Immune responses to polysaccharides: Lessons from humans and mice

Vaccine (2008) 26, 292—300

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journal homepage: www.elsevier.com/locate/vaccine

REVIEW

Immune responses to polysaccharides: Lessons from humans and mice ´ Africa Gonz´ alez-Fern´ andez a,1, Jose Faro a,b,∗,1, Carmen Fern´ andez c,1,2 a

Area of Immunology, Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain Estudos Avanc¸ados de Oeiras, Instituto Gulbenkian de Ciˆ encia, Oeiras, Portugal c Bio-007, Department of Immunology, WGI, The Arrhenius Laboratories F554, Stockholm University, S-106 91 Stockholm, Sweden b

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 pneumoniae, Haemophilus influenzae 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 discuss the long-lasting specific 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.............................................................................................................. Human PS vaccines to encapsulated bacteria. Facts and paradoxes ...................................................... Young children do not respond well to PSs........................................................................... TI-2 vaccines ........................................................................................................ Paradoxical effects of TI-2 vaccines .......................................................................... TD vaccines ......................................................................................................... Paradoxical effects of TD PS vaccines......................................................................... Immune responses to polysaccharides: lessons from animal models ......................................................

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∗ Corresponding author at: Area of Immunology, Faculty of Biology, University of Vigo, Campus Lagoas-Marcosende, 36310 Vigo, Spain. Tel.: +34 986812625; fax: +34 986812556. ´ Gonz´ E-mail addresses: [email protected] (A. alez-Fern´ andez), [email protected] (J. Faro), [email protected] (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

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Young mice do not respond well to PSs .............................................................................. Immune responses to TI-2 antigens in adult mice .................................................................... Immune responses to TD polysaccharide antigens in mice. Some key observations................................... The effect of polysaccharide epitopes ........................................................................ Priming with TI-2 PS can induce negative memory to TD responses ........................................... Differential effects of distinct adjuvants on the immune response to PSs ..................................... Concluding remarks ...................................................................................................... Conflict of interest ....................................................................................................... Acknowledgments ...................................................................................................... References .............................................................................................................

Introduction Vaccines made from protein antigens, such as secreted toxoids 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 influenza or Streptococcus pneumoniae, has in the past encountered many difficulties. These difficulties include low immunogenicity 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 specific fashion in the absence of T-cell help; they are thus representative of the class of antigens referred to as T-cellindependent type 2 (TI-2) [2]. Although some exceptions have been documented [3], these TI-2 antigens are in general poor inducers of immunological memory [1], but are able to induce rapid responses characterized by the dominant 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-celldependent (TD) responses) and are able to induce Abs of several isotypes (with an IgG component), long-lasting memory and affinity maturation [4]. Those differences in immune responses that depend on the antigen are crucial when considering vaccines, particularly those for encapsulated bacteria [5]. In an attempt to produce more efficient vaccines, PSs have been conjugated to various protein carriers making them TD antigens. Indeed, some PS—protein conjugate vaccines are much more effective than plain PS vaccines, eliciting long-term immunologic memory in infants and toddlers as well as in adults [6,7]. Yet, in some cases, the proteinconjugated 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 models, 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-

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gest some potential mechanism(s) responsible for these responses, which could help to gain insight into how to circumvent 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 deficient in IgG2 and IgG4 isotypes [9]. Although IgG1 and IgG3 reach normal adult levels by 2—3 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 people of 13—17 years of age, IgG2 levels are still significantly 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 system, which is unable to mount optimal humoral responses to bacterial PSs [13]. In addition to this natural deficiency in TI-2 responses, IgG2 subclass deficiency is, together with that of IgG3, one of the most frequent IgG subclass immunodeficiencies 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 dysfunction or by splenectomy) increases the risk of infection by encapsulated pathogens, such as S. pneumoniae or H. influenzae [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 adulttype immunophenotype and morphology within the first 5 months after birth, the infant MZ shows essentially different features to the adult situation [19]. There is a remarkable timing coincidence between the first 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].

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294 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 CD21—C3d 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 receptor. 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 considerably 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. meningitidis and H. influenzae type b, were based on their capsular PS. For example, the first 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 2—6 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 5—10 years [27], and the immunogenicity does not seem to be influenced by its route of administration (intramuscular or subcutaneous) [28]. Paradoxical effects of TI-2 vaccines Unlike the relatively high levels of bactericidal antibody generated 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 vaccine [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 first immunization with a meningococcal C PS vaccine [31]. The absence of induction of memory by conjugated meningococcal vaccines in adults previously vaccinated 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 first dose of this vaccine was immunogenic in adults, a second dose of meningococcal A/C PS, either plain or conjugated to diphtheria toxoid, induced lower bactericidal titers

to N. meningitidis group C than those seen following a first dose [33]. Hence, PSs such as those from meningococcal serogroups A/C seem to induce a long-lasting unresponsiveness 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, PS—protein conjugate vaccines have been recently developed for all three of the encapsulated pathogens mentioned above [34]. For this purpose, a number of protein and conjugation 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 vaccination programmes [35—38]. Since the development of these conjugated vaccines, some have been included in the routine immunization schedules 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 unconjugated PS vaccines, especially in young children <2 years of age. This is the case for the H. influenzae type b vaccine. This microorganism was once the most common cause of bacterial meningitis in children, but vaccination with TD conjugate vaccines has almost eliminated cases of meningitis 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. influenzae 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 PS—protein conjugate vaccines for S. pneumoniae, the heptavalent pneumococcal vaccine has had a strong impact in the prevention of invasive disease by vaccine serotypes, reducing, as was intended, the incidence of bacteraemia and meningitis by 82—97% [40,43]. Moreover, several studies have shown that the vaccine moderately reduced (10—50%) the pneumococcal otitis media in children [44—46]. 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 significantly 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 helping to reduce the number of deaths and clinical cases by more than 90%. Nevertheless, bacterial meningitis and septicaemia 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 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 PS—protein 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 2—4 months of age) of the conjugated meningococcal C vaccine did not show protection 2—4 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 first 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 allow TD 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 significant differences in the magnitude of the response between young adults vaccinated with unconjugated 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 highbactericidal 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 PS—protein conjugates have been extensively analysed in mouse models, because of their simple and convenient nature [56—59] and because results in mice are relatively well correlated with efficacy 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 affinity, 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 differences 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 homologue in humans has not yet been found. In this section, we discuss some aspects of murine immunity to several PSs that are relevant to the immune response

295 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 ((10—100) × 106 Da), and it has a very simple structure 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-specific antibodies [65]. This is in agreement with the general findings for other TI-2 antigens, such as Ficoll [66] or many bacterial capsular PSs [56—60]. The response of C57BL/6 mice to Dx is oligoclonal, with a predominant expression of the VH B512 and the VK OX1 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 findings in young children, deficient 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 epitopes 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 factors in the neonates, as cells transferred from young animals into irradiated adults have a similar behaviour. Our interpretation 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 compatible with the established fact that the expression of different V families follows a developmental programme that continues in neonates [71]. Nevertheless, these early findings could also be explained by the concept postulated by Timens et al. in humans [19] of a deficient 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 deficient population in the transferred B cells or a disruption of the normal splenic architecture 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 comparable 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-specific IgG [74]. A similar effect has been observed with other TI-2 antigens, such as Ficoll [3] and inactivated meningococcus serogroup B (NmB) [75].

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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 × 105 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 Dx—protein 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 Dx—protein 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 specific, but also carrier specific, 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 modifies posterior immune responses to TD forms of Dx [80—82], in a similar 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 specific 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 antibody responses when later immunized with a Dx—protein 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-specific germinal centres (GCs) were detected [84]. Nevertheless, 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 conjugate 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-specific B cells or regulation mediated by auto-anti-idiotypic Abs. The first 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 antibodies 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 Dx—protein 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 similar finding 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 antigen 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 Freund’s complete and incomplete adjuvants, as well as alum precipitate, do not have a major effect in the immunogenicity of the TI-2 form of 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 conjugate. 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]. Furthermore, CTB—Dx, 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 findings 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 CTinduced GC formation in the spleen, to the same extent as a Dx—protein 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-specific B cells located in the GC. The CT also enhanced the IgM response to plain Dx, but the global isotype profile 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 vaccines for some of the most common capsulated bacterial pathogens (pneumococcus, meningococcus and H. influenza) (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 Table 1

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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: Priming: PS TI-2; challenge: PS Previous contact with encapsulated bacteria??

Lack of memory to PS TD when: TD Priming: PS TI-2; challenge: PS TD 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 first 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 specific B cells, and that the reason for their unresponsiveness 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 existence in humans of an already pre-diversified 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 follicular B cells in them, to generate memory responses. These authors suggest that in older children and adults, a functional 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 adequately 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 form of the PS towards an enhanced TI-2-like immune response. In relation to the second problem, studies in mice suggest two non-mutually exclusive explanations in the human case: the PS—protein 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 PS—protein 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 profile. This type of response is similar to that observed in adult mice immunized with heat-killed whole pneumoccocal bacteria [87], or with Borrelia hermsii [88]. In such cases, a long-lasting IgM memory response was reported and could be specifically 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) [89—91] with moderate to low proliferation. Furthermore, regulatory T cells could become involved through their toll-like receptors (TLRs) [92], where their activation in TI-2 PSprimed 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 contrast 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 suggest 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 partially, 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 understanding of the mechanisms that underlie the immune responses to TI-2 PS antigens, and to develop stronger adjuvants for human immunization with acceptably low levels of toxicity [85,93,94].

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Conflict of interest The authors declare no conflict of interest.

Acknowledgments This work was supported by Stockholm University, the Ministerio 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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