Title Niespodziana.pdf · 2018-11-12 · ARTICLE PreDicta chip-based high resolution diagnosis of...

Title:

“PreDicta chip-based high resolution

diagnosis of rhinovirus-induced

wheeze”

Niespodziana, K., Stenberg-Hammar, K., Megremis, S., Cabauatan, C.R., Napora-

Wijata, K., Vacal, P.C., Gallerano, D., Lupinek, C., Ebner, D., Schlederer, T.,

Harwanegg, C., Söderhäll, C., van Hage, M., Hedlin, G., Papadopoulos, N.G., and

R. Valenta

Nat Commun. 2018 Jun 18;9(1):2382.

Applicant’s full address: Katarzyna Niespodziana, PhD Division of Immunopathology Department of Pathophysiology and Allergy Research Center of Pathophysiology, Infectiology, and Immunology Medical University of Vienna Waehringer Guertel 18-20, 1090 Vienna, Austria

Phone: +43-1-40400-51110 Fax: +43-1-40400-51300 Email: [email protected]

ARTICLE

PreDicta chip-based high resolution diagnosis ofrhinovirus-induced wheezeKatarzyna Niespodziana1, Katarina Stenberg-Hammar2,3, Spyridon Megremis4, Clarissa R. Cabauatan1,

Kamila Napora-Wijata1, Phyllis C. Vacal 1, Daniela Gallerano1, Christian Lupinek1, Daniel Ebner5,

Thomas Schlederer5, Christian Harwanegg5, Cilla Söderhäll3, Marianne van Hage6, Gunilla Hedlin2,3,

Nikolaos G. Papadopoulos4,7 & Rudolf Valenta1

Rhinovirus (RV) infections are major triggers of acute exacerbations of severe respiratory

diseases such as pre-school wheeze, asthma and chronic obstructive pulmonary disease

(COPD). The occurrence of numerous RV types is a major challenge for the identification of

the culprit virus types and for the improvement of virus type-specific treatment strategies.

Here, we develop a chip containing 130 different micro-arrayed RV proteins and peptides and

demonstrate in a cohort of 120 pre-school children, most of whom had been hospitalized due

to acute wheeze, that it is possible to determine the culprit RV species with a minute blood

sample by serology. Importantly, we identify RV-A and RV-C species as giving rise to most

severe respiratory symptoms. Thus, we have generated a chip for the serological identifi-

cation of RV-induced respiratory illness which should be useful for the rational development

of preventive and therapeutic strategies targeting the most important RV types.

DOI: 10.1038/s41467-018-04591-0 OPEN

1 Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, MedicalUniversity of Vienna, A-1090 Vienna, Austria. 2 Astrid Lindgren Children’s Hospital, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.3 Department of Women’s and Children’s Health, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 4Division of Infection, Immunity & RespiratoryMedicine, University of Manchester, Manchester M13 9NT, UK. 5 Phadia Austria GmbH, Part of Thermo Fisher Scientific ImmunoDiagnostics, A-1220 Vienna,Austria. 6 Immunology and Allergy Unit, Department of Medicine Solna, Karolinska Institutet and University Hospital, SE-171 77 Stockholm, Sweden. 7 AllergyDepartment, 2nd Pediatric Clinic, University of Athens, 106 79 Athens, Greece. These authors contributed equally: Katarina Stenberg-Hammar, SpyridonMegremis. Correspondence and requests for materials should be addressed to N.G.P. (email: [email protected])or to R.V. (email: [email protected])

NATURE COMMUNICATIONS | (2018) 9:2382 | DOI: 10.1038/s41467-018-04591-0 |www.nature.com/naturecommunications 1

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http://orcid.org/0000-0003-3846-3812

http://orcid.org/0000-0003-3846-3812

http://orcid.org/0000-0003-3846-3812

http://orcid.org/0000-0003-3846-3812

http://orcid.org/0000-0003-3846-3812

mailto:[email protected]

mailto:[email protected]

www.nature.com/naturecommunications


Respiratory viral infections are among the most commontriggers of acute exacerbations of pre-school wheeze,asthma, and chronic obstructive pulmonary disease

(COPD)1–3. Asthma and COPD are severe and disabling diseasesof the respiratory tract and hence represent a serious global healthproblem affecting different age groups. Acute pre-school wheezeand community-acquired pneumonia (CAP) are other commoncauses of emergency visits with possible viral etiology. There is anincreasing prevalence of these airways diseases, rising treatmentcosts, and therefore virus-induced respiratory illnesses are a heavyburden for patients and the community4,5. Respiratory viralinfections, mainly due to rhinovirus (RV), are responsible forapproximately 80% of wheeze and asthma exacerbations inchildren6,7. Moreover, infants with rhinovirus-induced wheezehave a significantly increased risk for subsequent development ofrecurrent wheeze and childhood asthma8. Since exposure to RVdoes not lead to wheezing illness in all children, additional factorssuch as the host genotype, defects of the respiratory epithelialbarrier, and/or atopic predisposition have been suggested to playimportant roles in asthma9–11.

RV is genetically a highly diverse virus group with more than160 distinct RV types which have been divided into three distinctRV species, RV-A, RV-B, and RV-C12,13. Rhinoviruses can alsobe classified according to which cellular receptor on humanrespiratory epithelial cells they use for entry14. RV-B and mostRV-A variants bind to the intercellular adhesion molecule-1(ICAM-1) (i.e., major RV group), while a subset of RV-A speciesbinds to the low-density lipoprotein receptor (i.e., minor RVgroup)15,16. More recently, a cadherin-related family member 3protein (CDHR3) has been reported as a one of the probablereceptors for the RV-C species17.

The identification of the culprit rhinovirus species responsiblefor severe exacerbations of respiratory disease is an extremelyimportant topic as certain RV species (e.g., RV-C) are suspectedto be associated with more severe wheezing illnesses and acuteasthma exacerbations in infants and children compared toothers18,19. In fact, there are also several preventive and ther-apeutic strategies for RV infections under development whichrequire a precise knowledge of the clinically relevant RV speciesto be targeted. For example, several approaches for developingvaccines based on polyvalent inactivated RV, synthetic RV-derived peptides and recombinant RV proteins have beenreported20–25. The formulation of a broadly protective vaccineobviously requires the inclusion of the clinically most relevantand common RV species. Furthermore, it has been shown thatblocking of the viral receptor on respiratory epithelial cells (e.g.,ICAM-1) can prevent RV infection26. Again therapeuticapproaches targeting the viral receptors require knowledge whichRV species are the most frequent and relevant ones. Finally, it isimportant to investigate the role of the different RV species forexacerbations of severe bronchial obstruction in different popu-lations and for different age groups and manifestations ofrespiratory illness (e.g., pre-school wheeze, asthma, COPD,asthma-COPD overlap: ACO, CAP). While RV is well establishedas an important trigger factor for childhood wheeze and asthma,less is known regarding the role of RV infections in exacerbationsof COPD and in respiratory disease exacerbations of older sub-jects3. Furthermore, the causal relationship of RV with CAP isalso unknown27.Currently, the detection of RV in the course of respiratory

infections is mainly based on reverse transcription of viral RNAand DNA amplification by polymerase chain reaction (PCR)28.Such tests can demonstrate the presence of virus-derived nucleicacid but they do not necessarily indicate that the particular virushad caused an infection and is indeed responsible for clinicalsymptoms in the patient29. In fact, rhinovirus RNA has been

found in a high proportion of asymptomatic infants and chil-dren30–32. Furthermore, little is known about levels and epitope-specificities of natural antibody responses capable of neutralizingrhinoviruses, and thus protecting individuals against RV infec-tions. Such information would be helpful for the development ofnew immunological strategies for the treatment and prevention ofRV-induced exacerbations of respiratory diseases. Therefore,there is a huge and so far unmet need for high-resolution ser-ological detection of rhinovirus infections. We have previouslyidentified the capsid protein VP1 and an N-terminal VP1 peptideas major target for the natural antibody response of RV-infectedsubjects33. Then we have demonstrated that in vivo inoculation ofsubjects with RV16 indeed induced increases of VP1-specificantibody responses which were best detected with the VP1 pro-tein of the corresponding species33. Similar increases of RV-specific antibodies were found in pre-school children after asthmaattacks using complete recombinant RV capsid proteins34. In arecent study performed in pre-school children with acute asthmaattacks, we observed that increases of RV-specific antibodyresponses reflected the severity of respiratory symptoms35.

In the present study funded by the European Union project“PreDicta” (https://cordis.europa.eu/project/rcn/96868_en.html),we investigated if it is possible to generate a microarray-basedserological test which can discriminate RV-A, RV-B, and RV-C asculprit species involved in childhood asthma attacks.

ResultsDevelopment of a high-resolution PreDicta microarray. Fig-ure 1 shows the arrangement of RV-derived proteins, peptides aswell as control proteins on the PreDicta chip and the selectionprocedure for the N-terminal VP1 peptides. Recombinant capsidproteins (VP1-VP4) and fragments thereof representing RV-A, B,C species as well as non-structural proteins from RV89 wereincluded (Supplementary Tables 1–3). Based on our previousfinding that antibodies from RV-infected patients react pre-ferentially with the N-terminus of VP1 (ref. 33), we includedsynthetic N-terminal VP1 peptides from 30 RV strains whichwere selected in a rational, multistep process to represent distinctRV strains (Fig. 1a, Supplementary Table 2). Starting with 107VP1 sequences retrieved from the NCBI database (RV-A: 76; RV-B: 25; RV-C: 6), multiple sequence alignments were performed toidentify clusters of peptides with high degrees of sequence iden-tities (Fig. 1a, Supplementary Table 2: Clusters A1–A17; B1–B9;C1–C3). In a next step, peptides were re-clustered into groupstaking the chemical properties of amino acids into consideration(Supplementary Table 2: AI–AXVIII; BI–BIX; CI–CIII). Fromthese groups 30 peptides with the most distantly relatedsequences were selected (Fig. 1b, c; Supplementary Tables 2 and3). For a further refinement of VP1, VP2, and VP3 antibodyresponses, VP2 and VP3 fragments as well as peptides spanningthe complete VP1 sequence from RV89 were included (Fig. 1d,Supplementary Tables 4 and 5). For control and calibrationpurposes we added recombinant allergens and control proteins(Fig. 1d, Supplementary Table 6) to the PreDicta chip.Proteins and peptides were spotted in triplicates on a pre-

activated glass slide containing six microarrays surrounded by aTeflon frame so that one chip allows testing of six serum samples(Fig. 1d). Identities and purities of recombinant proteins andpeptides were tested by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) followed by Coomassie BrilliantBlue staining, western-blotting, and by mass spectrometry,respectively (Supplementary Tables 3–5, Supplementary Fig. 1).Supplementary Table 7 shows that the PreDicta microarrayallows reproducible measurement of IgG levels to the antigensaccording to intra- and inter-assay variations.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04591-0

2 NATURE COMMUNICATIONS | (2018) 9:2382 | DOI: 10.1038/s41467-018-04591-0 |www.nature.com/naturecommunications

https://cordis.europa.eu/project/rcn/96868_en.html


RV strain recognition is broader in older wheezing children.The PreDicta chip was tested with sera from 120 pre-schoolchildren who were admitted to the hospital due to an acutewheezing episode35. Table 1 summarizes demographic and clin-ical data of the children investigated in this study. To investigateif the spectrum of recognized RV peptides varies by age, children

were grouped according to age (Group I: <1 year, n= 35; GroupII: 1–2 years, n= 53; Group III: >2 years, n= 32). Figure 2a, bshow the frequency and intensity of IgG and IgA antibodyreactivity of the children to the 30 N-terminal VP1 peptidesrepresenting RV-A, RV-B, and RV-C species. The most frequentand highest IgG responses were directed to RV-C-derived

a

Retrieval of VP1 amino acid sequences from the NCBI databaseRV-A: n=76; RV-B: n=25; RV-C: n=6; EV=1

Multiple sequence alignment of VP1 N-terminal peptides (VP1-p1: aa 1–40) using Clustal W2

b

RV-25RV-29

RV-78RV-11RV-02RV-1BRV-18RV-43

RV-59RV-16RV-19

RV-12RV-20RV-28

RV-68RV-89

RV-YPRV-QPM

RV-c25RV-45

RV-08RV-17RV-70

RV-69RV-04RV-27

RV-84RV-05RV-14

RV-86EV-68

RV-A

RV-B

RV-C

EV

[%]01p1 : NPVENYIDEVLNEVLVVPNIKESHHTTSNSAPLLDAAETG : 100.002p1 : ....................NS.NP.......A....... : 87.518p1 : ...................VN...AI......A....... : 87.529p1 : ......V.............R...PS......I....... : 87.516p1 : ....R.V.............N...P....A..V....... : 85.025p1 : ..I...V.Q...............PS......I....... : 85.0

11p1 : ....D.V.GI.............QA.......A....... : 82.559p1 : ......VND...........Q...P....A..A....... : 82.543p1 : ......V..I..Q......TV... S....A..A.......: 80.078p1 : ....E.V.Q..............KPQS.....V....... : 80.019p1 : ....K.V.TI..........N...PS...A..A....... : 77.512p1 : ....R.V.............NK.NGQL..A..A....... : 75.0

89p1 : ........S...........QP.TSVS.HA..A....... : 75.068p1 : ....K.TEA...........PA.NTQ...A..A....... : 72.520p1 : ....R.TEAI..........TS.NSQ...A..A....... : 70.028p1 : ....K.TEAL..........NP.NAQ.T.A..A....... : 67.508p1 : ..I.QFTEA..........TQA.NGSIA....A....... : 62.545p1 : ....QFAEA..DQ......TRP.DGLIA....A....... : 60.0

YPp1 : ....D...K.VDT..Q...TQP.GPQH.IQPSA.G.M.I. : 47.5c025p1 : ....QFV.N..E.......TQP.GPIHTTKPTA.S.M.I. : 47.5QPMp1 : ....EFVEHT.K......DTQA.GPVHTTKPQA.G.V.I. : 40.0

84p1 : GLEDVLEEVIVDKAKQTIASIN.NSKYTQQV.T.S.S... : 35.069p1 : GLG.ELEEV.IDKMKQ.TASVQ.GSKHTQKV.A.S.S... : 25.017p1 : GFEDELEEV.IDKMKQ.TASSQ.GPKHTQKV.A.S.N... : 22.570p1 : GFEGELEEV.IDKMKQ.TASSQ.GPKYTQKV.A.S.N... : 22.527p1 : GLG.ELEEVIVDKAKQTIASVS.NSKHTQKV.T.S.S... : 20.005p1 : GLEDDLVEVIVDKAQQTLASIK.DSKHTQKV.S.T.N... : 17.504p1 : GLG.DLMEVIVDKTHQTLASVK.DSKHTQKV.A.T.N... : 20.014p1 : GLGDELEEVIVEKTKQTVASIS.GPKHTQKV.I.T.N... : 15.086p1 : GLGDELEEVIVEKTKQTLASVATGSKYTQKV.S.S.N... : 15.0

68p1 : LDHLHGAEAAYQVESIIKTATDTVKSEIDAELGVVPSLNA : 12.5

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Re-clustering of VP1-p1 peptides according to chemical properties

Selection of one representative peptidesequence from each re-cluster

Fig. 1 Composition of the PreDicta chip. a Selection process of VP1 peptides representing different RV strains as summarized in Supplementary Table 2. bMultiple sequence alignment of selected VP1 peptides. Left margin: Strain numbers (red circle: RV-A; blue triangle: RV-C, green square: RV-B, black circle:Enterovirus 68). Dots in the alignment represent identical amino acids. Sequence identities (%) with peptide 1 from RV 1 (01p1; top line) are shown for eachpeptide on the right margin. c Phylogenetic tree of the VP1 peptide sequences in b. d RV microarray layout depicting the positions of recombinant RVproteins/protein fragments, synthetic peptides (VP1-p1, 89VP1-derived peptides), MBP (maltose binding protein), and other control proteins which werespotted in triplicates. Proteins and peptides are designated as described in Supplementary Tables 3–6

NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04591-0 ARTICLE




peptides followed by RV-A peptides whereas IgG reactivity toRV-B peptides was less frequent and intense (Fig. 2a). Similarresults were obtained for IgA responses which in general werelower than the IgG responses (Fig. 2b). The analysis of IgGreactivity to structural and non-structural proteins and torecombinant fragments and synthetic peptides spanning VP1,VP2, and VP3 from RV89 is shown in Supplementary Fig. 2a forall 120 children and in Supplementary Fig. 2b for those children(n= 41) who had shown increases of RV89-specific antibodyresponses in follow-up serum samples taken after recovery.Results obtained thus confirmed our earlier observations showingthat the majority of RV-specific antibody responses are directedagainst the N-terminus of VP1 as represented by the 30 peptidesfrom the N-terminus of VP1 proteins from the different RVspecies33. Some children showed an IgG response against VP2and in particular to a fragment representing the middle portion ofVP2 whereas VP3 and VP4 showed no relevant IgG reactivity(Supplementary Fig. 2a, b). Among the non-structural proteins,2C, 3A, and 3C showed some IgG reactivity (SupplementaryFig. 2).The analysis of IgG reactivity according to the age of children

at the acute visit showed that children <1 year of age had muchlower RV-specific IgG levels compared to children who wereolder than 1 year. There was almost no change of the RV strain-specificity pattern among the three age groups (Fig. 2c). However,we found a positive correlation between the number ofrecognized VP1 peptides and the age of the children (Fig. 2d).Children between 6 and 12 months of age recognized asignificantly lower number of N-terminal VP1 peptides thanolder (13–42 months) children. Children older than 2 yearsreacted with significantly more peptides than both groups ofyounger children (Fig. 2e).The patterns of antibody response against different RV

peptides were also analyzed using an independent bioinformaticsapproach (Supplementary Fig. 3). A phylogenetic clustering of allpeptides was prepared and used to generate peptide groupsaccording to sequence homology which represented to a largeextent the RV subgroups A, B, and C. Then an unsupervisedcomputer algorithm was used to cluster the patterns of antibody

responses. Finally, the results of these analyses were super-imposed. It turned out that there was a very strong correlationbetween the two groupings, one based on antibody reactivity andthe other based on sequence identities among peptides madethrough the unsupervised analysis. Thus antibody responsepatterns reflected very closely the peptide sequence similarities(Supplementary Fig. 3).

Identification of RV species-specific antibody increases. Basedon our previous observations that antibody increases specific forthe N-terminal portion of VP1 can be detected in serum samplesobtained from subjects after RV infection36, the PreDicta chipwas equipped with a VP1 peptide set which should allowdetecting species-specific immune responses at high resolution(Fig. 1). Supplementary Figure 4 shows images of RV-specificantibody responses measured in sera from six representativechildren obtained at the time of the acute episode of wheeze andin sera at a follow-up visit 2–3 months later. In sera from thefollow-up visit increased antibody responses to RV-A (sera #33,#108), RV-C (sera #119, #84), and RV-B (sera #66, #89) peptidescould be detected (Supplementary Fig. 4).We then compared the peptide-specific IgG antibody levels

in sera obtained from the 120 children at the time of the acutewheeze and at the follow-up 2–3 months later (i.e., median11 weeks later) (Table 1). Supplementary Figure 5 shows acolor-coded map of the peptide-specific antibody responses forthe acute phase and follow-up of each of the childrendemonstrating species-specific IgG increases. Next, we com-pared the peptide-specific increases in relative numbers for eachchild (Fig. 3a). According to these peptide-specific IgGincreases, children could be identified who responded prefer-entially to RV-A-derived peptides (n= 41), RV-C-derivedpeptides (n= 33), RV-B-derived peptides (n= 23), and somewith a mixed response pattern (n= 7) (Fig. 3a). For 16 childrenno increases of peptide-specific IgG responses were found(Fig. 3a, bottom). The same analysis was performed forincreases of antibody responses against complete recombinantVP1 proteins from the three RV-A strains (i.e., RV89, 16, 2),

Table 1 Demographic and clinical characterization of wheezing children

Characteristic Group I(N=35) Group II(N=53) Group III(N=32) Total(N=120)

Age (months)Median 10 17 32 18Range (Min–Max) 6–12 13–24 25–42 6–42

GenderMale:female ratio 26:9 31:22 19:12a 76:43Male (%) 74 58 61 63

Ever wheeze before, n (%) 23 (66) 44 (83) 25 (83)a 92 (77)Allergic sensitizationb

Food allergens, n (%) 5 (14) 7 (13) 10 (31) 22 (18)Respiratory allergens, n (%) 1 (3) 4 (7) 4 (12.5) 9 (7.5)

Hospitalized at the acute visit, n (%) 26 (74) 44 (83) 26 (87)a 96 (80)Weeks until follow-up visit

Median 11a 11.5a 12.5 11Range (Min–Max) 7–27 7–24 9–30 7–30

Presence:absence of cold symptoms 35:0 50:3 30:2 115:5Days with respiratory symptoms (%)

Median 13 11a 6.5 11Range (Min–Max) 1–85 0–100 1–63 0–100

Days with β2-agonists (%)Median 20 22a 29.5 22Range (Min–Max) 0–100 0–100 0–100 0–100

a1 or 2 values are missing for this variablebAllergen-specific IgE to food and/or respiratory allergens determined by MEDALL allergen chip for the acute and follow-up visits (≥0.3 ISU-E was considered positive)




the RV-C strain YP, and the RV-B strain 14 (Fig. 3a) butthe results were less clear and negative for several childrenbecause only few strains were covered with the recombinantproteins. We have also included in Fig. 3a (right column)results from the PCR testing performed using VP4–VP2-specific primers in 108 of the 120 children35, which showed thatthe nucleic acid-based detection of virus strains was negativefor approximately 25% of children with increases of RVpeptide-specific IgG levels which may indicate a higher

sensitivity of serology vs. PCR in these children. Moreover,PCR results did not correspond well with the specificities of theantibody responses. There were also 14 children withoutincreases of RV peptide-specific antibody responses who hadpositive PCR results (Fig. 3a, bottom).Figure 3b provides a summary of the absolute increases of

peptide-specific IgG responses showing that RV-A and RV-C-specific antibody responses dominated over RV-B-specificincreases.

a

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Fig. 2 RV-specific antibody responses in sera from children with acute wheeze. Frequencies and levels of a IgG and b IgA responses (y-axes: n, number ofreactive sera) to the N-terminal VP1 peptides from 30 RV strains (Supplementary Tables 2 and 4) (x-axes: red: RV-A species; green: RV-B species; blue:RV-C species). Antibody levels are color-coded and expressed as ISAC standardized units, ISU-G and ISU-A, respectively. cMedian IgG levels (y-axis: ISU-G) to VP1 peptides (x-axis) in children grouped according to age (6–12 months: squares; 13–24 months: triangles; 25–42: circles). d Spearman’s rankcorrelation between the number of IgG-reactive peptides (n, y-axis; median IgG >15 ISU) and age (x-axis: months). Correlation coefficient (ρ) and p-valueare shown. e Comparison of the number of IgG-reactive VP1 peptides (n, y-axis; median IgG >15 ISU) in children according to age (x-axis). Horizontal linesindicate medians. Statistically significant differences between groups are indicated (**p < 0.01, ****p < 0.0001) (Mann–Whitney U-test)





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Fig. 3 Increases of IgG antibodies to N-terminal VP1 peptides detect culprit RV species. a A map showing IgG antibody increases (gradient color scaleranging from minimal to maximal value for each of the subject: white to dark red) to recombinant VP1 proteins and N-terminal VP1 peptides from three RVspecies (top lines RV-A: red; RV-C: blue; RV-B: green) measured by the microarray and PCR data for the wheezing children (white squares: PCR negative;black squares: PCR positive; no squares: not tested due to lack of samples). Shown are children for which RV-A (n= 41), RV-C (n= 33), and RV-B (n= 23)were identified as culprit species according to chip analysis, as well as children with mixed (n= 7) and no responses (n= 15). b Absolute increases of IgGantibody levels between the acute and follow-up visit for the wheezing children (y-axes: n, number of reactive sera) to the VP1 peptides(x-axes: red: RV-A species; green: RV-B species; blue: RV-C species) are shown. Antibody increases are color-coded and expressed as ISAC standardizedunits (ISU-G)




Antibody signatures associated with severity of wheeze. Next,we investigated whether antibody responses to certain RV specieswere associated with the severity of RV-induced wheeze. For thispurpose, we determined the number of days with respiratorysymptoms and the number of days when medication was requiredin the period between the acute and follow-up visit. The numberof days with respiratory symptoms but not with medication wassignificantly higher in subjects with an increase in RV-A >Mixed> RV-C > RV-B specific signal (Fig. 4, top and middle panels). Wethen analyzed the sum of days with symptoms and medicationand found that RV-A and RV-C antibody increases were

associated with the highest number of days with symptoms andmedication (Fig. 4, bottom panel). Children with RV-A- or RV-C-specific antibody increases had significantly more days withsymptoms and medication than children with RV-B-specificantibody increases (Fig. 4, bottom panel).

DiscussionWithin the European Union-funded project PreDicta (https://cordis.europa.eu/project/rcn/96868_en.html) we developed thePreDicta chip which is based on 130 micro-arrayed RV-derivedproteins and peptides selected to represent the three RV species(RV-A, RV-B, and RV-C). Using the PreDicta chip we coulddemonstrate in a cohort of 120 pre-school children with acutewheeze and a follow-up visit that the RV-specific antibodyresponse (IgG > IgA) is directed against an N-terminal peptideof the major capsid protein VP1 which confirms earlier resultsobtained by the mapping of RV89-specific antibody respon-ses33. Since the PreDicta chip was designed to contain a panelof 30 synthetic peptides which represented the most diverse RVstrains of the three genetic RV species in terms of sequenceidentity and physicochemical properties we were able to per-form a high-resolution mapping of RV species-specific antibodyresponses by serology. In a cohort of clinically well-describedSwedish pre-school children from whom sera were availablefrom an episode of acute wheeze requiring an emergency roomvisit and from a follow-up visit after convalescence, peptidesfrom RV-A and RV-C species were most frequently recognizedwhereas RV-B species were much less commonly recognized.Interestingly, we found that older children (i.e., children olderthan 2 years of age) recognized peptides from more RV strainsthan younger children. This result would indicate that childrenencounter in their life different RV strains and thus maybroaden their IgG reactivity profiles later in life but this needsto be confirmed in longitudinal studies with samples taken fromthe same children at different ages, as has recently been done inthe analysis of the evolution of IgE reactivity profiles in allergicchildren in birth cohorts37–40. One of the important findings ofour study was that we could demonstrate that IgG reactivity topeptides from certain RV strains increased in the childrenwhich may allow identifying the culprit RV species responsiblefor the acute wheeze by serology. Furthermore, it turned outthat increases of IgG responses to RV-A and RV-C species weresignificantly associated with more severe illness as compared toIgG increases to RV-B. The PreDicta chip thus seems to be notonly suitable for identifying the culprit RV species responsiblefor an exacerbation of respiratory illness by simple serology butalso allows to determine those RV species giving rise to severesymptoms. Since we also had results from PCR-based testing ofnasal swab samples from the same children we could comparethe detection of strain-specific nucleic acid with antibodyresults. In fact, we found that all children without species-specific antibody increases were also negative in the PCR test.However, there was poor correlation between the PCR resultsfrom the nasal samples and the chip-based serological results.Furthermore, with the exception of four children (#3, #61, #65,#72) for which a positive PCR result has been obtained at thefollow-up visit, all PCR results were negative at both the acuteand follow-up visit in approximately 25% of the children forwhom species-specific antibody increases could be clearlydemonstrated. Several possibilities for the discrepancies may beconsidered. For example, it may be possible that the timeinterval after acute infection chosen for serology was notoptimal. However, we have previously investigated the timeinterval required for the appearance of VP1-specific antibodyincreases in a controlled infection study36 and it is therefore

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Fig. 4 Days with respiratory symptoms and medication use in childrenwithout and with RV-A, RV-B, RV-C, or mixed peptide-specific IgGincreases. Days with respiratory symptoms (top panel, y-axis), withmedication use (middle panel, y-axis), and with symptoms and medicationuse (bottom panel, y-axis) are shown for children without (negative),mixed, RV-A, RV-C, or RV-B increases of IgG (x-axes). Horizontal linesshow median values. Statistically significant differences between groups areindicated (***p < 0.001; **p < 0.01, *p < 0.05) (Mann–Whitney U-test)







very unlikely that the time interval used in our study was toolong and responsible for discrepancies between PCR testing andserology. We also do not think that cross-reactivity amongstrains or the original antigenic sin41 is responsible for theobserved differences because we have taken care to include onthe chip sequences from several different RV strains and theresults in Fig. 3a clearly show that the serological resultsobtained with the chip allow a bona fide discrimination of RV-A, RV-B, and RV-C infections because we have used a largepanel of RV peptides on the chip. Finally, we found that olderchildren recognized more RV peptides from different strainsthan younger children (Fig. 2c–e), which indicates that thechildren develop antibodies against new viruses and thus theconcept of the original antigenic sin does not seem to applyhere. One limitation of our study is, however, that we cannotexclude that infections with additional RV strains have occur-red in the time window between the first and second bloodsampling and thus are responsible for the discrepancy betweenPCR results and serology. Nevertheless we think that the dis-crepancy between PCR results and serology is rather due to thefact that not every virus detected by PCR causes an infectionwith a consecutive immune response. In fact, studies performedin young children report that up to 35% of asymptomaticsubjects have positive PCR results30,42. One more likely possi-bility for the poor correlation of PCR results and antibodyresults could be that we used a PCR strategy based on primersspecific for VP4- and VP2-encoding regions of the viral gen-ome, which may be less specific than PCR strategies based onthe amplification and sequencing of the VP1-encoding regionor of the complete RV genome12,28,43,44. In general, nucleicacid-based strategies for virus detection only demonstrate thepresence of virus-specific nucleic acid but provide no evidencethat an infection has taken place which gave rise to a specificimmune response. We therefore think that the PreDicta chipand future versions of it containing an even larger repertoire ofN-terminal VP1 peptides from more RV strains will be acomplementary tool in addition to PCR testing and eventuallyturn out to be even superior. The antibody test is actually fastand economical: It takes only few hours and requires onlymicroliter amounts of serum. Furthermore, serological analysisis robust and can be easily performed without need for PCRcyclers and subsequent sequencing. However, more prospectivestudies will be needed to investigate the diagnostic sensitivityand specificity of the RV chip. The PreDicta chip may beextremely useful to determine RV species-specific antibodyresponses in serum samples from existing cohorts world-wideto define the most common and relevant RV species involvedin respiratory illness. Chip-based measurements will allowexploring in prospective studies the role of RV infections in avariety of respiratory disease exacerbations. For example, it willbe possible to discriminate whether asthma exacerbations havebeen triggered by an RV infection or by allergen exposurebecause it has been shown that both factors (i.e., RV infectionsas well as allergen exposure) induce increases of specific anti-body responses when serum samples collected at the acute visitand during a follow-up visit are compared35,36,45,46. Theidentification of the culprit factors triggering asthma attacksbecomes increasingly important in respiratory medicine due toavailability of selective treatments of allergic asthma such asanti-IgE antibodies and a variety of other biologics such as anti-cytokine antibodies targeting different forms of asthma47,48.Furthermore, it will be possible to use the PreDicta chip tostudy by serological analysis the possible contribution of RVinfections in exacerbations of other respiratory diseases such asCOPD and ACO. Further studies are also necessary to performa multiple monitoring of the presence of RV strains and other

respiratory viruses by PCR and the immune reaction by serol-ogy in close intervals and for extended periods afterexacerbation.The reliable determination of the most common RV species

involved in triggering severe respiratory illness will ultimatelyprovide a rational basis for the development of RV vaccines andRV species-targeting therapeutic approaches20–26.

In conclusion, we developed and evaluated a high-resolutionantibody assay based on micro-arrayed peptides and recombinantantigens from the most common RV strains to identify antibodysignatures discriminating RV infections at the levels of differentRV species and allowed to point towards the culprit speciesresponsible for the triggering of acute pre-school wheezing. ThePreDicta chip has the potential to be useful for a serological globalmapping of RV infections, the identification of RV speciesinvolved in triggering different forms of severe respiratory illness,and for paving the road for RV-specific therapeutic and pro-phylactic treatment strategies, such as vaccines.

MethodsSelection and production of N-terminal VP1 peptides. VP1 amino acidsequences of RV strains representing the three RV species (RV-A: n= 76; RV-B:n= 25; RV-C: n= 6) were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/) (Supplementary Table 1). Multiple sequence alignments of the VP1N-terminal peptides (aa 1–40) were performed using ClustalW2 software availableat the EMBL-EBI website (http://www.ebi.ac.uk/tools/clustalw2) to determineamino acid sequence identities among the peptides. Peptide sequences showingsequence identities greater than 87.5% (i.e. differences of ≤5 aa) were groupedtogether into clusters (Fig. 1). Sequences among the clusters (SupplementaryTables 1 and 2, A1–A17, B1–B9, C1–C3) were re-aligned using GeneDoc software(http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html) and eachamino acid mismatch was analyzed regarding physicochemical properties of theamino acids. This procedure led to re-clustering of the peptides (SupplementaryTable 2, AI–XVIII, BI–BIX; CI–CIII). From each re-clustered group one repre-sentative RV strain peptide was selected for printing onto the chip (Fig. 1b, Sup-plementary Tables 2 and 3: RV-A: n= 18; RV-B: n= 9; RV-C: n= 3). Anenterovirus-derived peptide was also included (Fig. 1). For the set of peptides to beprinted a multiple sequence alignment was performed by ClustalW2 and a phy-logenetic tree was constructed by the Neighbor-Joining (N-J) method using MEGA6 software (www.megasoftware.net) (Fig. 1c). The evolutionary distances betweensequences were computed using the Kimura 2-parameter model with bootstrapvalues calculated from 1000 replicates. Additional 36 peptides spanning theRV89VP1 protein (Fig. 1, Supplementary Table 4) were selected to detect anti-bodies towards VP1 epitopes other than the N-terminal portion.

The peptides as well as the non-structural 3B protein from strain 89 (VPg:GPYSGEPKPKSRAPERRVVTQ) were produced by solid-phase synthesis with the9-fluorenyl-methoxy carbonyl (Fmoc)-method (CEM-Liberty, Matthews, NC, USAand Applied Biosystems, Carlsbad, CA, USA) on PEG-PS preloaded resins(Applied Biosystems). After synthesis, peptides were washed withdichloromethane, cleaved from the resins using 19 ml trifluoroacetic acid (TFA),0.5 ml silane, and 0.5 ml H2O and precipitated into pre-chilled tert-butylmethylether. Peptides were purified by reversed-phase high-performanceliquid chromatography in a 10–70% acetonitrile gradient using a Jupiter 4 μmProteo 90 Å, LC column (Phenomenex, Torrance, CA, USA) and an UltiMate 3000Pump (Dionex, Sunnyvale, CA, USA) to a purity >90%. Their identities andmolecular weights were verified by mass spectrometry (Microflex MALDI-TOF,Bruker, Billerica, MA, USA)49.

For the unsupervised analysis of antibody responses to RV peptides andproteins, unsupervised K-means clustering (K= 4) was used to define clusters ofpeptides with similar antibody response measurements. The K number of clusterswas pre-determined in order to match the number of the peptide homology groups.The peptides’ amino acid sequences were aligned with a Gap open cost of 10.0 anda Gap extension cost of 1.0. Based on the alignment, a homology distancecladogram was built using the Neighbor-Joining algorithm and 1000 bootstrapreplicates. The peptides were then color-coded based on the antibody responsecluster that they belonged to. Data were processed using the CLC GenomicsWorkbench (CLC, CLCbio, Qiagen, Hilden, Germany). The heat map representingRV-specific antibody responses was generated by Qlucore Omics Explorer(Qlucore, Lund, Sweden).

Expression and purification of recombinant RV proteins. Recombinant his-tagged structural (VP1–4) proteins from five representative RV strains (RV-A2,-A16, -A89, -B14, and -CYP) and MBP fusion proteins containing fragmentsthereof (VP1–3) as well as non-structural (2A, 2C, 3A, 3C, and 3D) proteins fromRV strain 89 were expressed in Escherichia coli as previously described33,36. DNAsequences coding for the complete genes or fragments thereof (accession numbers



https://www.ncbi.nlm.nih.gov/

https://www.ncbi.nlm.nih.gov/

http://www.ebi.ac.uk/tools/clustalw2

http://iubio.bio.indiana.edu/soft/molbio/ibmpc/genedoc-readme.html

http://www.megasoftware.net


are shown in Supplementary Table 5) were codon optimized for bacterialexpression, synthesized with the addition of the 3′ sequence coding for a C-terminal hexa-histidine tag and cloned into the NdeI and EcoRI sites of plasmidpET27b (Genscript, Piscataway, NJ, USA). Transformed E. coli BL21 (DE3) cells(Agilent Technologies, Santa Clara, CA, USA) were induced with 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and cells were harvested at time-points of max-imal expression. Recombinant proteins were purified by Nickel-affinity chroma-tography under denaturing conditions as previously described (Qiagen, Hilden,Germany). Refolding of recombinant proteins was achieved by a stepwise dialysisagainst 10 mM NaH2PO4 for structural and non-structural proteins and 20 mMTris-HCl, 200 mM NaCl, 1 mM EDTA for MBP fusion proteins, respectively. Thepurity of recombinant proteins was verified by SDS-PAGE followed by CoomassieBrilliant Blue staining and the identity by immunoblotting using a monoclonalmouse anti-His-tag antibody 1:1000 diluted (Cat: DIA-900, Dianova, Hamburg,Germany). Bound antibodies were detected with 1:1000 diluted alkalinephosphatase-coupled rat anti-mouse IgG antibodies (Cat: 557272; BD Biosciences,Erembodegem, Belgium). Protein concentrations were determined using BCAProtein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). The secondarystructure of the proteins was measured by circular dichroism spectroscopy on aJasko J-810 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan) at a proteinconcentration of 0.1 mg/ml in 10 mM NaH2PO4.

Detection antibodies and printing of microarrays. Anti-huIgG (Cat: 309-005-008; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and anti-huIgA (Cat.: 555885; Becton Dickinson, Franklin Lakes, NJ) were labeled withDyLight 650 (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). Customizedprinting of RV microarrays was done by Phadia-ThermoFisher using ImmunoCAPISAC (Immuno Solid-phase Allergen Chip) technology50,51. Spotting was per-formed by slow pin mode printing using the Aushon 2410 Printer (Aushon, Bill-erica, MA, USA). Stock solutions of peptides (5 mg/ml) were diluted 1:4 in aphosphate buffer, pH 8.4 and then used for spotting. Antigens were spotted intriplicates on a glass surface coated with an amino-reactive organic polymer, eachspot containing 50–200 fg of microarray component, corresponding to 1–5 atto-mol. Allergens used for the calibration and other control proteins spotted on themicroarray are listed in Supplementary Table 6.

Cohort of pre-school children with acute wheeze. Serum samples examined inthis study were from a cohort of 120 pre-school children who had been admitted tothe Paediatric Emergency Ward as a result of acute wheeze, at Astrid LindgrenChildren’s Hospital, Stockholm, Sweden (Table 1). This cohort and the genotypingof RV strains in the nasopharyngeal swab samples of 108 of the 120 children bynested PCR and sequencing have been previouslydescribed35. A molecular diagnostic platform for the rapid detection of 15respiratory strains was used in 118 of the 120 children. The following respiratoryviruses were found: Adenovirus: 7 children; Bocavirus: 8 children; Coronavirus: 6children; Influenza A/B: 1 child; Metapneumovirus: 3 children; Parainfluenzavirus:4 children: RSV: 22 children52. For 108 of the 120 children nasopharyngeal swabshad been available for the RV PCR targeting VP4/2 sequences. Written informedconsent was obtained from the parents or by the legal guardians and the study wasapproved by the Regional Ethics Committee of Karolinska Institutet, Stockholm,Sweden. Peripheral blood samples had been obtained within 24 h of presentation inthe emergency unit and sera were stored at −80 °C. In addition to blood samples,nasopharyngeal swab samples were obtained at the acute visit and again at thefollow-up visit by the research nurse and stored in the biobank at the Departmentof Clinical Microbiology, Karolinska University Hospital35. Follow-up sampleswere obtained between 6 and 30 weeks after the initial recruitment (median11 weeks) at a scheduled visit after recovery. Although this study was not plannedas a prospective study for the assessment of increases of RV-specific antibodies, thetime interval of 6–30 weeks was suited for this purpose because we found in anearlier study that increase of RV-specific IgG responses emerge 42 days afterexperimental inoculation36. At the follow-up visit, the guardians also filled out astandardized questionnaire concerning the number of days the child had sufferedfrom respiratory symptoms at home (i.e., ‘cough and/or wheeze’), use of medica-tion (i.e., β2-agonists, inhaled corticosteroids, leukotriene receptor antagonist), aswell as about any emergency visits between the acute and follow-up visits. The chipanalysis of the anonymized sera was performed with the approval of the ethicscommittee of Medical University of Vienna (EK1721/2014)37,50.

Microarray-based determination of antibody profiles. Microarrays were washedin a washing buffer (Phadia-Thermo Fisher) for 5 min by stirring. After drying bycentrifugation (1 min, 1000 g, RT), 35 µl of serum samples were applied on eachmicroarray and the slides were incubated for 2 h at gentle rocking (RT). For thedetection of RV-specific IgG and IgA antibodies, serum samples were diluted 1:300and 1:20 in a sample dilution buffer (Phadia-Thermo Fisher), respectively.Microarrays were then rinsed with washing buffer and washed for 5 min asdescribed above. After centrifugation, 30 µl of fluorescence-labeled antibodies (1µg/ml) was added and the slides were incubated 30 min at gentle rocking (in dark,RT). After further rinsing, washing and drying, microarrays were scanned using aconfocal laser scanner (LuxScan-10K microarray scanner, Capital-Bio, Beijing,

People’s Republic of China) and the image analysis was evaluated by MicroarrayImage Analyzer v3.1.2 software (Phadia-Thermo Fisher)50. For calibrationand determination of background signals, a calibrator serum (i.e., a pool ofallergic patients sera, diluted 1:10) and sample diluent were included in eachanalysis run.

Calibration and variability of the microarray. The Phadia Microarray ImageAnalysis software was used to process images, to calculate the mean fluorescenceintensities (FI) of triplicate analyses and to calibrate the results. A calibration curvewas generated by relating fluorescence intensities obtained by scanning the Pre-Dicta microarray with allergen-specific antibody levels measured by ImmunoCAP.Results were reported in ISAC standardized units(ISU)50,52. Background reactivities of fluorescence-labeled α-huIgG antibodiestowards all microarray components were determined by testing six replicates of asample diluent alone. For the characterization of the assay variability, one cali-brator serum and three normal sera were profiled at 1:100, 1:200, 1:500, and 1:1000in order to find a dilution at which the broadest spectrum of reactivity levels werecovered. To assess intra-assay (i.e. within experiment) variability, six replicates offour serum samples were used to measure IgG reactivities towards all microarraycomponents on the same day. To assess inter-assay (i.e. between experiments)variability, four serum samples were evaluated in experiments conducted on fiveconsecutive days. The evaluation of four different samples allowed determiningwhether the inter-assay variability was sample-dependent. For each microarraycomponent, the mean ISU-G, standard deviation (SD), coefficient of variation (CV%= SD/mean ISU-G) across the six replicates and five different experiments,respectively, were calculated for each sample. Microarray components were clas-sified according to the ISU-G level (>50, 25–50, 1–25) and averages across allcomponents within these groups on the array were also calculated for each of thesequantities.

Data analysis. Initial data processing was performed with Microsoft Excel. Fre-quencies (i.e., the number of reactive sera) and intensities (i.e., ISU-G levels) ofpeptide-specific IgG and IgA antibody levels were calculated using IBM SPSSStatistics (version 24; IBM Corp., Armonk, NY, USA). Median values of peptide-specific IgG levels (ISU-G) were calculated using GraphPad Prism 6 (La Jolla, CA,USA). The cut-off for a positive IgG reactivity was set at 1 ISU-G. The numbers ofIgG-reactive peptides in Fig. 2d, e were calculated for specific IgG levels >15 ISU-G.Increases of RV-specific antibody responses were calculated as differences betweenISU-G values measured at the follow-up (F) and the acute (A) visits (ΔISU-G=ISU-GF− ISU-GA) followed by subtraction of the double coefficient of variation(2 × CV%) calculated from the baseline values (ISU-GA) for each of the antigens.For IgG levels of 1–5 ISU-G and >5ISU-G, 25% and 12.5% were determined as CV% for intra-assay variability andsubtracted from the data, respectively. RV-specific IgM antibodies were not mea-sured because we found in an earlier study that no increases of RV-specific IgMantibody levels were detectable on days 4, 7, and 42 after experimental RVinoculation36.

Statistical analysis. GraphPad Prism 6 (La Jolla, CA, USA) was used to evaluateall statistical parameters. Correlation between the number of reactive peptides andage was evaluated by calculating the Spearman’s rank correlation coefficient (ρ).Differences between groups in number of reactive peptides and in number of daysthe children spent with respiratory symptoms, medication use, or with both wereassessed by Mann–Whitney U-test (two-tailed). Values of p < 0.05 were consideredstatistically significant.

Data availability. Primary data that support the findings of this study are availablefrom the corresponding author on request.

Received: 3 January 2018 Accepted: 7 May 2018

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33. Niespodziana, K. et al. Misdirected antibody responses against an N-terminalepitope on human rhinovirus VP1 as explanation for recurrent RV infections.FASEB J. 26, 1001–1008 (2012).

34. Iwasaki, J. et al. Comparison of rhinovirus antibody titers in children withasthma exacerbations and species-specific rhinovirus infection. J. Allergy Clin.Immunol. 134, 25–32 (2014).

35. Stenberg-Hammar, K. et al. Rhinovirus-specific antibody responses inpreschool children with acute wheeze reflect severity of respiratory symptoms.Allergy 71, 1728–1735 (2016).

36. Niespodziana, K. et al. Rhinovirus-induced VP1-specific antibodies are group-specific and associated with severity of respiratory symptoms. EBioMedicine 2,64–70 (2014).

37. Westman, M. et al. Mechanisms for the development of allergies consortium.Early childhood IgE reactivity to pathogenesis-related class 10 proteinspredicts allergic rhinitis in adolescence. J. Allergy Clin. Immunol. 135,1199–1206 (2015).

38. Posa, D. et al. Evolution and predictive value of IgE responses toward acomprehensive panel of house dust mite allergens during the first 2 decades oflife. J. Allergy Clin. Immunol. 139, 541–549 (2017).

39. Westman, M., Asarnoj, A., Hamsten, C., Wickman, M. & van Hage, M.Windows of opportunity for tolerance induction for allergy by studying theevolution of allergic sensitization in birth cohorts. Semin. Immunol. 30, 61–66(2017).

40. Wickman, M. et al. Detection of IgE reactivity to a handful of allergenmolecules in early childhood predicts respiratory allergy in adolescence.EBioMedicine 26, 91–99 (2017).

41. Henry, C., Palm, A. E., Krammer, F. & Wilson, P. C. From original antigenicsin to the universal influenza virus vaccine. Trends Immunol. 39, 70–79(2018).

42. Jartti, T. et al. Serial viral infections in infants with recurrent respiratoryillnesses. Eur. Respir. J. 32, 314–320 (2008).

43. Wisdom, A., Leitch, E. C., Gaunt, E., Harvala, H. & Simmonds, P. Screeningrespiratory samples for detection of human rhinoviruses (HRVs) andenteroviruses: comprehensive VP4-VP2 typing reveals high incidence andgenetic diversity of HRV species C. J. Clin. Microbiol. 47, 3958–3967 (2009).

44. Ledford, R. M. et al. VP1 sequencing of all human rhinovirus serotypes:insights into genus phylogeny and susceptibility to antiviral capsid-bindingcompounds. J. Virol. 78, 3663–3674 (2004).

45. Niederberger, V. et al. Antigens drive memory IgE responses in human allergyvia the nasal mucosa. Int. Arch. Allergy Immunol. 142, 133–144 (2007).

46. Egger, C., Horak, F., Vrtala, S., Valenta, R. & Niederberger, V. Nasalapplication of rBet v 1 or non-IgE-reactive T-cell epitope-containing rBet v 1fragments has different effects on systemic allergen-specific antibodyresponses. J. Allergy Clin. Immunol. 126, 1312–1315 (2010).

47. Heck, S., Nguyen, J., Le, D. D., Bals, R. & Dinh, Q. T. Pharmacological therapyof bronchial asthma: the role of biologicals. Int. Arch. Allergy Immunol. 168,241–252 (2015).

48. Gauthier, M., Ray, A. & Wenzel, S. E. Evolving concepts of asthma. Am. J.Respir. Crit. Care Med. 192, 660–668 (2015).

49. Focke-Tejkl, M. et al. Development and characterization of a recombinant,hypoallergenic, peptide-based vaccine for grass pollen allergy. J. Allergy Clin.Immunol. 2, 1207–1207 (2015).

50. Lupinek, C. et al. Advances in allergen-microarray technology for diagnosisand monitoring of allergy: the MeDALL allergen-chip. Methods 66, 106–119(2014).

51. Gallerano, D. et al. HIV microarray for the mapping and characterization ofHIV-specific antibody responses. Lab Chip 15, 1574–1589 (2015).

52. Stenberg-Hammar, K. et al. Subnormal levels of vitamin D are associated withacute wheeze in young children. Acta Paediatr. 103, 856–861 (2014).

AcknowledgementsThis study was funded by PreDicta, a FP7-funded EU project (No. 260895), by the FWF-funded projects F4605 and P29398 of the Austrian Science Fund, by research grants fromBiomay AG and Viravaxx, Vienna, Austria, by the Swedish Research Council, theSwedish Heart-Lung Foundation, Stockholm County Council (ALF project), the SwedishAsthma and Allergy Association´s Research Foundation, the King Gustaf V´s 80-yearFoundation, the Centre for Allergy Research at Karolinska Institutet and the SwedishCancer and Allergy Foundation.

Author contributionsConception and design: K.N., S.M., N.G.P., and R.V.; analysis and interpretation: K.N.,K.S.-H., S.M., C.R.C., K.N.-W., P.C.V., D.G., C.L., D.E., T.S., .C.H., C.S., M.v.H., G.H.,N.G.P., and R.V. Drafting and or reading the manuscript for important intellectualcontent: K.N., K.S.-H., S.M., C.R.C., K.N.W., P.C.V., D.G., C.L., D.E., T.S., .C.H., C.S.,M.v.H., G.H., N.G.P., and R.V.

Additional informationSupplementary Information accompanies this paper at https://doi.org/10.1038/s41467-018-04591-0.

Competing interests: C.L. and M.v.H. report personal fees from Thermo FisherScientific, outside the submitted work. M.v.H. serves as a consultant for Biomay AG,Vienna, Austria and Hycor Biomedical LLC, CA, US. N.G.P. reports personal fees from



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https://doi.org/10.1038/s41467-018-04591-0


Novartis, Faes Farma, Biomay AG, Vienna, Austria, HAL, Nutricia Research, Menarini,MEDA, Abbvie, MSD, Omega Pharma, Danone, grants from Menarini, outside thesubmitted work. R.V. reports grants from European Union, grants and personal feesfrom Biomay AG, Vienna, Austria, grants and personal fees from Viravaxx, Vienna,Austria, during the conduct of the study; grants from Austrian Science Fund (FWF),grants and personal fees from Biomay AG, Vienna, Austria, grants and personal feesfrom Viravaxx AG, Vienna, Austria, outside the submitted work. In addition, R.V. andK.N. are co-inventors in a patent application (PCT/AT2010/000416) regarding therhinovirus diagnosis reported in this paper. The remaining authors declare no competinginterests.

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…………………………………………………………….………………………………….

Hiermit erkläre ich mich einverstanden, dass Fr. Dr. Katarzyna Niespodziana unsere Arbeit

„PreDicta chip-based high resolution diagnosis of rhinovirus-induced wheeze” für den

Clemens von Pirquet-Preis für Allergieforschung der Österreichische Gesellschaft für

Allergologie und Immunologie einreicht.

I hereby declare to agree that Dr. Katarzyna Niespodziana applies with our paper “PreDicta

chip-based high resolution diagnosis of rhinovirus-induced wheeze” for the Clemens von

Pirquet Prize of the Austrian Society of Allergology and Immunology.

20/06/2018 Dr Spyridon Megremis Datum/Date Unterschrift/Signature


PreDicta chipbased high resolution diagnosis of rhinovirus-induced wheeze“ film den

Clemens von PirquetPreis für Allergieforschung der Österreichische Gesellschaft flur


1 hereby deciare to agree that Dr. Katarzyna Niespodziana applies with our paper “PreDicta

chipbased high resolution cliagnosis of rhinovirus4nduced wheeze‘ for the Clemens on

Pirquet Prize ofthe Austiian Society ot Allergology and Imrnunology

O7June2O18 C a sält Ca,4atn, PhD

JJatum f3ak LJnters chri/i Signatui c

DANIELA GALLERANO

…………………………………………………………….………………………………….








18/06/2018

…………………………………… ……………………………………… Datum/Date Unterschrift/Signature

…………………………………………………………….………………………………….








……………18/6/2018……… ……………………………………… Datum/Date Unterschrift/Signature

CURRICULUM VITAE

Katarzyna NIESPODZIANA, PhD

Date of birth

Place of birth

Nationality

Address

Mobile

Email

4th

November 1983 Koszalin, Poland Polish Satzingerweg 10/3/12 1210 Vienna, Austria 0043 699 190 31638 [email protected]

2007-2011

2005-2007

2003-2005

2002-2003

1998-2002

1991-1998

PhD Thesis at the Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria PhD-Program: “Inflammation and Immunity (IAI)” headed by Prof. Maria Sibilia Title: “Development of viral carrier-based allergy vaccines” Supervision: Prof. Dr. Rudolf Valenta Master Thesis at the Department of Forensic Medicine, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland Bachelor Thesis at the Department of Forensic Medicine, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Poland Szczecin University, Subject of study: Biology Grammar School, Liceum Ogólnokształcące nr II im. W. Broniewskiego, Koszalin, Poland Primary School, Koszalin, Poland

Personal data

Education

CURRICULUM VITAE

Since 2012

October 2014 – January 2016

August 2013 – September 2013

2011-2012

Postdoc position at the Division of Immunopathology (Head: Prof. Rudolf Valenta), Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria Postdoc position at the CD laboratory for the Development of Allergen Chips (Head: Dr. Susanne Vrtala), Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria Postdoctorial training at the Karolinska Institutet, Department of Medicine (Head: Prof. Marianne van Hage), Stockholm, Sweden Maternity Leave (Son Jakob, 22.04.2011)

Since 2016

2010-2016

Stand-alone Project Title: “High resolution mapping of human rhinovirus-specific antibody responses” Function: Project Leader Funding organization: Fonds zur Förderung der Wissenschaftlichen Forschung (FWF): P29398-B30 Granted amount of money: 344,053.50 € EU-Project “PreDicta” Title: “Post-infectious immune reprogramming and its association with persistence and chronicity of respiratory allergic diseases” Function: Co-Principal Investigator Funding organization: The European Commission, in the context of the 7

th Framework Program (FP7)

Granted amount of money: 644.400,00 € Duration: 2010 – 2016

Postdoctoral Training

Funded research projects

CURRICULUM VITAE

Since 2017

January 2016 – October 2016

June 2014 – Mai 2015

2014

September 2011 – October 2012

2010

Mentee at the Women’s Network Mentoring Program, Gender Mainstreaming and Diversity, Medical University of Vienna, Austria Leadership - Curriculum at the Medical University of Vienna, Austria Certified Program “Medical Education Vienna”, Medical University of Vienna, Austria Biometry II, Human resource development, Medical University of Vienna, Vienna, Austria “Schrittweise - Curriculum for Junior Scientists”, Gender Mainstreaming and Diversity, Medical University of Vienna, Austria Biometry I, Human resource development, Medical University of Vienna, Vienna, Austria

Since 2014

Since 2012

2013-2014

2013

“BL 1 - Gesunde und kranke Menschen”, MUV, assigned “Problemorientiertes Lernen – 1.Semester”, MUV, assigned “Viral Infections in Allergy”, PhD Program: “Molecular, Cellular and Clinical Allergology” Thesis Seminar, assigned “Data Visualization Tools”, PE-Seminar, MUV, assigned “BL 8 – Krankheit, Krankheitsursachen und Krankheitsbilder”, MUV, assigned Tutor of students during laboratory courses Co-Supervisor of the Diploma Thesis of Kristina Borochova Title: “Expression and purification of recombinant surface proteins G and F of human respiratory syncytial virus (HRSV)” “Immunologische Übungen”, assigned

Since 2017

Since 2016

Since 2013

International Archives of Allergy and Immunology World Allergy Organization Journal Vaccine

Scientific Education

Teaching activities

Reviewer for the following journals

CURRICULUM VITAE

2016

2016

2015

2014

2012

2012

2011

2011

2010

Poster Prize at the 7

th Retreat of the Center for Pathophysiology,

Infectiology and Immunology, September 20, 2016, Vienna, Austria Travel Grant from the Austrian Society for Allergology and Immunology (ÖGAI) for the International Congress of Immunology 2016”, Melbourne, 2016 Poster Prize at the 6

th Retreat of the Center for Pathophysiology,

Infectiology and Immunology, September 22, 2015, Vienna, Austria

“64

th Lindau Nobel Laureate Meeting”, Lindau, 29 June – 4 July, 2014;

Nominated for participation by the Austrian Academy of Sciences

“Young Investigators Award 2012”, American Academy of Allergy, Asthma and Immunology, AAAAI Dr. Maria Schaumayer Foundation Award, PhD thesis award “Award of Excellence 2011” of the Austrian Federal Minister for Science and Research “Clemens von Pirquet Award” of the Austrian Society for Allergology and Immunology (ÖGAI) “Travel grant for the FEBS & EFIS Workshop: Inflammatory Diseases and Immune Response, Basic Aspects, Novel Approaches and Experimental Models”, September 28 - October 1, 2010, Vienna, Austria

2010

2012

PCT/EP2012/061040: "Hepatitis B allergen carrier" PCT/AT2010/000416:“Pharmaceutical composition for the treatment and prevention of a rhinovirus infection”

Since 2015

Since 2013

Since 2007

Austrian Society for Molecular Biosciences and Biotechnology Junior Member of The European Academy of Allergy and Clinical Immunology (EAACI) Austrian Society of Allergology and Immunology (ÖGAI)

Awards and Honours

Patents

Memberships

CURRICULUM VITAE

1. Valenta, R., Focke-Tejkl. M., Niespodziana, K., Marth, K., and V. Niederberger. Recombinant allergens: What does the future hold? J Allergy Clin Immunol 2011; 127(4):860-4.

2. Edlmayr, J., Niespodziana, K., Focke-Tejkl, M., Linhart, B., and R. Valenta Allergen-specific immunotherapy: Towards combination vaccines for allergic and infectious diseases. Curr Top Microbiol Immunol 2011; 820:121-140.

1. “BIT’s 5th World Congress of Vaccine”, Hangzhou, China, March 18-20, 2013 Title: “Rhinovirus vaccine: Mission possible? 2. “EAACI – WAO Congress 2013”, Milan, June 22-26, 2013 Title: “The role of antibody response in rhinovirus infections” 3. “64th Lindau Nobel Laureate Meeting”, Lindau, 29 June – 4 July, 2014 Science Breakfast: “Predicting Phenotypes from Genotypes - a Brave New World?”

4. “EAACI – WAO Congress 2015”, Barcelona, June 6-10, 2015 Title: “New generation molecular diagnosis of rhinovirus infections” 5. Pirquet Club of the Austrian Society for Allergology and Immunology (ÖGAI), Vienna, Austria February the 15th, 2016 Title: “Die Bedeutung der Messung von Rhinovirus-spezifischen Antikoerperantworten in Asthmapatienten“, Vienna, Austria 6. PreDicta Consortium Meeting, Athens, Greece, March 21-22, 2016 Title: “Antibody responses to RV infection for discrimination of virus and allergen-induced respiratory diseases”

7. XI World Congress on COPD, Asthma & Immunopathology, Moscow, Russia, October 5-8, 2017 Title: “Risk factors for wheezing in preschool children: role of rhinovirus and allergen exposure” Title: “Rhinovirus diagnosis by blood testing with the “PreDicta” chip”

Invited speaker (conferences, seminars)

Reviews and book contributions

CURRICULUM VITAE

1. Szczerkowska-Dobosz, A., Niespodziana, K., K. Rebala, J. Garstecka, M. Lange, and W. Baranska-Rybak. Lack of association of HLA-C alleles with late-onset psoriasis in the northern Polish population. J Appl Genet 2007; 48:273-275.

2. Edlmayr, J., Niespodziana K., Linhart, B., Focke-Tejkl, M., Westritschnig, K., Scheiblhofer, S., Stoecklinger, A., Kneidinger, M., Valent, P., Campana, R., Thalhamer, J., Popow-Kraupp, T., and R. Valenta. A combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived surface protein VP1 and a nonallergenic peptide of the major timothy grass pollen allergen Phl p 1. J Immunol 2009; 182: 6298-306.

3. Rebala, K., Szczerkowska-Dobosz, A., Niespodziana, K., and J. Wysocka. Simple and rapid screening for HLA-Cw*06 in Polish patients with psoriasis. Clin Exp Dermatol 2010; 35:431-6.

4. Weiss, V.W., Bilek, G., Pickl-Herk, A., Subirats, X., Niespodziana, K., Valenta, R., et al. Liposomal leakage induced by virus-derived peptides, viral proteins and entire virions: Rapid analysis by chip electrophoresis. Anal Chem 2010; 82: 8146–8152.

5. Edlmayr, J.*, Niespodziana, K.*, Popow-Kraupp, T., Krzyzanek, V., Focke-Tejkl, M., Blaas, D., Grote, M., and R. Valenta. Recombinant rhinovirus-derived VP1 for vaccination against common cold infections. Eur Respir J 2011; 37:44-52. *contributed equally to the study

6. Niespodziana, K., Linhart, B., Focke-Tejkl, M., Civaj, V., Blatt, K., Valent, P., van Hage, M., Grönlund, H., and R. Valenta. A cat allergy vaccine based on Fel d-1 derived peptides fused to a PreS domain of Hepatitis B Virus (HBV) J Allergy and Clin Immunol 2011; 127(6):1562-70.

7. Niespodziana K., Napora K., Cabauatan C., Focke-Tejkl M., Keller W., Niederberger V., Tsolia M., Christodoulou I., Papadopoulos NG and R. Valenta. Misdirected antibody responses against an N-terminal epitope on human rhinovirus VP1 as explanation for recurrent RV infections. FASEB J 2012; 26: 1001–1008.

8. McLean G., Walton a R., Shetty S., Paktiawal N, Kebadze T., Gogsadze L., Niespodziana K., Valenta R., Bartlett N., Johnston SL. Rhinovirus infections and immunisation induce cross-serotype reactive antibodies to VP1. Antiviral Research 2012; 95: 193–201.

9. Pfanzagl B., Andergassen D., Edlmayr J., Niespodziana K., Valenta R. and Blaas D. Entry of human rhinovirus 89 via ICAM-1 into HeLa epithelial cells is inhibited by actin skeleton disruption and by bafilomycin. Arch Virol 2014; 159: 125-140.

10. Focke-Tejkl M., Weber M., Niespodziana K., Neubauer A., Huber H., Henning R., Stegfellner G., Maderegger B., Hauer M., Stolz F., Niederberger V., Marth K., Eckl-Dorna J., Weiss R., Thalhamer J., Blatt K., Valent P., Valenta R. Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergyJ Allergy Clin Immunol 2015; doi: 10.1016/j.jaci.2014.09.012.

11. Niespodziana, K., Cabauatan, C.R., Jackson, D.J., Gallerano, D., Trujillo-Torralbo, B., del Rosario, A., Mallia, P., Valenta, R., and Johnston, S.L. Rhinovirus-induced VP1-specific Antibodies are Group-specific and Associated With Severity of Respiratory Symptoms. EBioMedicine 2015; 2: 64-70; doi:10.1016/j.ebiom.2014.11.012.

Publications in peer-reviewed journals

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Szczerkowska-Dobosz%20A%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstract

http://dx.doi.org/10.1016/j.ebiom.2014.11.012

CURRICULUM VITAE

12. Gallerano, D., Wollmann, E., Lupinek, C., Schlederer, T., Ebner, D., Harwanegg, C.,

Niespodziana, K., Schmetterer, K., Pickl, W., Puchhammer-Stöckl, E., Sibanda, E. Valenta, R. HIV microarray for the mapping and characterization of HIV-specific antibody responses. Lab on a Chip 2015; 15(6): 1574-89. doi: 10.1039/c4lc01510j.

13. Gangl, K., Waltl, E., Vetr, H., Cabauatan, C., Niespodziana, K., Valenta R., and Niederberger V. Infection with rhinovirus facilitates allergen penetration across a respiratory epithelial cell layer. Int Arch Allergy Immunol 2015; 166(4):291-6. doi: 10.1159/000430441.

14. Stenberg-Hammar, K.*, Niespodziana, K.*, Söderhäll, C., James, A., Cabauatan, C.R., Melén, E., van Hage, M., Valenta, R., and Hedlin, G. Rhinovirus species and specific antibody response in preschool children with acute wheeze. Allergy 2016; 71(12):1728-1735. doi: 10.1111/all.12991. *contributed equally to the study

15. Cornelius, C., Schöneweis, K., Georgi, F., Weber, M., Niederberger, V., Zieglmayer, P., Niespodziana, K., Trauner, M., Hofer, H., Urban, S., Valenta, R. Immunotherapy With the PreS-based Grass Pollen Allergy Vaccine BM32 Induces Antibody Responses Protecting Against Hepatitis B Infection. EBioMedicine 2016; 11:58-67. doi: 10.1016/j.ebiom.2016.07.023.

16. Cabauatan, C.R., Campana, R., Niespodziana, K., Reinisch, C., Lundberg, U., Meinke, A., Henning, R., Neubauer, A., Valenta, R. Heat-labile Escherichia coli toxin enhances the induction of allergen-specific IgG antibodies in epicutaneous patch vaccination. Allergy 2017; 72:164-168. doi: 10.1111/all.13036.

17. Weber, M., Niespodziana, K., Linhart, B., Neubauer, A., Huber, H., Henning, R., Valenta, R., Focke-Tejkl, M. Comparison of the immunogenicity of BM32, a recombinant hypoallergenic B cell epitope-based grass pollen allergy vaccine with allergen extract-based vaccines. J Allergy Clin Immunol 2017; 31. pii: S0091-6749(17)30852-7. doi: 10.1016/j.jaci.2017.03.048.

18. Stenberg-Hammar, K., Niespodziana, K., van Hage, M., Kere, J., Valenta, R., Hedlin, G., and C. Söderhäll. Reduced CDHR3 expression in children wheezing with rhinovirus. Pediatr Allergy Immunol 2018; 29(2):200-206. doi: 10.1111/pai.12858.

19. Niespodziana, K., Stenberg-Hammar, K., Megremis, S., Cabauatan, C.R., Napora-Wijata, K., Vacal, P.C., Gallerano, D., Lupinek, C., Ebner, D., Schlederer, T., Harwanegg, C., Söderhäll, C., van Hage, M., Hedlin, G., Papadopoulos, N.G., and R. Valenta. PreDicta chip-based high resolution diagnosis of rhinovirus-induced wheeze. Nat Commun 2018; 9:2382. doi: 10.1038/s41467-018-04591-0.

20. Waltl, E.E., Selb, R., Eckl-Dorna, J., Müller, C.A., Cabauatan, C.R., Eiwegger, T., Resch-Marat,

Y., Niespodziana, K., Vrtala, S., Valenta, R., and V. Niederberger. Betamethasone prevents human rhinovirus- and cigarette smoke- induced loss of respiratory epithelial barrier function. Scientific Reports 2018; in press.

https://www.ncbi.nlm.nih.gov/pubmed/27444786



https://www.ncbi.nlm.nih.gov/pubmed/?term=Niespodziana%20K%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Linhart%20B%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Neubauer%20A%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Huber%20H%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Henning%20R%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Valenta%20R%5BAuthor%5D&cauthor=true&cauthor_uid=28576673

https://www.ncbi.nlm.nih.gov/pubmed/?term=Focke-Tejkl%20M%5BAuthor%5D&cauthor=true&cauthor_uid=28576673



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