Comparison of rhinovirus antibody titers in children withasthma exacerbations and species-specific rhinovirusinfection

Jua Iwasaki, BSc (Hons),a Wendy-Anne Smith, PhD,a Siew-Kim Khoo, BSc (Hons),b Joelene Bizzintino, PhD,b

Guicheng Zhang, PhD,b,c Des W. Cox, MD,b Ingrid A. Laing, PhD,b Peter N. Le Sou€ef, MD,b Wayne R. Thomas, PhD,a and

Belinda J. Hales, PhDa,b Perth, Australia

Background: Asthma exacerbations are associated with humanrhinovirus (HRV) infections, and more severe exacerbations areassociated with HRV-C. We have previously shown that theHRV-C–specific antibody response is low in healthy adult seraand that most of the antibody to HRV-C is cross-reactive withHRV-A.Objectives: To compare the antibody response to each HRVspecies in asthmatic and nonasthmatic children in whom thetype of HRV infection was known.Methods: Total and specific IgG1 binding to HRV viral capsidprotein antigens of HRV-A, -B, and -C were tested in the plasmafrom nonasthmatic children (n 5 47) and children presenting tothe emergency department with asthma exacerbations (n 5 96).HRV, found in most of the children at the time of theirexacerbation (72%), was analyzed using molecular typing.Results: Asthmatic children had higher antibody responses toHRV. The titers specific to HRV-A, and to a lesser extent

From aTelethon Institute for Child Health Research, and bthe School of Paediatrics and

Child Health, University of Western Australia, and cthe School of Public Health, Cur-

tin University.

This work was supported by the National Health and Medical Research Council of

Australia. J.I. was supported by an Australian Postgraduate Award, a University of

Western Australia Scholarship, a Stan and Jean Perron Scholarship, and an Asthma

Foundation of Western Australia Scholarship.

Disclosure of potential conflict of interest: J. Iwasaki has received research support from

the National Health and Medical Research Council of Australia, has received travel

support from Asthma Foundation WA, Friends of the Institute for Child Health

Research, the Australian Postgraduate Award, the European Academy of Allergy

and Clinical Immunology, and the Stan and Jean Perron Scholarship. S.-K. Khoo

has received research support from the National Health andMedical Research Council

of Australia and the University ofWestern Australia and is employed by the University

ofWestern Australia. J. Bizzintino and I. A. Laing have received research support from

the National Health and Medical Research Council of Australia and are employed by

the University of Western Australia. G. Zhang has received research support from

Brightspark. D. W. Cox has received research support from the National Health and

Medical Research Council of Australia. P. N. Le Sou€ef has received research support

from the National Health and Medical Research Council of Australia; is on advisory

boards for Teva and GlaxoSmithKline; is employed by the University of Western

Australia; and has received payment for lectures from GlaxoSmithKline Saudi Arabia.

W. R. Thomas has received research support from the National Health and Medical

Research Council of Australia; has received writing assistance, medicines, equipment,

or administrative support from the Health Department of Western Australia; and has

received royalties for use of allergen patents. B. J. Hales has received research support

from the National Health and Medical Research Council of Australia and is employed

by the Telethon Institute for Child Health Research. W.-A. Smith declares no relevant

conflicts of interest.

Received for publication November 1, 2013; revised February 14, 2014; accepted for

publication March 19, 2014.

Corresponding author: Belinda J. Hales, PhD, Telethon Institute for Child Health

Research, University of Western Australia, 100 Roberts Rd, Subiaco, WA 6008,

Australia. E-mail: belinda@ichr.uwa.edu.au.

0091-6749/$36.00

� 2014 American Academy of Allergy, Asthma & Immunology

http://dx.doi.org/10.1016/j.jaci.2014.03.014

HRV-B, were higher than in nonasthmatic controls. The species-specific responses to HRV-C were markedly lower than titers toHRV-A and HRV-B in both asthmatic and nonasthmaticchildren (P < .001). The titers both at presentation and afterconvalescence were not associated with the HRV genotypedetected during the exacerbation.Conclusions: The higher total anti-HRV antibody titers ofasthmatic children and their higher anti–HRV-A and -B titersshow their development of a heightened antiviral immuneresponse. The low species-specific HRV-C titers found in allgroups, even when the virus was found, point to a different andpossibly less efficacious immune response to this species. (JAllergy Clin Immunol 2014;nnn:nnn-nnn.)

Key words: Human rhinovirus, VP1, IgG1 antibody, asthmaexacerbation, children

Exacerbations of asthma in children are frequently associatedwith human rhinovirus (HRV) infections,1,2 and HRV-inducedwheeze in infancy is a predictor of the development of asthma.3,4

The newly recognized HRV-C species reportedly accounts formost of the attacks in children presenting to hospital with asthmaand is associated with more severe attacks than are HRV-A andHRV-B.5-7

Recent studies of antirhinovirus immunity have been directedto the innate immune responses of epithelial cells and to adaptiveimmunity. For the latter, T cells from adult atopic asthmaticpatients taken outside a period of exacerbation have been shownto produce less IFN-g in response to virion antigen compared withcells from nonatopic subjects.8,9 Reduced production of immuno-regulatory IL-10 was also reported although the antigen speci-ficity of its induction was not ascertained.9 It was proposed8,9

that asthmatic patients could have a reduced TH1 component,leading to poor viral clearance and immunopathology, withboth contributing to disease.

We have previously undertaken a detailed analysis of thespecies-specific and cross-reactive IgG1 antibody response toeach HRV species in healthy adults using the viral capsid protein1 (VP1).10 It found high IgG1 antibody-binding titers for all 3 spe-cies, but titers specific to HRV-Cwere low, with a large proportionof the anti–HRV-C antibody being cross-reactive to HRV-A. Thiswas despite their low amino acid sequence identity (;35%). Theaims here were to examine anti-HRVantibody responses to iden-tify features that might be important for children prone to asthmaexacerbation and determine whether there were differences in re-sponses to HRV-C than to HRV-A and B. Because children whodevelop asthma,11,12 especially those susceptible to exacerba-tion,13 have decreased antibody responses to the protein antigensof bacteria that colonize the respiratory mucosa, this could occur

1

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Abbreviations used

E30: E

chovirus 30

ED: E

mergency department

GST: G

lutathione-S-transferase

HPV: H

uman poliovirus

HRV: H

uman rhinovirus

VP1: V

iral capsid protein 1

for viral infection and concur with the increased prevalence ofbacterial and viral infections found in asthmatic children.14-16 Toexplore this, antibody binding to antigens of each HRV specieswas measured in children presenting to the emergency depart-ment (ED) for an asthma exacerbation, and again in convales-cence. These responses were then compared with those ofnonasthmatic controls.

METHODS

Study populationPaired acute and convalescent plasma from 96 children who presented to

the ED of Princess Margaret Hospital for Children with acute asthma

exacerbation was examined. Peripheral blood samples were obtained within

24 hours of presentation, and plasma were stored at 2808C. Samples in

convalescence were obtained either 6 to 26 weeks after the initial recruitment

(median 12weeks) or more than 26weeks after the initial recruitment (median

34 weeks) at a scheduled visit when the children were clinically well. Plasma

from 47 nonasthmatic control children was also examined. The control

children had no history of doctor-diagnosed asthma and were either

community controls (n 5 33) or siblings or friends of ED asthmatic children

recruited for the study (n5 14). The demographic characteristics for thewhole

study population are described in Table I, and a description of clinical charac-

teristics of the children presenting to hospital with asthma exacerbations in

Table II.

Most of the ED asthmatic children required hospital admission (95.6%)

with moderate to severe acute attacks (Table II). The study was approved by

the Ethics Committee at Princess Margaret Hospital for Children, and

parental/guardian written informed consent was obtained for each participant.

HRV detection and typingNasal secretion specimens collected from each child were tested for HRV

by direct fluorescent antibody testing and also analyzed with an HRV

molecular typing assay5 to determine which HRV strains were present at the

time of recruitment. As shown in Table I and reported previously,5,7 most of

the asthmatic children presenting to the ED had detectable HRV and the

majority HRV-C (68%).

AntigensHRV VP1 were produced as fusion polypeptides with a glutathione-

S-transferase (GST) at the N-terminus and a hexa-histidine tag on the

C-terminus as described.10 Briefly, DNA constructs representing VP1 antigens

from 2 genotypes of each species (see Table E1 in this article’s Online Repos-

itory at www.jacionline.org) were obtained by gene synthesis and expressed as

recombinant proteins in Escherichia coli (E coli). Recombinant VP1 antigens,

assembled as discrete multimers, were purified by glutathione-agarose affinity

chromatography (Sigma-Aldrich, St Louis, Mo) and high-resolution size

exclusion chromatography (GE Healthcare, Uppsala, Sweden). Secondary

structure similar to that of the natural antigens was demonstrated by circular

dichroism. VP1 of the related enteroviruses, human poliovirus (HPV) Sabin

VP1, and echovirus 30 (E30; AF128087.1) were similarly produced.

In addition to the representative VP1 antigens for each species, 2 isolate-

specific HRVVP1 antigens were produced from cDNA encoding the VP1 of 2

HRVisolates infecting ED asthmatic children at the time of recruitment. Semi-

nested PCR amplification was conducted using specifically designed primers

flanking the VP1 region. PCR products were then sequenced by the Australian

Genome Research Facility to obtain the VP1 nucleotide sequences, which

were subsequently engineered for expression as GST-fusion proteins.

IgG1-binding assayAs described in detail,10 IgG1 antibody titers were quantitated with a

dissociation-enhanced immunofluorescence assay using microtiter plate wells

coated with GST-fusion antigens standardized by titrating the coating concen-

trations with monoclonal anti-GST antibody (Sigma-Aldrich). Human

myeloma IgG1 (Sigma-Aldrich) was used as a negative control as well as sub-

sequently identified negative control sera. Three negative controls and a 2-fold

titration of reference sera (starting from a 1:50 dilution) for each antigen were

included on every plate to construct a standard curve. The absolute binding ti-

ters of the reference sera were calibrated by interpolating the results from a

side-by-side titration curve of humanized anti-Der p 2 chimeric IgG1 (Indoor

Biotechnologies, Charlottesville, Va) of known concentration. The same

molar coating of a recombinant GST-Der p 2 as the VP1, as first determined

with the monoclonal anti-GST, was used and the equivalence of the

slopes of the titrations demonstrated.10 Antibody values below the limit of

detection (500 ng/mL) were assigned 50% of the lower limit of detection

(250 ng/mL).

As well as quantifying the total IgG1 binding to VP1 antigens, the species-

specific titers were determined by absorbing plasma with the VP1 antigens

from the other HRV species, HPV Sabin and E30. The absorption was con-

ducted by first incubating the plasma in lysate mixtures of E coli, producing

the nontest VP1 antigens using concentrations shown to be effective.10

Tomeasure isolate-specific IgG1 binding, the test sera were preincubated in

a lysate mixture of E coli producing all HRV species, including the same spe-

cies that the isolate belongs to, as well as HPV Sabin and E30 VP1. They were

then assayed for binding to the VP1.

Statistical analysisAntibody titers to different antigens were compared by using the related-

samples Wilcoxon signed-rank test. Differences in antibody binding by

selected groups (ED asthmatic children/nonasthmatic control children) were

compared by using theMann-Whitney test. Differences in prevalence between

selected groups were compared by using the x2 test. After log-transformation,

there was an approximately normal distribution of antibody titers to HRV-A;

thus, the general linear model was used to compare differences between

selected groups when accounting for possible confounding factors.When sub-

jects with titers below the assay limit of 500 ng/mL were excluded from the

analysis, differences in antibody binding were compared with an independent

Student t test after log-transformation because there was an approximately

normal distribution for all antigens. Correlations were studied using Spearman

rho test. A P value of less than .05 was considered significant. All analyses

were performed using SPSS version 15.0 (Chicago, Ill) and GraphPad Prism

Software (La Jolla, Calif).

RESULTS

Total and species-specific antibody binding to

different HRV speciesAs determined for the 96 ED asthmatic children and 47

nonasthmatic controls, the total IgG1 binding to the VP1 antigensshowed high titers for all species (Fig 1, A). The median total IgGtiters to the HRV-A genotypes were typically 106 ng/mL, whereasthe titers to HRV-B and HRV-C ranged from 104 to 5 3 105

ng/mL. The species-specific titers obtained after immunoabsorp-tion showed that a large proportion of the total binding to each an-tigen was directed at epitopes shared with other HRV species orwith other enteroviruses, with titers being 10-fold lower than

TABLE II. Clinical data of ED asthmatic children at recruitment

Clinical information n (%)

Admitted (n 5 91) 87 (95.6)

Overnight observation ward 36 (39.6)

Hospital ward 50 (54.9)

Pediatric intensive unit 1 (1.1)

Severity group (n 5 92)*

Mild, score 0-3 16 (17.4)

Moderate, score 4-7 51 (55.4)

Severe, score 8-10 25 (27.2)

Upper respiratory tract infection (n 5 91) 78 (85.7)

Detection of viruses other than HRV 6 (6.25)

Received oral steroids at recruitment (n 5 75) 59 (78.6)

*Severity score assigned using a modified National Institute of Health score.5,7

TABLE I. Characteristics of the study population

Characteristic

ED asthmatic

children* (n 5 96)

Nonasthmatic

control children (n 5 47)

Mean age 6 SD at

recruitment (y)

6.4 6 3.4 4.5 6 3.6

Age range (y) 0.8-15.6 0.8-15.5

Male, n (%) 60 (62.5) 23 (48.9)

Atopic�, n (%) 77 (80.2) 11 (23.4)

HRV positive, n (%) 69 (71.9) 18 (38.3)

HRV-A, n 19 12

HRV-B, n 1 4

HRV-C, n 47 2

Dual infection�, n 2 0

*Doctor-diagnosed asthma exacerbation at recruitment. Three children were younger

than 2 years presenting as viral-induced wheeze. Analyses with and without these 3

subjects gave the same conclusion.

�Atopy was defined as at least 1 positive skin-prick test result to 11 common allergens.

A positive reaction was defined as a wheal size that was larger than the negative

control and greater than or equal to 3 mm in diameter.

�Dual infection was with HRV-A and HRV-C.

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the total antibody (Fig 1, B). The species-specific titers to HRV-Awere significantly higher than to HRV-B andHRV-C (P <.001) forboth the ED asthmatic children and control children, and re-mained significant after excluding children with titers below theassay limit (P < .001). The correlation of species-specific IgG1

binding between the 2 representative antigens for HRV-A washigh, being 0.982 and 0.924, respectively, for the ED asthmaticchildren and for the control children. The correlation ofspecies-specific binding between the 2 antigens representingeither HRV-B or HRV-C was significant but lower (0.779 forED asthmatic children, 0.495 for control children for HRV-B;0.765 for ED asthmatic children, 0.403 for control children forHRV-C).

Antirhinovirus antibody and asthma exacerbationsThe total and species-specific titers to HRV-A, -B, -C, and

HPV Sabin from children with an asthma exacerbation recruitedon presentation to the ED (ED asthmatic children) weremeasured and compared with those from nonasthmatic controlchildren.

Comparisons of total IgG titers to HRV VP1 between EDasthmatic children and nonasthmatic control children (Fig 1, A)showed that ED asthmatic children had higher titers to HRVanti-gens, which were significant for HRV-A34 (P5 .013), HRV-A1B

(P5 .002), HRV-B14 (P5 .021), andHRV-C5 (P5 .007) and thatthere was a trend for HRV-C3 (P 5 .084).

The specific anti–HRV-A titers of the ED asthmatic childrenwere higher than that of nonasthmatic children (Fig 1, B; P 5.007 for HRV-A34, P 5 .001 for HRV-A1B). The prevalenceof IgG1 binding was 92% (88 of 96) for HRV-A34 and 95%(91 of 96) for HRV-A1B in ED asthmatic children comparedwith 77% (36 of 47) for both these HRV-A genotypes in non-asthmatic children (P 5 .013 for HRV-A34; P 5 .001 forHRV-A1B). ED asthmatic children also had higher HRV-B–spe-cific titers, which was significant for HRV-B14 (P 5 .002) and atrend for HRV-B69 (P 5 .086). The prevalence of binding toHRV-B14 was also higher (P 5 .002) for ED asthmatic children(41 of 96, 43%) than for nonasthmatic control children (8 of 47,17%). Excluding the 3 children younger than 2 years presentingto hospital with virus-induced wheeze from the analysis did notaffect the significant difference in the titers of HRV-A34 (P 5.004), HRV-1B (P < .001), and HRV-B14 (P < .001) betweenthe 2 groups. Comparisons of antibody binding between EDasthmatic children and nonasthmatic children excluding childrenwith titers below the assay limit showed the differencesremained significant for HRV-A1B (P 5 .018), HRV-B14(P 5 .018), and HRV-B69 (P 5 .001) and a trend for HRV-A34 (P 5 .059). The increased response to HRV-A in EDasthmatic children was not associated with a current infectionwith HRV-A. As shown in Fig 2, ED asthmatic children whohad no detectable HRV-A at asthma exacerbation still had signif-icantly higher anti–HRV-A titers than did nonasthmatic children.There was no association between the severity of respiratorysymptoms at the time of recruitment and antibody titers to thedifferent antigens (data not shown).

HRV-C–specific titers were low in both nonasthmatic and EDasthmatic children (Fig 1, B), including those known to haveHRV-C at the time of hospital presentation (Fig 2). This is shownby the low anti–HRV-C titers found in ED asthmatic children withdetectable HRV-C, which were not significantly different to EDasthmatic children without a known HRV-C infection. ED asth-matic children with the HRV-C virus still had high titers toHRV-A (P < .001, data not shown).

There were no consistent changes found when the antibodybinding was measured in convalescence (median, 12 weeks). Asillustrated for HRV-C in asthmatic children with a known HRV-Cinfection (Fig 3), the anti–HRV-C titers of different subjects couldeither decrease or increase, or remain the same and were low. Thetiters measured with the HRV-A antigen (HRV-A34) remainedhigh with little variation (Fig 3). The same results were foundfor children who were followed up at a later time point (median,34 weeks) and in asthmatic children with a known HRV-A infec-tion at the time of hospital admission (data not shown).

High anti-HRV titers (106-107 ng/mL) were found in childrenfrom an early age (Fig 4), with both ED asthmatic and nonasth-matic children younger than 3 years having high titers toHRV-A. The higher anti–HRV-A titers in the ED asthmatic chil-dren were also significant (P < .001; Mann-Whitney) when thecomparisons were made with children younger than 6 years,and with the subgroup of this from age 3 to 6 years, showingthat it is an early event. The anti–HRV-C titers were low acrossthe whole age range for both controls and ED asthmatic children(Fig 4). Age did, however, have a positive correlation with IgG1

titers for HRV-A (r 5 .250, P 5 .014 for HRV-A34; r 5 .252,P 5 .013 for HRV-A1B) and HRV-B (r 5 0.523, P < .0001 for

FIG 1. IgG1 antibody binding to VP1 antigens representing HRV-A (HRV-A34 and HRV-A1B), HRV-B

(HRV-B14 and HRV-B69), and HRV-C (HRV-C3 and HRV-C5) as well as HPV Sabin in 96 ED asthmatic children

(filled circles) and 47 nonasthmatic control children (open circles).A, Total IgG1 binding (ng/mL). B, Species-

specific IgG1 binding (ng/mL). The median and interquartile range are indicated. Species-specific titers to

HRV-A were significantly higher than other antigens in both ED asthmatic children and nonasthmatic con-

trol children (P < .001) as determined using the related-samples Wilcoxon signed-rank test. Comparisons

between ED asthmatic children and nonasthmatic controls were analyzed using the Mann-Whitney test.

The prevalence (%) of subjects with titers below the assay limit of 500 ng/mL is indicated for each antigen.

Prevalence between the 2 groups was analyzed using the x2 test. *P < .05.

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4 IWASAKI ET AL

HRV-B14; r 5 .487, P < .0001 for HRV-B69) in ED asthmaticchildren. No significant correlation between age and anti-HRVIgG1 titers was found in nonasthmatic control children.

There were more male admissions than female admissions tothe ED with asthma exacerbations (Table I). To determine

whether this affected the results, children were stratified by sex,which showed sex differences in ED asthmatic children but notin nonasthmatic control children (data not shown). Despite thedifferences with age and sex, antibody titers to HRV-A in ED asth-matic children remained significantly different from those in

FIG 3. Species-specific IgG1 binding to HRV-C (HRV-C3) and HRV-A (HRV-

A34) antigens for plasma from 30 ED asthmatic children with a known

HRV-C infection at the time of hospitalization. The IgG1 titers for each indi-

vidual immediately following exacerbation from asthma (acute) and at

convalescence (median, 12 weeks) are indicated.

FIG 2. IgG1 antibody binding to HRV-A and HRV-C in children with a known HRV infection. ED asthmatic

children infected with HRV-A at presentation to hospital (n 5 19, pointed circles) did not have significantly

different titers to ED asthmatic children with no detectable HRV-A (n5 54, closed circles). ED asthmatic chil-

dren with no detectable HRV-A had significantly higher anti–HRV-A titers than did nonasthmatic control chil-

dren with no detectable HRV-A (n 5 35, open circles) with the P value indicated. ED asthmatic children

infected with HRV-C (n 5 47, pointed squares) have low species-specific IgG1 titers to HRV-C, which were

not significantly different to ED asthmatic children with no detectable HRV-C at the time of their acute

asthma exacerbation (n 5 45, filled squares). The anti–HRV-C response in ED asthmatic children was not

significantly different from that in nonasthmatic control children with no detectable HRV-C at the time of

recruitment (n 5 34, open squares). The median and interquartile range are indicated.

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nonasthmatic children after adjusting for age and sex (P 5 .019for HRV-A34; P 5 .002 for HRV-A1B).

Isolate-specific response to HRV VP1The VP1 of the exact infecting HRV-A genotype of 2 ED

asthmatic children were produced by amplifying the VP1 regionsfrom cDNA made from their nasal swabs. Immunoabsorptionused to compare the species- and isolate-specific responses foreach subject demonstrated that the isolate-specific response was8.4% of the species-specific response for one subject andnegligible for the other subject (Fig 5). At convalescence, theisolate-specific response increased marginally to 9.3% (subject1) and 0.2% (subject 2) of the species-specific response.

DISCUSSIONTwo observations reported here provide information that could

be of particular importance for elucidating the mechanisms ofHRV-induced asthma exacerbation. The first is that the totalantibody titers to the VP1 antigens of HRV, particularly HRV-Aand HRV-C, were higher in children with asthma exacerbationsthan in nonasthmatic control children. The IgG1 titers specific toHRV-A, and to a lesser extent HRV-B, were also higher in asth-matic children. This was evident at the time of exacerbationand, probably because of the high existing titers, did not revealan increase after convalescence. The higher titers were not asso-ciatedwith the presence or type of virus infection foundwhen pre-senting to the ED. The second is that although asthmatic andnonasthmatic children had high titers to the HRV-C antigens,they were mostly antibodies cross-reactive with the other HRVspecies and not specific for HRV-C. The species-specific titers

FIG 4. Specific IgG1 binding to HRV-A (HRV-A34), HRV-B (HRV-B14), and HRV-C (HRV-C3) in relation to age

in ED asthmatic children following acute asthma exacerbation (n 5 96) (A) and nonasthmatic control

children (n 5 47) (B). As determined by the Spearman rho test, there was a significant positive correlation

between age and IgG1 titers for HRV-A and HRV-B in ED asthmatic children.

FIG 5. Isolate-specific IgG1 titers in 2 HRV-infected subjects following asthma exacerbation and at convales-

cence. Results are represented as the percentage of isolate-specific response to species-specific response.

Both HRV isolates were HRV-A. The species-specific titer of subject 1 to the isolate was 2,687,584 ng/mL at

asthma exacerbation and 1,976,063 ng/mL at convalescence. The species-specific titer of subject 2 to the

isolate was 110,739 ng/mL at asthma exacerbation and 1,086,187 ng/mL at convalescence.

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to HRV-C were not even elevated for subjects presenting with anHRV-C infection and remained low without a consistent increasein convalescence. In contrast, antibodies specific for HRV-A andHRV-B could be readily measured and both the species-specificand total binding was higher in the ED asthmatic children.

The range of binding of specific titers at 250 to 13 107 ng/mL issimilar to that in adults10 and values obtained for other microbialand vaccine antigens.12,17,18 The high titers of capsid-binding an-tibodies, presumably accumulated from responses to repeatedinfection, highlight the pertinence of antibodies acting in concert

with inflammatory cells, mediating the resolution of infec-tions19,20 and producing immunopathology,21 as found for otherviruses such as influenza. The results here with matched virus iso-lates and plasma from infected hosts show that only a tiny percent-age of the antibody binding to the VP1 can be attributed toantibodies specific to the isolate and imply that the high titers per-sisted from previous infections. The high anti-HRV titers and thehigher titers of ED asthmatic children were found at a very earlyage, with HRV-B showing the highest increase with age, whichis possibly a reflection of its low prevalence in the community.

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Adult data showed that HRV-B titers eventually reach levelssimilar to those of HRV-A.10 There was a strong concordance oftiters between the 2 genotypes of HRV-A that was lower forHRV-B and HRV-C. The low concordance for HRV-B could alsoreflect the lower incidence of infectionwith this less prevalent spe-cies. The titers of the ED asthmatic children were initiallycompared with titers of controls that were not collected during acold episode. This was done on the basis that the IgG titer shouldnot have been elevated during the acute phase. It was subsequentlyfound that therewas little change in the titers of the asthmatic chil-dren that could be attributed to the infecting type of virus or thespecific binding to the infecting HRV genotype, and that the titerschanged little even for subjects after extended convalescence. Thedata accordingly show that the overall differences in the titers didnot just result from the infection on recruitment.

T-cell responses have provided the principal estimates ofoverall immune responses to HRV and have demonstrated thatisolates of HRV-A show considerable cross-reactivity.22,23 Nooverall increase in the T-cell proliferative response, however,was found after experimental HRV infection, and although therewas a positive relationship between virus shedding and in vitroT-cell proliferation, the same occurred for tetanus toxoid.24 Theresolution of infection has been linked to IFN-g rather than theoverall response.8,9,25 Evidence for increased HRV-inducedIL-10 has also been reported.8 Because inactivated HRV did notinduce high IFN-g responses8 and the studies of asthmatic pa-tients have used live virus,8,9,22 it needs to be determined whetherthe phenomena reported were due to the known ability of liverhinovirus to modify innate immune functions and antigen pre-sentation in vitro.26

The IgG1 antibody isotype examined in this study is the predom-inant antibody subclass usually found in responses of humans tobacteria and viruses and is the main subclass associated with pro-tection from viruses.27 The follicular central memory T cells thatare critical for isotype switching and B-cell expansion produce abroad range of cytokines.28 Because the cytokines prominent forhelping IgG1 production include IL-21, IL-10, and IL-4,29 theheightened antibodies foundhere are not inconsistentwith reducedresponses by IFN-g–releasing cells. The higher titers could resultfrom higher viral loads produced during infection or a heightenedaspect of immune responsiveness of asthmatic patients. They alsocontrast markedly with the IgG1 antibody responses found to theprotein antigens of the colonizingbacteriaHaemophilus influenzaeand Streptococcus pneumoniae11,12 that are reduced in children re-cruited from the ED.13 This is in the face of the known increasedbacterial colonization found in asthmatic patients14 and thefrequent bacterial detection during viral infection, including withHRV.30

The controls were not stratified for a formal analysis of atopybecause it is a difficult undertaking for this study, given the highassociation of asthma and atopy31 and that nearly all the ED chil-dren were highly atopic. There was nevertheless a significant cor-relation of total IgE and anti–HRV-A titers for the whole studypopulation (data not shown).

The low species-specific antibody titers to HRV-C have previ-ously been described in healthy adults10 occurring despite the factthat HRV-C infections are as prevalent as HRV-A and more prev-alent than HRV-B.32,33 The advance here is to show that they arenot associated with the susceptibility to asthma exacerbation orlinked to the detection of HRV-C in the nasopharynx. The preva-lence can be variable, and studies of young children report that up

to 35% of asymptomatic subjects have PCR-detectable HRV.33,34

Our data show a similar prevalence in the asymptomatic controls,and as found previously,5,7 higher rates of HRV-C infection in theED asthmatic group. An ‘‘original antigenic sin’’ phenomenonmight occur whereby the high cross-reactivity with HRV-A coulddirect responses to the cross-reactive determinants and thesemight not be optimal for immunity to the lower respiratory tractinfections frequently found for HRV-C. Susceptibility to HRV-A associated with an epitope-binding profile has been describedby others.35 The low specific responses could, however, equallyresult from the different biology of HRV-C including its interac-tion with different cell receptors and a priori different activationof innate immunity pathways.

In conclusion, this study provides a comparison of the bindingof antibodies from children to the VP1 antigens of 3 species ofHRV. The high binding of the total HRV and species-specificHRV-A and HRV-B antibodies of asthmatic children showevidence for persistent heightened immune reactivity. Thiscontrasts with the paucity of species-specific antibodies toHRV-C in both asthmatic and nonasthmatic children.

We thank the participants who took part in the study, L. A. Hazell and T. L.

Y. Chai for assistance with protein antigen preparation, and S. R. Stone for

assistance with circular dichroism analysis.

Key messages

d Children with asthma exacerbations have higher totalIgG1 antibody titers to HRV than do nonasthmatic con-trols, showing a heightened aspect of their immuneresponse to HRV.

d All specific anti–HRV-C titers were low, even when in-fected with HRV-C, with only anti–HRV-A and anti–HRV-B being higher in the asthmatic group.

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TABLE E1. HRV and HPV genotypes used in this study

Species Genotype Prototype Accession no.

HRV-A A34 FJ445189.1

HRV-A A1B D00239.1

HRV-B B14 NC001490

HRV-B B69 FJ445151

HRV-C C3 QPM EF186077

HRV-C C5 025 EF582386

HEV-C HPV Sabin AY184219.1

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