway microbial dysbiosis in asthma

167 patients8-12 with some focusing on airway microbiome profiles in asthmatics with different

168 inflammatory phenotypes.13-17 Inconsistencies in the reported results between the studies have

3
169 been observed which might hinder direct clinical applicability.18 In addition, most of the studies

170 conducted have used amplicon sequencing of bacterial ribosomal RNA (rRNA) which has a

171 limited ability to identify bacteria at species level, and therefore limits its clinical relevance.

172 Studying the induced sputum microbiome within the context of a large scale multicenter asthma

173 cohort study, such as the U-BIOPRED, using both 16s rRNA sequencing and metagenomics could

174 lead to more conclusive results. In this study, we hypothesized that the sputum microbiome,

175 through its host and environment interaction, can reveal distinct severe asthma phenotypes.

176 Specifically, we aimed to (1) identify severe asthma phenotypes through unsupervised unbiased

177 clustering of sputum microbiome profiles of severe asthmatics, (2) assess within-patient

178 longitudinal stability of the identified clinical clusters after 12-18 months and (3) evaluate the

179 robustness of the clinical clusters by subsequently analyzing the microbiome of patients from an

180 independent mild-to-moderate asthma cohort in the analysis.

4
181 Methods

182 Study design

183 The U-BIOPRED project is a multi-center prospective observational pan-European cohort study,

184 comprising 3 asthma sub-cohorts defined by standard clinical criteria: severe non-smoking

185 asthmatics (cohort A), severe previous or current smoking asthmatics (cohort B), mild-to-

186 moderate non-smoking asthmatics (cohort C), described in detail previously.19 All recruited

187 participants provided written informed consents and each study center obtained local medical

188 ethics committee approval. The study was registered under identifier NCT01976767 at

189 ClinicalTrials.gov.

190 The study involved 2 research visits: screening and baseline for the mild-moderate and severe

191 asthma patients and an additional longitudinal visit (12-18 months after baseline) for the severe

192 asthmatics.19 At the baseline and longitudinal visits, several questionnaires and biological

193 measurements were obtained from the recruited participants as described in detail elsewhere.19

194 Participants

195 One hundred severe and 24 mild-moderate U-BIOPRED adult asthmatics from 13 study centers

196 spanning 9 different European countries, provided induced sputum samples at baseline that

197 passed quality-control, with 46 severe asthmatics providing single additional follow-up samples

198 after 12-18 months (Figure E1) . Mild-to-moderate and severe asthmatics were defined

199 according to criteria of the Innovative Medicines Initiative and Global Initiative for Asthma

200 (GINA) guidelines.20, 21 They completed standard asthma control and quality of life

5
201 questionnaires,22, 23 underwent spirometry24 and were assessed for inflammatory biomarkers25,
26
202 and for atopy27 (for details see online supplement). All participants were considered as non-

203 smokers if they had not smoked for at least 12 past months and had <5 pack-year smoking

204 history.

205 Sputum induction, 16s rRNA amplicon sequencing and shotgun metagenomics processing

206 Sputum at baseline and longitudinal visits was induced by inhaling hypertonic (0.9% to 4.5%)

207 saline according to standardized protocols.26, 28 Induced sputum samples were prepared for

208 microbiome and metagenomics profiling, as described in details in the online supplement and

209 elsewhere.29

210 Data Analysis

211 The general data analysis workflow is shown in Figure E2 and is further described below:

212 Clustering protocol

213 Cluster benchmarking was based on the analysis performed by Brinkman et al.7 with

214 modifications to suit the microbiome data. To assess the patient variability in the microbiome

215 profiles, the Bray-Curtis β-diversity dissimilarity measure was computed separately on

216 numerical count data of 16s rRNA microbiome operational taxonomic units (OTUs) and

217 metagenomics species. Clustering was then performed using hierarchical ward2 agglomerative

218 clustering on the Bray-Curtis measure.30 The optimum number of clusters was determined

219 based on several indices such as; optimum average silhouettes width,31 total within-cluster sum

220 of square (WCSS),32 and Calinski-Harabasz33 indices. Cluster assignment of the patients was

6
221 internally validated by using partition around the medoids (PAM).34 Agreement in the clustering

222 of patients’ assignments between hierarchical Ward, and PAM clustering was quantified by

223 means of Pearson Chi-Square test or Fisher’s Exact test and Rand index.35 This was also

224 performed to assess if the clustering would differ in 16s rRNA sequencing as compared to

225 metagenomics approach. Clustering was further validated visually using topological data

226 analysis (TDA) with the Ayasdi workbench (version 7.15.0; Ayasdi, Menlo Park, Calif) as reported

227 previously.6, 7 In TDA, visual depiction of the shape of patients’ metagenomics data was

228 performed, where nodes represent patients’ points that are highly similar and connected by

229 edges (lines) to nodes that have data in common. The neighborhood lenses (filter functions) 1

230 and 2 were used that generate a graph of patients’ metagenomics data into two-dimensional

231 space by k-nearest neighbors algorithm. The TDA graph, thus, shows connections of each

232 patient point to its nearest neighbors by only information driven from the metagenome.

233 Cluster-wise stability was evaluated by consensus cluster distribution36 and by resampling the

234 data (1000 iterations) using bootstrapping, jittering and replacement of points by noise with

235 subsequent calculation of the Jaccard similarity indices.37

236 Metagenomics data was used to reveal the bacterial profiles of the identified clusters up to

237 species level.

238 Cluster migration

239 The same clustering protocol was re-performed for the longitudinal samples. Longitudinal

240 cluster migration and stability was assessed by cross-tabulating baseline and longitudinal

241 patient clusters assignments and assessed visually by a Sankey diagram.

7
242 Cluster robustness

243 Robustness of the clustering was evaluated by including mild-moderate asthmatics (U-BIOPRED

244 cohort C) to test whether their inclusion would fit the previous clustering solution or result in

245 cluster disintegration.

246 Statistical analysis

247 Patient cluster distribution according to the inclusion country and season of sample collection

248 was tested using Chi-Square test with Monte Carlo simulation (2000 permutations) and later

249 visualized by principal co-ordinate analysis (PCoA) on Bray-Curtis dissimilarity measure.

250 Differences in patients’ demographic and clinical characteristics between the baseline and

251 longitudinal visits and between the revealed clusters were compared using Wilcoxon signed-

252 rank and McNemar's tests for paired data, and Pearson Chi-Square, Fisher’s exact or Mann-

253 Whitney U tests for independent data as appropriate. Results are considered as significant at

254 alpha level < 0.05.

255 Differences in microbiome profiles between the clusters and between baseline and longitudinal

256 visits were compared using a Mann-Whitney U test and Wilcoxon signed-rank test, respectively.

257 P-values for metagenomics species differences were adjusted for multiple testing using

258 Benjamini-Hochberg false discovery rate correction (FDR).38 Results are considered significant at

259 FDR alpha level < 0.05.

8
260 A correlation heatmap (spearman and point-biserial correlations) was depicted between

261 bacterial species and asthma clinical characteristics that were found to be statistically significant

262 different between the baseline clusters.

263 All analyses were performed using R studio (version 1.1.453) with R software (version 3.5.1)

264 supported with the following packages; phyloseq, vegan, stats, cluster, factoextra,

265 ConsensusClusterPlus, fpc, fossil, metacoder, and SIAMCAT.

9
266 Results

267 The baseline and follow-up characteristics for the included participants are summarized in Table

268 1.

269 Unsupervised unbiased clustering of microbiome profiles of severe asthmatics at baseline

270 identified two main clusters

271 The 16s rRNA microbiome sequencing identified a total of 2777 OTUs, while the metagenomics

272 approach identified a total of 251 bacterial species. Bray-Curtis beta microbiome diversity

273 suggested 2 optimum clusters as evaluated by multiple indices (Figure E3). Applying hierarchical

274 ward clustering revealed two severe asthmatic groups which were driven only by the

275 microbiome profiles at baseline visit (Figure 1A). Partition around medoids (PAM) clustering also

276 revealed two isolated clusters (Figure 1B). Quantitative assessment of similarity in patients’

277 assignment between hierarchical ward and PAM clustering was performed by means of Pearson

278 Chi-Square test (χ2= 51.85, p < 1 x 10-11) and Rand index (RI= 0.82) suggesting great similarity.

279 The 16s rRNA microbiome generated clusters were highly concordant with the metagenomics

280 generated clusters as indicated by Pearson Chi-Square test (χ2= 63.659, p < 1.48 x 10-15) and

281 Rand index (RI= 0.85). Visual representation by the TDA analysis shows that two patient groups

282 can be driven by the microbiome profiles (Figure 1C) when the metagenomics species were

283 mapped to the color coding of the two hierarchical clusters.

284 There were no statistically significant associations between the patients’ clusters assignments

285 and the 9 countries from which samples were collected (p-values were 0.110 and 0.229 for

286 Hierarchical and PAM clustering, respectively) or the season of sample collection (p-values were

10
287 0.633 and 0.702 for hierarchical and PAM clustering, respectively). This was visually confirmed

288 by running PCoA on Bray-Curtis dissimilarity measure showing random patient allocations

289 (Figure E4).

290 Baseline clusters show distinct demographic and clinical characteristics

291 Table 2 shows the demographic and clinical characteristics of the two baseline severe asthma

292 clusters. Cluster 1 represent 75 % (n=75) of the severe asthmatics. More than half of them lived

293 in urban areas. As compared to cluster 2, cluster 1 patients had significantly lower percentages

294 of sputum neutrophils, higher percentages of sputum macrophages, and higher values for FEV1

295 and FEV1/FVC % predicted pre- and post- salbutamol (Figure E5A). In contrast, cluster 2 patients

296 had significantly younger age of asthma onset, were mostly non-smokers (84%), and were more

297 likely to live in suburban areas. In addition, a higher percentage of cluster 2 patients were

298 prescribed theophylline.

299 Microbial profiles of the two baseline severe asthma clusters

300 Cluster 2 patients had lower microbial richness and alpha-diversity indices when compared to

301 cluster 1 (Figure 2). Figure E6 shows the bacterial phylogenetic map of the two baseline clusters.

302 Statistical testing showed that a total of 28 species remained significantly different between the

303 two clusters after FDR correction. All of them were more abundant in cluster 1 as compared to

304 cluster 2 patients (Figure 3 and Figure 4). Those species were related to three dominant phyla;

305 Firmicutes, Bacteriodetes and Actinobacteria, and to Proteobacteria to a lesser extent which

306 comprise the following main genera; Veillonella, Prevotella, Alloprevotella, Streptococcus,

307 Porphyromonas, Rothia, Haemophilus, Neisseria, Megasphaera, and few others. In contrast,

11
308 there was a trend in increased relative abundances of few pathogenic species, such as

309 Haemophilus influenza, Moraxella catarrhalis, and Streptococcus pseudopneumoniae in cluster 2

310 compared to cluster 1 patients, however these results were not statistically significant (Figure

311 E7). Correlations between individual bacterial species and asthma characteristics were of weak

312 or moderate (r <0.50) strength (Figure E8) and reflected the findings revealed by the clustering.

313 Unsupervised clustering of the microbiome profiles of severe asthmatics at follow-up

314 (longitudinal clusters)

315 Microbiome data were available for 46 (out of 100) severe asthma patients after 12-18 months

316 from baseline inclusion. Similar to the baseline visit, different indices suggested that the

317 microbiome profiles of severe asthma patients at the longitudinal visit allocated the patients

318 into two main clusters (Figure E9). Hierarchical ward clustering had a great similarity with PAM

319 (Figure E10) as quantified by Fisher’s Exact test (p < 1 x 10-6) and Rand index (RI= 0.875).

320 Demographic and clinical characteristics of the longitudinal sputum microbiome clusters

321 Table 3 shows the demographic and clinical characteristics of the two longitudinal severe

322 asthma clusters. Longitudinal cluster 2 patients were more likely to live in rural areas (55.6%) as

323 compared to cluster 1 patients who were more likely to live in urban areas (70.3%). Similar to

324 the baseline analysis, longitudinal cluster 2 patients had higher percentages of sputum and

325 blood neutrophils, lower percentages of sputum macrophages, and lower values for FEV1 %

326 predicted pre- and post- salbutamol (Figure E5B). In addition, higher percentage of cluster 2

327 patients were prescribed long-acting anticholinergics compared to cluster 1.

328

12
329 Microbial profiles of the longitudinal severe asthma clusters

330 Longitudinal cluster 2 patients had lower microbial richness and diversity compared to

331 longitudinal cluster 1 patients estimated by multiple indices as shown in Figure E11. Figure E12

332 shows the bacterial phylogenetic map of the two longitudinal clusters. A total of 13 species

333 remained significantly different between the two clusters after FDR correction. All of them were

334 more abundant in cluster 1 compared to cluster 2 patients (Figure E13, and Figure E14). Those

335 species were related to the following main genera; Veillonella, Prevotella, Streptococcus, Rothia,

336 Haemophilus, and Neisseria.

337 Stability of the longitudinal clusters over time in severe asthmatics

338 Patients who had both baseline and longitudinal microbiome samples were cross-tabulated to

339 check similarity in clusters assignment between the two visits (Figure 5). Out of 46 patients, 39

340 (84.7%) remained cluster-stable longitudinally. Quantitative assessment resulted in statistically

341 significant Fisher’s Exact test (p <.01) and relatively high Rand index (RI = 0.74) suggesting

342 relative cluster stability after 12-18 months. No significant differences in microbial richness and

343 diversity (Figure E15) or in species relative abundances after FDR correction between baseline

344 and longitudinal visits have been observed. Figure E16 shows a bar chart of mean percentage in

345 relative abundance of bacterial taxa at baseline and longitudinal visits.

346 Cluster robustness when including mild-to-moderate asthmatics in the analysis

347 Repeating the baseline clustering including both the data from the severe asthmatics (n=100)

348 and those with mild-moderate asthma (n=24) also resulted in two main clusters (Figure E17); 23

13
349 of 24 patients (95.8%) of the mild-moderate asthmatics were assigned to cluster 1, with only 1

350 mild-moderate asthmatic being assigned to cluster 2 using hierarchical clustering. This patient

351 displayed clinical characteristics like those of severe asthmatics in cluster 2 in respect of young

352 age of asthma onset, sputum neutrophilia, and decreased percentage of sputum macrophages

353 and FEV1 values (Table E1). A cross-table to asses if the severe asthma patients had changed

354 their initial cluster assignment after inclusion of the mild-moderate asthma group is shown in

355 Table E2. High cluster stability is indicated by Pearson Chi-Square test (χ2= 73.397, p < 1 x 10-15)

356 and Rand index (RI= 0.904), suggesting robustness of the clustering model.

14
357 Discussion

358 This is the first study to investigate microbiome-driven phenotypes and their stability overtime

359 in severe asthmatics. Using unbiased clustering based on microbiome profiles we show that,

360 severe asthma can be stratified into two phenotypic clusters. These clusters significantly

361 differed by age of asthma onset, patient residential locations, smoking history, percentage of

362 blood and/or sputum neutrophils and macrophages, spirometry and used asthma medications.

363 At both baseline and longitudinal visits, the severe asthmatics in cluster 2 had worse lung

364 function, with associated blood and/or sputum neutrophilia and decreased sputum

365 macrophages compared to cluster 1 patients. In addition, they were more likely to receive add-

366 on asthma medications, such as theophylline or long acting anticholinergics, possibly indicating

367 greater needs to control their more severe airway obstruction.

368 The two phenotypic clusters were associated with markedly distinct microbiome profiles; cluster

369 1 had higher bacterial richness and diversity compared to cluster 2, both at baseline and

370 longitudinal visits. Cluster 2 was characterized by a clear deficiency of several bacterial species

371 including Veillonella, Prevotella, Alloprevotella Streptococcus, Porphyromonas, Rothia,

372 Haemophilus, Neisseria, and Megasphaera genera. Most of these species are considered as

373 commensals inhabiting the oropharyngeal region and the airways. This microbial dysbiosis was

374 associated with blood and/or sputum neutrophilia, deceased sputum macrophages and worse

375 lung function outcomes at baseline and longitudinal visits. A study by the Severe Asthma

376 Research Program (SARP) has shown that sputum neutrophilia (with or without eosinophilia) is a

377 characteristic of more severe asthma phenotypes.39 In our study, the neutrophilia in cluster 2

378 could be attributed to the presence of either “subclinical infection” or modulation of the airway
15
379 “immune tone” by the microbiota. Thus, deficiency of commensal bacteria leads to increased

380 risk of infections with pathogenic ones, as manifested by increased abundance of Haemophilus

381 influenza, Moraxella catarrhalis, and Streptococcus pseudopneumoniae. Consequently, these

382 bacteria could be responsible for the observed blood and/or sputum neutrophilia. Our results

383 are complementary to another U-BIOPRED study which showed that adult severe asthmatics

384 had lower sputum microbiome α-diversity compared to mild-moderate asthmatics and healthy

385 controls, and this diversity was inversely correlated with sputum neutrophils.29 This was in-line

386 with previously reported studies showing that neutrophilic asthma is characterised by low

387 airway bacterial diversity and high abundances of the Proteobacteria phylum, especially the

388 Moraxella and Haemophilus genera.14-16 In addition, a decrease in the percentage of sputum

389 macrophages may suggest a defective innate immune response with impaired macrophage

390 phagocytosis of these pathogenic bacteria. This finding was in agreement with a previous study

391 that showed severe asthmatics have a reduced macrophages phagocytic capacity for certain

392 pathogenic bacteria such as H. influenzae, compared to non-severe asthmatics and healthy

393 subjects.40

394 A study in COPD patients showed that survivors of 1-year mortality had higher relative

395 abundances of Veillonella compared to non-survivors.41 This was partly in agreement with the

396 finding that the less severe cluster 1 patients in our study had higher relative abundances of

397 Veillonella bacteria among others as compared to cluster 2 patients, which may suggest these

398 bacteria might have protective role in chronic respiratory diseases. Although few species, such

399 as Haemophilus parainfluenzae, can become opportunistic pathogens in some situations, their

400 increased abundances in cluster 1 patients was not associated with asthma severity

16
401 characteristics such as exacerbation frequency. These findings imply that the crosstalk of several

402 species within the airway microbial community and their interplay with innate immunity may

403 provide a better clinical relevance instead of looking at the roles of single/few bacterial species.

404 A striking finding in our study is that approximately 85% of the patients remained cluster stable

405 after 12-18 months. In addition, the bacterial dysbiosis was not related to either current or

406 previous antibiotic intake (ever), suggesting that the microbial profiles of these patients were

407 not a main consequence of short-term intake of antibiotics. Rather, they were probably shaped

408 over a long-time period and might have a genetic background42 and/or resulted from a life-long

409 exposure to environmental factors.43

410 Tobacco smoking has been reported previously to be associated with sputum microbiome

411 diversity of asthmatic patients, 44 in addition, it has been reported to induce neutrophilia in

412 chronic airway disease including asthma.45, 46 However, in our study, the more severe cluster 2

413 patients were more likely to be non-smokers (84.0%). Since microbial dysbiosis and neutrophilia

414 were more often observed in cluster 2 patients, it seems unlikely that this is mainly attributed to

415 smoking and possibly denoting the interplay between microbial dysbiosis and innate immunity

416 altering the airway immune tone. In another study investigating the airway microbiome in

417 asthmatics, neutrophilia was the strongest predictor for microbiota variance, while smoking was

418 not a predictor14 supporting the findings observed in cluster 2. This warrants further

419 investigation to gain more insight on the trajectories of neutrophilic asthma and possible

420 overlap/delineation between microbial-associated and smoking-associated neutrophilic asthma.

421 Cluster 2 patients were more likely to live in suburban or rural areas, in contrast to cluster 1

422 patients who were more likely to live in urban areas. We could speculate that there is relatively
17
423 more traffic in or around suburbs and longer driving times due to commuting to work.

424 Therefore, more exposure to car gases/particulate air pollution may influence the airway

425 microbiome profiles.47 In addition, exposure to fine particulate air pollution can increase

426 neutrophils,48 which may contribute to the neutrophilia we see in cluster 2 patients.

427 The relative cluster stability and robustness we found in our study is an ideal criterion for

428 personalized diagnostic or therapeutic decisions in asthmatics. In addition, the inclusion of mild-

429 moderate asthma group fitted well to our previous clustering scheme where most of this

430 patient group (> 95 %) were assigned to the “milder” asthma cluster except for one patient who

431 also showed close characteristics to the severe asthma cluster 2 patients regarding young age of

432 asthma onset, neutrophilia and low values of FEV1. This suggests that using the criteria of the

433 observed microbiome profiles may enable better diagnosis and/or prediction of more severe

434 phenotypes and hence tailor therapeutic decisions. In addition, the relative stability of the

435 clusters after 12-18 months indicates that management approaches aiming at correcting the

436 pathophysiology of these patients should be sought instead of just symptoms control which is

437 not sufficient. This may include approaches that aim to reshape the lung microbiome, for

438 example using long term pre-, pro- or synbiotics. Moreover, therapeutic strategies directed

439 towards the neutrophilic corticosteroid-resistant asthma phenotype and innate immunity may

440 be tried in these patients, such as macrolide antibiotics,49, 50 low dose theophylline,51 and other

441 anti-neutrophilic compounds.52, 53

442 Our study has many strengths. First, the analysis protocol is unbiased and its clinical significance

443 is driven only by the microbiome. Second, the pan-European nature of this study makes our

444 results more generalizable and valid than previously reported single country/center studies with
18
445 lower sample sizes. Third, the utilization of both 16s rRNA sequencing and metagenomics

446 further increase the reliability and generalizability to other studies. In addition, the limited, but

447 affordable 16s rRNA method provided highly concordant clustering results to the metagenomics

448 approach, which further extend it potential applicability in clinical practice in sittings where

449 metagenomics could not be performed. Fourth, we internally validated our finding by applying

450 different clustering algorithms that were highly consistent. Although adding an independent

451 mild-moderate asthma cohort is not considered as a validation, its perfect harmonization with

452 the clustering solution prove the robustness and clinical relevance of the clustering technique

453 used. Fifth, shotgun metagenomics allowed us to reveal bacterial associations up to species

454 levels and hence increasing the clinical relevance, unlike most previous studies that used 16s

455 rRNA based-methods (limited in identifying bacterial species). Finally, this study is regarded as

456 one of the first attempts to uncover microbiome-driven phenotypes and elaborate targets for

457 precision medicine within severe asthma patients.

458 However, there are also limitations. First, we sampled only one airway compartment (induced

459 sputum). Whether other sampling compartments would provide additional microbiome-related

460 information needs to be determined. Second, only two time points were measured over a

461 follow-up design of 12-18 months, which may not be sufficient to adequately assess longitudinal

462 shifts of microbiome clusters in asthmatics. Third, some patients were lost to follow-up which

463 may create further bias in the assessment. However, we are the first study to investigate the

464 microbiome clusters in severe asthmatics which might serve as a basis for future investigations.

465 Finally, using an external cohort outside the U-BIOPRED for validation is still needed to confirm

466 our findings.

19
467 In conclusion, our study has shown that microbiome-driven clustering can be used as an

468 unbiased way to detect phenotypes in severe asthmatics. Furthermore, they are relatively

469 stable after 12-18 months of follow-up and demonstrated robustness after inclusion of

470 independent asthma cohort. Asthma patients with the more severe, microbiome-driven

471 phenotype, comprised approximately 25% of the severe asthmatics and need to be considered

472 targets for personalized medicine decisions targeting the airway microbiome.

473 Acknowledgment

474 Some of the drawn objects in the graphical abstract were adapted from Servier Medical Art

475 (https://smart.servier.com) in accordance with a Creative Commons Attribution 3.0 Unported

476 License (https://creativecommons.org/licenses/by/3.0/).

20
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23
596 Tables
597
598 Table 1: Baseline and longitudinal patient characteristics for the severe and mild-moderate asthmatics.
Severe asthmatics Mild-moderate
Characteristic asthmatics
Baseline Longitudinal p-value* Baseline
(n=100) (n=46) (n=24)
Age (years), median (IQR) 55 (46.0- 57 (51.3-63.5) 4.3 x 10-11 40.50 (25.75-
62.0) 51.00)
Age of onset (years), median (IQR) 27 (7-46) 37 (14-49) NA 9 (3-22)
Females (%) 58 (58.0%) 25 (54.3%) NA 11 (45.8%)
BMI (kg/m2), median (IQR) 27.72 (24.67- 27.71 (24.37- NA 24.02 (21.80-
32.45) 31.04) 30.18)
Race (white Caucasian, %) 92 (92.0%) 43 (93.5%) NA 23 (95.8%)
Residential Location (%) NA
• Rural 26 (26.0%) 10 (21.7%) 6 (25%)
• Suburban 25 (25.0%) 8 (17.4%) 8 (33.3%)
• Urban 49 (49.0%) 28 (60.9%) 10 (41.7%)
Atopy (%) 70 (70.0%) 31 (67.4%) NA 23 (95.8%)
Non-smoking patients (%) 66 (66.0%) 30 (65.2%) NA 24 (100%)
Eosinophils % in sputum, median (IQR) 2.75 (0.37- 2.19 (0.76- .838 0.72 (0.21-1.81)
19.27) 17.12)
Neutrophils % in sputum, median 57.98 (39.59- 62.95 (51.19- .096 42.17 (26.16-
(IQR) 81.98) 78.24) 75.18)
Macrophages % in sputum, median 22.82 (10.15- 21.57 (10.01- .068 42.97 (21.62-
(IQR) 39.55) 37.84) 66.69)
Eosinophils % in blood, median (IQR) 3.43 (1.58- 3.50 (1.86- .024 3.55 (1.81-4.46)
6.51) 4.97)
Neutrophils % in blood, median (IQR) 58.48 (53.96- 60.55 (54.88- .190 58.75 (53.35-
67.45) 76.83) 64.82)
FEV1 % predicted pre salbutamol, 62.78 (45.31- 61.42 (49.03- .642 92.00 (87.34-
median (IQR) 74.16) 74.75) 104.45)
FEV1 % predicted post salbutamol, 73.08 (52.77- 72.79 (55.24- .468 103.13 (88.51-
median (IQR) 86.92) 85.61) 114.07)
FEV1/FVC % predicted pre salbutamol, 73.46 (61.03- 75.21 (61.35- .282 89.99 (79.80-
median (IQR) 83.66) 83.33) 96.54)
FEV1/FVC % predicted post 76.49 (65.03- 78.70 (64.03- .461 96.91 (87.42-
salbutamol, median (IQR) 87.28) 85.90) 100.83)
FENO in ppb, median (IQR) 23.50 (13.25- 27.50 (16.90- .708 30.25 (19.50-
45.00) 50.00) 58.13)
Number of exacerbations per year, 2 (1-3) 2 (0-3) .737 0 (0-1)
median (IQR)
ACQ5 score average, median (IQR) 2.3 (1.60- 1.9 (0.95- .589 1.00 (0.45-1.55)
3.20) 3.00)
AQLQ score average, median (IQR) 4.46 (3.50- 4.59 (3.67- .245 5.59 (4.84-6.56)
5.50) 5.60)

24
 Current asthma medication used (%):
• ICS 100 (100%) 42 (91.3%) .125 24 (100%)
• SABA 63 (63.0%) 27 (58.7%) .999 19 (79.2%)
• LABA 99 (99.0%) 43 (93.5%) .250 1 (4.2%)
• OCS 87 (87.0%) 14 (30.4%) .070 0 (0.0%)
• Short-acting Anticholinergics 9 (9.0%) 3 (6.5%) .999 1 (4.2%)
• Long-acting Anticholinergics 29 (29.0%) 15 (32.6%) .999 0 (0.0%)
• Leukotriene antagonists 43 (43.0%) 19 (41.3%) .999 0 (0.0%)
• Theophylline 21 (21.0%) 7 (15.2%) .999 0 (0.0%)
Antibiotic usage (%):
• Current intake 18 (18.0%) 7 (15.2%) .500 0 (0.0%)
• Current and previous (ever) 22 (22.0%) 26 (56.5%) <.001 1 (4.2%)
intake
599 Variables described as n (% of n) unless specified, IQR: interquartile range, BMI: body mass index, FEV1:
600 forced expiratory volume in 1 Second, FVC: forced vital capacity, FENO: fraction of exhaled nitric oxide,
601 ACQ5: 5-item asthma control questionnaire, AQLQ: asthma quality of life questionnaire, ICS: inhaled
602 corticosteroids, SABA: short acting beta agonist, LABA: long acting beta agonist, OCS: oral
603 corticosteroids. *p-values were computed for paired differences for only patients who have both
604 baseline and longitudinal sputum samples.

25
605 Table 2: Demographic and clinical characteristics of the baseline clusters.

Characteristics Baseline P-
Cluster 1 (n=75) Cluster 2 (n=25) value
Age (years), median (IQR) 55 (46-62) 57 (49-63) .327
Age of onset (years), median (IQR) 30.50 (14.00- 16.00 (5.00-33.50) .012
48.25)
Females (%) 44 (58.7%) 14 (44.0%) .815
BMI (kg/m2), median (IQR) 27.73 (24.60- 27.47 (24.48- .591
32.61) 30.82)
Race (white Caucasian, %) 70 (93.3%) 22 (88.0%) .409
Residential Location (%) .009
• Rural 21 (28.0%) 5 (20.0%)
• Suburban 13 (17.3%) 12 (48.0%)
• Urban 41 (54.7%) 8 (32.0%)
Atopy (%) 54 (72.0%) 16 (64.0%) .359
Non-smokers (%) 45 (60.0%) 21 (84.0%) .028
Eosinophils % in sputum, median (IQR) 3.81 (0.19-21.92) 2.28 (0.39-7.08) .446
Neutrophils % in sputum, median (IQR) 53.40 (32.40- 86.90 (57.32- <.0001
70.74) 92.73)
Macrophages % in sputum, median (IQR) 26.69 (14.60- 9.21 (3.89-19.49) <.0001
47.30)
Eosinophils % in blood, median (IQR) 3.50 (1.52-6.53) 3.28 (1.74-6.42) .987
Neutrophils % in blood, median (IQR) 58.16 (53.93- 58.80 (53.48- .678
66.54) 70.57)
FEV1 % predicted pre salbutamol, median (IQR) 65.24 (52.38- 47.66 (38.36- .035
74.15) 74.18)
FEV1 % predicted post salbutamol, median (IQR) 74.88 (61.52- 51.44 (42.90- .009
86.94) 87.62)
FEV1/FVC % predicted pre salbutamol, median 74.62 (65.60- 65.99 (51.82- .030
(IQR) 83.97) 77.86)
FEV1/FVC % predicted post salbutamol, median 79.25 (67.02- 68.19 (52.31- .012
(IQR) 89.11) 83.86)
FENO in ppb, median (IQR) 26.25 (12.63- 22.00 (14.00- .328
53.00) 26.75)
Number of exacerbations per year, median (IQR) 2 (1-3) 2 (1-4) .416
ACQ5 score average, median (IQR) 2.40 (1.40-3.20) 2.20 (1.65-3.15) .783
AQLQ score average, median (IQR) 4.53 (3.52-5.53) 4.09 (3.33-5.14) .417
Current asthma medication use (%):
• ICS 75 (100%) 25 (100%) NA
• SABA 45 (60.0%) 18 (72.0%) .282
• LABA 74 (98.7%) 25 (100%) .999
• OCS 33 (44.0%) 14 (56.0%) .298
• Short-acting Anticholinergics 8 (10.7%) 1 (4.0%) .444
• Long-acting Anticholinergics 20 (26.7%) 9 (36.0%) .373
• Leukotriene antagonists 32 (42.7%) 11 (44.0%) .907
• Theophylline 11 (14.7%) 10 (40.0%) .007

26
 OCS normalized dose (mg) 10.00 (8.44-16.25) 10.00 (5.00-14.38) .363
(n=33) (n=14)
Antibiotic usage (%):
• Current intake 14 (18.7%) 4 (16.0%) .999
• Current and previous (ever) intake 18 (24%) 4 (16.0%) .403
606 Variables described as n (% of n) unless specified, IQR: interquartile range, BMI: body mass index, FEV1:
607 forced expiratory volume in 1 Second, FVC: forced vital capacity, FENO: fraction of exhaled nitric oxide,
608 ACQ5: 5-item asthma control questionnaire, AQLQ: asthma quality of life questionnaire, ICS: inhaled
609 corticosteroids, SABA: short acting beta agonist, LABA: long acting beta agonist, OCS: oral
610 corticosteroids. P-values for testing statistical significance between the two longitudinal clusters were
611 calculated using Pearson Chi-Square or Fisher’s Exact tests as appropriate for categorical variables and
612 Mann-Whitney U test for continuous variables.

27
613 Table 3: Demographic and clinical characteristics of the longitudinal clusters.
Characteristics Longitudinal P-
Cluster 1 (n=37) Cluster 2 (n=9) value
Age (years), median (IQR) 57 (48-64) 55 (53-65) .957
Age of onset (years), median (IQR) 37.00 (17.50- 29.50 (3.50-49.75) .716
48.50)
Females (%) 22 (59.5%) 3 (33.3%) .264
BMI (kg/m2), median (IQR) 27.96 (24.25- 29.56 (24.83- .744
31.87) 30.36)
Race (white Caucasian, %) 35 (94.6%) 8 (88.9%) .488
Residential Location (%) .012
• Rural 5 (13.5%) 5 (55.6%)
• Suburban 6 (16.2%) 2 (22.2%)
• Urban 26 (70.3%) 2 (22.2%)
Atopy (%) 25 (67.6%) 6 (66.7%) .872
Non-smokers (%) 23 (62.2%) 7 (77.8%) .463
Eosinophils % in sputum, median (IQR) 2.65 (0.88-16.45) 0.99 (0.51-18.69) .651
Neutrophils % in sputum, median (IQR) 59.40 (45.03- 83.76 (72.58- .002
72.64) 95.66)
Macrophages % in sputum, median (IQR) 27.24 (15.23- 5.68 (1.37-12.45) <.0001
38.99)
Eosinophils % in blood, median (IQR) 3.65 (2.07-5.29) 2.07 (0.81-4.13) .103
Neutrophils % in blood, median (IQR) 59.81 (53.72- 76.61 (65.31- .023
65.82) 82.98)
FEV1 % predicted pre salbutamol, median (IQR) 63.76 (50.55- 56.17 (40.00- .036
77.23) 62.28)
FEV1 % predicted post salbutamol, median (IQR) 74.33 (63.23- 64.22 (44.64- .036
86.27) 78.08)
FEV1/FVC % predicted pre salbutamol, median 77.35 (62.08- 65.50 (51.22- .116
(IQR) 83.38) 76.02)
FEV1/FVC % predicted post salbutamol, median 80.40 (67.68- 65.04 (50.82- .081
(IQR) 86.22) 78.93)
FENO in ppb, median (IQR) 25.00 (15.90- 28.25 (19.00- .999
54.50) 35.88)
Number of exacerbations per year, median (IQR) 2 (0-3) 2 (0-3) .663
ACQ5 score average, median (IQR) 2.0 (0.9-3.1) 1.8 (1.0-2.7) .957
AQLQ score average, median (IQR) 4.59 (3.86-5.53) 4.48 (3.44-6.09) .807
Current medication use (%):
• ICS 33 (89.2%) 9 (100%) .571
• SABA 20 (54.1%) 7 (77.8%) .270
• LABA 34 (91.9%) 9 (100%) .999
• OCS 10 (27.0%) 4 (44.4%) .423
• Short-acting Anticholinergics 2 (5.4%) 1 (11.1%) .488
• Long-acting Anticholinergics 7 (18.9%) 8 (88.9%) <.001
• Leukotriene antagonists 15 (40.5%) 4 (44.4%) .999
• Theophylline 4 (10.8%) 3 (33.3%) .124

28
 OCS normalized dose (mg) 10.00 (8.13-12.50) 13.75 (5.63-20.00) .999
(n=10) (n=4)
Antibiotics usage (%):
• Current intake 4 (10.8%) 3 (33.3%) .124
• Current and previous (ever) intake 20 (54.1%) 6 (66.7%) .711
614 Variables described as n (% of n) unless specified, IQR: interquartile range, BMI: body mass index, FEV1:
615 forced expiratory volume in 1 Second, FVC: forced vital capacity, FENO: fraction of exhaled nitric oxide,
616 ACQ5: 5-item asthma control questionnaire, AQLQ: asthma quality of life questionnaire, ICS: inhaled
617 corticosteroids, SABA: short acting beta agonist, LABA: long acting beta agonist, OCS: oral
618 corticosteroids. P-values for testing statistical significance between the two longitudinal clusters were
619 calculated using Pearson Chi-Square or Fisher’s Exact tests as appropriate for categorical variables and
620 Mann-Whitney U test for continuous variables.

29
621 Figure Legends

622 Figure 1: A; Hierarchical cluster dendogram in a tree-like structure, where patients’ nodes (leaves at the
623 bottom of the dendogram) that are statistically closely connected are joined together by edges (small
624 branches). The small branches are further joined up by larger branches (bottom-up), to the upper part of
625 the dendogram representing the two main branches (clusters) originating from the severe asthma
626 cohort. B; partition around the medoids (PAM) clusering show two relatively detached ellipses. Similarity
627 in patients’ assignment between the two clustering algorithms was assessed by Pearson Chi-Square test
628 (χ2= 51.85, p << 1 x 10-11) and Rand index (RI= 0.802) suggesting great similarity. Bootstrapping, jittering
629 and replacement of points by noise schemes (1000 iterations) resulted in Jaccard similarity indices
630 ranged from 0.82-1 for both clusters by either hierarchical clustering or PAM suggesting highly stable
631 clusters. C; Topological data analysis (TDA) graph, where nodes were colored in accordance with baseline
632 hierarchical clustering of patients. Two distinct patient clusters based on the metagenome profile were
633 observed, where blue nodes represent cluster 1 patients, while red nodes represent cluster 2 patients.
634 Yellow nodes represent less matched patient cluster assignment by TDA compared to the hierarchical
635 clustering.

636 Figure 2: Upper panel; Venn diagram represents the metagenomics species distribution between the two
637 clusters. Lower panel; different alpha diversity measures reveal that cluster 2 patients had much lower
638 microbial diversity compared to cluster 1 patients (all p-values < .0001).

639 Figure 3: Metagenomics phylogenetic map shows statistically significant differential bacterial taxa
640 between the two baseline clusters after false discovery rate (FDR) correction. Nodes color corresponds
641 to the median difference in relative abundances of the bacterial taxa. The darker the color of the
642 phylogenetic branches, the higher median differences, while grey nodes and branches indicate no
643 significant differences. The magenta color indicates that all significant taxa were more abundant in
644 cluster 1 compared to cluster 2, while absence of cyan color in the phylogenetic map indicate that no
645 significant taxa were more abundant in cluster 2 as compared to cluster 1.

646 Figure 4: Statistically significant differentially abundant species after false discovery rate (FDR) correction
647 between baseline cluster 1 and cluster 2 patients by metagenomics. All of them were more abundant in
648 cluster 1 compared to cluster 2 patients.

649 Figure 5: Cross-tabulated and Sankey diagram of patients’ clusters assignments among 46 patients with
650 both baseline and follow-up visits: 39 patients are cluster stable (in boldface), and 7 patients migrate
651 between clusters. Cells described as n (% of baseline clusters n). p-value was generated using Fisher’s
652 exact test.

653

30
A Hierarchical cluster dendogram C Topological data analysis (TDA)

Cluster 2

Metric: normalized correlation

Lenses: neighborhood lens 1
and neighborhood lens 2

Baseline clusters
B Partition around the medoids (PAM)

Cluster 1
 Cluster 1

Cluster 2

4 53 144
Alpha diversity measures

Baseline clusters

Selenomonas_flueggei
Selenomonas_sputigena
Eubacterium_yurii
Peptostreptococcaceae_noname
unclassified Peptostreptococcus_unclassified
Peptostreptococcus
Clostridiales_Family_XIII_Incertae_Sedis_unclassified
Eubacterium_infirmum
Peptostreptococcus_stomatis
Clostridiales_Family_XIII_Incertae_Sedis_noname
Clostridiales_Family_XIII_Incertae_Sedis
Eubacteriaceae_bacterium_CM5
Eubacteriaceae_bacterium_OBRC8
Eubacteriaceae_noname
Eubacteriaceae
Eubacteriaceae_bacterium_ACC19a
Eubacteriaceae_noname_unclassified Eubacterium
Eubacterium_saphenum
Eubacterium_brachy
Lachnospiraceae_bacterium_oral_taxon_082
Lachnospiraceae_noname
Shuttleworthia
Lachnoanaerobaculum
Lachnospiraceae_bacterium_ICM7
Shuttleworthia_satelles
Lachnoanaerobaculum_saburreum
Granulicatella_unclassified Granulicatella
Lactococcus_lactis
Lactococcus
Streptococcus_tigurinus
Streptococcus_vestibularis
Streptococcus_salivarius
Streptococcus_australis
Streptococcus_thermophilus
Streptococcus_peroris
Streptococcus
Streptococcus_oligofermentans
Streptococcus_sp_BS35b
Streptococcus_sanguinis
Streptococcus_mitis_oralis_pneumoniae
Streptococcus_gordonii
Streptococcus_anginosus
Streptococcus_parasanguinis
Streptococcus_infantis
Lactobacillus_fermentum
Lactobacillus_ultunensis
Neisseria_unclassified
Neisseria_sicca Haemophilus_influenzae
Neisseria_elongata Haemophilus_sputorum
Neisseria_polysaccharea Haemophilus_parainfluenzae
Neisseria_flavescens
Kingella_oralis Haemophilus_paraphrohaemolyticus
Neisseria_bacilliformis Neisseria_sp_oral_taxon_014
Haemophilus
Polaromonas_unclassified Kingella_unclassified Neisseria_cinerea Haemophilus_parahaemolyticus
Fusobacterium_necrophorum Neisseria Haemophilus_haemolyticus Aggregatibacter_unclassified
Aggregatibacter_segnis
Neisseria_meningitidis Neisseria_lactamica
Fusobacterium_nucleatum Kingella
Fusobacterium_periodonticum Variovorax_unclassified Aggregatibacter
Polaromonas Neisseria_subflava Haemophilus_pittmaniae Actinobacillus_unclassified
Veillonella_dispar Aggregatibacter_aphrophilus
Neisseria_macacae Neisseria_gonorrhoeae Pantoea_agglomerans
Veillonella_unclassified Fusobacterium Kingella_denitrificans Actinobacillus
Veillonella_atypica Eggerthia_catenaformis Variovorax Pantoea_unclassified
Alicycliphilus_denitrificans Pasteurellaceae
Neisseriaceae Simonsiella_muelleri
Moraxella_catarrhalis Cardiobacterium_hominis
Veillonella Eggerthia Leptotrichia_unclassified
Alicycliphilus
Simonsiella Acinetobacter_lwoffii Pantoea
Veillonella_parvula
Solobacterium_moorei Comamonadaceae Acinetobacter_unclassified
Comamonas Pseudomonas_unclassified
Moraxella Escherichia_unclassified
Selenomonas_infelix Eikenella
Solobacterium Lautropia_mirabilis Acinetobacter Cardiobacterium_valvarum Agrobacterium_unclassified
Leptotrichia_shahii
Megasphaera_micronuciformis Leptotrichia Comamonas_unclassified Pseudomonas_aeruginosa
Selenomonas_artemidis Lautropia unclassified Eikenella_corrodens
Pseudomonas Cardiobacterium Escherichia
Mitsuokella_unclassified
Erysipelotrichaceae Fusobacteriaceae Moraxellaceae Enterobacteriaceae
Agrobacterium_tumefaciens
Centipeda_periodontii Gallionellaceae_unclassified Agrobacterium
Megasphaera_unclassified Leptotrichia_wadei
Pseudomonadaceae
Selenomonas Megasphaera
Burkholderiaceae Stenotrophomonas
Mitsuokella
Centipeda
Neisseriales unclassified
Brevundimonas_unclassified
Bulleidia
Burkholderiales Pasteurellales Cardiobacteriaceae_unclassified
Cardiobacteriaceae Shinella_zoogloeoides
Selenomonas_noxia Gallionellaceae Stenotrophomonas_maltophilia
Leptotrichiaceae Pseudomonadales Shewanella_baltica Hyphomicrobiaceae_unclassified Shinella
Veillonellaceae Xanthomonadaceae unclassified
Brevundimonas_diminuta
Dialister_micraerophilus Fusobacteriales Rhizobiaceae
Bulleidia_extructa Gallionellales Hyphomicrobiaceae
Sinorhizobium_unclassified
Brevundimonas
Dialister Ochrobactrum_anthropi
Erysipelotrichales Xanthomonadales Cardiobacteriales Shewanella
Leptotrichiaceae_unclassified Sinorhizobium
Enterobacteriales
Anaeroglobus Betaproteobacteria Ochrobactrum Campylobacter_showae
Campylobacter_rectus
Dialister_invisus Brucellaceae
Gammaproteobacteria Caulobacteraceae
Peptostreptococcaceae unclassified
Anaeroglobus_geminatus Shewanellaceae Campylobacter Treponema_denticola
Filifactor
Parvimonas_micra Selenomonadales Alteromonadales RhizobialesCaulobacterales Campylobacter_concisus
Campylobacteraceae Treponema_maltophilum
Campylobacter_curvus
Filifactor_alocis Fusobacteriia
Alphaproteobacteria Campylobacter_gracilis
Treponema_socranskii
Parvimonas Treponema
Clostridiales_Family_XI_Incertae_Sedis
Erysipelotrichia Proteobacteria Campylobacterales Desulfovibrio_desulfuricans
Desulfovibrio
Treponema_lecithinolyticum
Epsilonproteobacteria
Parvimonas_unclassified
Spirochaetaceae Treponema_medium
Clostridiales Treponema_vincentii
DesulfovibrionaceaeBilophila_unclassified
Catonella_morbi Negativicutes Deltaproteobacteria
Desulfovibrionales Bilophila
Fusobacteria candidate_division_TM7_single_cell_isolate_TM7c
Catonella Clostridia Candidatus_Saccharibacteria_noname Jonquetella_unclassified unclassified
Spirochaetales Candidatus_Saccharibacteria_noname_unclassified
Spirochaetia candidate_division_TM7_single_cell_isolate_TM7b
Lachnospiraceae
Stomatobaculum
Stomatobaculum_longum
Firmicutes Spirochaetes
Candidatus_Saccharibacteria_noname
Candidatus_Saccharibacteria_noname
Synergistales
Jonquetella
Candidatus_Saccharibacteria Jonquetella_anthropi
Synergistaceae Actinomyces_cardiffensis
Bacteria
Oribacterium Synergistia Candidatus_Saccharibacteria_noname
Oribacterium_sinus Synergistetes Actinomyces_odontolyticus
Pyramidobacter
Fretibacterium Actinomyces_viscosus
Oribacterium_sp_oral_taxon_078 Abiotrophia_defectiva
Tenericutes Fretibacterium_fastidiosum
Actinomyces Actinomyces_graevenitzii
Pyramidobacter_piscolens
Granulicatella_adiacens Abiotrophia
Mollicutes Mycoplasma
Mycoplasma_pneumoniae Actinomycetaceae Actinomyces_oris
Mycoplasmatales Mycoplasmataceae Actinomyces_naeslundii Actinomyces_sp_ICM47
Aerococcaceae
Carnobacteriaceae Actinobacteria Gordonia_terrae
Granulicatella_elegans
Bacilli Gordonia
Gordoniaceae Rothia_aeria
Lactobacillales
Streptococcaceae Bacillus_cereus_thuringiensis Actinobacteria Actinomycetales Micrococcaceae
Rothia_mucilaginosa
Streptococcus_constellatus Atopobium_parvulum Rothia
Actinomycetales_noname
Streptococcus_agalactiae Atopobium_vaginae
Bacillus Bacteroidetes_noname unclassified
Rothia_dentocariosa
Streptococcus_intermedius
Bacteroidetes Atopobium_rimae
Nocardioidaceae Propionibacteriaceae Rothia_unclassified
Nodes
Bacteroidetes_noname Tropheryma Propionibacteriaceae_unclassified
Bacillaceae
Atopobium
Streptococcus_pseudopneumoniae Coriobacteriales Corynebacteriaceae Propionibacterium_acidipropionici
Lactobacillaceae Flavobacteriia Bifidobacteriales
Streptococcus_cristatus Bacillales Bacteroidetes_noname
Tropheryma_whipplei Corynebacterium_durum
Nocardioides Propionibacterium_granulosum
1.00
−0.3500
%DFWHULDOVSHFLHV count
Bacillales_noname Cryptobacterium_curtum
Corynebacterium Propionibacterium
Bacteroidetes_oral_taxon_274
Gemella_morbillorum Coriobacteriaceae Corynebacterium_tuberculostearicum
Bacteroidetes_noname Cryptobacterium Nocardioides_unclassified Corynebacterium_matruchotii
Propionibacterium_propionicum
Flavobacteriales Bacteroidia
median difference
Scardovia_wiggsiae
Corynebacterium_pseudodiphtheriticum Propionibacterium_acnes
Gemella_haemolysans
Gemella Bacteroidetes_bacterium_oral_taxon_272 Slackia
Lactobacillus
Gemella_unclassified
Staphylococcaceae
Gemella_sanguinis
Chryseobacterium_gleum
Scardovia Bifidobacteriaceae
Scardovia_unclassified Alloscardovia
Slackia_exigua
Slackia_unclassified
−0.1560 7.94
Lactobacillus_salivarius Chryseobacterium Alloscardovia_omnicolens
Chryseobacterium_unclassified Olsenella
Flavobacteriaceae Parascardovia
Staphylococcus_epidermidis
Staphylococcus_saprophyticus Riemerella
Parascardovia_denticolens Bifidobacterium
Olsenella_unclassified
Olsenella_uli
−0.0389 28.80
Staphylococcus Tannerella_forsythia Bacteroidales Prevotella_sp_C561
Prevotella_dentalis
Tannerella Prevotella_oris
Riemerella_unclassified
Staphylococcus_aureus Bifidobacterium_dentium
Staphylococcus_haemolyticus
Staphylococcus_hominis
Capnocytophaga_ochracea
Porphyromonadaceae
Prevotella_oulorum
Prevotella_maculosa
Prevotella_pallens
Prevotella_salivae
Prevotella_marshii
Prevotella_denticola
Bifidobacterium_longum
0.0000 63.50
Capnocytophaga_granulosa Prevotella_loescheii Prevotella_histicola
Porphyromonas_asaccharolytica Prevotella_multiformis
Capnocytophaga Prevotella_buccae
Capnocytophaga_unclassified
Porphyromonas_uenonis
Capnocytophaga_sp_oral_taxon_329
Porphyromonas_sp_oral_taxon_279
Porphyromonas
Prevotellaceae
Prevotella_micans
Prevotella_intermedia
Prevotella Prevotella_bivia
Prevotella_veroralis
Prevotella_melaninogenica
0.0389 112.00
Capnocytophaga_gingivalis Porphyromonas_gingivalis Prevotella_multisaccharivorax Candidatus_Prevotella_conceptionensis
Prevotella_sp_oral_taxon_473
Porphyromonas_endodontalis Prevotella_baroniae
Porphyromonas_gulae
Porphyromonas_catoniae
Alloprevotella
Alloprevotella_tannerae
Prevotella_nigrescens Prevotella_disiens
Prevotella_nanceiensis
Prevotella_pleuritidis
0.1560 175.00
Porphyromonas_sp_oral_taxon_278 Alloprevotella_rava Prevotella_saccharolytica
Prevotella_oralis
Alloprevotella_unclassified
0.3500 251.00
 Differentially abundant features Significance Fold change Prevalence shift

Veillonella atypica
Haemophilus parainfluenzae

Prevotella melaninogenica

Rothia mucilaginosa

Veillonella unclassified

Veillonella parvula

Prevotella histicola

Veillonella dispar

Megasphaera micronuciformis

Streptococcus parasanguinis

Prevotella salivae

Prevotella pallens

Alloprevotella tannerae

Streptococcus salivarius
Baseline cluster 1
Baseline cluster 2
Porphyromonas endodontalis

Prevotella nigrescens

Granulicatella unclassified

Prevotella veroralis

Prevotella oris

Neisseria flavescens

Prevotella nanceiensis

Campylobacter concisus

Fusobacterium nucleatum

Actinomyces graevenitzii

Capnocytophaga unclassified

Streptococcus infantis

Alloprevotella rava

Haemophilus haemolyticus

(í (í (í (í (í (í (  2 4  í í         

Abundance ORJíVFDOH íORJDGMS YDOXH Generalized fold change Prevalence [%]
 Cluster 1 Baseline Cluster 2 Baseline p-value Rand index
Cluster 1 Longitudinal 33 (91.7%) 4 (40%) .0014 0.736
Cluster 2 Longitudinal 3 (8.3%) 6 (60%)
'( '+

•••••• !"!#$•••%&• •••••• !!,-&.%••/%&$•

"* 0
•••••• !)!#$•••%&• •••••• !)!,-&.%••/%&$•
 Subjects screened
Screen failure n= 94
n=730 Adverse events n= 4
Lost to follow up n=2
Withdrew consent n=7
Participated in another study
n=1
Subjects enrolled Other reasons n=12
n=610

Severe asthma Mild/moderate asthma Healthy controls

n=421 n=88 n=101

Induced sputum Induced sputum

n=181 n=43

Quality suitable for Quality suitable for

metagenomic analysis metagenomic analysis
n=100 n=24

Baseline Baseline
n=100 n=24

Follow-up
n=46
 Hierarchical cluster dendogram

Longitudinal clusters
Partition around the medoids (PAM)
 Alpha diversity measure

Longitudinal clusters
 Longitudinal cluster 1

Longitudinal cluster 2
Bacterial species count Bacterial species count

Selenomonas_flueggei
Selenomonas_sputigena
Eubacterium_yurii
Peptostreptococcaceae_noname
unclassified Peptostreptococcus_unclassified
Peptostreptococcus
Clostridiales_Family_XIII_Incertae_Sedis_unclassified
Eubacterium_infirmum
Peptostreptococcus_stomatis
Clostridiales_Family_XIII_Incertae_Sedis_noname
Clostridiales_Family_XIII_Incertae_Sedis
Eubacteriaceae_bacterium_CM5
Eubacteriaceae_bacterium_OBRC8
Eubacteriaceae_noname
Eubacteriaceae
Eubacteriaceae_bacterium_ACC19a
Eubacteriaceae_noname_unclassified Eubacterium
Eubacterium_saphenum
Eubacterium_brachy
Lachnospiraceae_bacterium_oral_taxon_082
Lachnospiraceae_noname
Shuttleworthia
Lachnoanaerobaculum
Lachnospiraceae_bacterium_ICM7
Shuttleworthia_satelles
Lachnoanaerobaculum_saburreum
Granulicatella_unclassified Granulicatella
Lactococcus_lactis
Lactococcus
Streptococcus_tigurinus
Streptococcus_vestibularis
Streptococcus_salivarius
Streptococcus_australis
Streptococcus_thermophilus
Streptococcus_peroris
Streptococcus
Streptococcus_oligofermentans
Streptococcus_sp_BS35b
Streptococcus_sanguinis
Streptococcus_mitis_oralis_pneumoniae
Streptococcus_gordonii
Streptococcus_anginosus
Streptococcus_parasanguinis
Streptococcus_infantis
Lactobacillus_fermentum
Lactobacillus_ultunensis
Neisseria_unclassified
Neisseria_sicca Haemophilus_influenzae
Neisseria_elongata Haemophilus_sputorum
Neisseria_polysaccharea Haemophilus_parainfluenzae
Neisseria_flavescens
Kingella_oralis Haemophilus_paraphrohaemolyticus
Neisseria_bacilliformis Neisseria_sp_oral_taxon_014
Haemophilus
Polaromonas_unclassified Kingella_unclassified Neisseria_cinerea Haemophilus_parahaemolyticus
Fusobacterium_necrophorum Neisseria Haemophilus_haemolyticus Aggregatibacter_unclassified
Aggregatibacter_segnis
Neisseria_meningitidis Neisseria_lactamica
Fusobacterium_nucleatum Kingella
Fusobacterium_periodonticum Variovorax_unclassified Aggregatibacter
Polaromonas Neisseria_subflava Haemophilus_pittmaniae Actinobacillus_unclassified
Veillonella_dispar Aggregatibacter_aphrophilus
Neisseria_macacae Neisseria_gonorrhoeae Pantoea_agglomerans
Veillonella_unclassified Fusobacterium Kingella_denitrificans Actinobacillus
Veillonella_atypica Eggerthia_catenaformis Variovorax Pantoea_unclassified
Alicycliphilus_denitrificans Pasteurellaceae
Neisseriaceae Simonsiella_muelleri
Moraxella_catarrhalis Cardiobacterium_hominis
Veillonella Eggerthia Leptotrichia_unclassified
Alicycliphilus
Simonsiella Acinetobacter_lwoffii Pantoea
Veillonella_parvula
Solobacterium_moorei Comamonadaceae Acinetobacter_unclassified
Comamonas Pseudomonas_unclassified
Moraxella Escherichia_unclassified
Selenomonas_infelix Eikenella
Solobacterium Lautropia_mirabilis Acinetobacter Cardiobacterium_valvarum Agrobacterium_unclassified
Leptotrichia_shahii
Megasphaera_micronuciformis Leptotrichia Comamonas_unclassified Pseudomonas_aeruginosa
Selenomonas_artemidis Lautropia unclassified Eikenella_corrodens
Pseudomonas Cardiobacterium Escherichia
Mitsuokella_unclassified
Erysipelotrichaceae Fusobacteriaceae Moraxellaceae Enterobacteriaceae
Agrobacterium_tumefaciens
Centipeda_periodontii Gallionellaceae_unclassified Agrobacterium
Megasphaera_unclassified Leptotrichia_wadei
Pseudomonadaceae
Selenomonas Megasphaera
Burkholderiaceae Stenotrophomonas
Mitsuokella
Centipeda
Neisseriales unclassified
Brevundimonas_unclassified
Bulleidia
Burkholderiales Pasteurellales Cardiobacteriaceae_unclassified
Cardiobacteriaceae Shinella_zoogloeoides
Selenomonas_noxia Gallionellaceae Stenotrophomonas_maltophilia
Leptotrichiaceae Pseudomonadales Shewanella_baltica Hyphomicrobiaceae_unclassified Shinella
Veillonellaceae Xanthomonadaceae unclassified
Brevundimonas_diminuta
Dialister_micraerophilus Fusobacteriales Rhizobiaceae
Bulleidia_extructa Gallionellales Hyphomicrobiaceae
Sinorhizobium_unclassified
Brevundimonas
Dialister Ochrobactrum_anthropi
Erysipelotrichales Xanthomonadales Cardiobacteriales Shewanella
Leptotrichiaceae_unclassified Sinorhizobium
Enterobacteriales
Anaeroglobus Betaproteobacteria Ochrobactrum Campylobacter_showae
Campylobacter_rectus
Dialister_invisus Brucellaceae
Gammaproteobacteria Caulobacteraceae
Peptostreptococcaceae unclassified
Anaeroglobus_geminatus Shewanellaceae Campylobacter Treponema_denticola
Filifactor
Parvimonas_micra Selenomonadales Alteromonadales RhizobialesCaulobacterales Campylobacter_concisus
Campylobacteraceae Treponema_maltophilum
Campylobacter_curvus
Filifactor_alocis Fusobacteriia
Alphaproteobacteria Campylobacter_gracilis
Treponema_socranskii
Parvimonas Treponema
Clostridiales_Family_XI_Incertae_Sedis
Erysipelotrichia Proteobacteria Campylobacterales Desulfovibrio_desulfuricans
Desulfovibrio
Treponema_lecithinolyticum
Epsilonproteobacteria
Parvimonas_unclassified
Spirochaetaceae Treponema_medium
Clostridiales Treponema_vincentii
DesulfovibrionaceaeBilophila_unclassified
Catonella_morbi Negativicutes Deltaproteobacteria
Desulfovibrionales Bilophila
Fusobacteria candidate_division_TM7_single_cell_isolate_TM7c
Catonella Clostridia Candidatus_Saccharibacteria_noname Jonquetella_unclassified unclassified
Spirochaetales Candidatus_Saccharibacteria_noname_unclassified
Spirochaetia candidate_division_TM7_single_cell_isolate_TM7b
Lachnospiraceae
Stomatobaculum
Stomatobaculum_longum
Firmicutes Spirochaetes
Candidatus_Saccharibacteria_noname
Candidatus_Saccharibacteria_noname
Synergistales
Jonquetella
Candidatus_Saccharibacteria Jonquetella_anthropi
Synergistaceae Actinomyces_cardiffensis
Bacteria
Oribacterium Synergistia Candidatus_Saccharibacteria_noname
Oribacterium_sinus Synergistetes Actinomyces_odontolyticus
Pyramidobacter
Fretibacterium Actinomyces_viscosus
Oribacterium_sp_oral_taxon_078 Abiotrophia_defectiva
Tenericutes Fretibacterium_fastidiosum
Actinomyces Actinomyces_graevenitzii
Pyramidobacter_piscolens
Granulicatella_adiacens Abiotrophia
Mollicutes Mycoplasma
Mycoplasma_pneumoniae Actinomycetaceae Actinomyces_oris
Mycoplasmatales Mycoplasmataceae Actinomyces_naeslundii Actinomyces_sp_ICM47
Aerococcaceae
Carnobacteriaceae Actinobacteria Gordonia_terrae
Granulicatella_elegans
Bacilli Gordonia
Gordoniaceae Rothia_aeria
Lactobacillales
Streptococcaceae Bacillus_cereus_thuringiensis Actinobacteria Actinomycetales Micrococcaceae
Rothia_mucilaginosa
Streptococcus_constellatus Atopobium_parvulum Rothia
Actinomycetales_noname
Streptococcus_agalactiae Atopobium_vaginae
Bacillus Bacteroidetes_noname unclassified
Rothia_dentocariosa
Streptococcus_intermedius
Bacteroidetes Atopobium_rimae
Nocardioidaceae Propionibacteriaceae Rothia_unclassified
Nodes
Bacteroidetes_noname Tropheryma Propionibacteriaceae_unclassified
Bacillaceae
Atopobium
Streptococcus_pseudopneumoniae Coriobacteriales Corynebacteriaceae Propionibacterium_acidipropionici
Lactobacillaceae Flavobacteriia Bifidobacteriales
Streptococcus_cristatus Bacillales Bacteroidetes_noname
Tropheryma_whipplei Corynebacterium_durum
Nocardioides Propionibacterium_granulosum
1.00
−0.3500
%DFWHULDOVSHFLHV count
Bacillales_noname Cryptobacterium_curtum
Corynebacterium Propionibacterium
Bacteroidetes_oral_taxon_274
Gemella_morbillorum Coriobacteriaceae Corynebacterium_tuberculostearicum
Bacteroidetes_noname Cryptobacterium Nocardioides_unclassified Corynebacterium_matruchotii
Propionibacterium_propionicum
Flavobacteriales Bacteroidia
median difference
Scardovia_wiggsiae
Corynebacterium_pseudodiphtheriticum Propionibacterium_acnes
Gemella_haemolysans
Gemella Bacteroidetes_bacterium_oral_taxon_272 Slackia
Lactobacillus
Gemella_unclassified
Staphylococcaceae
Gemella_sanguinis
Chryseobacterium_gleum
Scardovia Bifidobacteriaceae
Scardovia_unclassified Alloscardovia
Slackia_exigua
Slackia_unclassified
−0.1560 7.94
Lactobacillus_salivarius Chryseobacterium Alloscardovia_omnicolens
Chryseobacterium_unclassified Olsenella
Flavobacteriaceae Parascardovia
Staphylococcus_epidermidis
Staphylococcus_saprophyticus Riemerella
Parascardovia_denticolens Bifidobacterium
Olsenella_unclassified
Olsenella_uli
−0.0389 28.80
Staphylococcus Tannerella_forsythia Bacteroidales Prevotella_sp_C561
Prevotella_dentalis
Tannerella Prevotella_oris
Riemerella_unclassified
Staphylococcus_aureus Bifidobacterium_dentium
Staphylococcus_haemolyticus
Staphylococcus_hominis
Capnocytophaga_ochracea
Porphyromonadaceae
Prevotella_oulorum
Prevotella_maculosa
Prevotella_pallens
Prevotella_salivae
Prevotella_marshii
Prevotella_denticola
Bifidobacterium_longum
0.0000 63.50
Capnocytophaga_granulosa Prevotella_loescheii Prevotella_histicola
Porphyromonas_asaccharolytica Prevotella_multiformis
Capnocytophaga Prevotella_buccae
Capnocytophaga_unclassified
Porphyromonas_uenonis
Capnocytophaga_sp_oral_taxon_329
Porphyromonas_sp_oral_taxon_279
Porphyromonas
Prevotellaceae
Prevotella_micans
Prevotella_intermedia
Prevotella Prevotella_bivia
Prevotella_veroralis
Prevotella_melaninogenica
0.0389 112.00
Capnocytophaga_gingivalis Porphyromonas_gingivalis Prevotella_multisaccharivorax Candidatus_Prevotella_conceptionensis
Prevotella_sp_oral_taxon_473
Porphyromonas_endodontalis Prevotella_baroniae
Porphyromonas_gulae
Porphyromonas_catoniae
Alloprevotella
Alloprevotella_tannerae
Prevotella_nigrescens Prevotella_disiens
Prevotella_nanceiensis
Prevotella_pleuritidis
0.1560 175.00
Porphyromonas_sp_oral_taxon_278 Alloprevotella_rava Prevotella_saccharolytica
Prevotella_oralis
Alloprevotella_unclassified
0.3500 251.00
 Differentially abundant features Significance Fold change Prevalence shift

Veillonella atypica

Prevotella melaninogenica

Haemophilus parainfluenzae

Rothia mucilaginosa

Veillonella parvula

Streptococcus mitis oralis pneumoniae

Longitudinal cluster 1
Veillonella unclassified Longitudinal cluster 2

Neisseria unclassified

Veillonella dispar

Prevotella salivae

Streptococcus parasanguinis

Streptococcus salivarius

Rothia dentocariosa

(í (í (í4 (í3 (í (í (     í í         

$EXQGDQFH ORJíVFDOH íORJDGMS YDOXH *HQHUDOL]HGIROG FKDQJH 3UHYDOHQFH [%]

Alpha diversity measure

Time points (12-18 months window)

 H
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Longitudinal
 Hierarchical cluster dendogram
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Baseline clusters
Partition around the medoids (PAM)

Mild-
-mo
Mild-moderate group
 Severe
asthmatics

Induced
sputum

V4 16s rRNA Shotgun

sequencing metagenomics

ɴ-diversity ɴ-diversity
Unsupervised approach

Clustering with Check clustering agreement Clustering with

estimating optimum estimating optimum
number of clusters Would the limited 16s rRNA method provide number of clusters
similar patient clustering information as
compared with metagenomics?

Clustering validation

Obtained clusters Check clusters differences

and phenotype In what clinical and microbiota aspects do
discovery these clusters differ?

Follow-up Incorporate mild-

induced sputum to-moderate
samples after asthmatics
12-18 months
Check clusters robustness
Does inclusion of mild-moderate asthmatics
fall within the same clustering solution or
result in clusters disintegration?
Repeated analysis
as baseline samples

Check overtime clusters stability

Do the patients migrate between baseline
and longitudinal clusters?
Within-cluster sum of squares Average silhouettes width Calinski-Harabasz index

2 optimum number 2 optimum number 2 optimum number

of clusters of clusters of clusters

Number of clusters Number of clusters

Number of clusters
Consensus matrix = 2 Consensus cumulative distribution function (CDF)

Metric: baseline microbiome

Bray-Curtis dissimilarity measure
 Clusters ● Cluster 1 ● Cluster 2
A Baseline clusters
Sputum neutrophils (%) Sputum macrophages (%) FEV1 pre−salbutamol (% pred) FEV1 post−salbutamol (% pred)
100 ● ● 1.6e−05 ●
●● ●●● ● 4.9e−05 125 ●● 0.03 125 ● ● 0.0087
● ●
●●
● ●● ● ● ●
● ● ●●
●● ● ● ●● ● ●
● ●
75 ●●
● ●
● ●
●●
●
●
75 ●
●● ● 100 ●
● 100 ●
●●
●
●●●
● ●
● ● ● ● ● ●● ● ●● ● ●
●
●● ● ●
● ● ● ● ●● ●
● ●● ● ●●
● ● ●●● ● ●●●
● ●●
● ● ● ●●
●●● ●●● ●● ●● ● ●●● ●
● ●
● ● ●● ●
●
● ●
●
50 ●●
●● ●
● ● ● ● ● 75 ●● ●
●●●●●●
●●
● ●●
●● ● 75 ● ●● ●●● ●
●
●
●

50 ● ●●●●●●
● ●
●
●
●
●●
●
●
●●●●●
●
●
●●
● ●●
●●
● ●●● ●●
●● ●
● ●
● ● ●● ● ● ●● ● ●●● ●● ●● ●
●● ●● ●● ●●
● ● ● ● ●●●
● ●● ●● ●
● ● ●●
25
●
●●
●
●
● ●●
● ●● 50 ● ●●
●
● ●
●
● 50 ●●
● ●●
● ● ●●
Clinical characteristics

● ● ● ● ●● ●
● ●● ● ● ● ●● ● ●●
25 ●
●●● ●
● ●
●
●●●●● ● ●●●
●● ● ●
●
●● ●
●●● ●
●
● ●
● ●
●●
● ●
● ●●
●
● ● ●● ●● ● ● ● ● ●
● ● ●● ●
●●●
●● ●●● ●●
●
● ●
●●
●●
●●
●
●●
● 25 ●●
●
25 ● ●
●
0 ● ● ● ● ●

Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 1 Cluster 2

Value

FEV1/FVC pre−salbutamol (% pred) FEV1/FVC post−salbutamol (% pred) Age of asthma onset (years)
0.03 ●
● ●
0.012 ● ●●
● ● 0.012
● ● ● ● ●
100 ● 100 60 ●●
● ●● ●
●● ● ● ●●
● ●●
●
● ● ● ●●● ● ● ●
●●
●
● ● ● ●
● ●
●●●● ●●● ●● ●● ●●●● ●● ●
● ● ● ● ●
●● ●● ● ● ●● ●●●
●● ● ●● ● ●
80 ●●
●●●●●
● ●
80 ●●●● ●●● ●
●
●
● 40 ●● ●
●● ●
● ● ●
●
●
●
●
●
●● ● ● ●●
● ● ●●
● ● ● ●● ●
● ● ● ●
● ●● ●● ● ● ● ● ● ●●
●
●
● ●
● ●
● ●● ● ● ● ● ●
● ●●●
●● ● ● ●● ● ●
●
● ●
● ● ●●
●●
● ●● ● ●● ●
●
●● ●
● ● ●● ●
60 ●●●
●●● ●
●● ●● ● ●
60 ●
● ● ●
20 ●●
●
●
●
●
●
● ●
● ●
●
● ● ● ● ●● ● ●
● ●
● ● ● ● ● ●
● ●
● ● ● ● ● ● ●● ●
●
● ● ●●
40 ●
●● ● ● 40 ●●
●
0
●● ●● ●●
●
● ● ●●
●

Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 1 Cluster 2

B Longitudinal clusters
Sputum neutrophils (%) Sputum macrophages (%) Blood neutrophils (%) FEV1 pre−salbutamol (% pred)
100 ● 0.0017 80 ● ● 3e−05 0.02 ● ● 0.036
●
●
●
90 ●
● ●● ● ●
●● ●● ● ● ● ●
●

75 ●
●
●●
●
● 60 ●
●
● 80 ● ● ●
●
● ●
● ● 80 ●
●
●
● ●
●●

●● ● ● ● ●● ● ●
● ● ● ● ● ●● ●
●●
●
●
● ●
40
● ● 70 ● ● ●
● ●
●

50
●● ●
●● ● ● ● ●● ●
● ●●
●
●
● ●
●
60 ● ●
● ●●●● ●● ●
● ● ●●
● ●
●●
●● ● 60 ● ● ●
●
● ● ● ● ●● ● ● ● ●
●● ● ●
●●● ● ●●
20 ●
Clinical characteristics

● ● ● ● ● ●
25 ●
●
●
●● ● ●
● ● ●
●
●●
50 ●
●
●
● 40 ●
● ● ●● ● ●
●
●● ● ● ● ●
●● 0 ● ● ●●
40
● ● ●

Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 1 Cluster 2 Cluster 1 Cluster 2

Value

FEV1 post−salbutamol (% pred)

●
●
0.036
100 ●
●●
● ● ●
●● ●●
● ●●
● ●●
75 ●●
●
●●
● ●
●● ●
● ● ● ●
●

●● ●
50 ●● ●
● ●
●
●
●
●

Cluster 1 Cluster 2

Microbiome−driven clustersDWEDVHOLQHDQGIROORZXSYLVLWV
 Baseline cluster 1

Baseline cluster 2
Bacterial species count Bacterial species count
 0.26 0.24 0.25 0.23 Veillonella atypica
* * * *
−0.20 0.22 0.25 0.27 0.25 0.22 Prevotella histicola
* * * ** * *
−0.23 0.21 0.24 0.26 0.24 0.22 0.28 Megasphaera micronuciformis
* * * ** * * **
−0.29 0.42 0.26 Streptococcus parasanguinis
** **** **
−0.38 0.27 0.22 0.30 0.26 −0.36 Prevotella melaninogenica
*** ** * ** ** **
−0.30 0.38 0.25 0.27 0.20 0.23 Veillonella parvula
** **** * ** * *
−0.21 0.24 0.22 0.24 0.25 0.27 Actinomyces graevenitzii
* * * * * **
−0.26 0.34 0.20 0.25 0.20 0.27 Prevotella salivae
** *** * * * **
−0.27 0.37 0.23 0.25 Fusobacterium nucleatum
** *** * *
0.33 0.21 Campylobacter concisus
*** *
−0.25 0.22 0.27 Veillonella dispar
* * **
−0.39 0.30 0.21 0.23 Granulicatella unclassified
**** ** * *
−0.32 0.26 0.32 0.25 Veillonella unclassified
** ** ** *
0.20 0.23 Prevotella veroralis
* *
0.22 0.25 Alloprevotella rava
* *
−0.25 0.30 0.21 0.25 0.29 Prevotella pallens
* ** * * **
−0.33 0.26 0.26 0.23 Haemophilus parainfluenzae
*** ** ** *
−0.43 0.44 0.23 0.25 0.28 −0.20 Rothia mucilaginosa
**** **** * * ** *
−0.37 0.43 0.21 0.22 Prevotella nigrescens
*** **** * *
−0.33 0.37 Prevotella nanceiensis
*** ***
−0.38 0.38 0.20 Porphyromonas endodontalis
*** **** *
−0.41 0.43 Alloprevotella tannerae
**** ****
−0.24 0.25 −0.25 Streptococcus infantis
* * *
0.25 −0.24 Prevotella oris
* *
0.30 0.30 Streptococcus salivarius
** **
−0.21 0.31 Neisseria flavescens
* **
0.27 Haemophilus haemolyticus
**
−0.34 0.35 Capnocytophaga unclassified
*** ***
0.23 −0.25 −0.29 Haemophilus influenzae
* * **
0.27 Moraxella catarrhalis
**
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Within-cluster sum of squares Average silhouettes width Calinski-Harabasz index

2 optimum number 2 optimum number 2 optimum number

of clusters of clusters of clusters

Number of clusters Number of clusters

Number of clusters
Consensus matrix = 2 Consensus cumulative distribution function (CDF)

Metric: longitudinal microbiome

Bray-Curtis dissimilarity measure
 U-BIOPRED
The U-BIOPRED consortium wishes to acknowledge the help and expertise of the following
individuals and groups without whom, the study would not have been possible:
------------------
Definition for the U-BIOPRED Study Group Supplementary authors
Clinical site research leads
Platform leads
Data cleaning team
Data analysis team
Scientific Board and Management Board members
Core project management staff

Definition for the U-BIOPRED Study Group Contributors list

Significant involvement in the clinical study

Use of list:
This list is to be used for all non-core clinical papers.

Instructions
Follow up clinical papers should re-use the baseline cohort description paper lists, in order to recognize
the clinical staff involved in the study.

U-BIOPRED Supplementary authors

Name Affiliation
Adcock I M National Heart and Lung Institute, Imperial College, London, UK;
Ahmed H European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France;
Auffray C European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France;
Bakke P Department of Clinical Science, University of Bergen, Bergen,
Norway;
Bansal A T Acclarogen Ltd, St. John’s Innovation Centre, Cambridge, UK;
Baribaud F Janssen R&D, LLC, Spring House, PA, USA
Bates S Respiratory Therapeutic Unit, GSK, London, UK;
Bel E H Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Bigler J Previously Amgen Inc
Bisgaard H COPSAC, Copenhagen Prospective Studies on Asthma in
Childhood, Herlev and Gentofte Hospital,
University of Copenhagen, Copenhagen, Denmark
Boedigheimer M J Amgen Inc.; Thousand Oaks, USA
Bønnelykke K COPSAC, Copenhagen Prospective Studies on Asthma in
Childhood, Herlev and Gentofte
Hospital, University of Copenhagen, Copenhagen, Denmark;
Brandsma J University of Southampton, Southampton, UK
Brinkman P Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Bucchioni E Chiesi Pharmaceuticals SPA, Parma, Italy
Burg D Centre for Proteomic Research, Institute for Life Sciences,
University of Southampton, Southampton, UK
Bush A National Heart and Lung Institute, Imperial College, London, UK;
Royal Brompton and Harefield NHS trust, UK
Caruso M Dept. Clinical and Experimental Medicine, University of Catania,
Catania, Italy;
Chaiboonchoe A European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France;
Chanez P Assistance publique des Hôpitaux de Marseille - Clinique des
bronches, allergies et sommeil, Aix Marseille Université, Marseille,
France
Chung F K National Heart and Lung Institute, Imperial College, London, UK;
Compton C H Respiratory Therapeutic Unit, GSK, London, UK
Corfield J Areteva R&D, Nottingham, UK;
D’Amico A University of Rome ‘Tor Vergata’, Rome Italy;
Dahlén B Karolinska University Hospital & Centre for Allergy Research,
Karolinska Institutet, Stockholm, Sweden
Dahlén S E Centre for Allergy Research, Karolinska Institutet, Stockholm,
Sweden
De Meulder B European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France;
Djukanovic R NIHR Southampton Respiratory Biomedical Research Unit and
Clinical and Experimental Sciences, Southampton, UK;
Erpenbeck V J Translational Medicine, Respiratory Profiling, Novartis Institutes for
Biomedical Research, Basel, Switzerland;
Erzen D Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Fichtner K Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Fitch N BioSci Consulting, Maasmechelen, Belgium;
Fleming L J National Heart and Lung Institute, Imperial College, London, UK;
Royal Brompton and Harefield NHS trust, UK
Formaggio E Previously CROMSOURCE, Verona Italy
Fowler S J Division of infection, immunity and respiratory medicine, School of
biological sciences, University of Manchester, Manchester
University NHS Foundation Trust, Manchester Academic Health
Science Centre, Manchester, United Kingdom
Frey U University Children’s Hospital, Basel, Switzerland;
Gahlemann M Boehringer Ingelheim (Schweiz) GmbH,Basel, Switzerland;
Geiser T Department of Respiratory Medicine, University Hospital Bern,
Switzerland;
Goss V NIHR Respiratory Biomedical Research Unit, University Hospital
Southampton NHS Foundation Trust, Integrative Physiology and
Critical Illness Group, Clinical and Experimental Sciences, Sir Henry
Wellcome Laboratories, Faculty of Medicine, University of
Southampton, Southampton, UK;
Guo Y Data Science Institute, Imperial College, London, UK;
Hashimoto S Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Haughney J International Primary Care Respiratory Group, Aberdeen, Scotland;
Hedlin G Dept. Women’s and Children’s Health & Centre for Allergy
Research, Karolinska Institutet, Stockholm, Sweden;
Hekking P W Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Higenbottam T Allergy Therapeutics, West Sussex, UK;
Hohlfeld J M Fraunhofer Institute for Toxicology and Experimental Medicine,
Hannover, Germany
Holweg C Respiratory and Allergy Diseases, Genentech, San Francisco, USA
Horváth I Semmelweis University, Budapest, Hungary
Howarth P NIHR Southampton Respiratory Biomedical Research Unit, Clinical
and Experimental Sciences and Human Development and Health,
Southampton, UK
James A J Centre for Allergy Research, Karolinska Institutet, Stockholm,
Sweden;
Knowles RG Knowles Consulting Ltd, Stevenage. UK;
Knox A J Respiratory Research Unit, University of Nottingham, Nottingham,
UK;
Krug N Fraunhofer Institute for Toxicology and Experimental Medicine,
Hannover, Germany;
Lefaudeux D European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France;
Loza M J Janssen R&D, LLC, Spring House, PA, USA
Lutter R Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Manta A Roche Diagnostics GmbH, Mannheim, Germany
Masefield S European Lung Foundation, Sheffield, UK;
Matthews J G Respiratory and Allergy Diseases, Genentech, San Francisco, USA;
Mazein A European Institute for Systems Biology and Medicine, CNRS-ENS-
UCBL-INSERM, Lyon, France
Meiser A Data Science Institute, Imperial College, London, UK
Middelveld R J M Centre for Allergy Research, Karolinska Institutet, Stockholm,
Sweden
Miralpeix M Almirall, Barcelona, Spain;
Montuschi P Università Cattolica del Sacro Cuore, Milan, Italy;
Mores N Università Cattolica del Sacro Cuore, Milan, Italy;
Murray C S Division of infection, immunity and respiratory medicine, School of
biological sciences, University of Manchester, Manchester
University NHS Foundation Trust, and Manchester Academic Health
Science Centre, Manchester, United Kingdom
Musial J Dept. of Medicine, Jagiellonian University Medical College, Krakow,
Poland
Myles D Respiratory Therapeutic Unit, GSK, London, UK;
Pahus L Assistance publique des Hôpitaux de Marseille, Clinique des
bronches, allergies et sommeil
Espace Éthique Méditerranéen, Aix-Marseille Université, Marseille,
France;
Pandis I Data Science Institute, Imperial College, London, UK
Pavlidis S National Heart and Lung Institute, Imperial College, London, UK
Postle A University of Southampton, UK
Powel P European Lung Foundation, Sheffield, UK;
Praticò G CROMSOURCE, Verona, Italy
Puig Valls M CROMSOURCE, Barcelona, Spain
Rao N Janssen R&D, LLC, Spring House, PA, USA
Riley J Respiratory Therapeutic Unit, GSK, London, UK;
Roberts A Asthma UK, London, UK;
Roberts G NIHR Southampton Respiratory Biomedical Research Unit, Clinical
and Experimental Sciences and Human Development and Health,
Southampton, UK;
Rowe A Janssen R&D, UK;
Sandström T Dept of Public Health and Clinical Medicine, Umeå University,
Umeå, Sweden;
Schofield JPR Centre for Proteomic Research, Institute for Life Sciences,
University of Southampton, Southampton, UK
Seibold W Boehringer Ingelheim Pharma GmbH, Biberach, Germany
Selby A NIHR Southampton Respiratory Biomedical Research Unit, Clinical
and Experimental Sciences and Human Development and Health,
Southampton, UK;
Shaw D E Respiratory Research Unit, University of Nottingham, UK;
Sigmund R Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Singer F Pediatric Respiratory Medicine, Department of Pediatrics,
Inselspital, Bern University Hospital, University of Bern, Bern,
Switzerland.
Skipp P J Centre for Proteomic Research, Institute for Life Sciences,
University of Southampton, Southampton, UK
Sousa A R Respiratory Therapeutic Unit, GSK, London, UK;
Sterk P J Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
Sun K Data Science Institute, Imperial College, London, UK
Thornton B MSD, USA
van Aalderen W M Academic Medical Centre, University of Amsterdam, Amsterdam,
The Netherlands;
van Geest M AstraZeneca, Mölndal, Sweden;
Vestbo J Centre for Respiratory Medicine and Allergy, Institute of
Inflammation and Repair, University of Manchester and University
Hospital of South Manchester, Manchester Academic Health
Sciences Centre, Manchester, United Kingdom
Vissing N H COPSAC, Copenhagen Prospective Studies on Asthma in
Childhood, Herlev and Gentofte Hospital,
University of Copenhagen, Copenhagen, Denmark;
Wagener A H Academic Medical Center Amsterdam, Amsterdam, The
Netherlands
Wagers S S BioSci Consulting, Maasmechelen, Belgium
Weiszhart Z Semmelweis University, Budapest, Hungary;
Wheelock C E Centre for Allergy Research, Karolinska Institutet, Stockholm,
Sweden;
Wilson S J Histochemistry Research Unit, Faculty of Medicine, University of
Southampton, Southampton, UK;

Contributors

Aliprantis Antonios, Merck Research Laboratories, Boston, USA;

Allen David, North West Severe Asthma Network, Pennine Acute Hospital NHS Trust, UK
Alving Kjell, Dept Women’s & Children’s Health, Uppsala University, Uppsala, Sweden
Badorrek P, Fraunhofer ITEM; Hannover, Germany
Balgoma David, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Ballereau S, European institute for Systems Biology and Medicine, University of Lyon, France
Barber Clair, NIHR Southampton Respiratory Biomedical Research Unit and Clinical and
Experimental Sciences, Southampton, UK;
Batuwitage Manohara Kanangana, Data Science Institute, Imperial College, London, UK
Bautmans An, MSD, Brussels, Belgium
Bedding A, Roche Diagnostics GmbH, Mannheim, Germany
Behndig AF, Umeå University, Umea, Sweden
Beleta Jorge, Almirall S.A., Barcelona, Spain;
Berglind A, MSD, Brussels, Belgium
Berton A, AstraZeneca, Mölndal, Sweden
Bochenek Grazyna, II Department of Internal Medicine, Jagiellonian University Medical
College, Krakow, Poland;
Braun Armin, Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover,
Germany;
Campagna D, Department of Clinical and Experimental Medicine, University of Catania,
Catania, Italy;
Carayannopoulos Leon, Previously at: MSD, USA;
Casaulta C, University Children’s Hospital of Bern, Switzerland
Chaleckis Romanas, Centre of Allergy Research, Karolinska Institutet, Stockholm, Sweden
Davison Timothy Janssen R&D, LLC, Spring House, PA, USA
De Alba Jorge, Almirall S.A., Barcelona, Spain;
De Lepeleire Inge, MSD, Brussels, BE
Dekker Tamara, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Delin Ingrid, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Dennison P, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and
Experimental Sciences, NIHR-Wellcome Trust Clinical Research Facility, Faculty of Medicine,
University of Southampton, Southampton, UK;
Dijkhuis Annemiek, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Dodson Paul, AstraZeneca, Mölndal, Sweden
Draper Aleksandra, BioSci Consulting, Maasmechelen, Belgium;
Dyson K, CROMSOURCE; Stirling, UK
Edwards Jessica, Asthma UK, London, UK;
El Hadjam L, European Institute for Systems Biology and Medicine, University of Lyon
Emma Rosalia, Department of Clinical and Experimental Medicine, University of Catania,
Catania, Italy;
Ericsson Magnus, Karolinska University Hospital, Stockholm, Sweden
Faulenbach C, Fraunhofer ITEM; Hannover, Germany
Flood Breda, European Federation of Allergy and Airways Diseases Patient’s Associations,
Brussels, Belgium
Galffy G, Semmelweis University, Budapest, Hungary;
Gallart Hector, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Garissi D, Global Head Clinical Research Division, CROMSOURCE, Italy
Gent J, Royal Brompton and Harefield NHS Foundation Trust, London, UK;
Gerhardsson de Verdier M, AstraZeneca; Mölndal, Sweden;
Gibeon D, National Heart and Lung Institute, Imperial College, London, UK;
Gomez Cristina, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Gove Kerry, NIHR Southampton Respiratory Biomedical Research Unit and Clinical and
Experimental Sciences, Southampton, UK;
Gozzard Neil, UCB, Slough, UK;
Guillmant-Farry E, Royal Brompton Hospital, London, UK
Henriksson E, Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden
Hewitt Lorraine, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Hoda U, Imperial College, London, UK
Hu Richard, Amgen Inc. Thousand Oaks, USA
Hu Sile, National Heart and Lung Institute, Imperial College, London, UK;
Hu X, Amgen Inc.; Thousand Oaks, USA
Jeyasingham E, UK Clinical Operations, GSK, Stockley Park, UK
Johnson K, Centre for respiratory medicine and allergy, Institute of Inflammation and repair,
University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK
Jullian N, European Institute for Systems Biology and Medicine, University of Lyon
Kamphuis Juliette, Longfonds, Amersfoort, The Netherlands;
Kennington Erika J., Asthma UK, London, UK;
Kerry Dyson, CromSource, Stirling, UK;
Kerry G, Centre for respiratory medicine and allergy, Institute of Inflammation and repair,
University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK
Klüglich M, Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Knobel Hugo, Philips Research Laboratories, Eindhoven, The Netherlands;
Kolmert Johan, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Konradsen J R, Dept. Women’s and Children’s Health & Centre for Allergy Research,
Karolinska Institutet, Stockholm, Sweden
Kots Maxim, Chiesi Pharmaceuticals, SPA, Parma, Italy;
Kretsos Kosmas, UCB, Slough, UK
Krueger L, University Children's Hospital Bern, Switzerland
Kuo Scott, National Heart and Lung Institute, Imperial College, London, UK;
Kupczyk Maciej, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Lambrecht Bart, University of Gent, Gent, Belgium;
Lantz A-S, Karolinska University Hospital & Centre for Allergy Research, Karolinska Institutet,
Stockholm, Sweden
Larminie Christopher, GSK, London, UK
Larsson L X, AstraZeneca, Mölndal, Sweden
Latzin P, University Children’s Hospital of Bern, Bern, Switzerland
Lazarinis N, Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden
Lemonnier N, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-
INSERM, Lyon, France
Lone-Latif Saeeda, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Lowe L A, Centre for respiratory medicine and allergy, Institute of Inflammation and repair,
University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK
Manta Alexander, Roche Diagnostics GmbH, Mannheim, Germany
Marouzet Lisa, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Martin Jane, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Mathon Caroline, Centre of Allergy Research, Karolinska Institutet, Stockholm, Sweden
McEvoy L, University Hospital, Department of Pulmonary Medicine, Bern, Switzerland
Meah Sally, National Heart and Lung Institute, Imperial College, London, UK;
Menzies-Gow A, Royal Brompton and Harefield NHS Foundation Trust, London, UK;
Metcalf Leanne, Previously at: Asthma UK, London, UK;
Mikus Maria, Science for Life Laboratory & The Royal Institute of Technology, Stockholm,
Sweden;
Monk Philip, Synairgen Research Ltd, Southampton, UK;
Naz Shama, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Nething K, Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Nicholas Ben, University of Southampton, Southampton, UK
Nihlén U, Previously AstraZeneca; Mölndal, Sweden;
Nilsson Peter, Science for Life Laboratory & The Royal Institute of Technology, Stockholm,
Sweden;
Niven R, North West Severe Asthma Network, University Hospital South Manchester, UK
Nordlund B, Dept. Women’s and Children’s Health & Centre for Allergy Research, Karolinska
Institutet, Stockholm, Sweden
Nsubuga S, Royal Brompton Hospital, London, UK
Östling Jörgen, AstraZeneca, Mölndal, Sweden;
Pacino Antonio, Lega Italiano Anti Fumo, Catania, Italy;
Palkonen Susanna, European Federation of Allergy and Airways Diseases Patient’s
Associations, Brussels, Belgium.
Pellet J, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM,
Lyon, France
Pennazza Giorgio, Unit of Electronics for Sensor Systems, Department of Engineering,
Campus Bio-Medico University of Rome, Rome, Italy
Petrén Anne, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Pink Sandy, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Pison C, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM,
Lyon, France
Rahman-Amin Malayka, Previously at: Asthma UK, London, UK;
Ravanetti Lara, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Ray Emma, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Reinke Stacey, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Reynolds Leanne, Previously at: Asthma UK, London, UK;
Riemann K, Boehringer Ingelheim Pharma GmbH & Co. KG; Biberach, Germany
Robberechts Martine, MSD, Brussels, Belgium
Rocha J P, Royal Brompton and Harefield NHS Foundation Trust
Rossios C, National Heart and Lung Institute, Imperial College, London, UK;
Russell Kirsty, National Heart and Lung Institute, Imperial College, London, UK;
Rutgers Michael, Longfonds, Amersfoort, The Netherlands;
Santini G, Università Cattolica del Sacro Cuore, Milan, Italy;
Santonico Marco, Unit of Electronics for Sensor Systems, Department of Engineering, Campus
Bio-Medico University of Rome, Rome, Italy
Saqi M, European Institute for Systems Biology and Medicine, CNRS-ENS-UCBL-INSERM,
Lyon, France
Schoelch Corinna, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
Scott S, North West Severe Asthma Network, Countess of Chester Hospital, UK
Sehgal N, North West Severe Asthma Network; Pennine Acute Hospital NHS Trust
Sjödin Marcus, Centre for Allergy Research, Karolinska Institutet, Stockholm, Sweden
Smids Barbara, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Smith Caroline, NIHR Southampton Respiratory Biomedical Research Unit, Southampton, UK
Smith Jessica, Asthma UK, London, UK;
Smith Katherine M., University of Nottingham, UK;
Söderman P, Dept. Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden
Sogbesan A, Royal Brompton and Harefield NHS Foundation Trust, London, UK;
Spycher F, University Hospital Department of Pulmonary Medicine, Bern, Switzerland
Staykova Doroteya, University of Southampton, Southampton, UK
Stephan S, Centre for respiratory medicine and allergy, Institute of Inflammation and repair,
University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK
Stokholm J, University of Copenhagen and Danish Pediatric Asthma Centre Denmark
Strandberg K, Karolinska University Hospital & Karolinska Institutet, Stockholm, Sweden
Sunther M, Centre for respiratory medicine and allergy, Institute of Inflammation and repair,
University Hospital of South Manchester, NHS Foundation Trust, Manchester, UK
Szentkereszty M, Semmelweis University, Budapest, Hungary;
Tamasi L, Semmelweis University, Budapest, Hungary;
Tariq K, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and Experimental
Sciences, NIHR-Wellcome Trust Clinical Research Facility, Faculty of Medicine, University of
Southampton, Southampton, UK;
Thörngren John-Olof, Karolinska University Hospital, Stockholm, Sweden
Thorsen Jonathan, COPSAC, Copenhagen Prospective Studies on Asthma in Childhood,
Herlev and Gentofte
Hospital, University of Copenhagen, Copenhagen, Denmark;
Valente S, Università Cattolica del Sacro Cuore, Milan, Italy;
van de Pol Marianne, Academic Medical Centre, University of Amsterdam, Amsterdam ,The
Netherlands;
van Drunen C M, Academic Medical Centre, University of Amsterdam, Amsterdam, The
Netherlands;
Van Eyll Jonathan, UCB, Slough, UK
Versnel Jenny, Previously at: Asthma UK, London, UK;
Vink Anton, Philips Research Laboratories, Eindhoven, The Netherlands;
von Garnier C, University Hospital Bern, Switzerland;
Vyas A, North west Severe Asthma Network, Lancashire Teaching Hospitals NHS Trust, UK
Wald Frans, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
Walker Samantha, Asthma UK, London, UK;
Ward Jonathan, Histochemistry Research Unit, Faculty of Medicine, University of
Southampton, Southampton, UK;
Wetzel Kristiane, Boehringer Ingelheim Pharma GmbH, Biberach, Germany
Wiegman Coen, National Heart and Lung Institute, Imperial College, London, UK;
Williams Siân, International Primary Care Respiratory Group, Aberdeen, Scotland;
Yang Xian, Data Science Institute, Imperial College, London, UK
Yeyasingham Elizabeth, UK Clinical Operations, GSK, Stockley Park, UK;
Yu W, Amgen Inc.; Thousand Oaks, USA
Zetterquist W, Dept. Women’s and Children’s Health & Centre for Allergy Research, Karolinska
Institutet, Stockholm, Sweden
Zolkipli Z, NIHR Southampton Respiratory Biomedical Research Unit, Clinical and
Experimental Sciences and Human Development and Health, Southampton, UK;
Zwinderman A H, Academic Medical Centre, University of Amsterdam, The Netherlands;

Partner organisations
Novartis Pharma AG University of Southampton, Southampton, UK

Academic Medical Centre, University of Imperial College London, London, UK

Amsterdam, Amsterdam, The Netherlands
University of Catania, Catania, Italy University of Rome ‘Tor Vergata’, Rome, Italy

Hvidore Hospital, Hvidore, Denmark Jagiellonian Univ. Medi.College, Krakow,

Poland
University Hospital, Inselspital, Bern, Semmelweis University, Budapest, Hungary
Switzerland
University of Manchester, Manchester, UK Université d’Aix-Marseille, Marseille, France

Fraunhofer Institute, Hannover, Germany University Hospital, Umea, Sweden

Ghent University, Ghent, Belgium Ctr. Nat. Recherche Scientifique, Lyon,

France

Università Cattolica del Sacro Cuore, Rome, University Hospital, Copenhagen, Denmark
Italy

Karolinska Institutet, Stockholm, Sweden Nottingham University Hospital, Nottingham,

UK

University of Bergen, Bergen, Norway Netherlands Asthma Foundation, Leusden,

NL

European Lung Foundation, Sheffield, UK Asthma UK, London, UK

European. Fed. of Allergy and Airways Lega Italiano Anti Fumo, Catania, Italy
Diseases Patients’ Associations, Brussels,
Belgium

International Primary Care Respiratory Philips Research Laboratories, Eindhoven,

Group, Aberdeen, Scotland NL

Synairgen Research Ltd, Southampton, UK Aerocrine AB, Stockholm, Sweden

BioSci Consulting, Maasmechelen, Belgium Almirall

AstraZeneca Boehringer Ingelheim

Chiesi GlaxoSmithKline
 Roche UCB

Janssen Biologics BV Amgen NV

Merck Sharp & Dome Corp

MEMBERS OF THE ETHICS BOARD

Name Task Affiliation e-mail
Jan-Bas Prins Biomedical research LUMC/the J.B.Prins@lumc.nl
Netherlands
Martina Gahlemann Clinical care BI/Germany Martina.Gahlemann@boehringer-
ingelheim.com

Luigi Visintin Legal affairs LIAF/Italy visintin@inrete.it

Hazel Evans Paediatric care Southampton/UK hazel.evans@uhs.nhs.uk

Martine Puhl Patient representation (co NAF/ the martine@puhl.nl

chair) Netherlands

Lina Buzermaniene Patient representation EFA/Lithuania lina.buzermaniene@pavb.lt

Val Hudson Patient representation Asthma UK hudsonval7@gmail.com

Laura Bond Patient representation Asthma UK lvbond22@googlemail.com

Pim de Boer Patient representation and IND deboer.pim@hetnet.nl

pathobiology

Research ethics VUMC/the g.widdershoven@vumc.nl

Guy Widdershoven Netherlands

Ralf Sigmund Research methodology and BI/Germany ralf.sigmund@boehringer-

biostatistics ingelheim.com

THE PATIENT INPUT PLATFORM

Name Country
UK
Amanda Roberts

UK
David Supple (chair)

The Netherlands
Dominique Hamerlijnck

Jenny Negus UK

Juliёtte Kamphuis The Netherlands

Lehanne Sergison UK

Luigi Visintin Italy

Pim de Boer (co-chair) The Netherlands

Susanne Onstein The Netherlands

MEMBERS OF THE SAFETY MONITORING BOARD

Name Task
William MacNee Clinical care

Renato Bernardini Clinical pharmacology

Paediatric care and

Louis Bont infectious diseases

Per-Ake Wecksell Patient representation

Pim de Boer Patient representation and

pathobiology (chair)

Martina Gahlemann Patient safety advice and

clinical care (co-chair)

Ralf Sigmund Bio-informatician