Development of immunologic memory

against tetanus toxoid and pertactin

antigens from the diphtheria-tetanus-
pertussis vaccine in atopic versus
nonatopic children
Patrick G. Holt, DSc,b Anna Rudin, PhD,d Claudia Macaubas, PhD,a Barbara J. Holt,
BSc,a Julie Rowe, PhD,a Richard Loh, FRACP,e and Peter D. Sly, FRACPc West Perth,
Australia

Background: Recent findings suggest that a hallmark of the Key words: Atopy, children, TH1/TH2, vaccines, cytokines
atopic phenotype is reduced capacity to respond to vaccine
antigens, as well as to environmental allergens, during infancy. The nature of the immunologic defect(s) associated
This deficiency, which is most marked for the cytokine IFN-γ, with genetically determined susceptibility to atopy, and
appears transient but can result in a long-lasting imbalance how it interacts with known environmental risk factors,
within T helper cell (TH) memory responses to allergens. Indi- represents an area of intensive research. An issue of par-
rect evidence suggests that parallel effects may occur within
ticular interest, which is becoming increasingly contro-
immunologic memory responses against vaccine antigens in
versial in both the biomedical and lay press, concerns
atopic children.
Objective: Our purpose was to compare vaccine antigen-spe- interactions between the developing immune system in
cific TH memory responses in atopic and nonatopic children. children genetically susceptible to atopy and microbial
Methods: We analyzed specific serum IgG and cytokine respons- stimulation through normal environmental contact or
es to pertactin and tetanus antigens as well as to mitogen (PHA) through deliberate exposure by vaccination.
and house dust mite (HDM) allergen in 25 HDM-sensitized This complex issue has arisen in the light of a series of
atopic and 25 nonatopic 6-year-old children who were vaccinated recent findings on postnatal maturation of adaptive immu-
and boosted with diphtheria-tetanus-pertussis (DTP) vaccine. nity in children and how these relate to the subsequent
Results: PBMCs from the atopic subjects produced higher lev- development of diseases such as atopic asthma. It has
els of TH1 and TH2 cytokines to HDM allergen and PHA. Vac-
been recognized for many years that certain aspects of
cine antibody titers were normal in the atopic subjects; vac-
immune function are poorly expressed in humans at birth
cine-specific TH2 responses were rarely detectable, yet TH1
(IFN-γ) responses, in particular against tetanus, were frequent and do not attain adult-equivalent levels of competence
and higher in the atopic subjects (121.5 [SE 64.3] vs 8.0 [3.5] for several years postnatally.1 Recently, it has become evi-
pg/mL culture fluid, P = .04). Corresponding pertactin dent that the principal functions that exhibit this matura-
responses were comparable in both groups. tional deficiency in early life are associated with the T
Conclusions: At the completion of the full primer-booster DTP helper type 1 (TH1) arm of the immune response.2 The
vaccination regimen, levels of vaccine-specific immunity in primary function of TH1 immunity involves antimicrobial
atopic 6-year-old children are at least equivalent to their defense, and the principal stimulus for postnatal matura-
nonatopic counterparts, indicating that the transient atopy- tion of the relevant TH1 functions is provided by contact
associated deficiency in TH1 function in childhood can be suc-
with microbial flora and pathogens in the extrauterine
cessfully overcome by appropriate vaccination and boosting
environment.3,4 Secondarily, TH1 immunity also provides
regimens. (J Allergy Clin Immunol 2000;105:1117-22.)
a protective counterbalance against the development of
excessive T helper type 2 (TH2)–mediated immune
responses, which through elaboration of cytokines such as
IL-4, IL-5, and IL-13 can potentially produce pathogenic
From the aTVW Telethon Institute for Child Health Research, the Depart-
ments of bMicrobiology and cPaediatrics, University of Western Australia,
allergic inflammation.5
the dDepartment of Medical Microbiology and Immunology, Gothenburg It has also been recently shown that key aspects of
University, and the eDepartment of Clinical Immunology, Princess Mar- long-term immunologic memory against environmental
garet Hospital for Children, Perth, Australia. allergens, in particular the balance within the T helper cell
Supported by the National Health and Medical Research Council of Australia
system between TH1 versus TH2 immunity, is frequently
and by GlaxoWellcome, United Kingdom.
Received for publication Nov 5, 1999; revised Jan 17, 2000; accepted for pub- established during early childhood,6,7 and hence the rate
lication Jan 18, 2000. at which postnatal maturation of TH1 function(s) proceeds
Reprint requests: P. G. Holt, DSc, Division of Cell Biology, TVW Telethon during this period is likely to be a significant determinant
Institute for Child Health Research, PO Box 855, West Perth WA 6872, of subsequent allergen responder phenotype.4
Australia.
Copyright © 2000 by Mosby, Inc.
In this context, we have recently demonstrated that
0091-6749/2000 $12.00 + 0 1/1/105804 generalized T helper cell activity in neonates and infants
doi:10.1067/mai.2000.105804 genetically at high risk of atopy is depressed relative to
1117
1118 Holt et al J ALLERGY CLIN IMMUNOL
JUNE 2000

The current study focused on one aspect of this ques-

Abbreviations used tion, the development of humoral and cellular immunity
CMI: Cellular immunity to the triple antigen (diphtheria-tetanus-pertussis [DTP])
DTP: Diphtheria-tetanus-pertussis vaccine vaccine in panels of atopic and nonatopic 6-year-old chil-
HDM: House dust mite dren who have undergone the full preschool immuniza-
mRNA: Messenger RNA tion-boosting program.
RT: Reverse transcriptase
SPT: Skin prick test
TH1: T helper type 1
METHODS
TH2: T helper type 2 Subjects
Two groups of 25 age-matched children, respectively, atopic
(mean 6.0 and range 5.8-6.3 years) and nonatopic (mean 5.9 and
range 5.6-6.1 years) were selected from a nested case-control sub-
the population at large and that the rate of postnatal mat- sample from a continuing cohort study. Skin prick test (SPT)
uration of T helper cell functions (in particular TH1-asso- responses were assessed to a panel of inhalant and food allergens
ciated functions) is intrinsically slower in these high-risk including house dust mite (HDM). The atopic subjects selected were
subjects.7,8 Furthermore, this in turn is reflected in pat- all HDM responsive and the nonatopic subjects were unresponsive to
terns of developing allergen-specific T-cell memory dur- the full panel. All subjects in the study had been vaccinated with cel-
ing early childhood, notably in the failure of high-risk lular DTP vaccine (CSL, Melbourne, Australia) at 2, 4, 6, and 18
months and at 5 years. The study was carried out with the approval
subjects to switch off (or “immune deviate”) the TH2
of the Princess Margaret Hospital for Children Ethics Committee.
component of their responses.7,9 Recent studies also sug-
gest that T-cell memory development against vaccine Cell preparation and culture
antigens may be compromised in high-risk children.
PBMCs were cryopreserved after collection and batch analyzed,
Notably, a retrospective study on 12-year-old children as per previous studies.6,7 After being thawed, the cells were resus-
has demonstrated that expression of atopy and asthma at pended at 1 × 106 viable cells per milliliter in serum-free medium
this age was strongly associated with a low capacity to supplemented with 2-mercaptoethanol, which has previously been
develop stable TH1-dependent cellular immunity (CMI) shown to be optimal for measuring responses to allergens or mito-
to BCG vaccination during infancy10; we have shown a gens, or RPMI 1640 medium (Gibco, Australia) supplemented with
comparable association in a pilot study on early develop- 5% pooled human AB serum, which is optimal for demonstrating
ment of CMI to the “triple” antigen vaccine during infan- responses to vaccine antigens.17 Aliquots of 0.5 to 1.0 mL from
cy,11 and a generalized deficiency has also been reported each subject were cultured at 37°C with 5% carbon dioxide for 48
hours in medium alone or medium containing 2 µg/mL pertactin
in numbers of circulating CD45+RO+ T helper memory
from B pertussis (SmithKline Beecham, Belgium), tetanus toxoid
cells in older atopic children.12
(0.5 Lf/mL; CSL Laboratories, Australia), PHA (HA16, 1 µg/mL;
Collectively, these latter findings suggest that chil- Murex Australia), or HDM allergen (10 µg/mL; aqueous extract of
dren at genetic risk of atopy, which with current figures Dermatophagoides pteronyssinus from CSL, Australia).
constitute 35% to 45% of the population, may be intrin- After culture the cells were centrifuged and used immediately
sically hyporesponsive to vaccines given during infancy, for RNA extraction; the culture supernatants were stored at –20°C
and if so this has important public health implications in for ELISA.
relation to pediatric vaccination programs. To further
complicate this picture, however, it has also been sug- Analysis of cytokine responses
gested on the basis of indirect epidemiologic evidence Semiquantitative reverse transcriptase (RT)–PCR was used for
that early childhood vaccination may in some circum- detection in cell pellets of messenger RNA (mRNA) specific for IL-
4 and IL-9 because IL-4 levels were below the limits of detection by
stances increase the risk for subsequent development of
ELISA, and reagents for IL-9 ELISA were not available to us.
atopy.13,14 Such unexpected effects might conceivably
mRNA specific for β-actin was also determined. Details of primer
derive from direct influences of components of the vac- sequences, conditions for RT-PCR reactions, and detection of PCR
cine (eg,TH2 “adjuvants” in Bordetella pertussis cell products by slot blotting were as described previously.6,9 Production
walls15) or indirectly by lowering of the overall micro- levels of mRNA for IL-4 and IL-9 above background controls were
bial burden during childhood and thus reduction in the expressed as ratios to the β-actin control as described previously.6,9
level of stimulation provided to the developing immune Quantitation of IL-5, IL-10, IL-13 and IFN-γ protein in super-
system.16 natants used ELISA assays as detailed.6,9 Data were expressed as
These issues are increasingly being debated in the picograms of cytokine protein per milliliter of culture after subtrac-
public arena, particularly those relating to infections and tion of backgrounds from unstimulated cultures.
vaccination programs, and the implications of this debate Antibody assays
provide an imperative for more focused research on the
B pertussis IgG was measured with use of a commercial ELISA
underlying immunologic processes in early life. In par-
kit (PanBio, Australia) and expressed as arbitrary units per milliliter.
ticular, there is an urgent need for detailed comparative Tetanus- and diphtheria toxoid–specific IgG was measured in the
information on the nature of immune responses to vac- clinical immunology laboratory at Princess Margaret Hospital with
cines commonly used during infancy in atopic and use of in-house ELISA assays with World Health Organi-
nonatopic children, especially in relation to T helper cell zation–derived standards provided by CSL (Melbourne, Australia)
function. and expressed as international units per milliliter.
J ALLERGY CLIN IMMUNOL Holt et al 1119
VOLUME 105, NUMBER 6, PART 1

FIG 1. Cytokine responses to mitogen and allergen in PBMCs from 6-year-old children. PBMCs were stimulated
with either HDM (top) or PHA (bottom) as detailed in Methods, and culture supernatants were harvested at 48
hours for ELISAs. Data shown are group means ± SE.

Statistical analyses measured by IL-5, IL-10, or IL-13 protein or mRNA spe-

Data were analyzed by the Mann-Whitney U test with Statview cific for IL-4 or IL-9 were comparable. IFN-γ responses
(SAS Institute, Cary, NC). against the tetanus antigen were significantly higher
within the atopic group.
RESULTS Cytokine responses against pertactin were less fre-
Cytokine production patterns in atopic quent and of lower magnitude. The most consistent were
versus nonatopic children IFN-γ, and group mean responses were not significantly
different (atopic 13.1 [SE 5.9] vs nonatopic 10.6 [SE 5.2]
Cytokine responses in cultures of HDM-stimulated pg/mL culture fluid, P = .9).
PBMCs clearly distinguished between atopic populations
who were responsive to HDM and their nonatopic coun- DISCUSSION
terparts (Fig 1, top). Notably, HDM-induced production
of the TH2 cytokines IL-5 and IL-13 and also the TH1 Previous studies from our laboratory on inhalant aller-
cytokine IFN-γ were markedly increased in the SPT+ gen-specific T helper cell function during early life have
atopic group. demonstrated that responses in neonates follow the typi-
This pattern was mirrored in cytokine responses cal fetal pattern, being dominated by TH2 cytokines,
induced by the T-cell mitogen PHA (Fig 1, bottom). In together with a very low and variable TH1 component
particular, polyclonal production of IL-5 and IL-13 were that is usually detectable only at the level of mRNA.18
significantly increased in the atopic group; mean levels of During early infancy the contribution of TH2 cytokines to
production of IFN-γ were also higher among the atopic these responses rapidly wanes in nonatopic subjects, with
subjects, but the differences were not statistically signifi- concomitant up-regulation of allergen-specific TH1 reac-
cant because of the large variations within the groups. tivity as exemplified by IFN-γ.18 In contrast, allergen-
specific18 and generalized TH1 activity8 up-regulate
Vaccine-specific antibody production more slowly in atopic subjects, who instead consolidate
IgG antibody to all 3 vaccine antigens was detected in their fetally primed TH2 immunity,7,9 possibly as a direct
all subjects, and no significant differences were observed result of the relative paucity of cross-regulatory signals
between the 2 groups (Fig 2). from the TH1 arm of their responses.4
As noted above, there is some evidence to suggest that
Cytokine responses to vaccine antigens capacity to develop sustained vaccine immunity in
The most consistent responses detected were against response to priming during infancy may also be compro-
the tetanus toxoid antigen. As shown in Fig 3, mean mised in atopic subjects. Two recently described exam-
response levels within the atopic and nonatopic groups as ples are in vivo TH1-dependent tuberculin responsiveness
1120 Holt et al J ALLERGY CLIN IMMUNOL
JUNE 2000

FIG 2. Vaccine antigen-specific IgG antibody in serum. Antibody levels were determined as detailed in Meth-
ods. Data shown are group means ± SE.

FIG 3. Cytokine responses to tetanus toxoid antigen in PBMCs from 6-year-old children. PBMCs were stimulat-
ed with tetanus toxoid antigen, and ensuing cytokine responses were determined as cytokine protein (by
ELISA) or cytokine-specific mRNA relative to mRNA specific for the housekeeping gene β-actin (by semiquan-
titative RT-PCR), as detailed in Methods. Data shown are group means ± SE.

to BCG vaccination given at ages <3months10 and in of both TH2 and especially TH1 cytokines in atopic
vitro lymphoproliferation (a surrogate marker for CMI) neonates7,19-24 and infants,8 and the findings in Fig 1
in response to tetanus antigen measured at outcome age suggest that this maturational deficiency is redressed by
2 years in children vaccinated during infancy.11 the age of 6 years.
In the current study we focused on panels of atopic and Fig 1 additionally demonstrates that IFN-γ responses
nonatopic 6-year-old children who had been fully vacci- against HDM are also well developed in T helper memory
nated during infancy with the DTP vaccine and then boost- cells from 6-year-old HDM-SPT+ atopic children, and in
ed at age 5 years. We sought information on their specific fact exceed those of nonatopic children. We have recently
T helper cell cytokine and antibody responses to antigens reported similar findings in a cohort of children of com-
in the vaccine and additionally their overall T-cell respon- parable age.6 The atopic children in this study additional-
siveness with use of a polyclonal T-cell stimulant. ly displayed vigorous production of IL-5 and IL-13 to
As shown in Fig 1, at age 6 years the atopic group dis- HDM and thus exhibited the mixed (or TH0-like) cytokine
played enhanced polyclonal production of the TH2 pattern typical of many atopic adults.25,26 The increased
cytokines IL-5 and IL-13, and their IFN-γ production production levels of IFN-γ manifested by the atopic sub-
capacity was at least equivalent to that of nonatopic sub- jects at this age may be indicative of an underlying com-
jects. Earlier studies from several laboratories have pensatory mechanism that is associated with redressing
demonstrated reduced capacity for polyclonal production their earlier TH1/TH2 imbalance; additional cross-section-
J ALLERGY CLIN IMMUNOL Holt et al 1121
VOLUME 105, NUMBER 6, PART 1

al studies are required to determine how long this appar- ly used. In addressing this issue, continuing prospective
ent IFN-γ overshoot persists in this group and whether it studies in our laboratory are focusing on DTP
plays a role in disease pathogenesis. This latter possibili- vaccine–specific T helper cell function in birth cohorts
ty is plausible, given recent findings from human27,28 and spanning the period of 3 to 18 months of age. Specific
experimental animal systems29,30 that suggest that TH1 studies are also urgently required on T helper cell
immunity may contribute to tissue damage at sites of responses to mucosal vaccines over this age range
allergic inflammation. In relation to the overall allergic because even less information is available on the nature
sensitization process, these findings also imply that the of the immune responses that these induce in infants.
delayed postnatal upsurge of TH1 function in the atopic
subjects may occur too late to provide effective feedback
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