Influence of Frequent Infectious Exposures on General and Varicella-Zoster Virus-Specific Immune Responses in Pediatricians

Benson Ogunjimi,a,b,c Evelien Smits,d,e Steven Heynderickx,d,f Johan Van den Bergh,d Joke Bilcke,a Hilde Jansens,g Ronald Malfait,g

Jose Ramet,c Holden T. Maecker,h Nathalie Cools,d Philippe Beutels,a,i Pierre Van Dammej

Centre for Health Economics Research and Modeling Infectious Diseases (CHERMID), Vaccine and Infectious Disease Institute (VAXINFECTIO), University of Antwerp,Antwerp, Belgiuma; Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt, Belgiumb; Department of Pediatrics, AntwerpUniversity Hospital, Edegem, Belgiumc; Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (VAXINFECTIO), University of Antwerp, Antwerp,Belgiumd; Center for Oncological Research Antwerp, University of Antwerp, Antwerp, Belgiume; Center for Cell Therapy and Regenerative Medicine, Antwerp UniversityHospital, Edegem, Belgiumf; Department of Laboratory Medicine, Antwerp University Hospital, Edegem, Belgiumg; Institute for Immunity, Transplantation, and Infection,Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USAh; School of Public Health and Community Medicine, TheUniversity of New South Wales, Sydney, Australiai; Centre for the Evaluation of Vaccination (CEV), Vaccine and Infectious Disease Institute (VAXINFECTIO), University ofAntwerp, Antwerp, Belgiumj

Reexposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studiedthe effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive expo-sures to varicella-zoster virus (VZV) elicited persistently high immune responses. Blood samples from 11 pediatricians andmatched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity bymeans of measuring T-cell subset percentages, antibody titers and gamma interferon (IFN-�)/interleukin 2 (IL-2)-producingT-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), tetanus toxin (TT), and VZV were determined.Pediatricians had lower levels of circulating CD4�-naive T cells and showed boosting of CD8� effector memory T cells. Althoughno effect on humoral immunity was seen, repetitive exposures to VZV induced persistently higher percentages of IFN-�-positiveT cells against all VZV antigens tested (VZV glycoprotein E [gE], VZV intermediate-early protein 62 [IE62], and VZV IE63) thanin controls. T cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Althoughno differences in cellular or humoral immunity were found between the pediatricians and controls for AdV-5 or TT, we did findlarger immune responses against CMV antigens in pediatricians. Despite the high infectious burden, we detected a robust anddiverse immune system in pediatricians. Repetitive exposures to VZV have been shown to induce a stable increased level of VZV-specific cellular but not humoral immunity. Based on our observations, VZV IE63 can be considered a candidate for a zostervaccine.

The secondary immune response in humans, elicited after reex-posure to a virus or other pathogens, can reinforce the quan-

tity and quality of the immune response against the challengingpathogen. However, the existence of a saturation level or evenexhaustion after repetitive natural exposures has not been suffi-ciently studied in humans. Also, some authors have discussed theexistence of competition between pathogens in regard to humoralimmunity (1, 2). The question thus remains how individuals withfrequent exposures to different pathogens and repetitive expo-sures to some pathogens maintain a healthy immune system inbalance. In particular, the ubiquitous varicella-zoster virus (VZV)presents a challenging dilemma, as stated by Hope-Simpson (3),who hypothesized that reexposure to VZV might postpone thereactivation of VZV, herpes zoster. As such, simulation programshave predicted temporary increases in herpes zoster incidence af-ter the introduction of a childhood widespread VZV vaccinationprogram (4–6). Although a live attenuated VZV vaccine is cur-rently universally given to children in several countries (e.g.,United States, Germany, and Australia), much debate regardingthe suitability of such a program remains (5, 7–46), and a recentmultidisciplinary systematic review has concluded that so-calledexogenous boosting exists, but the true extent of this is yet to bedetermined (47).

In this exploratory study, we have set forth the following goals.First, we aimed to describe the general effects of frequent infec-

tious exposures in pediatricians on their humoral and cellularimmune responses. Second, we set out to examine virus-specificeffects of repeated exposures, particularly for VZV.

MATERIALS AND METHODSStudy design and subjects. Eleven pediatricians (age range, 33 to 60 years;mean age, 42 years; 7 women) comprising a high-exposure (HE) groupdonated venous blood samples at three different time points: winter (24February to 16 March 2012) (HE-WIN), spring (11 May to 8 June 2012)(HE-SPR), and summer (3 to 19 July 2012) (HE-SUM). Information re-garding chickenpox exposure frequencies in pediatricians is shown inTable 1 (as recorded in their study journals). Eleven age (�1 year)- andgender-matched normally exposed healthy control individuals (CO) withno known exposure to chickenpox during the past 2 years donated venousblood samples at a single time point (2 to 20 July 2012). Following recently

Received 29 December 2013 Accepted 9 January 2014

Published ahead of print 15 January 2014

Editor: R. L. Hodinka

Address correspondence to Benson Ogunjimi, benson.ogunjimi@uantwerp.be.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00818-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00818-13

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proposed guidelines, venipuncture was performed at fixed sampling sites(48). This study was approved by the ethics board of the University Hos-pital Antwerp, Antwerp, Belgium. Written informed consent was ob-tained from all study participants.

White blood cell count determination. One fresh EDTA tube persample was analyzed using an automated hemocytometer (Sysmex XS-1000i) in order to obtain the total white blood cell and differential counts.

Blood processing and flow cytometric analyses. Peripheral bloodmononuclear cells (PBMC) were isolated by Ficoll-Paque Plus gradientseparation (Amersham Biosciences, Uppsala, Sweden) from freshly ob-tained heparinized blood. The freezing of PBMC was performed in 90%fetal bovine serum (FBS) (Gibco Invitrogen, Ghent, Belgium) supple-mented with 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Stein-heim, Germany). Filled cryovials were put into Nalgene cryoboxes beforefreezing at �80°C, and afterwards, the cells were stored in liquid nitrogen.Thawing of the cells for batch analysis was done in a water bath at 37°C.After washing with preheated (37°C) medium supplemented with 10%FBS, flow cytometric analysis was performed. For this, PBMC were leftunstimulated or stimulated overnight in the presence of GolgiPlug (BDBiosciences) with (i) phytohemagglutinin (PHA) plus ionomycin, (ii)VZV intermediate-early protein 62 (IE62) PepMix (JPT, Berlin, Ger-many), (iii) VZV IE63 PepMix (JPT), (iv) VZV glycoprotein E (gE) Pep-Mix (JPT), (v) tetanus toxin (TT) mix (PANATecs, Heilbronn, Ger-many), (vi) cytomegalovirus (CMV) pp65 peptide mix (NIH, Bethesda,MD), or (vii) human adenovirus type 5 (AdV-5) penton PepMix (JPT).Per condition, 1 � 10E6 PBMC per ml Iscove’s modified Dulbecco’s me-dium (Gibco Life Technologies, Ghent, Belgium) plus 10% FBS werestimulated using 1 �g peptide/ml. Afterwards, the cells were washed andstained using the antibody panel as described in Table 2. In doing so, thecell subsets were determined based on membrane expression levels ofCD3, CD4, CD8, CD45RA, and CCR7 (see Fig. S1 in the supplementalmaterial for the gating strategy). Next, the cells were fixed and permeab-ilized using fluorescence-activated cell sorting (FACS) lysing solution andFACS permeabilizing solution 2 (BD Biosciences), according to the man-ufacturer’s protocol, before staining to determine intracellular expressionlevels of gamma interferon (IFN-�) and interleukin 2 (IL-2). Nonreactiveisotype-matched antibodies were used as controls for IL-2, IFN-�, andCCR7. Flow cytometric analysis was performed on a BD FACSAria II flowcytometer (BD Biosciences). If possible, 1,000,000 events were measured.The FlowJo software version 9.6.2 (Tree Star, Inc., Ashland, OR) was usedfor cytometric data analysis. As suggested recently, absolute cell countswere obtained by multiplying the ratio of marker-positive cells to thawed,viable, and single cells with the number of lymphocytes plus monocytesper volume unit (49). However, we noted no major differences betweenthe analyses based on the relative and absolute data.

Determination of antibody responses. Blood serum samples werefrozen at �80°C until batch analysis. All serological analyses were per-formed for both IgM and IgG. For the analysis of VZV-specific antibodies,two methods were used, a VZV IgM/G glycoprotein-based enzyme-linkedimmunosorbent assay (gpELISA) (Serion, Germany) and VZV-infectedcell lysate on XL Liaison using a DiaSorin kit (USA). IgG directed againstCMV pp150, pp28, p38, and p52 and IgM against CMV pp150, p52(RoCO, Basel, Switzerland), AdV-5 hexon-specific IgM/G (Demeditec,Germany), and tetanus toxin-specific IgM/G (Demeditec) titers were de-

termined as well. The positivity thresholds used were those given by themanufacturers.

Data management and statistics. Hematocytological cell counts andIgG titers against the different pathogens were compared between thedifferent sampling points (HE-WIN, HE-SPR, HE-SUM, and CO). Forthe flow cytometric analysis, only the time points HE-SPR and HE-SUMwere retained for further analysis due to a low viability in the PBMC fromHE-WIN.

First, T-cell subset percentages of CD3� T cells were compared be-tween the different sampling groups (HE-SPR, HE-SUM, and CO). TheCD3� CD4� and CD3� CD8� subsets were further subtyped as CCR7�

CD45RA� (naive T cells [Tna]), CCR7� CD45RA� (central memory Tcells [Tcm]), CCR7� CD45RA� (effector memory T cells [Tem]), andCCR7� CD45RA� (effector T cells [Tef]). Next, the percentages ofIFN-�- or/and IL-2-producing T-cell subtypes were calculated perstimulus by subtracting the percentage from the unstimulated sam-ples. Fisher’s exact test for contingency tables, comparing stimulatedwith unstimulated samples, was used to retain positive differences, witha P value of �0.05 (one-sided). Comparisons with a P value of 0.05 butwith �2,000 cells in the parent group were left blank due to the low cellcount (465/4,752 blanks with 144 due to one unusable PBMC sample inthe HE-SUM group). The proportions of antigen-responsive individualsper sampling group and per antigen were compared with the seropreva-lence per sampling group and per pathogen. The comparison was notformally made between AdV-5 hexon antibody and penton cytokine re-sponses due to the broader range of responses against AdV-5 hexon IgGby different adenovirus types compared to those against the AdV-5 pen-ton peptide mix. Individuals were considered immune to VZV or CMV ifthey had a positive serological or cytokine response against VZV or CMV,respectively. All individuals were considered immune against TT (due tovaccination), and for the cellular analyses against AdV-5 penton, onlyindividuals with positive cytokine responses were considered immune toAdV-5. The proportions of antigen-responsive individuals were com-pared per antigen between the three sampling groups. Next, antigen-stim-ulus-induced IFN-�-producing total T-cell and T-cell subset percentageswere calculated per pathogen for the pathogen-immune individuals.Comparisons were made between antigens and between the samplinggroups. Spearman’s correlations were calculated between the VZV IgGtiters and VZV IE62, VZV IE63, and VZV gE responses. Although ourmain focus was directed toward the antiviral cytokine IFN-�, a sensitivityanalysis included the combination of IFN-�- and IL-2-producing cells aswell.

All comparative statistical analyses were performed using either a two-sided t test (paired or unpaired) when the variable was deemed normallydistributed, or a nonparametric test (Wilcoxon signed-rank or Mann-Whitney rank-sum test) if not. A nonparametric test was used if nonnor-mality was concluded by the Shapiro-Wilk statistic for at least one of thedistributions, and otherwise both a t test and a nonparametric test wereused. However, if the nonparametric test proved to have more power todetect differences than did the t test (an indication of nonnormality), theresults from the nonparametric test were used. In addition, all statisticalanalyses based on cytokine-producing cell counts were performed using

TABLE 2 Antibody panel for PBMC staining

Target Fluorochromea Clone Company

IFN-� FITC B27 BDCCR7 PE 150503 BDCD3 PE-Texas Red S4.1 Life TechnologiesCD45RA PE-Cy7 HI100 BDLIVE/DEAD Violet Life TechnologiesIL-2 APC 5344.111 BDCD8 Pacific orange 3B5 Life TechnologiesCD4 APC-H7 RPA-T4 BDa FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin.

TABLE 1 Monthly chickenpox exposure frequencies in pediatriciansa

Data

No. of exposures in:

February March April May June

Range 1–5 0–10 2–15 2–23 1–12Median 2 5 5 9 5No. of respondents 4 7 7 7 9a All pediatricians had an exposure to chickenpox within, at most, 11 weeks prior to thefirst sampling point, but mostly within a few weeks.

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nonparametric tests due to the skewed distributions of these counts. Mat-lab 2012b and SPSS 20 were used for the analyses. The results were con-sidered significant if there was a P value of �0.05 or tended to be signifi-cant if the P value was 0.05 and �0.10.

RESULTSComparison of white blood cell counts. The total white blood cellcount tended to be higher in the HE-SUM (summer sampling ofpediatricians) group than in the HE-SPR (spring sampling of pe-diatricians), HE-WIN (winter sampling of pediatricians), and CO(control group) groups (factor 1.1, P 0.066; factor 1.1, P 0.079; factor 1.2, P 0.034, respectively). The neutrophil countwas higher in the HE-SUM group than in the HE-SPR group (fac-tor 1.2, P 0.028). The monocyte count tended to be higher in theHE-SUM group than in the HE-WIN group (factor 1.2, P 0.053).

Comparison of T-cell subset percentages (Fig. 1). The CD4�

CCR7� CD45RA� (CD4� Tna) percentage was lower in the HE-SUM group than in the CO group (factor 1.6, P 0.041). TheCD4� Tna percentage also tended to be lower in the HE-SPR

group than in the CO group (factor 1.4, P 0.076). In addition,we found that the CD8� Tna percentage tended to be lower in theHE-SUM group versus the HE-SPR group (factor 1.1, P 0.092).The CD8� CCR7� CD45RA� (CD8� Tem) percentage tended tobe higher in the HE-SPR and HE-SUM groups than in the COgroup (factor 1.3, P 0.076; factor 1.4, P 0.099, respectively).No differences were found for the total CD4�, total CD8�,CCR7� CD45RA� (Tcm), or CCR7� CD45RA� (Tef) percent-ages.

Antibody detection against common pathogens. Pathogenseropositivity remained constant at all time points in the HEgroups. All study participants were VZV IgG seropositive (forboth the VZV gpELISA and VZV cell lysate). All HE samples wereAdV-5 hexon seropositive, whereas the IgG titer was negative forone CO participant and unequivocal for another. For CMV only,a tendency (P 0.085) toward more IgG positive individuals wasshown in the HE samples (9/11) than in the CO samples (4/11)(Fig. 2 and Table 3).

None of the pathogens’ IgG titers indicated statistically mean-

FIG 1 Comparison of T-cell subset percentages (of CD3� PBMC). Distribution of T-cell subsets is presented per sampling as a box plot (with medians ashorizontal lines, interquartile range as boxes, ranges as vertical lines, and outliers as dots). Data for CD4� Tna, CO versus HE-SPR (P 0.076); for CD4� Tna,CO versus HE-SUM (P 0.041); for CD8� Tna, HE-SPR versus HE-SUM (P 0.092); for CD8� Tem, CO versus HE-SPR (P 0.076); and for CD8� Tem, COversus HE-SUM (P 0.099). All other P values are 0.10.

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ingful differences between the sampling groups, although theVZV cell lysate titer had a tendency to be higher in the HE-WINgroup than in the HE-SUM group (factor 1.1, P 0.090), and theCMV IgG titer showed a trend toward higher values in the HEgroups (Fig. 2). We note that several CMV IgG titers in the HEgroups reached the experimental upper limit.

Pathogen-specific T cells. Although CMV-specific IgG titerswere detected in only 4/11 CO samples, we detected CMV pp65-specific T cells in 10/11 CO samples when using IFN-� or/and IL-2intracellular cytokine staining upon stimulation (P 0.024). Inaddition, we observed that CMV positivity in the HE samplesreached 100% while using cellular immunology instead of serol-ogy as a measurement tool. We also noted only 6/11 CO sampleshaving VZV IE63-specific T cells (P 0.036), whereas all CO andHE samples had responses to VZV IE62. The percentage of sam-ples with cellular responses to VZV gE T cells ranged between 73and 90%. The percentage of individuals with TT-specific T cellswas significantly lower in CO (6/11) samples than in the HE-SPR(11/11, P 0.036) and HE-SUM (10/10, P 0.036) samples.CD4� and CD8� responses were observed against all antigenstested (Table 3).

Comparing IFN-� T-cell percentages between antigens andbetween groups. Altogether, we noted that CMV pp65- and VZV

IE62-specific T-cell responses were the most abundant, althoughsome differences existed between the CO and HE samples (Fig. 3and 4).

The control individuals had more CMV pp65-specific T cellsthan T cells against VZV gE (P � 0.001), VZV IE63 (P 0.020), orTT (P � 0.001). Also, specific T cells against AdV-5 penton andVZV IE62 were more prevalent than those against VZV gE (P 0.0039 and P 0.079, respectively). Similarly, pediatricians hadhigher CMV pp65-specific T-cell responses both during springand summer than against VZV gE (P 0.042 and P 0.0052,respectively) or TT (P 0.0024 and P 0.015, respectively).Comparable to the controls, VZV IE62 responses were more fre-quent than VZV gE responses in both the HE-SPR (P 0.020) andHE-SUM (P 0.044) groups. However, VZV IE62 responses werealso more frequent than TT responses in both the HE-SPR (P 0.0031) and HE-SUM (P 0.024) groups and more frequent thanAdV-5 penton responses during the spring (P 0.049). We alsonoted that during the spring, TT-specific T-cell responses wereless frequent than those against AdV-5 penton (P 0.011) andVZV IE63 (P 0.0063).

Overall, we found positive correlations between the percent-ages of T cells against VZV IE62 and VZV IE63 (data not shown).Aggregating the different groups also showed a minor correlation

FIG 2 Antibody detection against common pathogens. IgG titers per pathogen (AdV-5 hexon [IU/ml], CMV [IU/ml], TT [IU/100 ml], VZV cell lysate[mIU/ml], VZV gp [mIU/ml]) and per sampling group are presented using a box plot (with medians as horizontal lines, interquartile ranges as boxes, ranges asvertical lines, and outliers as dots). Data for VZV cell lysate, HE-WIN versus HE-SUM (P 0.090). All other P values are 0.10.

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between the percentages of T cells against VZV gE, VZV IE62, andVZV IE63 (data not shown).

The antigen-specific analysis between the sampling groups(Fig. 4) found the percentage of CMV-specific T cells to be higherin the HE-SUM group than in the CO (P 0.024) group. AllVZV-specific T-cell percentages were lower in controls than inhighly exposed individuals (both spring and summer): VZV IE62was lower in the CO group than in the HE-SPR (P 0.023) andHE-SUM (P 0.034) groups, VZV IE63 was lower in the COgroup than in the HE-SPR (P 0.064) and HE-SUM (P 0.030)groups, and VZV gE was lower in the CO group than in the HE-SPR (P 0.028) and HE-SUM (P 0.0019) groups. No differ-ences between the sampling groups were found for AdV-5 pentonor TT.

Analysis of within-individual distribution of antigen-spe-cific T-cell subsets. The CO, HE-SPR, and HE-SUM antigen-spe-cific T-cell subset proportions showed for all antigens tested thatIFN-�-producing T cells mainly had a CD4� Tem, CD8� Tem, orcombined Tem subset. Noteworthy are the CD4� Tem domi-nance for VZV gE and the CD8� Tem dominance for VZV IE62(Fig. 5; see also Table S1 in the supplemental material).

Combining IFN-�- and IL-2-specific T-cell responses. Ingeneral, we noted that the inclusion of IL-2 did not have a major

effect on the results obtained with IFN-� alone. However, theproportion of CD4� Tcm responses was increased with the inclu-sion of IL-2 for all antigens tested (see Fig. S2 and Table S1 in thesupplemental material).

DISCUSSION

This exploratory study was designed to analyze the cellular andhumoral immune responses at different time points (winter,spring, and summer) in a group with high exposure to infections(represented by pediatricians) and to compare these to single timepoint responses (summer) in a group with what we considered anormal exposure load (non-MDs, nonteachers, etc.). The aim wasto analyze both the general indices of immune and antigen-spe-cific responses, particularly against VZV but also against CMV,adenovirus type 5, and tetanus toxin.

The total white blood cell count was about 10% to 20% higherfor the high-exposure group during the summer than in the win-ter and spring and than in the normal-exposure group. The neu-trophil and monocyte counts in the high-exposure group werehigher during the summer than during the spring and winter,respectively. Importantly, we did not find any differences for thelymphocyte counts. These results might imply that highly exposedindividuals have an upregulated innate immune system during

TABLE 3 Humoral and cellular immunity against common pathogensa

Pathogen Cellular antigen Group

No. ofindividualswith positiveresponses/totalno. ofindividuals for:

P for IgGvs CMI

CMI

P for:

No. ofindividuals withresponsive cells/total no. ofindividuals for:

P for CD4�

vs CD8�IgG CMICO vsHE-SPR

CO vsHE-SUM

HE-SPR vsHE-SUM

CD4�

cellsCD8�

cells

AdV-5 AdV-5 penton CO 9/10 9/11 NSb NS NS 6/11 6/10 NSHE-SPR 11/11 11/11 7/11 9/11 NSHE-SUM 11/11 10/10 9/10 6/10 NS

CMV CMV pp65 CO 4/11 10/11 0.024 NS NS NS 8/10 6/10 NSHE-SPR 9/11 11/11 NS 10/11 9/11 NSHE-SUM 9/11 10/10 NS 9/10 9/10 NS

Tetanus TT CO 10/11 6/11 NS 0.036 0.036 NS 6/11 3/11 NSHE-SPR 11/11 11/11 NS 8/11 5/11 NSHE-SUM 11/11 10/10 NS 10/10 5/10 0.033

VZV VZV gE CO 11/11 8/11 NS NS NS NS 6/11 3/10 NSHE-SPR 11/11 9/11 NS 9/11 3/11 0.016HE-SUM 11/11 9/10 NS 9/10 4/10 0.058

VZV VZV IE62 CO 11/11 11/11 NS NS NS NS 10/11 7/11 NSHE-SPR 11/11 11/11 NS 9/11 9/11 NSHE-SUM 11/11 10/10 NS 10/10 9/10 NS

VZV VZV IE63 CO 11/11 6/11 0.036 0.036 NS NS 5/11 5/11 NSHE-SPR 11/11 11/11 NS 8/11 9/11 NSHE-SUM 11/11 9/10 NS 8/10 6/10 NS

a Shown is memory against pathogens using either humoral (IgG) or cell-mediated immunity (CMI) (IFN-�- or/and IL-2-producing T cells) as proxy. Fisher’s exact test was usedto compare immunity as assessed by IgG or CMI (against an antigen) for three sampling groups (P values of �0.10 are shown). CMI is compared among the three groups (P valuesof �0.10 are shown). For each antigen, the presence of CD4� and CD8� IFN-� or/and IL-2 responses are assessed and compared with each other per antigen and per group (Pvalues of �0.10 are shown).b NS, nonsignificant.

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seasons with frequent infectious challenges that is reflected inhigher white blood cell counts in the peripheral circulation (on“standby”) at moments with less infectious challenges.

We found that the highly infectious burden was associated witha loss of CD4� CCR7� CD45RA� (naive) T cells in pediatricians.This was contrasted with a trend toward a higher prevalence ofCD8� CCR7� CD45RA� (effector memory) T cells during thesummer, possibly still due to the antigen-specific proliferationduring the winter and spring.

CMV IgG seroprevalence was higher in pediatricians than inthe controls, which was in accordance with another study thatdocumented higher CMV seroprevalence in child caretakers (50).The CMV IgG titer suggested higher values for the high-exposuregroups than the normal-exposure group. Both natural (8, 9, 16,38) and vaccine-based reexposures (51–53) to VZV have beenshown to majorly increase VZV-specific antibody titers. The pe-diatricians in our study, however, did not have large differences in

VZV-specific antibody titers. Both higher VZV-specific cellularimmunity and higher secretory immunoglobulin A (sIgA) levelsin saliva (13) might explain these observations. We note, however,that more specific VZV antibody titrations (see Jenke et al. [54])focused on VZV IE62 or VZV IE63 epitopes might show the dif-ferences between pediatricians and controls that are currently av-eraged out by the large VZV glycoprotein response.

Previous studies have identified several VZV antigens capableof eliciting cellular immune responses (55–57). VZV gE, the mostabundant VZV glycoprotein, and VZV IE63, the immediate-earlyphosphoprotein most likely involved in VZV reactivation (58–60), have been shown to elicit CD4� T-cell responses (57, 58). Theimmediate-early phosphoprotein VZV IE62, however, is knownto elicit CD8� T-cell responses as well (55). Our study identifiedboth CD4� and CD8� responses against all antigens, albeit not inall samples (which also might be influenced by HLA types). Note-worthy was our finding of a relatively low prevalence (6/11 sam-

FIG 3 Comparing IFN-�-producing T-cell percentages (of CD3� PBMC) between antigens. Box plot representations (with medians as horizontal lines,interquartile ranges as boxes, ranges as vertical lines, and outliers as dots) are used for the six different antigens (AdV-5, adenovirus type 5 penton; CMV,cytomegalovirus; TT, tetanus toxin; VZV, varicella-zoster virus) per sampling condition (aggregated, aggregate of all sampling groups; CO, control group forhigh-exposure group; HE-SPR, high-exposure group in spring; HE-SUM, high-exposure group in summer). For P values, see text.

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ples) of T-cell responses against VZV IE63 in the normal-exposuregroup but not in the high-exposure group (11/11 and 9/10 sam-ples, respectively). Overall, both VZV IE62- and VZV IE63-spe-cific IFN-�-producing T-cell responses were more frequent thanthose against VZV gE. However, VZV-specific T-cell responseswere modestly correlated with each other although most pro-nounced between VZV IE62 and VZV IE63. We noted that theVZV-specific cellular and humoral immune responses were notcorrelated with each other.

Terada and colleagues (11) documented higher responder cellfrequencies in pediatricians after stimulation with VZV cell cul-ture antigens. Using state-of-the-art techniques, such as flow cy-tometry and peptide mixes, we found that all three VZV-specificantigens tested consistently elicited higher IFN-�-producing T-cell percentages in the high-exposure groups than in the normal-exposure group. In general, CD4� and CD8� IFN-�-producing Tcells were dominated by the absence of the expression of CCR7and CD45RA markers giving them an effector memory pheno-type. Our combined results imply that frequent reexposure toVZV stimulates the proliferation of T cells against all VZV anti-gens. These findings further support the exogenous boosting hy-pothesis formulated by Hope-Simpson (3), suggesting that reex-posure to VZV has a protective effect against herpes zoster. Thelow prevalence of VZV IE63-specific cellular responses in the nor-mal-exposure group also suggests a faster decline in the memoryresponse against this protein, which has been implicated in VZVreactivation (58–60). The lower incidence of herpes zoster in pe-

diatricians found in some studies (12), although not all (34),might thus be caused by more frequent boosting of the cellularimmune responses against VZV IE63. Our results also emphasizethe existence of VZV-specific CD8� responses, including a CD8�

dominance against VZV IE62. We note that our antigens con-sisted of overlapping 15-mer peptide mixes designed to stimulateboth CD4� and CD8� T-cell responses, particularly when used onthawed PBMC (61). In contrast, many studies in the past, andimportantly, the Zostavax vaccine immunogenicity studies, usedVZV cell lysate as a stimulus (11, 16, 38, 53, 62–65). Thus, anychange in the CD8� component against VZV might have beenmissed. Also, the more recently published GlaxoSmithKline(GSK) VZV gE subunit vaccine immunogenicity study used 20-mer peptide mixes that might be too long to detect CD8� T-cellresponses (52).

When identifying CMV pp65-responsive individuals, intra-cellular detection of IFN-� or IL-2 production by T cells uponstimulation with CMV pp65 peptides resulted in more individ-uals with a memory response against CMV than the serologicalmethod. Although the greatest improvement of detection sensi-tivity was seen in the normal-exposure group (going from 4/11 to10/11 samples), the proof of concept was noteworthy for the pe-diatricians, as CMV positivity increased from only 9/11 sampleswhen using IgG as the gold standard to 100% when using intra-cellular cytokine staining as a diagnostic tool. The increased sen-sitivity of cellular immunity for memory detection compared tohumoral immunity, an important finding for at-risk groups such

FIG 4 Comparing IFN-�-producing T-cell percentages (of CD3� PBMC) between sampling groups. Box plot representations (with medians as horizontal lines,interquartile ranges as boxes, ranges as vertical lines, and outliers as dots) are used for the three different sampling groups for each antigen (AdV-5, adenovirustype 5 penton; CMV, cytomegalovirus; TT, tetanus toxin; VZV, varicella-zoster virus). Data for CMV pp65, CO versus HE-SUM (P 0.024); for VZV gE, COversus HE-SPR (P 0.028); for VZV gE, CO versus HE-SUM (P 0.019); for VZV IE62, CO versus HE-SPR (P 0.023); for VZV IE62, CO versus HE-SUM(P 0.034); for VZV IE63, CO versus HE-SPR (P 0.064); and for VZV IE63, CO versus HE-SUM (P 0.030). All other P values are 0.10.

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as transplant patients and pregnant women, was recently alreadyshown for CMV (66) and had already been shown for variouspathogens, such as hepatitis C virus (67). CMV pp65-specificIFN-�-producing T cells were more frequent than those againstVZV IE63, VZV gE, TT, and AdV penton. The relatively highprevalence of CMV-specific T cells is consistent with the resultsfrom other studies (68, 69). The CMV pp65-specific T-cell fre-quencies were also higher in samples from pediatricians than inthose from the controls. We found no systematic differences in theT-cell frequencies against TT or AdV-5 penton between the pedi-atricians and controls, although TT-specific IFN-�- or IL-2-pro-ducing CD4� CCR7� CD45RA� (effector memory) cells weremore frequent at both sampling points for pediatricians than forcontrols.

This exploratory study was limited by its sample size, and conse-quently, its lower power for finding significant differences that mightexist (e.g., in regard to antibody responses). Despite this, several sys-tematic and significant results were found. The limited sample sizemade it impossible to assess immunity dysregulation caused byCMV, as was recently proposed for VZV (70). Nonetheless, someimportant conclusions can be made based on this study.

Future work should focus on a reanalysis of the VZV vaccine im-munogenicity studies by making use of 15-mer peptide mixes. Inaddition, our (albeit indirect) support for the importance of VZVIE63 in VZV reactivation might stimulate the development of a her-

pes zoster vaccine directed at VZV IE63. Future work should analyzethe long-term effects of frequent exposures on the specific and non-specific immune system, keeping in mind that the population in thecurrent study consisted mainly of young individuals.

In conclusion, while comparing immune responses betweenhighly exposed and normally exposed individuals, we observed atransient depletion of naive CD4� T cells and boosting of CD8�

effector memory T cells. Although no effect on humoral immu-nity was seen, we found that all VZV-specific antigens tested hadpersistently higher IFN-�-producing T-cell percentages afterstimulation in the highly exposed than in the normally exposedindividuals, thereby illustrating the stimulatory effects of the sec-ondary immune response.

ACKNOWLEDGMENTS

We thank all the participants for their participation and M. Claeys for hisconstructive comments.

The human CMV pp65 peptide pool was obtained through the NIHAIDS Research and Reference Reagent Program (Division of AIDS,NIAID, NIH). This work was supported by grants of the Research Foun-dation Flanders (project grant, predoctoral fellowship to B.O., postdoc-toral fellowships to J.B. and N.C.), the Hercules Foundation-Belgium, andthe University of Antwerp (predoctoral fellowship to J.V.D.B.). Thefunders had no role in study design, data collection or analysis, the deci-sion to publish, or preparation of the manuscript.

FIG 5 Comparing differences in IFN-�-producing T-cell subsets between sampling groups. Box plot representations (with medians as horizontal lines,interquartile ranges as boxes, ranges as vertical lines, and outliers as dots) are used to compare T-cell subset percentages (of CD3� PBMC) for the three differentsampling groups. See Table S1 in the supplemental material for P values.

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