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Newborns Develop a Th1-Type Immune Response to Mycobacterium bovis Bacillus Calmette-Guérin Vaccination This information is current as 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 Subscription Permissions Email Alerts This article cites 28 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/163/4/2249.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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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Montgomery, A. M. Yawman, R. R. Deck, C. M. DeWitt, I. M. Orme, S. Baldwin, C. D’Souza, A. Drowart, E. Lozes, P. Vandenbussche, J. P. Van Vooren, M. A. Liu, and J. B. Ulmer. 1996. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat. Med. 8:893. 17. Barnes, P. F., V. Mehra, B. Rivoire, S. J. Fong, P. J. Brennan, M. S. Voegtline, P. Minden, R. A. Houghten, B. R. Bloom, and R. L. Modlin. 1992. Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis. J. Immunol. 148: 1835. 18. Lindblad, E. B., M. J. Elhay, R. Silva, R. Appelberg, and P. Andersen. 1997. Adjuvant modulation of immune responses to tuberculosis subunit vaccines. Infect. Immun. 65:623. 19. Bennett, S., and E. Riley. 1992. The statistical analysis of data from immunoepidemiological studies. J. Immunol. Meth. 146:229. 20. Verhagen, C. E., T. C. van der Pouw Kraan, A. A. Buffing, M. A. Chand, W. R. Faber, L. A. Aarden, P. K. Das. 1998. Type 1- and type 2-like lesional skin-derived Mycobacterium leprae-responsive T cell clones are characterized by coexpression of IFN-g/TNF-a and IL-4/IL-5/IL-13, respectively. J. Immunol. 160:2380. 21. Inaba, K., M. Inaba, M. Naito, and R. M. Steinman. 1993. Dendritic cell progenitors phagocytose particulates, including bacillus Calmette-Guérin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178:479. Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017 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 Th2 clones were shown to produce IL-4 when stimulated by both B cells or macrophages, but only B cells could promote a proliferative response (27). The mechanism involved in the induction of the Th2 response in unvaccinated infants is unknown. The Ag specificity of the response suggests that it could be related to exposure to environmental mycobacteria. Indeed, in The Gambia, 2to 4-mo-old infants are likely to be exposed to environmental mycobacteria through their mucosa. Gastrointestinal immunization was shown to induce preferentially Th2-type responses in animals (28). Further studies are needed to evaluate whether a similar Th2type response is triggered by other microorganisms colonizing the 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 The Journal of Immunology 22. Henderson, R. A., S. C. Watkins, and J. L. Flynn. 1997. Activation of human dendritic cells following infection with Mycobacterium tuberculosis. J. Immunol. 159:635. 23. Flamand, V., V. Donckier, F. X. Demoor, A. Le Moine, P. Matthys, M. L. Vanderhaegen, Y. Tagawa, Y. Iwakura, A. Billiau, D. Abramowicz, and M. Goldman. 1998. CD40 ligation prevents neonatal induction of transplantation tolerance. J. Immunol. 160:4666. 24. Ohshima, Y., and G. Delespesse. 1997. T cell-derived IL-4 and dendritic cellderived IL-12 regulate the lymphokine-producing phenotype of alloantigenprimed naive human CD4 T cells. J. Immunol. 158:629. 25. Siegrist, C. A., F. Saddallah, C. Tougne, X. Martinez, J. Kovarik, and P. H. Lambert. 1998. 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