Special Editors: Stefan Ehlers & Stefan H. E. Kaufmann
A. H. J. van den Biggelaar, Division of Cell Biology, Telethon Institute for Child Health Research, PO Box 855, West Perth, WA 6872, Australia. E-mail Anitav@ichr.uwa.edu.au
There is increasing evidence that the functional state of the immune system at birth is predictive of the kinetics of immune maturation in early infancy. Moreover, this maturation process can have a major impact on early vaccine responses and can be a key determinant of risk for communicable and non-communicable diseases in later life. We hypothesize that environmental and genetic factors that are often typical for poor-resource countries may have an important impact on prenatal immune development and predispose populations in low-income settings to different vaccine responses and disease risks, compared to those living in high-income countries. In this paper we aimed to summarize the major differences between neonatal and adult immune function and describe what is known so far about discrepancies in immune function between newborns in high- and low-income settings. Further, we discuss the need to test the immunological feasibility of accelerated vaccination schedules in high-risk populations and the potential of variation in disease specific and non-specific vaccine effects.
The neonatal immune system differs in many aspects from that of an adult. Here we summarize differences in neonatal and mature immune function that have been reported predominantly by studies involving populations from industrialized settings, focusing in particular upon innate and T cell responses. Each section is followed by what is known about variation in neonatal immune responses between children born under different environmental conditions, or the identification of potential prenatal risk factors.
Neonatal compared to adult innate immune function
Compared to that of an adult, the neonatal innate immune system is characterized by an impaired production of proinflammatory cytokines such as interleukin (IL)-12p70, IL-27, interferon (IFN)-γ and type I IFN, that depend upon the transcription factor interferon regulatory factor 3 (IRF-3), while the production of IL-6, IL-10 and IL-23 that require activation of nuclear factor kappa B (NF-κB) is intact [1–4]. As an exception, agonists signalling through Toll-like receptor (TLR)-7/8 can induce robust proinflammatory immune responses in neonatal innate immune cells, at least as far as the production of tumour necrosis factor (TNF)-α and IL-12p40 is concerned .
Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) are deficient in number and function in early life [6,7], maintain a relatively immature phenotype [8,9] and provide suboptimal antigen presentation/co-stimulation [9,10]. Interestingly, in neonatal mice proinflammatory DC function has been shown to be inhibited by neonatal CD5+ B cells producing IL-10 [11,12].
Differences in neonatal innate immune function
There is increasing evidence that neonatal innate immune function varies between children born under different environmental conditions. Recently we identified differences in TLR-mediated innate immune responses in cord blood samples of Papua New Guinean (PNG) versus Australian (AUS) newborns . Compared to AUS newborns, cord blood mononuclear cells (CBMC) from PNG newborns produced lower IL-6 and type-I IFN responses to lipoteichoic acid (LTA), lower TNF-α responses to lipopolysaccharide (LPS), but higher bacillus Calmette-Guérin (BCG)-induced IL-10 and IFN-γ responses; the expression of TLR-2 and TLR-9 (mRNA) on resting CBMC was higher but that of TLR-4 lower; and in response to exogenous IFN-γ priming the enhancement of IL-12p70, IFN-γ and TNF-α production was considerably restricted. Notably, in contrast to AUS newborns, natural killer (NK) cells of PNG newborns did not produce IFN-γ in response to IFN-γ-primed BCG stimulation . More recent preliminary findings indicate further that the capacity of antigen-presenting cells (APC) to induce T cell proliferation is reduced for PNG compared to AUS CBMC (Fig. 1).
Other studies, including one in rural Europe, have shown that TLR-2 expression is higher in newborns whose mothers were exposed to environments rich in microbial compounds . Findings that giving probiotic bacteria to mothers in pregnancy is more effective in preventing allergic diseases  than giving these supplements to infants in the postnatal period  provides important evidence for a role of bacterial products in modulating immune development prior to birth. In accordance, malarial parasites can modulate innate immune function prenatally, as demonstrated by elevated TLR-mediated IFN-γ responses and reduced TNF-α responses in Gabonese (Central African) newborns whose mothers had malaria during pregnancy compared to those born to mothers who were either not infected or treated for infections during pregnancy .
Although microbial burden is the most striking difference between populations in developed and developing countries, there are many environmental and genetic factors that could lead to potential differences in neonatal early immune function. For example, prenatal exposure to environmental tobacco smoke has been associated with reduced neonatal TLR function , but this is in contrast with findings that in adult smokers TLR-mediated inflammatory responses to bacterial challenges are enhanced via a mechanism that involves elevated serum oxidant levels [19,20]. Premature birth, which is more common in developing than developed societies, has been associated with reduced TLR-4 and myeloid differentiation primary response gene 88 (MyD88) expression , whereas the increase in births by caesarean section in the developing world may contribute further to differences in neonatal innate immune function, such as increased expression of TLR-2 and TLR-9, reduced expression of TLR-4 and elevated TLR-mediated IFN-γ responses . Finally, innate immune function is, to a certain extent, regulated genetically  and the frequency of functional mutations in TLR-related genes is known to vary between populations, possibly as a result of genetic drift  or differences in infectious pressure [24,25].
Neonatal T cell function
Neonatal compared to adult T cell function
There is some contention in the literature regarding the potential of in utero priming of memory T helper type 1 cell (Th1) responses, such as has been shown for malaria infections in pregnancy , or whether neonatal T helper cells are naive recent thymic emigrants with low-affinity T cell receptor (TCR)–major histocompatibility complex (MHC)/peptide interactions that lack the potential for conventional T memory development . Overall, Th1 responses are impaired in newborns as a result of deficient IL-12 responses and hypermethylation of cytosine–guanine dinucleotide (CpG) and non-CpG sites within and adjacent to the IFN-γ promoter , which results in a relative skewing of T helper cell responses towards type 2.
The discovery of a new lineage of CD4+ effector T helper cells that selectively produce IL-17 (Th17 cells) has provided exciting new insights into immune regulation, host defence and the pathogenesis of inflammatory disorders, the function of which appears to be impaired at birth .
Differences in neonatal T cell function
Our preliminary data show that the proliferative capacity of CD4+ T cells derived from PNG cords is lower than that of AUS cords, which is not attributable solely to differences in the APC compartment (Fig. 1). In contrast, PNG CBMC produce significantly higher type 1 and type 2 cytokines in response to T cell mitogen stimulation compared to AUS CBMC (Fig. 2), which we believe is explained by a higher frequency of CD4+ T cells in CBMC of PNG compared to AUS CBMC. Our preliminary data suggest that, based on a similar expression of CD45RO and T cell receptor excision circles (TRECs), there is no difference in the maturity or activation status of resting CD4+ T cells of PNG or AUS newborns.
In contrast to our findings, another study reported a lower percentage of CD4+ T cells and elevated expression of maturation and activation markers on CD4+ T cells derived from Gabonese (African) compared to Austrian (European) CBMC .
Studies that have investigated the effect of infections with helminth or malarial parasites in pregnancy have provided the most compelling evidence for a prenatal environmental effect on T cell development. In addition to priming T cell responses [26,31], there is increasing evidence that in utero parasite exposure can induce immunological tolerance in some newborns through the induction of T cell anergy and enhanced IL-10 production . This hyporesponsiveness may have a spillover effect on non-specific T cells . Finally, although CD8+ are not discussed further here, it is important to mention that in utero exposure to human cytomegalovirus infection has been shown to lead to the expansion and differentiation of a mature and functional response of specific CD8+ T cells .
There is increasing evidence that the effect of environmental factors such as diet, tobacco smoke and microbial infections on T cell function may be mediated through epigenetic effects. This may include the activation of silencing of cytokine loci such as IFN-γ and IL-4 that are fundamental for the differentiation of naive T cells into Th1 and Th2 .
Neonatal regulatory T cell responses
Whereas some studies have reported that regulatory T cells (Treg) are abundant before and at the time of birth and have strong immunosuppressive properties , others have reported that compared to adults cord (CD4+ CD25high) Treg cells are reduced in number, have a reduced expression of forkhead box P3 (FoxP3) and limited or no suppressive activity [37,38].
Differences in neonatal regulatory T cells
Our preliminary data indicate a lower frequency of CD25high FoxP3+ CD4+ Tregs in CBMC derived from PNG compared to AUS newborns. This is in line with findings that CD4+CD25high cells were lower in number and showed a reduced expression of FoxP3 and CTLA-4 in cords derived from Gabonese compared to Austrian newborns .
In children born to mothers with placental Plasmodium falciparum infection at delivery, ex vivo percentages of CD4+CD25hi and CD4+CD25+CTLA-4+ cells were reported to be higher compared to newborns whose mothers' placentas were negative for malaria parasites . Apart from malaria infection in pregnancy [26,32], there is as yet no evidence that infections with other pathogens or environmental exposures such as biomass fuel exposure can influence prenatal neonatal Treg responses.
One strategy to reduce the high susceptibility to and mortality from (vaccine preventable) infectious diseases in early life is to develop vaccines that can safely induce protective immune responses in young infants. For high-risk populations this involves the consideration of accelerated immunization schedules, including neonatal vaccination, to induce the earliest possible protection. The immaturity of T cells in early life is a main impediment to administering vaccines to neonates; however, this T cell hyporesponsiveness may be overcome by addition of vaccine adjuvant enhancing innate immune responses, as has been shown for BCG . Considering the differences in neonatal immune function that we and others are starting to identify, it is likely that the efficacy and potential side effects of such adjuvant vaccines may vary between children born in genetically and environmentally diverse populations. Below we discuss an example of a neonatal safety and immunogenicity immunization trial we performed to prove the immunological feasibility of such a strategy. Furthermore, we present a series of studies arguing that specific and non-specific vaccine effects may vary between populations due to variation in neonatal immune function.
A neonatal pneumococcal conjugate vaccination trial in Papua New Guinea
Despite the efficacy of pneumococcal conjugate vaccines, Streptococcus pneumoniae remains an important cause of serious morbidity and mortality in young infants in developing countries, where the age of onset of disease is often younger than the recommended vaccination age of 6 weeks . To prove the feasibility of neonatal 7-valent pneumococcal conjugate vaccination (7-PCV), we performed a safety and immunogenicity trial in which PNG newborns were randomized to receive three doses of the 7-valent (7-v) pneumococcal conjugate vaccine, either at birth, 1 month and 2 months (neonatal group), at 1, 2 and 3 months (infant group) or no 7vPCV at all (control group). In line with findings for vaccinated adults in the United States , T cell responses to the carrier protein CRM197 were of a mixed Th1/Th2 phenotype in all 7vPCV-vaccinated PNG children at 3 months of age . However, children primed with a neonatal dose of 7vPCV produced significantly higher Th2, but not Th1, cytokine responses to CRM197, compared to children who had received 7vPCV only at 1 and 2 months of age . This difference in recall Th cell responses between children first vaccinated at birth or 1 month of age was no longer detectable at 9 months of age, when all children had completed their three-dose 7vPCV vaccination schedule (unpublished preliminary findings). Elevated type-2 T cell cytokine responses to CRM197 were not associated with reactogenicity to 7v-PCV, but were associated with increased local reactivity to a combined Haemophilus influenza B–diphtheria–tetanus–whole cell pertussis (DTwP/Hib) vaccine , as shown previously for Australian infants immunized with a diphtheria–tetanus–acellular pertussis (DTaP) vaccine . Neonatal 7vPCV immunization did not interfere with T cell responses to concomitant vaccines or the overall T cell development; however, TLR-mediated IL-6 and IL-10 responses were enhanced in both the neonatal and infant 7v-PCV vaccination groups . Altogether, these findings show that despite the relative unresponsiveness of the neonatal immune system of PNG compared to AUS newborns, neonatal 7vPCV vaccination is feasible in this high-risk population. Nevertheless, it is important to keep in mind that outcomes of neonatal immunization may vary between populations and are vaccine-dependent. For example, neonatal immunization with DTaP at birth has been shown to reduce immune responses to subsequent vaccines as a result of vaccine interference [45,46].
Non-specific effects of vaccines in high-risk populations
An important series of studies in Guinea Bissau have reported non-specific beneficial and harmful effects of routine vaccinations on childhood survival [47–51]. Although in response to a call by the World Health Organization (WHO) observational studies from other countries, including one in PNG , have challenged this finding, the possibility that these observations may be explained by vaccine interference – that may vary depending on immunization schedules and environmental and genetic population differences – needs to be considered. For example, the efficacy of BCG is known to vary between populations  as well as that any capacity of BCG to prevent atopic disease appears to be limited to children in developing countries who are vaccinated at a very young age [54,55]. Indeed, as we have shown recently, a possible explanation for this variation may be differences between populations in neonatal innate immune responses to BCG predicting recall T cell responses in later infancy .
There is increasing evidence that neonatal immune responses vary between geographically diverse populations as a consequence of differences in environmental and genetic risk factors. Considering the importance of the functional state of the neonatal innate immune system in the kinetics of immune maturation, this implies that the prenatal period provides an important window of opportunity to influence immune responses in later life. By studying populations in different geographic settings concurrently, it may be possible to elucidate critical differences in the ontogeny of immune regulation in the neonatal period that cannot be found when populations are studied in isolation. Important unanswered questions include how quickly the immune system matures and how this may differ between populations, and the nature of host factors responsible for variation within and between populations in neonatal immune function, including within the intrauterine environment.
The authors would like to thank the parents and guardians of the study children for their participation, and A/Professor D. Lehmann, A/Professor P. Richmond, Professor P. Siba, Mr W. Pomat, Dr S. Phuanukoonnon, Professor S. Prescott, Dr D. Strickland, Ms J. Lisciandro and other research staff members from the University of Western Australia Centre of Child Health Research and the Papua New Guinean Institute for Medical Research for their contributions to the work that has been described here. This work has been funded by an International Collaborative Research grant from the Wellcome Trust and Australian National Health and Medical Research Council (NHMRC) (071613/Z/03/Z) and a NHMRC Project Grant (513836). Dr A. H. J. van den Biggelaar is supported by a NHRMC R. Douglas Wright Biomedical Career Development grant (458780).