Vaccine (2008) 26, 292300

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REVIEW
Immune responses to polysaccharides:
Lessons from humans and mice

Africa Gonzalez-Fernandez
a,1
, Jose Faro
a,b,,1
, Carmen Fernandez
c,1,2
a
Area of Immunology, Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain
b
Estudos Avanc ados de Oeiras, Instituto Gulbenkian de Ci encia, Oeiras, Portugal
c
Bio-007, Department of Immunology, WGI, The Arrhenius Laboratories F554,
Stockholm University, S-106 91 Stockholm, Sweden
Received 17 July 2007; received in revised form 26 September 2007; accepted 18 November 2007
Available online 3 December 2007
KEYWORDS
Negative memory;
Polysaccharides;
Vaccines
Summary This review focuses on the immune response to non-conjugated and conjugated
polysaccharide vaccines derived from encapsulated pathogens, such as Streptococcus pneumo-
niae, Haemophilus inuenzae and Neisseria meningitidis. Special attention is paid to a number
of side effects observed following the use of some of these vaccines. For example, we dis-
cuss the long-lasting specic refractoriness induced by unconjugated polysaccharides, and the
absence of an effective immune response in adults vaccinated with some conjugated vaccines.
We argue that studies performed in the mouse model can help to understand those paradoxical
effects observed in humans, and the mechanisms underlying such processes.
2007 Elsevier Ltd. All rights reserved.
Contents
Introduction.............................................................................................................. 293
Human PS vaccines to encapsulated bacteria. Facts and paradoxes ...................................................... 293
Young children do not respond well to PSs........................................................................... 293
TI-2 vaccines ........................................................................................................ 294
Paradoxical effects of TI-2 vaccines .......................................................................... 294
TD vaccines ......................................................................................................... 294
Paradoxical effects of TD PS vaccines......................................................................... 294
Immune responses to polysaccharides: lessons from animal models ...................................................... 295

Corresponding author at: Area of Immunology, Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain.
Tel.: +34 986812625; fax: +34 986812556.
E-mail addresses: africa@uvigo.es (

A. Gonz alez-Fern andez), jfaro@uvigo.es (J. Faro), carmen.fernandez@imun.su.se (C. Fern andez).
1
These authors share leadership and contributed equally to this work.
2
Tel.: +46 8 16 4599; fax: +46 8 612 9542.
0264-410X/$ see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2007.11.042
Immune responses to polysaccharides 293
Young mice do not respond well to PSs .............................................................................. 295
Immune responses to TI-2 antigens in adult mice.................................................................... 295
Immune responses to TD polysaccharide antigens in mice. Some key observations................................... 296
The effect of polysaccharide epitopes ........................................................................ 296
Priming with TI-2 PS can induce negative memory to TD responses ........................................... 296
Differential effects of distinct adjuvants on the immune response to PSs ..................................... 296
Concluding remarks ...................................................................................................... 296
Conict of interest....................................................................................................... 298
Acknowledgments ...................................................................................................... 298
References ............................................................................................................. 298
Introduction
Vaccines made from protein antigens, such as secreted tox-
oids or structural components of pathogens, have been very
successful in the induction of protective immune responses.
In contrast, the development of protective vaccines against
bacterial pathogens carrying polysaccharide (PS) capsules,
such as Neisseria meningitidis, Haemophilus inuenza or
Streptococcus pneumoniae, has in the past encountered
many difculties. These difculties include low immuno-
genicity in children, poor induction of memory responses
and only temporary protection [1]. Many of these problems
have been solved, at least partially, but others remain as
immunological paradoxes.
The PS molecules carry repeated carbohydrate residues
and can, thereby, activate directly B cells in a spe-
cic fashion in the absence of T-cell help; they are thus
representative of the class of antigens referred to as T-cell-
independent type 2 (TI-2) [2]. Although some exceptions
have been documented [3], these TI-2 antigens are in gen-
eral poor inducers of immunological memory [1], but are
able to induce rapid responses characterized by the dom-
inant production of IgM, even though other isotypes like
IgG2 antibodies (Abs) are also produced. However, protein
antigens require the collaboration of CD4+ T cells (T-cell-
dependent (TD) responses) and are able to induce Abs of
several isotypes (with an IgG component), long-lasting mem-
ory and afnity maturation [4]. Those differences in immune
responses that depend on the antigen are crucial when con-
sidering vaccines, particularly those for encapsulated bac-
teria [5]. In an attempt to produce more efcient vaccines,
PSs have been conjugated to various protein carriers making
them TD antigens. Indeed, some PSprotein conjugate vac-
cines are much more effective than plain PS vaccines, elic-
iting long-term immunologic memory in infants and toddlers
as well as in adults [6,7]. Yet, in some cases, the protein-
conjugated PS vaccines induce a response in young adults
similar to that obtained with the plain PS preparation [8].
Our aim here is to review this and other paradoxes
in human vaccination and then to compare them with
the corresponding knowledge obtained from murine mod-
els, as many aspects of the immunogenicity of PSs in
humans have also been observed in mice. These models
have been instrumental in the elucidation of cellular and
molecular mechanisms in ways that cannot be conducted
in human research. We compare and discuss the human
immune responses to some current PS vaccines in TI-2 and
TD forms, as well as their counterparts in mice, and sug-
gest some potential mechanism(s) responsible for these
responses, which could help to gain insight into how to cir-
cumvent the low success and paradoxical behaviour of these
vaccines.
Human PS vaccines to encapsulated bacteria.
Facts and paradoxes
Young children do not respond well to PSs
It has been shown that children under 2 years are decient
in IgG2 and IgG4 isotypes [9]. Although IgG1 and IgG3 reach
normal adult levels by 23 years of age, the levels of IgG2
and IgG4 rise more slowly and, also, there is a poor crossing
of the placenta by IgG2 [10], so that, even in young peo-
ple of 1317 years of age, IgG2 levels are still signicantly
lower than in adults [11]. As the IgG2 isotype is considered to
be the most effective/active immunoglobulin against some
carbohydrates [12], the long-known susceptibility of young
children to recurrent infections by encapsulated bacteria
may possibly be due to the immaturity of their immune sys-
tem, which is unable to mount optimal humoral responses
to bacterial PSs [13]. In addition to this natural deciency
in TI-2 responses, IgG2 subclass deciency is, together with
that of IgG3, one of the most frequent IgG subclass immun-
odeciencies in children [14]. In any case, young children
are much more impaired in their ability to make Abs to the
PS coatings of bacteria than in making Abs to proteins.
The immune response to many PS antigens requires a
functionally intact spleen [15]. Its absence (either by dys-
function or by splenectomy) increases the risk of infection
by encapsulated pathogens, such as S. pneumoniae or H.
inuenzae [16], and requires antibiotic prophylaxis [17].
This may be related to observations suggesting that the
splenic marginal zone (MZ), which includes MZ macrophages
and MZ B cells, is the site of the initiation of the immune
response to PS antigens. In humans, this is a crescent-shaped
lymphoid compartment covering follicles [18]. Interestingly,
while most cellular compartments of the human immune
system have completed their maturation to a near adult-
type immunophenotype and morphology within the rst 5
months after birth, the infant MZ shows essentially different
features to the adult situation [19]. There is a remarkable
timing coincidence between the rst appearance of MZ B
cells with adult features, and the acquisition of the ability
to mount an immune response to PSs, including encapsulated
bacteria [19].
294

A. Gonz alez-Fern andez et al.
Moreover, it has been shown that children under 2 years
of age have low or absent expression of CD21 (complement
receptor 2) on their MZ B cells [20]. The immune response
to PSs begins when they bind to the complement factor
C3d, and the interaction CD21C3d seems to be crucial for
an anti-PS immune response [20]. Therefore, the absence
of response to many PS antigens in children under 2 years
of age could be due to a combination of factors, such as
low levels of IgG2 and splenic immaturity for C3d recep-
tor. However, neither all immune responses to bacterial PSs
are humoral [21], nor do all PS antigens that induce a TI-2
response behave in the same way. For example, the primary
immune response to the meningococcal PSs A and C is con-
siderably different to that against other meningococcal PSs
(reviewed in 22).
TI-2 vaccines
Until very recently, the only vaccines available for some
encapsulated pathogens, such as S. pneumoniae, N. menin-
gitidis and H. inuenzae type b, were based on their
capsular PS. For example, the rst pneumococcal vaccine
carried 14 serogroups and was replaced in 1983 by a new
vaccine carrying the capsular PS of 23 serogroups [23],
while the meningococcal vaccine was either tetravalent for
serogroups A, C, Y and W-135 or bivalent for serogroups A
and C [24]. These vaccines have mostly been used to control
outbreaks caused by virulent strains. As previously stated,
they are not immunogenic in infants below 2 years of age
(who are therefore at increased risk of disease), they induce
short immune responses in older children and adults, and,
after repeated vaccinations, no memory immune responses
are generated at any age [25].
It has been reported for the meningococcal vaccine that,
among children of 26 years of age immunized with the
A/C PS vaccine, only 50% had serum bactericidal titers
1:8 1 month later, while after 12 months this percentage
fell to 20% [26]. In contrast, adults receiving unconjugated
meningococcal PS vaccines develop higher concentrations of
protective anticapsular antibodies that last for up to 510
years [27], and the immunogenicity does not seem to be
inuenced by its route of administration (intramuscular or
subcutaneous) [28].
Paradoxical effects of TI-2 vaccines
Unlike the relatively high levels of bactericidal antibody gen-
erated in adults after a single dose of meningococcal PS from
serogroup C, several authors have observed immunologic
refractoriness in children after repeated doses of this vac-
cine [29], similar to that observed with PSs from serogroup
A after revaccination [30]. This refractory state has even
been observed in adults receiving a conjugated TD vaccine
4 years after a rst immunization with a meningococcal C
PS vaccine [31]. The absence of induction of memory by
conjugated meningococcal vaccines in adults previously vac-
cinated with plain meningococcal PS has also been seen [32].
Lakshman et al. [33] reported a similar inhibitory effect
using a combination of meningococcal A/C PS. Although
a rst dose of this vaccine was immunogenic in adults, a
second dose of meningococcal A/C PS, either plain or conju-
gated to diphtheria toxoid, induced lower bactericidal titers
to N. meningitidis group C than those seen following a rst
dose [33]. Hence, PSs such as those from meningococcal
serogroups A/C seem to induce a long-lasting unrespon-
siveness or negative memory to further immunizations,
even to conjugated vaccines. This could have important
consequences, including an increased risk of meningococcal
infection. How can this negative effect be explained?
TD vaccines
To overcome some of the negative effects of TI vaccines,
PSprotein conjugate vaccines have been recently devel-
oped for all three of the encapsulated pathogens mentioned
above [34]. For this purpose, a number of protein and con-
jugation procedures have been tested. The use of proteins
such as tetanus or diphtheria toxoids has attracted much
attention, as they are probably more stimulatory to the
immune system than, for example, bovine serum albumin,
and also because they are routinely used in human vaccina-
tion programmes [3538].
Since the development of these conjugated vaccines,
some have been included in the routine immunization sched-
ules that are being implemented or adapted in various
countries [39,40]. These protein-conjugated PS vaccines
have been found to be much more effective than uncon-
jugated PS vaccines, especially in young children <2 years
of age. This is the case for the H. inuenzae type b vac-
cine. This microorganism was once the most common cause
of bacterial meningitis in children, but vaccination with TD
conjugate vaccines has almost eliminated cases of menin-
gitis produced by this bacteria [41]. The vaccine has been
particularly useful for prevention in high-risk children.
Although conjugate vaccines have been very successful
against H. inuenzae type b disease, they currently have
a more restricted use against multi-serotype/serogroup
pathogens like pneumococci and meningococci [42]. The
major reasons for this restricted use could be the high cost of
the vaccines and the fact that there are different prevalent
serotypes in different geographical areas.
Among the PSprotein conjugate vaccines for S. pneu-
moniae, the heptavalent pneumococcal vaccine has had a
strong impact in the prevention of invasive disease by vac-
cine serotypes, reducing, as was intended, the incidence of
bacteraemia and meningitis by 8297% [40,43]. Moreover,
several studies have shown that the vaccine moderately
reduced (1050%) the pneumococcal otitis media in chil-
dren [4446]. However, in one of these studies, undertaken
in two US cities, it was found that hospital visit rates for
pneumonia caused by this pathogen were not signicantly
reduced in children aged <2 years [46].
For meningococcal infections, one of the major causes of
bacterial meningitis in children and teenagers worldwide,
the conjugate vaccines have also been instrumental in help-
ing to reduce the number of deaths and clinical cases by
more than 90%. Nevertheless, bacterial meningitis and sep-
ticaemia caused by this pathogen have not been completely
eliminated [47].
Paradoxical effects of TD PS vaccines
TD vaccines for S. pneumoniae are able to induce a good
antibody titer in young children (<2 years old), although
Immune responses to polysaccharides 295
some studies have shown little protection 1 year after the
last scheduled dose, and low-vaccine effectiveness in older
children and adults [48]. Indeed, instead of inducing a good
memory response, there is some evidence suggesting that
the PSprotein conjugate vaccine may behave similarly to
a plain PS vaccine [49,50].
In a UK report about conjugated meningococcal vaccines,
children under 2 years of age receiving three doses (at 24
months of age) of the conjugated meningococcal C vaccine
did not show protection 24 years after immunization [51].
Another study in Spain with the same vaccine, showed that
the effectiveness (the percentage reduction in the attack
rate in vaccinated compared with unvaccinated children
in the same birth cohorts) dropped from 98% for the rst
year, after three immunizations (at 2, 4 and 6 months of
age), to 78% in the following 3 years [39]. Similar results
were reported in a recent paper, comparing these data from
the UK and Spain with data from a Canadian study [52]. As
a potential explanation, Weller et al. [53] have suggested
that, in adults, the ability of the MZ to trap TI-2 antigens may
also affect, at least partially, the TD PS antigens, thereby
reducing the triggering of a TD immune response. However,
they postulated that, in babies, a poorly developed MZ would
allowTD PS antigens to reach follicles and to trigger a normal
TD immune response.
There is a controversy about the effect of conjugated
meningococcal vaccines in adults. Some evidence suggests
that there are no signicant differences in the magnitude of
the response between young adults vaccinated with uncon-
jugated or with conjugated serogroup C PS vaccines [8]. In
adults, immune response failures to these vaccines have
been reported [54]. Yet, some authors have found high-
bactericidal titers and superior functional activity in serum
after immunization with a conjugate serogroup C vaccine
[55].
Immune responses to polysaccharides: lessons
from animal models
The immunogenic properties of PSs and PSprotein con-
jugates have been extensively analysed in mouse models,
because of their simple and convenient nature [5659]
and because results in mice are relatively well correlated
with efcacy in humans [60]. Moreover, in general, immune
responses elicited by PSs are similar in mice and humans, and
are characterized by oligoclonality [61,62], Abs with little
heterogeneity and low afnity, minor anamnestic increase
in Abs titers [2] and some degree of somatic hypermutation
[61,62].
As in humans, the murine spleen also has a very important
role in recognizing encapsulated bacteria, although differ-
ences in the B-cell compartment of the MZ between humans
and mice have been recently reported [18]. Moreover, mouse
MZ macrophages are phenotypically distinct from human MZ
macrophages. For example, murine MZ macrophages express
the C-type lectin SIGNR1 [63], which has been shown to bind
dextran and to play a relevant role in the immune response
against encapsulated bacteria [63,64]. However, its homo-
logue in humans has not yet been found.
In this section, we discuss some aspects of murine immu-
nity to several PSs that are relevant to the immune response
in humans. Although information is given about various TI-2
antigens, particular emphasis is placed on the carbohydrate
dextran B512 (Dx), as it is one of the most extensively
studied carbohydrates and it appears to be an appropriate
model of the immunogenicity of many PS antigens. Dx is
derived from Leuconostoc mesenteroides as a high-MW PS
antigen ((10100) 10
6
Da), and it has a very simple struc-
ture dominated by non-branched (1>6) glucose residues.
This makes it easy to work with, both in TI- and in TD-forms.
Immune responses to native Dx are characterized by the
predominant production of IgM followed by lower amounts
of IgG3, IgG2b and IgA-specic antibodies [65]. This is in
agreement with the general ndings for other TI-2 antigens,
such as Ficoll [66] or many bacterial capsular PSs [5660].
The response of C57BL/6 mice to Dx is oligoclonal, with
a predominant expression of the VH B512 and the VK OX-
1 genes. The same type of restricted gene expression is
observed independently of whether animals are immunized
with the plain PS (TI-2 form) or coupled to a protein (TD
form) [67,68].
Young mice do not respond well to PSs
As with the ndings in young children, decient responses
in young mice (under 1 month of age) have been observed
using Dx B512 [69,70]. The poor response in young mice was
restricted to the PSs, as the response to other antigenic epi-
topes from haptens or proteins coupled to Dx was found to
be more adult-type [69,70]. Unresponsiveness to Dx did not
seem to be due to the presence of soluble suppressive fac-
tors in the neonates, as cells transferred from young animals
into irradiated adults have a similar behaviour. Our interpre-
tation was that unresponsiveness to Dx could be due to a lack
of B cells expressing the required Ig receptor in the B-cell
compartment of young mice [69]. This possibility is compati-
ble with the established fact that the expression of different
V families follows a developmental programme that con-
tinues in neonates [71]. Nevertheless, these early ndings
could also be explained by the concept postulated by Timens
et al. in humans [19] of a decient population of mature
MZ cells in young individuals, or by the model of Weller et
al. [53], which postulates an immature, non-functional MZ
in young humans. Therefore, in the experiment mentioned
above, either a decient population in the transferred
B cells or a disruption of the normal splenic architec-
ture in the adult irradiated mice could explain the poor
response.
Immune responses to TI-2 antigens in adult mice
Secondary immune responses to native Dx are suppressed
in relation to primary responses [72], which is compa-
rable to that described in human capsular PS vaccines.
Potential explanations to this unresponsiveness include
clonal exhaustion of B cells in the absence of memory T
cells, regulation mediated by auto-anti-idiotypic Abs [72,73]
and feedback inhibition by Ag-specic IgG [74]. A similar
effect has been observed with other TI-2 antigens, such as
Ficoll [3] and inactivated meningococcus serogroup B (NmB)
[75].
296

A. Gonz alez-Fern andez et al.
Immune responses to TD polysaccharide antigens
in mice. Some key observations
Immunogenicity of Dx B512 decreases with a reduction in
MW, so that low-MW Dx (5 10
5
Da and below) are either
poor immunogens or are not immunogenic, unless coupled to
proteins (Fern andez C., unpublished results). The response
induced by the Dxprotein complex is then dependent
upon T-cell help. Nevertheless, the quality of that response
depends on a number of intrinsic and extrinsic factors, such
as the radicals of the PS involved in the coupling to protein,
the previous experience of the animal with the TI-2 form of
the PS, and adjuvant co-administration.
The effect of polysaccharide epitopes
Two types of response to Dx were observed, depending on
the methodology used to make the Dxprotein conjugates
[76,77]. Conjugates produced by random coupling of Dx to
protein, displayed immunological responses similar to the
native TI-2 form of Dx, i.e., with little immunoglobulin class
switch and with a secondary response of a similar magnitude
to the primary response. However, complexes obtained by
conjugating the low-MW PS to proteins via the reducing end
were found to induce optimal TD responses [77]. For the
meningococcal antigen NmB, the suppressive effect is not
only epitope specic, but also carrier specic, i.e., it applies
to haptens on the same NmB antigen, but not when coupled
to non-related carrier proteins [78,79].
Priming with TI-2 PS can induce negative memory to TD
responses
Immunization with the TI-2 form of Dx results not only in a
suboptimal immune response, but it also modies posterior
immune responses to TD forms of Dx [8082], in a simi-
lar way to that found for meningococcal TI-2 PS vaccines in
humans [83]. Studies by our group using meningococcal PS
commercial vaccines show that priming with a TI-2 form (PS
A and C vaccine) induces a negative effect in mice (lower
levels of specic IgG) to the further boosts with the PS C
vaccine in TD form (conjugated to tetanus toxoid) [Diaz et
al., unpublished results].
Native Dx-primed mice mount relatively high IgM anti-
body responses when later immunized with a Dxprotein
conjugate, but they produce very low, if any, IgG anti-Dx
[80]. This pattern of TI-2 response was also clearly evident,
when looking at the histology of the spleen where almost no
Dx-specic germinal centres (GCs) were detected [84]. Nev-
ertheless, the anti-protein antibody response was normal in
these mice, demonstrating that only the anti-Dx-responding
cells were affected. However, mice primed with the TD con-
jugate and repeatedly re-immunized with Dx in TI-2 form
generated a high-IgG anti-Dx response [84].
As mentioned above, possible explanations are clonal
exhaustion of Dx-specic B cells or regulation mediated
by auto-anti-idiotypic Abs. The rst explanation is very
unlikely, as mice primed with TI-2 Dx and challenged with
TD Dx produce relatively high titers of IgM anti-Dx, and
many of these antibodies have the same VH-VK expression
as those induced in the primary immune response to TI-2
Dx and to TD Dx [82]. Moreover, auto-anti-idiotypic antibod-
ies do not seem to play a role, as mice injected passively
with monoclonal anti-Dx Abs bearing the dominant idiotype
do not show a suppressed TD IgG response to Dxprotein
antigens [82]. Finally, suppression cannot be ascribed to
negative signals delivered through Fc receptor, because
mice lacking the inhibitory Fc receptor showed the same
pattern of suppression than wild type mice [82]. A simi-
lar nding has been reported for the suppression induced
by TI-2 Ficoll of posterior responses induced by a TD Ficoll
antigen [3]. It has been shown for the meningococcal anti-
gen NmB that suppression involves a radiation-resistant cell
with the likely participation of T cells [78,79], perhaps
regulatory T cells. Nevertheless, the issue of suppressed
secondary TD immune responses by priming with the plain
PS, at least for some common TI-2 antigens, is yet to be
solved.
Differential effects of distinct adjuvants on the immune
response to PSs
Freunds complete and incomplete adjuvants, as well as
alum precipitate, do not have a major effect in the immuno-
genicity of the TI-2 formof Dx or in improving the response to
the TD forms when used in young animals, or in the response
to suboptimal TD conjugates when used in adults [77 and
Fern andez C., unpublished results]. However, cholera toxin
(CT) was found to be a very potent adjuvant either for native
Dx (secondary IgM levels were enhanced eightfold), or for Dx
conjugated to protein in TD form, where a major increase in
IgM and IgG anti-Dx antibody production was detected [80].
The effect was most pronounced for the suboptimal TD con-
jugate. Interestingly, CT was also able to partially abrogate
the unresponsiveness to Dx in the TI-2 non-responder strain
CBA/N, and improve the response in young mice [66]. Fur-
thermore, CTBDx, which is a conjugate of the non-toxic
part of CT with a non-immunogenic low-MW Dx, elicited an
anti-Dx response in nude mice [85].
In agreement with the ndings cited above, CT was also
found to be important for the generation of splenic GC in
mice. Primary immunization of mice with native Dx and CT-
induced GC formation in the spleen, to the same extent as
a Dxprotein conjugate [80,84,86]. The suppression of the
secondary splenic response induced by native Dx (including
GC formation) was reverted by using CT in the immunizations
with TI-2 Dx. In these circumstances, a secondary splenic GC
response, similar to that induced by TD Dx, was generated,
with almost all the Dx-specic B cells located in the GC. The
CT also enhanced the IgMresponse to plain Dx, but the global
isotype prole of that response was not altered, that is, very
little, if any, IgG was produced. Therefore, the choice of
adjuvant may be very critical to optimize the responses to
at least some capsular PSs.
Concluding remarks
Currently, there are four major problems with human vac-
cines for some of the most common capsulated bacterial
pathogens (pneumococcus, meningococcus and H. inuenza)
(1) Lack of/or poor response to plain PSs by babies and
toddlers.
(2) Low immunogenicity of some PS TD vaccines compared
to pure protein TD vaccines.
Immune responses to polysaccharides 297
Table 1 Comparison of human and mouse immune responses to PSs in TI-2 and TD forms
Human Mouse
Young children do not respond to carbohydrates Young mice do not respond to carbohydrates
Partially solved: adjuvant
Decreased responses to repeated immunization with PS TI-2 Decreased responses to repeated immunization with PS TI-2
Solved: adjuvant
Weak immunogenicity of PS TD: low-IgG responses in adults
to PS TD vaccines
Weak (suboptimal) immunogenicity of PS TD (epitope)
Solved: adjuvant
Lack of memory to PS TD when: Lack of memory to PS TD when:
Priming: PS TI-2; challenge: PS TD Priming: PS TI-2; challenge: PS TD
Previous contact with encapsulated bacteria?? Previous contact with encapsulated bacteria
Could be solved by early PS TD immunization and adjuvant
(3) At least, partial immunologic refractoriness induced by
TI-2 PS vaccines to subsequently administered PS TD
vaccines.
(4) Lower immunogenicity of PS TD vaccines in adults than
in toddlers.
Importantly, similar observations have been made in
mice, which prompt potential lines of research to solve
these problems with the above-mentioned human vaccines
(Table 1).
In relation to the rst problem, it is remarkable that the
response to Dx in young and non-responder (CBA/N) mice
can be improved with the use of a proper adjuvant such as
CT [66]. This suggests that very young children could also
have specic B cells, and that the reason for their unre-
sponsiveness to PSs is that other cells or relevant histological
structures are still not in place or not fully mature. Recently,
Weller et al. [53] proposed a model postulating the exis-
tence in humans of an already pre-diversied repertoire of B
cells in the splenic MZ, which would behave as pre-activated
natural effector cells ready to respond to TI-2 antigens to
produce natural IgM antibodies. However, an immature and
non-functional MZ, like that of children under 2 years of age,
would lead to a poor response to plain PS vaccines, while
protein-conjugated PS vaccines could trigger naive follicu-
lar B cells in them, to generate memory responses. These
authors suggest that in older children and adults, a func-
tional MZ would trap both plain and protein-conjugated PS
antigens, leading to TI-2-like immune responses. Yet, if, as
suggested by our comparative analysis, mice and humans
respond similarly to PS antigens, this model does not ade-
quately explain how, when coupled to a protein, common
TI-2 PS antigens can induce, in both humans and mice, a
normal TD response, nor does it clarify how priming with
some common TI-2 PS antigens can re-direct the response
induced by a TD formof the PS towards an enhanced TI-2-like
immune response.
In relation to the second problem, studies in mice sug-
gest two non-mutually exclusive explanations in the human
case: the PSprotein coupling is not optimal, the adjuvant
is not optimal, or both. Nevertheless, at least in mice, a
proper adjuvant like CT can improve the response to a poor
coupling.
Thirdly, although puzzling, recent analysis of the
refractoriness to PSprotein vaccines in mice, previously
immunized with the TI-2 form of the PS [80,82], points
to a practical way of, at least partially, overcoming it.
Hence, although animals primed with TI-2 Dx show a very
poor TD-type response when challenged with the TD form
of the antigen, with or without a weak adjuvant (e.g.,
alum), similarly primed animals challenged with the TD
antigen plus a strong adjuvant (like CT) develop a high-
memory response, albeit with a TI-2 prole. This type
of response is similar to that observed in adult mice
immunized with heat-killed whole pneumoccocal bacte-
ria [87], or with Borrelia hermsii [88]. In such cases, a
long-lasting IgM memory response was reported and could
be specically ascribed to the activation of the subset of
B-1b cells. Another non-exclusive explanation is that the
responding B cells are of high avidity and differentiate
directly to plasma cells (high-Blimp-1 expression) [8991]
with moderate to low proliferation. Furthermore, regula-
tory T cells could become involved through their toll-like
receptors (TLRs) [92], where their activation in TI-2 PS-
primed individuals would prevent conventional T-helper
activity to drive the response to the challenging TD PS
antigen.
Finally, the lower immunogenicity of PS TD vaccines in
adults compared to young children could be due to this
same inhibitory mechanism for at least some capsular PSs,
and to the fact that it is very likely that adults, in con-
trast to young children, have experienced several times the
responses induced by contact with TI-2 capsular PS.
The comparison of the immune responses to PSs in TI-2
and TD forms in humans and mice reviewed in this paper
shows considerable similarities between the two species
(summarized in Table 1). Data from animal models sug-
gest that an early immunization with PS TD vaccines could
prevent negative effects induced by plain PSs. They also
indicate that a strong adjuvant could help to, at least par-
tially, overcome the low-immune response and the lack of
memory induced by PS vaccines. In short, more research is
required both to deepen and broaden our current under-
standing of the mechanisms that underlie the immune
responses to TI-2 PS antigens, and to develop stronger adju-
vants for human immunization with acceptably low levels of
toxicity [85,93,94].
298

A. Gonz alez-Fern andez et al.
Conict of interest
The authors declare no conict of interest.
Acknowledgments
This work was supported by Stockholm University, the Minis-
terio de Educaci on y Ciencia (Consolider-Ingenio 2010) and
the Xunta de Galicia. We thank Teresa Carretero and Ted
Cater for revising the English of the manuscript.
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