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Vaccination in early life: standing up to the challenges
Elodie Mohr and Claire-Anne Siegrist
The challenge for any vaccine design is to elicit protective
humoral and/or cytotoxic immunity against life threatening
pathogens while remaining innocuous. Neonatal vaccinology
faces additional challenges linked to intrinsic peculiarities of the
innate and adaptive neonatal immune system. These include
anti-inflammatory rather than pro-inflammatory responses to
innate signals, preferential Th2 differentiation limiting the
induction of Th1 and cytotoxic responses, trends to
immunoregulatory responses and weak plasma cell and
germinal centre B cell responses. Recent progresses in our
understanding of the molecular bases of these physiological
peculiarities and of the mode of action of novel adjuvants open
new opportunities to design vaccine formulations and
immunization strategies better adapted to the early life period.
Address
WHO Collaborative Center for Vaccine Immunology, Departments of
Pathology-Immunology and Pediatrics, University of Geneva,
Switzerland
Corresponding author: Siegrist, Claire-Anne
(Claire-Anne.Siegrist@unige.ch)
Current Opinion in Immunology 2016, 41:1–8
This review comes from a themed issue on Vaccines
Edited by Rino Rappuoli and Ennio De Gregorio
For a complete overview see the Issue and the Editorial
Available online 19th April 2016
http://dx.doi.org/10.1016/j.coi.2016.04.004
0952-7915/# 2016 Elsevier Ltd. All rights reserved.
Introduction
Despite the development of vaccines against a growing
spectrum of pathogens, neonates and infants pay a heavy
toll to infectious diseases. This burden is largely attrib-
utable to the unavailability and/or inadequate use of
vaccine formulations circumventing the intrinsic prop-
erties of the early life immune system. Indeed, few
vaccines (BCG, oral polio, hepatitis B) may already be
administered at birth. These prime for effective T cell
(BCG) or B cell (oral polio, hepatitis B) responses but
fail to elicit significant primary antibody responses.
Postponing immunization to the 2nd month of life
enhances immune response capacity, such that infant
immunization (against tetanus (T), diphtheria (D), per-
tussis (Pa), polio (IPV), hepatitis B (HBV), Hemophilus
Influenzae b (Hib), pneumococcus (PCV) and rotavirus) is
routinely initiated at 6–8 weeks of age. Antibody
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responses to this first infant dose are weak, requiring
the administration of repeat doses at 1 or 2 months
intervals and thus delaying the onset of protection.
Thus, the 2016 US infant immunization schedule
recommends 3 primary doses of DTPa-IPV-HBV/Hib
and PCV at 2-4-6 months. Infant responses are short-
lived, requiring a booster already in the 2nd year of life
[1]. The same limitations apply to new vaccines: as an
example, the protective efficacy of the novel RTS,S
malaria vaccine (although adjuvanted with MPL and
QS21) is higher in children than in infants [2].
To stand up to their challenges and protect against major
early life viral (influenza, Respiratory Syncytial Virus
(RSV)), bacterial (pertussis, streptococcus, meningococ-
cus) or parasite (malaria) pathogens, neonatal vaccines
should safely elicit strongly protective responses after a
single dose — and such responses should be sustained —
or easily boosted.
Thus, the kinetics, the magnitude and the duration of
protection induced by neonatal vaccines should all be
enhanced.
The neonatal immune system is adapted to the chal-
lenges of leaving abruptly the almost sterile uterine
environment for the external world, where it faces
constant antigenic stimulations. These adaptations re-
quire the implementation of immune tolerance to self-
antigens and vital foreign elements like food and com-
mensal bacteria, whereas pathogens require the rapid
development of potent immune responses to ensure
immediate and long-term survival. How these seeming-
ly colliding processes, involving a delicate balance be-
tween tolerogenic and pro-inflammatory responses, are
orchestrated towards the establishment of healthy ho-
meostasis [3] remains largely unclear. Hence, vaccina-
tion requires dedicated strategies to overcome neonatal
immaturity [4] and immunoregulatory mechanisms
[5,6] while avoiding the excessive inflammation that
could lead to tissue damage, allergies or autoimmune
disorders.
The neonatal immune system is characterized by anti-
inflammatory rather than pro-inflammatory responses to
danger signals and antigens, resulting into the prefer-
ential differentiation of CD4+ helper T cell (Th) to-
wards Th2 cells — antagonizing Th1 and cytotoxic
responses against intracellular pathogens [7], by the
propensity to differentiate into immunoregulatory cells
over effector/memory cells [5,8], by limited plasma cell
(PC) and germinal centre (GC) B cell responses [9] and
Current Opinion in Immunology 2016, 41:1–8
2 Vaccines
occasionally by the presence of maternal antibodies
with immunomodulatory properties [10]. These com-
plex features affect responses to both pathogens and
vaccines. Nonetheless, the ongoing deciphering of the
molecular mechanisms underlying the peculiar behav-
iour of neonatal immune cells is unveiling news
windows of opportunities: newly developed adjuvants
and immunization strategies have proven able to
improve neonatal responses in preclinical or in vitro
models, and may eventually apply to neonatal human
immunization.
Here we discuss recent progresses in the understanding of
the early life immune system and how some limitations
may be overcome by the use of specific adjuvants and
immunization strategies.
The early life immune system
Because of low exposure to foreign antigens in utero, the
newborn’s adaptive immune system is mainly composed
of naı̈ve lymphocytes [11,12,13]. Few effector memory
CD4+ T cells that readily produce cytokines have been
identified in human cord blood, but whether and how
they could be recruited into protective responses remains
to be determined [14]. Naive lymphocytes require sev-
eral divisions and differentiation steps, that is at least 1–2
weeks, to become protective effectors [15]. Therefore,
the paucity of antigen-experienced cells confers vulnera-
bility to fast replicating pathogens and leaves newborns
reliant on their sole innate immune system, equally
immature [16].
Innate cells express a range of receptors recognizing
conserved danger-associated molecules expressed by
pathogens or distressed cells. Upon engagement of these
innate receptors, adult dendritic cells (DC), neutrophils
and macrophages produce inflammatory cytokines and
increase their capacity to present antigens to T cells,
initiating and shaping adaptive responses. In neonates,
responses to two major danger pathways, the Toll-like
receptors (TLR) signalling and the IL-1/inflammasome
pathways, are dampened and fail to induce potent pro-
inflammatory responses — including IL-12p70, the mas-
ter cytokine for Th1 and cytotoxic responses [17,18,19]
(Figure 1a,b). Endosomal TLR ligands (such as viral
single-strand or double-strand RNA and bacterial CpG
oligonucleotides) binding TLR-3, 7/8 and 9, respectively,
trigger better responses in neonates and may offer a
strategy to enhance Th1 responses (see below and
Figure 1a,b) [20,21,22].
Likewise, recent studies with human cord blood cells
pinpointed that monocyte-derived DC readily respond
to co-activation of dectin-1 and TLR7/8 by producing
IL-12p70, and that neonatal plasmacytoid DC (pDC)
respond to the viral RNA content of trivalent inacti-
vated influenza vaccines by producing Type interferons
Current Opinion in Immunology 2016, 41:1–8
(IFN) and supporting the induction of IFN-g-secreting
Th1 cells [23,24,25]. Thus, C-lectins and/or neonatal
pDC represent potential targets to elicit Th1 antiviral
protection in human newborns (see below and
Figure 1c).
Intrinsic features of neonatal CD4+ T cells further in-
crease the difficulties to stimulate adequate immune
responses. Although high levels of miR-181a increases
TCR-induced calcium signalling in neonatal CD4+ T
cells compared to adult, this signal fails to induce AP-1-
dependent cytokines production and result in anergy
[26]. Neonatal human and murine CD4+ T cells differ-
entiate preferentially into Th2 effectors, partly through
epigenetic control involving hypermethylation of IFN-g
locus and hypomethylation of the Th2 locus [12]. Other
mechanisms underlying the resistance to IL-12 and
preferential Th2 neonatal differentiation include the
storage of a non-secreted IL-4 isoform in the cytoplasm
of human neonatal CD4+ T cells [27] and the expression
of the IL4Ra/IL13Ra heteromeric receptor by neonatal
Th1 cells, inducing their apoptosis upon IL-4 ligation [7].
That the neonatal T cell pool is composed of high
proportions of recent thymic emigrants [11] may con-
tribute to their lower responsiveness and preferential
differentiation into Th2 and regulatory T cells
[8,28,29]. It is also likely that the low responsiveness
of neonatal cells to the TLR and IL-1/inflammasome
pathways impacts on the intrinsic ability of T cells to
respond to vaccines and pathogens. Indeed, bacterial
components stimulate T cell cytotoxicity through
TLR and IL-1 signalling [30], and TLR engagement
is necessary for effector T cells to overcome Treg sup-
pression [31].
Humoral responses offer several layers of protection
differing by their efficacy and duration (Figure 2). In
early life, low antibody titres and the need for repeated
vaccine doses reflect the limitations of extrafollicular
(EF) and GC-derived plasma cell responses. This
results from B cell-intrinsic features, including weaker
BCR-mediated signals, the lower expression of co-stim-
ulatory molecules for T cells and the slow maturation of
marginal zone B cells, a subset of B cells recognizing
conserved pathogen-derived molecules to mount rapid
T-independent responses in extrafollicular foci [9]. But
early life humoral responses are mainly controlled by B-
cell extrinsic limitations. Follicular dendritic cells
(FDC) only develop slowly after birth, which delays
GC formation [32], and bone marrow stromal cells
provide insufficient survival factors (such as A Prolifer-
ation Inducing Ligand (APRIL)) to support post-GC
long-lived PC [33]. This insufficient accessory support
also affects the human gut, where low numbers of IgA+
PC correlate with poor expression of APRIL until one
month of life, when both IgA+ PC and APRIL appear
[34]. Most importantly, the expansion of T follicular
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 Vaccination in early life Mohr and Siegrist 3

Figure 1

(a) Cell death (b) (c)

MF59 Alum Uric acid dsDNA
CpG BCG Curdlan BCG CAF01
Zymosan

TLR1
R-848 TLR2 Dection-1 Mincle
TLR5 FcγR
PuR TLR4 CD14 TLR6 IL-1R

4. Syk Syk
IL-1 ASC/CARD Lipid rafts
cytokine family
3. TLR9
MyD88
TLR7/8
2. TLR3 CARD9
Inflammasome
TRIF 5. MALT1 Bcl10
ATP ENDOSOME
Cathepsin B
LYSOSOME
Pro-IL-1 Pro NOD-like NFκB NFκB
cytokine family IRF3 IRF7
caspase-1 receptors
1. 6.
CYTOSOL CYTOSOL
NUCLEUS NUCLEUS Inflammatory
Pro-IL-1 Pro-caspase-1 IL-12p70 TNFα Type I IFNs IL-12p70 Type I IFNs cytokines

7.
NLR pathway TLR pathway CLR pathway

Current Opinion in Immunology

Innate receptors signalling in neonates and adjuvant targets. Black arrows indicate signalling pathways and blue arrows adjuvant targets. Bold
arrows are confirmed effective pathways in neonates, while dashed arrows and elements represent suboptimal pathways. (a) The IL-1/
inflammasome and (b) the TLR signalling modules are two essential synergic components of the danger-sensing machinery, which trigger the
production of inflammatory cytokines such as IL-1b, TNF-a, Type I IFN (IFNa and IFNb), IL6 and IL-12p70. (a) The IL-1/inflammasome pathways
requires two signals: (1) from the TLR, inducing the expression of pro-forms of IL-1 family members (IL-1b, IL-18, IL-33) and caspase 1 by NFkB
activation, and (2) a second signal mediating the maturation of caspase-1 and inflammasome assembly (composed by a nucleotide-binding
oligomerization domain receptors or NOD-like receptors (NLRs), the adaptor apoptosis-associated speck-like protein containing a CARD domain
(ASC), and caspase-1). This second signal results from the ligation of intracellular NOD-like receptor sensing RNAs or the binding of crystals to
lipid rafts provoking lysosome disruption, and subsequent release of cathepsin B in the cytosol, and/or ATP triggering autocrine activation through
purine receptors (PuR). (3) The active inflammasome releases active IL-1 family members. Alum adjuvants likely act directly by binding to lipid
rafts, and indirectly by inducing cell death and release of natural TLR agonists and uric acid, providing signals 1 and 2 respectively [38]. MF59 is
thought to act through ATP/PuR to initiate activation of innate immune cells at the site of injection [63]. In neonates, the limited induction of pro-
IL-1 and pro-caspase 1 expression [48] and the limited cleavage of pro-IL1b by the inflammasome [18] blunt the IL1/inflammasome responses.
(b) Toll-like receptors (TLR) bind pathogen-derived components at the cell surface (TLR1-2, 4–6) or in the endosomes (TLR3, 7/8, 9) where they
recognize microbial-derived and host-derived nucleic acids. TLR4 can also be endocytosed (not depicted). TLR signalling (4) occurs through the
engagement of the intracellular Toll-IL-1 resistance domain (TIR) with the adaptor proteins MyD88 or TRIF (5). This activates IL-1R-associated
(IRAKs) and TNF-associated (TRAFs) factors (not depicted), leading to the activation of nuclear factor kB (NFkB) and Interferon-regulatory factor
(IRF) 3 and/or 7 (6) and IL-12, TNFa and type I IFN responses (7). Several steps limit neonatal TLR-mediated pro-inflammatory responses: TLR
signalling is antagonized by an inhibitory pathway triggered by high plasma ADP [68] and possibly blunted by lower expression of the TLR4/TLR2
co-receptor CD14 [20] and MyD88 [20]. Less effective IRF-7 nuclear translocation and DNA binding of IRF3 [17] limit IL-12 and type I IFN
responses. Interestingly, TLR7/8 agonists are refractory to ADP inhibition and thus active on neonatal cells [48]. (c) Dectin-1 and Mincle signalling.
Dectin-1 and Mincle are innate surface receptors of the C-lectin family. They bind b-glucans on the cell wall of fungi and bacteria and the cord
factor (TDB) expressed on the surface of M. tuberculosis, respectively. Dectin-1 signals directly through its cytoplasmic tyrosine-based activation
motif (ITAM), recruiting the Syk, while Mincle uses ITAM on the FcRg chain. Syk activates the Card9–Bcl10–MALT1 complex, triggering NF-kB and
subsequent production of pro-inflammatory cytokines. These pathways have proven functional in humans in cord blood monocyte-derived and
pDC stimulated with Curdlan [23], in neonatal and infant mice immunized with the TDB-including CAF01 adjuvant [56,57] and following BCG
immunization [60].

helper (TFH) cells, which are critical for effective GC formulations or adjuvants improving neonatal TFH
responses, was recently identified as a key limiting responses is a major challenge to induce the GC
factor for the development of early life GC responses responses required for the establishment of long-lived,
[35,36,37]. Therefore, the development of vaccine high affinity effectors (Figure 2).

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4 Vaccines

Figure 2

DURATION OF PROTECTION

Extrafollicular responses Germinal center responses Memory cells

Memory CXCR5
Short lived
plasma cells B cells Long lived
OX40 OX40L
plasma cells
ICOS ICOSL
TCR PP-MHC II
TFH CD40
CD40L
cells IL-21R
IL-21 BCR

Ag 2.
3. FDC 4.
BCR
GC B
Cytokines Receptors cells Memory
GC B B cells
TCR PP-MHC II
GC B GC B
CD40L CD40

cycling
CD4+ T 1.
B cells
helper cells

Current Opinion in Immunology

B cells differentiation towards plasma cell and memory B cells in early life vaccine responses. Dotted arrows/lines represent the pathways/cells
reduced in neonates. In the B cell follicles of the lymph nodes draining the injection site, B cells binding to their cognate antigen (Ag) through their
B Cell Receptor (BCR) migrate to the interface between the B cell and the T cell zones, where they interact with activated CD4 T helper (Th) cells
(1). Th/B cell interactions (involving MHC class II/Ag-derived peptides on B cells and TCRab/CD4 on T cells) deliver B cell survival and activation
signals through CD40/CD40L. Activated B cells proliferate and may differentiate either in situ (extrafollicular (EF) responses) (2) or into germinal
centres (3). In early life, most B cells differentiate along the EF pathway and into quiescent memory B cells (MBC) rather than plasma cells (PC).
This generates a low and transient antibody response of weak affinity for the vaccine Ag. Antigen-specific B cells blasts which receive appropriate
activation signals upregulate the BCL-6 transcription factor and migrate back to the B cell follicles, where they seed the germinal centre (GC)
reaction at the vicinity of follicular DC (FDC) retaining the Ag captured in immune complexes (3). In these structures, GC B cells proliferate and
undergo affinity maturation through somatic hypermutation, selection and proliferation. This process requires close interactions of B cells with T
follicular helper cells (TFH), a subset of T helper cells homing to the GC through CXCR5/CXCL13 chemotaxis and delivering specific helper and
survival signals to GC B cells, and with FDCs. In early life, the paucity of FDCs and suboptimal TFH/GC B cell interactions fail to deliver sufficient
B cell activation/survival signals, restricting the GC reaction. GC-derived B cells may differentiate into MBC or long-lived PC capable of surviving
in specific BM niches. In early life, GC-derived B cells preferentially differentiate into MBCs and away from long-lived PC, the early life BM failing
to provide sufficient PC survival signals. Adjuvants capable of inducing potent GC responses and long-lived BMPCs in early life remain to be
identified.

Immunization strategies for early life
protection
Understanding the factors and mechanisms underlying
the specific patterns of early life innate and adaptive
responses progressively paves the way towards the iden-
tification of novel vaccine and adjuvant formulations
which fail to be identified by studies performed on adults.
Alum, the adjuvant currently used in infant vaccines,
exacerbates the neonatal Th2 bias by promoting the
release of prostaglandins. A body of evidence in vitro
indicates that alum uses components of the inflamma-
some IL-1/caspase 1 pathways [38], the latest discovered
being the NEK kinase 7 which promotes inflammasome
assembly [39,40]. Alum is insufficient to promote effec-
tive plasma cell responses or the expansion of Tfh cells
and GC B cells in mouse neonates [35,37], but it
effectively induces memory B cells, likely along the
EF pathway, and triggers affinity maturation already in
neonates [41]. These limitations fit in well with the

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observation that inflammasome IL-1/caspase 1 pathways
are dampened in human neonates (Figure 1) [18]. How-
ever, the exact mechanisms of alum adjuvanticity in vivo
are still matter of discussions, may differ between innate
and adaptive responses, and could involve alternative
pathways as yet to be discovered and assessed in neonatal
life [38]. Nonetheless, alum may prove an appropriate
vehicle for the retention/sustained delivery of early life
immunomodulators.
TLR agonists have been extensively tested as immuno-
modulators (reviewed in [42]), and early life responsive-
ness to TLR agonists assessed in vitro and occasionally in
vivo [43,44]. They may mediate B cell adjuvanticity by
acting on multiple innate cells including DC (to promote
the activation of T helper cells) and B cells, which express
subsets of TLRs (TLR2, TLR4, TLR7 and TLR9 in
mouse and TLR2, TLR7 and TLR9 in human), and
respond to their agonists by activation, proliferation,
cytokine secretion and PC differentiation [45,46]. In
human, an aluminium-adsorbed TLR2 ligand-containing
Hib vaccine promoted antibody responses when given at
2 months of age — but not in neonates and to the
detriment of memory responses [43]. Its development
was thus not pursued. MPL (Monophosphoryl Lipid A), a
TLR4 ligand, synergizes with aluminium salts to improve
the magnitude and duration of B cell responses to hepa-
titis B and HPV vaccines in adults [47]. The ontogeny of
TLR4 responses has not been characterized other than in
vitro [43,48]. However, a combination of MPL and QS21
(quillaja saponaria 21) enhanced the immunogenicity of
the RTS,S malaria vaccine in infants [2]. Studies in
mouse suggest that its efficacy results from enhanced DC
and monocyte activation [49]. TLR7/8 agonists exert
potent adjuvanticity in adult mice [50], have entered
clinical development and have been identified as unique-
ly capable of activating human neonatal antigen-present-
ing cells [19,21,51], suggesting promising early life
adjuvanticity. TLR9 agonists have been extensively stud-
ied, mainly in addition to aluminium salts, and shown to
increase early life murine Ab responses [32,35,52,53].
Nucleotide oligomerization domain (Nod)-like-receptor
agonists elicit innate neonatal responses in whole-blood
assays, but were not yet tested in vivo [54].
C-type lectin agonists, which activate macrophages and
DC through the Syk-Card9 pathway [55] (Figure 1c), are
promising early life adjuvants. On human neonatal DC,
Mincle agonists (such as trehalose 6,6 dimycolate (TDB))
and Dectin-1 agonists (such as zymosan or curdlan) en-
hanced DC activation and Th1 polarization, in synergy
with TLR agonists [23]. In vivo, TDB induces potent
Th1/Th17 neonatal responses through the exquisite tar-
geting of a small number of DC when formulated in
cationic liposomes (CAF01) [56,57] which are being de-
veloped as a versatile vaccine adjuvant platform [58]
eliciting prolonged CD4+ T cell responses in human
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Vaccination in early life Mohr and Siegrist 5
adults [59]. Together with TLR ligands, TDB is a critical
adjuvant factor of the Bacillus Calmette–Guérin (BCG)
vaccine, which shows remarkable ability to promote Th1
and antibody responses when used concomitantly with
other vaccine antigens [60] (Figure 1b,c).
MF59, a squalene-based emulsion developed by Novartis
Vaccines (now GSK), markedly increased Ab responses
and protection against influenza in 6-36 months infants
[61]. A recent study using a system biology approach
unravelled this adjuvanticity to be mediated by the
induction of more homogenous, robust and more adult-
like gene signatures [62]. Part of the adjuvanticity of
MF59 relies on ATP release by muscle cells at the site
of injection and the subsequent strong recruitment of
CD11b+ innate cells encompassing neutrophils and
monocytes [63] (Figure 1a). Studies in mice demonstrat-
ed that MF59 mediates its B cell adjuvanticity through
the induction of potent TFH/GC responses in infants —
but fails to do so in neonates in which additional limita-
tions remain to be overcome [36].
Few other B cell adjuvants have been tested in early life.
A polysaccharide adjuvant based on delta inulin was
reported to increase mainly IgM Ab to influenza, with
no suggestion of an induction of GC responses [64]. The
nontoxic mutant of E. coli heat-labile enterotoxin LT-
K63 adjuvant enhanced anti-pneumococcal neonatal
responses, expanding the FDC network and inducing
some GC responses [52]. Its clinical development was
interrupted after the occurrence of facial nerve paralysis
following intranasal immunization, but this provided a
proof of concept that some GC responses may be elicited
through appropriate early life adjuvantation.
Conclusion
Past vaccine strategies relied on the empirical use of
attenuated pathogens and aluminium-based adjuvants,
whose mechanism of action remained obscure for more
than 80 years [38]. Today, the knowledge acquired in
microbiology and immunology allows the identification of
the specific pathways and factors that uniquely shape the
early life immune system and novel tools, including
system biology approaches, are being applied to study
these critical issues [62]. This enables hypotheses-driven
approaches towards the identification of vaccine formula-
tions specifically designed to stand up to the challenge of
eliciting more rapid, effective and sustained vaccine
responses as early in life as required by the risks of
exposure. Consequently, a growing number of vaccines
and adjuvants are being tested in early life animal models,
with human cord blood cells, and eventually in clinical
trials including children [65].
The road will be long until novel vaccines are demon-
strated sufficiently effective and safe to be used already in
neonates — when required by high risks of exposure.
Current Opinion in Immunology 2016, 41:1–8
6 Vaccines
Meanwhile, maternal immunization has re-emerged as a
safe and powerful strategy to protect against early life
death, be it from influenza [66] or pertussis [67], and
possibly in a near future from RSV or Group B Strepto-
coccus.
Acknowledgements
Research work in the Center for Vaccinology is supported by the Swiss
National Research Foundation, the European Union’s Seventh Framework
Programme (EU FP7) projects ADITEC (HEALTH-F4-2011-280873), the
EU Horizon 2020 project TBVAC 2020 (Grant No. 643381); the Innovative
Medicines Initiative (IMI) Joint Undertaking (JU) VSV-EBOVAC and the
Fondation Mérieux.
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