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Newborns Develop a Th1-Type Immune
Response to Mycobacterium bovis Bacillus
Calmette-Guérin Vaccination
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of June 16, 2017.
Arnaud Marchant, Tessa Goetghebuer, Martin O. Ota, Ingrid
Wolfe, Serign J. Ceesay, Donat De Groote, Tumani Corrah,
Steve Bennett, Jeremy Wheeler, Kris Huygen, Peter Aaby,
Keith P. W. J. McAdam and Melanie J. Newport
J Immunol 1999; 163:2249-2255; ;
http://www.jimmunol.org/content/163/4/2249
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References
Newborns Develop a Th1-Type Immune Response to
Mycobacterium bovis Bacillus Calmette-Guérin Vaccination1
Arnaud Marchant,2* Tessa Goetghebuer,* Martin O. Ota,* Ingrid Wolfe,* Serign J. Ceesay,*
Donat De Groote,† Tumani Corrah,* Steve Bennett,‡ Jeremy Wheeler,‡ Kris Huygen,§
Peter Aaby,¶ Keith P. W. J. McAdam,* and Melanie J. Newport*
E
arly immunization is required to protect infants from
pathogens to which they are exposed during early childhood, but is limited by the immaturity of the neonatal
immune system (1, 2). This immaturity affects the response of B
lymphocytes to T cell-independent Ags and restricts the use of
polysaccharide vaccines against bacteria like Streptococcus pneumoniae. Data obtained in animals have suggested that the quality
of the T lymphocyte response is also affected. Newborn mice preferentially develop Th2-type responses following immunization
and are deficient in Th1 responses (1–3). Th1 responses are characterized by the production of IFN-g and are considered to be
required for protection against mycobacteria and viruses whereas
Th2 responses, characterized by the production of IL-4, IL-5, and
IL-13, protect against helminths and are involved in atopic reactions (4). A Th2 bias in newborns would therefore impair their
response to vaccines against diseases caused by mycobacteria or
viruses. More recently, several groups have shown that newborn
mice can develop both Th1 and Th2 responses, depending on the
type of APCs, the presence of adjuvants, and the dose of Ags
administered (3, 5– 8).
Our knowledge about T lymphocyte responses in human newborns is limited. In vitro studies on cord blood mononuclear cells
have shown that newborn lymphocytes have a defective IFN-g
production in response to mitogens (9, 10). Recent work by P.G.
*Medical Research Council Laboratories, Fajara, The Gambia; †University of Liège,
Liège, Belgium; ‡London School of Hygiene and Tropical Medicine, London, United
Kingdom; §Pasteur Institute of Brussels, Brussels, Belgium; ¶Projecto de Saude de
Bandim, Bissau, Guinea-Bissau and Danish Epidemiology Science Center, Copenhagen, Denmark
Received for publication March 22, 1999. Accepted for publication May 19, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
S.B. is supported by the Medical Research Council (MRC), U.K. This investigation
received financial support from the MRC, U.K., the United Nations Development
Program (UNDP)/World Bank/World Health Organization (WHO) Special Program
for Research and Training in Tropical Diseases (TDR), and the WHO Global Program
for Vaccines and Immunization.
2
Address correspondence and reprint requests to Dr. Arnaud Marchant, Medical Research Council Laboratories, Post Office Box 273, Banjul, The Gambia, West Africa.
E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
Holt’s group indicates that allergens commonly cross the placenta
and induce the differentiation of fetal T lymphocytes into Th2 cells
(11). The ability of human newborns to develop a Th1 immune
response upon immunization has not been studied.
Following World Health Organization (WHO) recommendations, newborns are vaccinated with Mycobacterium bovis bacillus
Calmette-Guérin (BCG)3 in The Gambia as in other developing
countries (12). In adults, BCG triggers a Th1 response that was
shown to be necessary for the control of mycobacterial infections
(13–15). This prospective and randomized study was undertaken to
evaluate whether a similar response is induced in newborns and
whether age at vaccination influences immunogenicity. Immune
response to BCG was evaluated in vitro using mycobacterial Ags
that are current antituberculosis vaccine candidates (16 –18).
Materials and Methods
Study design
This study was a prospective and randomized trial approved by the Gambia
Government/Medical Research Council (MRC) Ethical Committee. Neonates were identified at birth by the study field worker at Fajikunda Health
Center. The study was explained to the mother, and she was invited to
enroll her child. The following were excluded from the study : neonates
born to mothers with evidence of systemic infection at the time of delivery,
neonates presenting with any congenital defect or a birth weight below 2.5
kg, and twin neonates. If the mother agreed, one of the study pediatricians
(T.G. or M.O.) visited the neonate in the family compound within the first
36 h of life. The pediatrician gave BCG immediately to neonates born in
compounds with a history suggestive of tuberculosis, and these children
were excluded from the study. Enrolled neonates were then randomly allocated in blocks of six to one of three vaccination groups (BCG given at
birth or 2 or 4 mo of age). The same dose of BCG vaccine (0.1 ml, lot
E61440A, Evans Medical, Leatherhead, England) was given to all infants.
BCG immunogenicity was measured 2 mo after vaccination. Prevaccination samples from infants randomized to receive BCG at 2 or 4 mo of age
were used as controls for infants vaccinated at birth or at 2 mo of age,
respectively. Induction of immunological memory was assessed at 1 yr of
age. For ethical reasons, BCG vaccination could not be delayed for more
than 4 mo. Therefore, no unvaccinated controls could be studied at one
3
Abbreviations used in this paper: BCG, Mycobacterium bovis bacillus CalmetteGuérin; PPD, purified protein derivative; ST-CF, short-term culture filtrate; KMTB, heat-killed Mycobacterium tuberculosis; SI, stimulation index; CI, confidence interval.
0022-1767/99/$02.00
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Data obtained in animals indicate that neonatal immune responses are biased toward Th2. This could reduce the efficacy of
vaccines against viral and mycobacterial diseases. The ability of human newborns to develop a Th1 immune response upon
immunization has not been studied. Since the vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers a Th1-type
response in adults, we investigated whether it induces a similar response in newborns and whether age at vaccination influences
immunogenicity. We found that BCG vaccination at birth induces a memory Th1-type response of similar magnitude to that when
given later in life. This study demonstrates that human newborns can be immunized against pathogens controlled by a Th1
immune response. The Journal of Immunology, 1999, 163: 2249 –2255.
2250
year of age. At vaccination and follow-up visits, all children were examined by one of the study pediatricians and found to be healthy.
In vitro lymphocyte stimulation with mycobacterial Ags
Cytokine release and lymphocyte proliferation assays
IFN-g, IL-5, and IL-13 concentrations were measured in culture supernatants collected on day 2 after PHA (5 mg/ml) stimulation and day 6 after
antigenic stimulation (PPD and ST-CF, 1 mg/ml; K-MTB, 100 mg/ml, 10
kDa and Ag 85 complex, 1 and 10 mg/ml) using commercially available
ELISAs (BioSource Europe, Fleurus, Belgium). Lymphocyte proliferation
was evaluated on day 7 after the addition of 1 mCi of [methyl-3H]thymidine per well (Amersham Life Science, Little Chalfont, U.K.) during the
final 15 h of culture. Thymidine incorporation was measured by liquid
scintillation using a Betaplate reader (LKB 1205, Turku, Finland).
IL-4 release assay
IL-4 production was measured using an Enzyme-Linked-Immuno-Trapping-Assay as described elsewhere (DW Groote, X. Gevaert, R. Gathy,
M. Lopez, S. Benyoucef, and M. Malaise, manuscript in preparation).
Briefly, diluted blood or PBMC suspension was incubated in sterile ELISA
plates (Maxisorp Immunoplates, Nunc, Roskilde, Denmark) coated with
anti-IL-4 capture F(ab9)2 mAb (clone 4B3) containing antigenic preparations (PHA, PPD, ST-CF, 10 kDa, and Ag 85 complex, 10 mg/ml; K-MTB,
100 mg/ml). After 4 days, plates were washed, and captured IL-4 was
detected using a secondary detecting HRP-conjugate F(ab9)2 anti-IL-4
mAb (clone 10H12). IL-4 concentrations were inferred from a recombinant
IL-4 standard curve that was included in every plate.
Statistical analysis
Proliferative responses were calculated as stimulation index (SI) dividing
geometric mean Ag-stimulated cpm by background cpm (19). For cytokines, background production was subtracted from Ag-stimulated production. We excluded from the analysis postvaccination data from infants who
showed evidence of sensitization to mycobacterial Ags before vaccination.
Sensitization was defined as proliferation SI above mean 1 2 SD of values
obtained in a series of 15 cord blood samples collected during the study, for
at least two of the following mycobacterial Ag preparations: PPD (SI .
2.6), ST-CF (SI . 2.6), and K-MTB (SI . 7.2). For comparisons between
groups, data were logarithm transformed and compared by the one way
ANOVA, or Kruskal Wallis nonparametric test, where there were many
nonresponders to the Ag. Despite randomization, infants vaccinated at 4
mo were more often males and from the Mandinka ethnic group while
infants vaccinated at birth or at 2 mo were more often firstborn and from
the Jola ethnic group. Multiple regression analysis was conducted to allow
for any confounding effects of these factors. Those measurements with
many nonresponders were grouped and analyzed by ordinal logistic regression. Tables and figures show adjusted regression p values. The unadjusted
p values are not shown because they were not materially different. Since a
total of 88 multiple comparisons were performed among the different immune responses to different mycobacterial Ags, it would be expected for
about four p values to be significant at p , 0.05 solely due to chance. All
statistical analysis was done using Stata (version 5; Stata Corporation, College Station, TX).
Results
Study population
One hundred and thirty-seven neonates were enrolled between November 1996 and April 1997. Fifty-one infants were studied at 2
mo of age, including 27 vaccinated at birth and 24 immediately
before vaccination. Forty-two infants were studied at 4 mo of age,
including 19 vaccinated at the age of 2 mo and 23 immediately
before vaccination. Fifty-eight infants were studied at 1 yr of age,
including 23 vaccinated at birth, 19 vaccinated at the age of 2 mo,
and 16 vaccinated at the age of 4 mo. Eighty-five infants were not
studied at some stage during the follow-up for the following reasons: mother traveling (n 5 9), blood sampling refused (n 5 31),
BCG vaccination given outside the study (n 5 23), death (n 5 4),
technical failure in the blood sampling or processing (n 5 12), and
evidence of sensitization to mycobacterial Ags prior to vaccination
(n 5 1 at 2 mo and n 5 5 at 4 mo of age, see Materials and
Methods).
BCG immunogenicity in infants vaccinated at birth or at 2 mo
of age
To evaluate immunogenicity of BCG vaccination at birth or at 2
mo of life, we measured proliferative and cytokine responses to
tuberculin PPD and compared them to the responses to the mitogen
PHA (Fig. 1). We observed that 2-mo-old infants who were vaccinated at birth displayed strong proliferative responses to PPD
whereas age-matched unvaccinated controls had only minimal responses (Fig. 1, left panel). Proliferative responses to PPD in
4-mo-old infants who were vaccinated at 2 mo were similar to
infants vaccinated at birth, while age-matched unvaccinated infants again had minimal responses. The proliferative response to
PPD was associated with the production of high levels of IFN-g
both in infants vaccinated at birth or at 2 mo of age (Fig. 1, middle
panel). The levels of IFN-g were lower in infants vaccinated at 2
mo than in infants vaccinated at birth, but this difference was not
significant. Very low levels of IFN-g were detected in unvaccinated controls at both ages.
In contrast to IFN-g, no significant IL-4 production was induced
by PPD in infants who received BCG at birth (Fig. 1, right panel).
Surprisingly, between the age of 2 and 4 mo, unvaccinated infants
acquired a significant IL-4 response to PPD. This IL-4 production
was partly, although not significantly, prevented by BCG vaccination at the age of 2 mo. The Ag specificity of these differences
is demonstrated by the similarity of the proliferative and cytokine
responses to PHA observed in all four groups (Fig. 1). In summary,
these data indicate that infants vaccinated at birth and at 2 mo of
age develop a Th1-type response to PPD while a Th2-type response predominates in unvaccinated infants.
Ag specificity of the immune response to BCG in infants
To further characterize the antigenic targets of the immune response induced by BCG in infants, we studied lymphocyte responses to extracellular mycobacterial Ags that are considered to
be antituberculosis vaccine candidates. These included an ST-CF,
the Ag 85 complex, and the 10-kDa Ag. In parallel, we studied the
response to intracellular Ags represented by a killed M. tuberculosis preparation (K-MTB). Table I shows that infants vaccinated
at birth or at 2 mo of age had significant proliferative responses to
all extracellular Ags tested when compared with unvaccinated
controls. The intracellular Ag preparation was also found to be an
important target. The proliferative responses to these Ags were
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Due to the limited amount of blood available, evaluation of BCG immunogenicity 2 mo after vaccination was performed on heparinized blood
diluted in 9 vol of RPMI 1640 medium (BioWittaker, Verviers, Belgium)
supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (Life
Technologies, Paisley, Scotland). Evaluation of immunological memory at
1 yr of age was performed on PBMC separated by density gradient centrifugation (Lymphoprep, Nycomed, Oslo, Norway) and resuspended in
RPMI 1640 medium supplemented with 100 U/ml penicillin and 100
mg/ml streptomycin, 10 mM HEPES (ICN Biomedicals, Costa Mesa, CA),
2 mM L-glutamine (Life Technologies), and 10% human AB serum (Sigma, St. Louis, MO). Diluted blood or PBMC suspension was incubated for
various periods of time in 96-well plates (200 ml/well) (Becton Dickinson,
Rutherford, NJ) containing Ag preparations. Mycobacterial Ags included
the purified protein derivative (PPD, RT48, Statens Serum Institut, Denmark), extracellular mycobacterial Ags including M. tuberculosis short
term culture filtrate (ST-CF, provided by Prof. Peter Andersen, Statens
Serum Institut, Copenhagen, Denmark) (18), the Ag 85 complex from BCG
(16), and the recombinant Mycobacterium tuberculosis 10-kDa Ag (provided by Dr. Mahavir Singh, WHO Recombinant Protein Bank) (17), as
well as an intracellular mycobacterial Ag preparation (K-MTB, M. tuberculosis H37Rv strain autoclaved for 30 min at 120°C). Wells containing
medium alone or PHA (PHA-L, Sigma Chemicals) were used as the negative and positive controls, respectively.
Th1-TYPE IMMUNE RESPONSE IN NEWBORNS
The Journal of Immunology
2251
associated with significant IFN-g production, although the levels
of IFN-g were generally lower in infants vaccinated at 2 mo (table
II). The 10-kDa Ag was found to be a weaker stimulus of IFN-g
production. As observed for PPD, between the age of 2 and 4 mo,
unvaccinated infants acquired a low but significant IL-4 response
to mycobacterial Ags, this response being less marked for the
10-kDa Ag (Table III). The production of IL-4 was not signifi-
cantly influenced by BCG vaccination either at birth or at 2 mo
of age.
Induction of immunological memory by BCG vaccination in
newborns
To evaluate whether the Th1-type response to BCG in newborns is
associated with the induction of immunological memory, the
Table I. Proliferative responses (SI) to extracellular and intracellular mycobacterial Ags after BCG
vaccination at birth or at 2 mo of age
Age at Vaccination
Stimulus
PHAb
ST-CF
Ag 85 complex
10 kDa
K-MTB
Group
Birtha
2 moa
pp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
25 (13–46)
18 (12–27)
0.31
1 (1–2)
56 (29–110)
,0.001
2 (2–3)
12 (7–19)
,0.001
2 (1–2)
5 (3–9)
0.001
3 (2–5)
97 (64–148)
,0.001
15 (9–26)
16 (9–30)
0.62
2 (1–3)
38 (20–72)
,0.001
3 (2–5)
8 (4–16)
,0.001
2 (1–3)
5 (3–9)
,0.001
4 (2–6)
65 (42–101)
,0.001
0.18
0.95
0.14
0.33
0.62
0.44
0.63
1.00
0.82
0.15
a
Proliferative responses (geometric mean, 95% CI) were measured at 2 mo of age in 27 infants vaccinated at birth and 24
age-matched unvaccinated controls and at 4 mo of age in 19 infants vaccinated at 2 mo and 23 age-matched unvaccinated
controls. The table shows the regression p values adjusted for sex, ethnic group, and birth order.
b
Responses to PHA included in the table are the same as the ones presented in Fig. 1.
p, p value for 2-mo-old compared to 4-mo-old vaccinated or control infants.
pp, p value for vaccinated compared to age-matched control infants.
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FIGURE 1. Immune responses to BCG vaccination at birth or at 2 mo of age. Proliferative responses (left panels), IFN-g production (middle panels),
and IL-4 production (right panels) were measured at 2 mo of age in 22 infants vaccinated at birth (hatched bars) and 22 age-matched unvaccinated controls
(open bars) and at 4 mo of age in 19 infants vaccinated at 2 mo (hatched bars) and 23 age-matched unvaccinated controls (open bars). Data are expressed
as geometric mean and 95% CI. The figure shows the regression p values adjusted for sex, ethnic group, and birth order. ppp, p , 0.001 as compared with
age-matched unvaccinated infants; pp, p 5 0.002 as compared with 2-mo-old unvaccinated infants; p, p 5 0.07 as compared with age-matched unvaccinated
infants.
2252
Th1-TYPE IMMUNE RESPONSE IN NEWBORNS
Table II. IFN-g production (pg/ml) in response to extracellular and intracellular mycobacterial Ags after
BCG vaccination at birth or at 2 mo of age
Age at Vaccination
Stimulus
b
PHA
ST-CF
Ag 85 complex
10 kDa
K-MTB
a
Group
Birth
2 moa
pp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
5521 (2772–10,995)
4479 (2848–7044)
0.42
6 (4–11)
887 (386–2034)
,0.001
51 (26–102)
326 (153–696)
0.001
46 (24–86)
149 (68–324)
0.03
29 (13–64)
3170 (1615–6220)
,0.001
4807 (2058–11,230)
6426 (3256–12,682)
0.36
4 (0–168)
217 (78–596)
,0.001
107 (39–290)
229 (61–855)
0.22
72 (31–164)
210 (77–575)
0.06
47 (16–134)
2400 (922–6247)
,0.001
0.58
0.38
0.25
0.05
0.54
0.79
0.89
0.64
0.99
0.46
cohort was followed up and studied at 1 yr of age. In these
experiments, in which the response of PBMC to extracellular and
intracellular mycobacterial Ags was evaluated, no significant IL-4
production could be detected (data not shown). As an alternative
method to assess Th2 responses, we measured the production of
IL-5 and IL-13, two cytokines that are, like IL-4, preferentially
produced by human Th2 cells (4, 20). The production of IL-5 and
IL-13 could not be measured retrospectively in 2- and 4-mo-old
infants due to lack of culture supernatants.
As shown in Fig. 2, a memory Th1-type response to PPD was
detected in infants vaccinated at birth. This response was characterized by the production of high levels of IFN-g but only low
levels of IL-5 and IL-13. A similar Th1 response to PPD was
detected in infants vaccinated at 2 or 4 mo of age. The lymphocyte
response to PHA was equivalent in the three study groups. Table
IV shows that infants vaccinated at birth also display a Th1-type
memory response to the intracellular K-MTB preparation. At the
concentration used in these experiments (1 mg/ml), the extracellular ST-CF preparation induced only a low and similar lymphocyte response in the three study groups. A similar phenomenon
was observed with the purified 10-kDa and 85 complex Ags, which
induced a detectable response in only a minority of the subjects
from the three study groups (data not shown).
Discussion
Animal studies have led to the concept that neonatal immune responses are biased toward the Th2 type (1–3, 5– 8). Only limited
Table III. IL-4 production (pg/ml) in response to extracellular and intracellular mycobacterial Ags after
BCG vaccination at birth or at 2 mo of age
Age at Vaccination
Stimulus
PHAb
ST-CF
Ag 85 complex
10 kDa
K-MTB
Group
Birtha
2 moa
pp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
Control
Vaccinated
ppp
78 (48–128)
92 (48–173)
0.48
13 (8–22)
9 (5–15)
0.51
1 (0–2)
1 (0–2)
0.44
1 (1–2)
1 (0–2)
0.41
2 (1–4)
3 (1–5)
0.29
93 (52–166)
104 (63–171)
0.59
29 (16–52)
25 (12–51)
0.92
8 (4–17)
3 (1–5)
0.23
5 (2–12)
1 (0–3)
0.12
10 (5–19)
11 (6–21)
0.40
0.60
0.83
0.04
0.06
,0.001
0.01
0.11
0.50
0.004
0.003
a
IL-4 production (geometric mean, 95% CI) was measured at 2 mo of age in 22 infants vaccinated at birth and 22 agematched unvaccinated controls and at 4 mo of age in 19 infants vaccinated at 2 mo and 23 age-matched unvaccinated controls.
The table shows the regression p values adjusted for sex, ethnic group, and birth order.
b
Responses to PHA included in the table are the same as the ones presented in Fig. 1.
p, p value for 2-mo-old compared to 4-mo-old vaccinated or control infants.
pp, p value for vaccinated compared to age-matched control infants.
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a
IFN-g production (geometric mean, 95% CI) was measured at 2 mo of age in 24 infants vaccinated at birth and 23
age-matched unvaccinated controls and at 4 mo of age in 19 infants vaccinated at 2 mo and 23 age-matched unvaccinated
controls. The table shows the regression p values adjusted for sex, ethnic group, and birth order.
b
Responses to PHA included in the table are the same as the ones presented in Fig. 1.
p, p value for 2-mo-old compared to 4-mo-old vaccinated or control infants.
pp, p value for vaccinated compared to age-matched control infants.
The Journal of Immunology
2253
Table IV. Memory immune response to BCG vaccination at birth or 2 or 4 mo of age
Age at Vaccination
Stimulus
b
PHA
ST-CF
K-MTB
a
Immune Response
Birth
2 moa
4 moa
pp
Proliferation (SI)
IFN-g (pg/ml)
IL-5 (pg/ml)
IL-13 (pg/ml)
Proliferation (SI)
IFN-g (pg/ml)
IL-5 (pg/ml)
IL-13 (pg/ml)
Proliferation (SI)
IFN-g (pg/ml)
IL-5 (pg/ml)
IL-13 (pg/ml)
11 (6–18)
264 (122–570)
12 (5–25)
532 (345–821)
7 (4–11)
9.2 (4–18)
1.4 (1–3)
1.1 (0–2)
18.8 (11–31)
222 (138–355)
1.8 (1–4)
4.2 (2–8)
8 (4–15)
181 (86–383)
16 (8–32)
392 (210–731)
9.2 (6–15)
11.7 (5–25)
2.1 (1–5)
1.5 (0–4)
27.5 (18–42)
190 (105–342)
2.3 (1–6)
4.3 (2–10)
11 (6–20)
202 (90–452)
23 (8–63)
458 (246–856)
7.7 (5–13)
9.3 (3–25)
5.8 (2–17)
3.3 (1–9)
20.8 (12–36)
116 (51–266)
4.8 (2–11)
7.7 (3–17)
0.70
0.63
0.13
0.80
0.47
0.93
0.29
0.22
0.29
0.49
0.48
0.15
a
Immune responses to extracellular and intracellular mycobacterial Ags were measured at 1 yr of age in 23 children
vaccinated at birth, 19 children vaccinated at 2 mo, and 16 children vaccinated at 4 mo of age. Data are expressed as geometric
mean and 95% CI.
b
Responses to PHA included in the table are the same as the ones presented in Fig. 2.
p, Regression p values adjusted for sex, ethnic group, and birth order.
FIGURE 2. Memory immune
responses to BCG vaccination at
birth or 2 or 4 mo of age. Proliferative responses as well as
IFN-g, IL-5, and IL-13 production, were measured at 1 yr of
age in 23 children vaccinated at
birth, 19 children vaccinated at 2
mo, and 16 children vaccinated
at 4 mo of age. Data are expressed as geometric mean and
95% CI. No significant difference was found among the three
study groups.
responses. Mycobacteria, including BCG, infect dendritic cells and
markedly increase their ability to present Ags to T cells (21, 22).
This higher Ag-presenting capacity is related to an increased
expression of costimulatory surface molecules and cytokines like
IL-12. In newborn mice, the use of external costimulatory signals,
such as IL-12, anti-CD40 mAbs, or adult dendritic cells at the time
of immunization, can prevent the differentiation of helper T lymphocytes into Th2 cells and promotes Th1 responses (3, 5, 23). In
the presence of IL-12 and optimal costimulatory signals, human
newborn T lymphocytes differentiate into Th1 cells whereas, under
suboptimal conditions, IL-4-producing cells are generated (24).
Similar mechanisms are probably involved in the ability of adjuvants, DNA vaccines, or replicating viruses to promote Th1 responses in newborn mice (7, 8, 25).
The immune response to vaccines can also be influenced by
other factors, including the route of vaccination and the dose of Ag
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data are available on the responses developed during early life in
humans. Exposure of fetal T lymphocytes to allergens crossing the
placenta was shown to induce their differentiation into Th2 cells
(11). Our study shows for the first time that human newborns can
develop a Th1-type response upon immunization. Infants vaccinated at birth with BCG developed a Th1 response of similar magnitude to infants immunized at 2 or 4 mo of age. The persistence
of the Th1 response 1 yr after vaccination indicates that BCG
vaccination at birth activates memory T cells. Current research on
new antituberculosis vaccines is focusing on extracellular mycobacterial Ags (16 –18). We observed that both extracellular and
intracellular Ags are important immune targets after BCG vaccination in infants.
The Th1-type immune response induced by BCG in newborns is
likely to be dependent on the activation of APCs. Dendritic cells
are the APCs involved in the initiation of primary immune
2254
Acknowledgments
We thank Prof. Peter Andersen and Dr. Mahavir Singh for mycobacterial
Ag preparations. We are grateful to Dr Claire-Anne Siegrist and Prof.
4
P. Aaby, S. O. Shaheen, C. B. Heyes, A. Goudiaby, A. J. Hall, A. W. Shiell, H.
Jensen, and A. Marchant. Early BCG vaccination and reduction in atopy in GuineaBissau. Submitted for publication.
Paul-Henri Lambert for their support, to Drs. Hilton Whittle, Johan Vekemans, and Christian Lienhardt and to Prof. Michel Goldman for helpful
comments and critical review of the manuscript, and to Ms. Isatou Drammeh for help with field work. This study would not have been possible
without the help and support of Mr. Kebba Jobe, EPI Programme, Dr.
Kebba Manneh, TB Control Programme, and the staff of Fajikunda Health
Centre, The Gambia. We are grateful to the mothers who accepted having
their babies enrolled into a study on the first day of their life.
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administered. The Th1 response induced by BCG in human newborns could have been promoted by the intradermal route of immunization. The poor Th1 response to BCG vaccination observed
by Barrios et al. in newborn mice could at least partly be related to
the fact that the immunization route they used was i.p. (1). In
newborn mice, high doses of Ag preferentially activate Th2 responses whereas Th1 responses are induced by low doses of Ags
(6). The dose of Ag is unlikely to have affected the immune
response to BCG in our study population since a relatively high
dose of BCG was administered, generally that used to immunize
individuals above the age of 1 yr.
Unvaccinated infants developed a Th2-type response to mycobacterial Ags between the age of 2 and 4 mo. The production of
IL-4 was associated with a detectable proliferative response in
only 5 of the 23 4-mo-old infants studied. Although this was not
specifically determined in our study, the defective proliferation is
likely to be related to a weak IL-2 production but could also be
dependent on the type of APCs. In newborn mice immunized with
alloantigens, D. Abramowicz et al. described a similar Th2 response associating IL-4 production with no proliferation and no
detectable IL-2 production (26). In the absence of IL-2, murine
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the Th2 response in unvaccinated infants is unknown. The Ag
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was shown to induce preferentially Th2-type responses in animals
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gastrointestinal tract during early childhood.
The observation that BCG induces a Th1-type response in neonates has several potential implications. Firstly, since neonatal
BCG vaccination represents one of the first infectious challenges
during life, it could have a strong impact on the development of the
immune system. It has been suggested that the Th1 response induced by mycobacteria could prevent the development of allergenspecific Th2 responses and atopy during childhood (29). We have
recently observed that BCG vaccination during the first week of
life is associated with a decreased risk of atopy in children in
Guinea-Bissau.4 Secondly, although protective immunity against
mycobacterial infections is not fully understood, a Th1-type response is known to be necessary (14, 15). The early IFN-g production detected in our study therefore plays a central role in the
ability of the newborns to control the infection with BCG and
allows the safe administration of the vaccine. In addition, the fact
that BCG induces a memory Th1-type response in newborns supports the recommendation of WHO to give BCG as soon as possible after birth to prevent tuberculosis during early childhood
(12). Finally, our data indicate that human newborns can be
immunized against pathogens controlled by a Th1 response.
In addition to DNA vaccines and adjuvants, immunization strategies could include the use of BCG as a vector expressing foreign
Ags (30, 31).
Th1-TYPE IMMUNE RESPONSE IN NEWBORNS
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