Differential Effect of TLR2 and TLR4 on the Immune Response after Immunization with a Vaccine against Neisseria meningitidis or Bordetella pertussis

Abstract

Neisseria meningitidis and Bordetella pertussis are Gram-negative bacterial pathogens that can cause serious diseases in humans. N. meningitidis outer membrane vesicle (OMV) vaccines and whole cell pertussis vaccines have been successfully used in humans to control infections with these pathogens. The mechanisms behind their effectiveness are poorly defined. Here we investigated the role of Toll-like receptor (TLR) 2 and TLR4 in the induction of immune responses in mice after immunization with these vaccines. Innate and adaptive immune responses were compared between wild type mice and mice deficient in TLR2, TLR4, or TRIF. TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. In contrast, immune responses were not lower in TLR2−/− mice but tended even to be higher after immunization. Together our data demonstrate that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required.

About the Authors:

Floris Fransen

Affiliations Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands, Department of Immunology and Infectious Diseases, Utrecht University, Utrecht, The Netherlands

Rachel M. Stenger

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Martien C. M. Poelen

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Harry H. van Dijken

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Betsy Kuipers

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Claire J. P. Boog

Affiliations Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands, Department of Immunology and Infectious Diseases, Utrecht University, Utrecht, The Netherlands

Jos P. M. van Putten

Affiliation: Department of Immunology and Infectious Diseases, Utrecht University, Utrecht, The Netherlands

Cécile A. C. M. van Els

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Peter van der Ley

* E-mail: [email protected]

Affiliation: Laboratory of Vaccine Research, Netherlands Vaccine Institute, Bilthoven, The Netherlands

Introduction

The innate immune system senses microbes through a number of receptors present on innate immune cells that can recognize a wide variety of microbial structures [1]. This group of receptors is often referred to as pattern recognition receptors (PRRs). There are several classes of PRRs, including Toll-like receptors (TLRs), C-type lectin like receptors, RIG-I like receptors, and Nod-like receptors. The TLR family is the best characterized class to date. In humans, 10 different TLRs have been described and each TLR recognizes distinct microbial structures [2]. For example, lipopolysaccharide (LPS), a major component of Gram-negative bacteria, activates TLR4, lipoproteins and several other structures activate TLR2, unmethylated CpG DNA of bacteria and certain viruses activate TLR9, and viral dsRNA is recognized by TLR3 [2]. Four adaptor proteins mediate TLR signalling: MyD88, TRIF, MAL, and TRAM [3]. All TLRs signal through MyD88, except TLR3, which signals solely through TRIF. Moreover, TLR4 is the only TLR which utilizes both MyD88 and TRIF [4]. Activation of these proteins eventually leads to induction of pro-inflammatory cytokines and type I interferon, respectively. Activation of TLR7/8 and TLR9 also leads to the induction of type I interferon, but in a MyD88-dependent manner [1].

The primary function of TLRs is to detect pathogens and activate innate immune cells to clear the infection immediately. However, TLRs also play an important role in the initiation of adaptive immune responses [5], [6]. Dendritic cells (DCs) are thought to play a central role in linking innate and adaptive immunity after TLR triggering, because of their superior capacity to stimulate T cells [7]. Which TLR is activated determines what types of cytokines and other factors are produced by the DCs, which in turn dictates whether the CD4⁺ T cells differentiate into Th1, Th2, Th17, or Treg [1],[8]. Because TLR ligands can both initiate and direct adaptive immunity, they have great potential as adjuvants. However, the claim that TLR activation always plays an important role in the induction of an adaptive immune response after vaccination has been challenged recently [9].

Many of the currently licensed vaccines are live attenuated strains or contain elements of killed microbes [5]. These vaccines likely contain structures that are recognized by TLRs and contribute to the immunogenicity. This has indeed been demonstrated for a number of vaccines [5], [10]–[13], but for the majority of vaccines this information is still lacking. It is important to identify the pathways induced by these successful vaccines for the rational design of new vaccines and/or adjuvants. Moreover, the human population is genetically very diverse and some individuals might have deficiencies in the pathways that are induced by the vaccine, which could explain why some individuals respond poorly after vaccination [14]. We decided to study the role of TLRs in the immunogenicity of two LPS-containing vaccines against the Gram-negative bacterial pathogens Neisseria meningitidis and Bordetella pertussis.

N. meningitidis is a leading cause of meningitis and sepsis worldwide [15]. The bacterium can be divided into several serogroups based on its capsule. For most serogroups (A, C, Y, and W-135) capsular polysaccharide vaccines are available, but not for serogroup B, because its capsular polysaccharide is not immunogenic. An attractive alternative for serogroup B is an outer membrane vesicle (OMV) vaccine [16]. OMV vaccines have been shown to be effective in controlling epidemics in Cuba, Norway, and New Zealand, where one particular clone of N. meningitidis serogroup B was causing high rates of meningococcal disease [16]. B. pertussis is the causative agent of whooping cough in humans. To prevent this disease, whole cell pertussis vaccines have been used for many decades in developed countries and are still used today in developing countries. However, due to adverse effects the whole cell vaccine has now been replaced in developed countries with a safer subunit vaccine consisting of a few B. pertussis antigens [17], [18]. The N. meningitidis OMV vaccine and whole cell pertussis vaccine both contain LPS and lipoproteins, which activate TLR4 and TLR2 respectively [19]–[23]. Ligands of these TLRs have been shown to have adjuvant activity in numerous studies in mice [11], [24]–[27].

Here we investigated the role of TLR2 and TLR4 in the induction of immune responses in mice after immunization with a N. meningitidis OMV vaccine and a whole cell pertussis vaccine. Innate cytokine induction, T cell responses, and antibody production were compared between wild type mice and mice deficient in either TLR2, TLR4, or TRIF. Surprisingly, TLR2−/− mice were not compromised in any of the responses after immunization. In contrast, TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. We conclude that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required.

Results

TRIF-deficient and TLR4-deficient mice have reduced innate cytokines levels after immunization

As a N. meningitidis OMV vaccine we chose OMVs derived from serosubtype P1.5-1,2-2. This serosubtype is one of the prevalent variants in The Netherlands. Moreover, serosubtype P1.5-1,2-2 is among the more immunogenic serosubtypes in humans and mice [28]–[30]. As a whole cell pertussis vaccine we used a mixture of the vaccine strains 134 and 509. In humans these vaccines are administered with alum as the adjuvant [16]–[18]. However, we did not include alum to study more specifically the intrinsic adjuvant activity of the vaccines.

The role of TLR2 and TRIF in vaccine-induced responses after immunization was examined by immunizing wild type C57BL/6 mice, TLR2−/− mice, and TRIF-deficient mice, which all have a C57BL/6 background [31], [32]. To investigate the role of TLR4, responses were compared between TLR4-deficient C3H/HeJ mice and wild type C3H/HeOuJ mice [33]. All mouse strains were immunized subcutaneously with either PBS, N. meningitidis OMVs, or whole cell pertussis vaccine. After two and four hours a blood sample was taken from all mice to analyze serum cytokine levels of IL-1β, IL-6, IL-10, IL-12p70, RANTES, and TNF-α. In wild-type mice N. meningitidis OMVs induced modest levels of IL-6 and RANTES measurable at 2 and 4 h compared to PBS (Fig. 1A and data not shown). OMV-induced IL-6 levels at 2 hours were significantly lower in TRIF-deficient and TLR4-deficient mice and tended to be higher in TLR2−/− mice. Levels of OMV-induced RANTES were not impaired in the deficient mouse strains. Whole cell pertussis vaccine induced measurable levels of IL-6, IL-12p70, RANTES, and IL-10 (Fig. 1B and data not shown). Again TRIF deficient mice and TLR4-deficient mice were clearly compromised in cytokine induction compared to wild type mice. TLR2−/− mice were not impaired in cytokine production, IL-6 even tended to be higher. These results suggest that early serum cytokine production in vivo after immunization with N. meningitidis OMVs or whole cell pertussis vaccine requires mainly LPS signalling and not TLR2 activation.

[Figure omitted. See PDF.]

Figure 1. Serum cytokine levels shortly after vaccination.

Two and four hours after immunization blood samples were taken from all mice and cytokine levels in the sera were analyzed with Luminex. Results for N. meningitidis OMVs are shown in panel A, results for whole cell pertussis vaccine are shown in panel B. There were 3 mice per group for the animals that received PBS and 6 mice per group for N. meningitidis OMVs and whole cell pertussis vaccine. The data are expressed as means, error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

https://doi.org/10.1371/journal.pone.0015692.g001

Comparison of antigen-specific antibody levels

The amount of antibodies was determined in all sera. It has been demonstrated previously that class switching to IgG2a/c and IgG2b depends on a Th1 response and class switching to IgG1 and IgE depends on a Th2 response [34]–[36]. Therefore, to analyze antibody production and the type of immune response that was induced, antigen-specific total IgG, antigen-specific IgG subclasses, and total IgE were determined in the serum. N. meningitidis OMVs induced a Th1-dependent antibody profile in wild type C57BL/6 mice, reflected by high levels of IgG2b and IgG2c, almost no IgG1, and no increase in total IgE compared to PBS injected mice (Fig. 2A). IgG3 levels were also very low. Strikingly, TLR2−/− mice immunized with N. meningitidis OMVs were not impaired at all in the antibody response, IgG1 and IgG3 levels even tended to be higher. On the other hand, N. meningitidis OMVs induced significant lower amounts of IgG in TRIF-deficient mice, which was mainly due to lower levels of IgG2b and IgG2c (Fig. 2A). In contrast, IgG1 and IgG3 levels to OMVs were significantly higher in TRIF-deficient mice compared to wild type mice. TLR4-deficiency did not significantly reduce antibody levels to OMVs, although for IgG, IgG2a, IgG2b, and IgG3 there was a tendency for reduced levels in TLR4-deficient mice (Fig. 2B).

[Figure omitted. See PDF.]

Figure 2. Antibody titers of mice after immunization with N. meningitidis P1.5-1,2-2 OMVs.

Mice were immunized with N. meningitidis P1.5-1,2-2 OMVs and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or N. meningitidis P1.5-1,2-2 OMVs was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Levels of serum bactericidal antibodies after immunization with N. meningitidis P1.5-1,2-2 OMVs are shown in panel C. Data are expressed as means of log₁₀ titers for 6 mice per group, except levels of serum bactericidal antibodies in C57BL/6 and TLR2−/− mice (n = 12). Error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.

https://doi.org/10.1371/journal.pone.0015692.g002

Bactericidal antibodies are currently considered to be the most important correlate of protection for meningococcal disease [37], [38]. Furthermore, IgG2a/c, IgG2b, and IgG3, but not IgG1, isotypes can activate complement [39]. We tested the bactericidal activity of the sera of all mice immunized with N. meningitidis OMVs. Consistent with the lower IgG2b and IgG2c levels, TRIF-deficient mice had significantly lower levels of bactericidal antibodies compared to wild type mice (Fig. 2C). Similar amounts of bactericidal antibodies were induced in wild-type and TLR2−/− mice. Finally, TLR4 deficient mice tended to have less bactericidal antibodies than wild-type mice (Fig. 2D). Together these results suggest that LPS signalling also contributes to the generation of bactericidal antibodies after immunization with N. meningitidis OMVs, whereas TLR2 activation is not required.

The differences between the different mouse strains in antibody levels were quite similar for the mice that received whole cell pertussis vaccine and the mice that received N. meningitidis OMVs. Again TLR2−/− mice did not show any defects in the induction of antibodies (Fig. 3A). Moreover, IgG3 and IgE levels tended to be higher in TLR2−/− mice compared to wild type mice. TRIF-deficient mice had significant lower amounts of IgG, which was mainly due to lower levels of isotypes IgG2b and IgG2c, as with N. meningitidis OMVs. However, in contrast to N. meningitidis OMVs, whole cell pertussis vaccine did not induce higher levels of IgG1 and IgG3 in TRIF-deficient mice. TLR4 deficient mice had lower amounts of all antibody isotypes compared to wild-type mice, except IgE (Fig. 4B). The differences were significant for IgG, IgG2a and IgG1. The whole cell pertussis vaccine also induced the production of total IgE, in contrast to N. meningitidis OMVs. In summary, our results demonstrate that LPS signalling contributes to the antibody response after immunization with N. meningitidis OMVs and whole cell pertussis vaccine. However, activation of TLR2 does not seem to contribute to the antibody response.

[Figure omitted. See PDF.]

Figure 3. Antibody titers of mice after immunization with whole cell pertussis vaccine.

Mice were immunized with whole cell pertussis vaccine (Bordetella pertussis) and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or whole cell pertussis vaccine was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Data are expressed as means of log₁₀ titers for 6 mice per group. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.

https://doi.org/10.1371/journal.pone.0015692.g003

[Figure omitted. See PDF.]

Figure 4. Proliferation of spleen cells after restimulation with antigen or peptide.

Spleens were taken from the mice after immunizations and spleen cells were incubated for 4 days with medium, antigen, or peptides, after which [³H]thymidine incorporation was determined. Spleen cells of C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice immunized with PBS (3 mice per group) or N. meningitidis P1.5-1,2-2 OMVs (6 mice per group) were restimulated with 1 µM of peptides 11–24/25 and 4–52/53 (A). Spleen cells of C3H/HeOuJ and C3H/HeJ mice immunized with PBS (3 mice per group) or N. meningitidis P1.5-1,2-2 OMVs (6 mice per group) were restimulated with peptide pool 6 (1 µM of each peptide, B). Spleen cells of C57BL/6, TLR2−/−, TRIF-deficient, C3H/HeOuJ, and C3H/HeJ mice immunized with PBS (3 mice per group) or whole cell pertussis vaccine (B. pertussis, 6 mice per group) were restimulated with 500 ng/ml of FHA antigen (C). Data are expressed as means of stimulation indices.

https://doi.org/10.1371/journal.pone.0015692.g004

Spleen cells of the different mouse strains proliferate equally after in vitro antigen restimulation

Also the spleen was taken from the mice immunized as described above. Spleen cells were restimulated with antigen or peptides for 4 days and proliferation of the cells was assessed by measuring [³H]thymidine incorporation. Spleen cells of mice with a C57BL/6 background and immunized with either N. meningitidis OMVs or PBS were restimulated with two PorA peptides, which were previously identified as two P1.5-1,2-2 PorA epitopes recognized by C57BL/6 mice (M. Poelen and C. van Els, unpublished data). Spleen cells from N. meningitidis OMV immunized mice clearly proliferated in response to the two peptides, suggesting that CD4⁺ T cells specific for those epitopes were generated after immunization (Fig. 4A). Interestingly, there was no difference between the three mouse strains in spleen cell proliferation. Which epitopes of P1.5-1,2-2 PorA were recognized by C3H/HeJ and C3H/HeOuJ mice was not known to us. Therefore, spleen cells of these mice immunized with either PBS or N. meningitidis OMVs were restimulated with different peptides pools spanning the entire P1.5-1,2-2 PorA protein sequence (data not shown). We found that one peptide pool induced the highest proliferation of spleen cells derived from N. meningitidis OMV immunized mice (Fig. 4B). There was also no difference in spleen cell proliferation between TLR4-deficient and wild-type mice. Together these results suggest that activation of TLR2 or TLR4 has no influence on CD4⁺ T cell proliferation after immunization with N. meningitidis OMVs.

To investigate CD4⁺ T cell proliferation after whole cell pertussis vaccine immunization, spleen cells of all the different mice immunized with whole cell pertussis vaccine or PBS were restimulated with a purified B. pertussis antigen, filamentous hemagglutinin (FHA, Fig. 4C). Clearly, only spleen cells of mice that received whole cell pertussis vaccine proliferated. Moreover, as with the PorA peptides, there were no significant differences between the mouse strains. Thus it seems that also for whole cell pertussis vaccine, TLR2 or TLR4 activation has little influence on CD4⁺ T cell proliferation.

LPS signalling and TLR2 activation influence quality of T cell responses

Naïve CD4⁺ T cells can differentiate into Th1, Th2, Th17, or Treg cells. Th1 cells produce IFN-γ, Th2 cells produce IL-4, IL-5, and IL-13, Th17 cell produce IL-17, and most Treg cell subsets produce IL-10 [40], [41]. These cytokines were measured in the supernatant of the spleen cells stimulated with antigen or peptides to identify the type of T cell responses that were induced. Spleen cells of mice with a C57BL/6 background immunized with either N. meningitidis OMVs or PBS were restimulated with two P1.5-1,2-2 PorA peptides. Cytokines were analyzed in the supernatant for both peptides. Cells from TLR2−/− mice immunized with N. meningitidis OMVs tended to produce more IL-5, IL-10, IL-13, and IFN-γ after stimulation with peptide 11–24/25 (Fig. 5A). The other peptide, 4–52/53, generally induced higher production of cytokines by spleen cells of all the mice immunized with N. meningitidis OMVs (Fig. 5B). Moreover, peptide 4–52/53 induced spleen cells of TRIF-deficient mice to produce significant higher levels of IL-4, IL-5, and IL-13 and tended to produce less IFN-γ compared to spleen cells of wild type C57BL/6 mice. Clearly, the response was skewed towards a Th2 response in the TRIF-deficient mice, consistent with what we noticed at the antibody level (Fig. 2A). Spleen cells of TLR2−/− mice also produced significant higher levels of IL-13 than wild type spleen cells (Fig. 5B). Furthermore, IL-5 and IL-17 tended to be higher. Spleen cells of TLR4-defcient and wild-type mice were restimulated with different peptide pools. Since peptide pool 6 induced the highest proliferation, cytokine production by cells stimulated with this pool was measured (Fig. 5C). Remarkably, spleen cells from TLR4-deficient mice immunized with N. meningitidis OMVs did not produce any of the cytokines analyzed, although the cells clearly proliferated as much as the cells from wild-type mice (Fig. 4B). In contrast, wild-type cells produced all cytokines, except IL-4. Overall these results show that after immunization of wild type mice with N. meningitidis OMVs, antigen-specific T cells mainly produce IFN-γ. This suggests that N. meningitidis OMVs mainly induce a Th1 response in wild type mice. We also demonstrate that some antigen-specific T cells produce IL-17. Furthermore, our results suggest that TLR4 activation is required for the generation of cytokine producing T cells and that TRIF signalling contributes to a Th1 response. Finally, TLR2 activation is not required for the generation of cytokine producing T cells, but in contrast seems somewhat inhibitory.

[Figure omitted. See PDF.]

Figure 5. Cytokines produced by spleen cells after restimulation with P1.5-1,2-2 PorA peptides.

Spleen cells from C57BL/6, TLR2−/−, or TRIF-deficient (TRIFdef) mice immunized with PBS or N. meningitidis P1.5-1,2-2 OMVs were incubated for 4 days with peptide 11–24/25 (A) or peptide 4–52/53 (B). Spleen cells from C3H/HeOuJ and C3H/HeJ mice immunized with PBS or N. meningitidis P1.5-1,2-2 OMVs were incubated for 4 days with peptide pool 6 (C). Cytokines IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (N. meningitidis OMVs), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.

https://doi.org/10.1371/journal.pone.0015692.g005

Spleen cells of the different mouse strains immunized with whole cell pertussis vaccine were restimulated with purified FHA antigen. The strong proliferation of cells of mice with a C57BL/6 background after antigen restimulation was associated with the presence of IL-2 and IL-17 (Fig. 6A). In addition, low amounts of IFN-γ and IL-5 were only produced by the spleen cells of TRIF-deficient mice immunized with whole cell pertussis vaccine. FHA stimulated spleen cells of wild-type C3H/HeOuJ mice immunized with whole cell pertussis vaccine produced all cytokines measured (Fig. 6B). Levels of IL-17 were especially high followed by IFN-γ. In contrast, reminiscent of the N. meningitidis OMVs model, FHA stimulated spleen cells of TLR4-deficient C3H/HeJ mice hardly produced any cytokines, even though they proliferated as much as wild-type C3H/HeOuJ spleen cells. Together, our results demonstrate that whole cell pertussis vaccine immunization induced a Th17/Th1 response, which was impaired in TLR4-deficient mice.

[Figure omitted. See PDF.]

Figure 6. Cytokines produced by spleen cells after restimulation with FHA.

Spleen cells from C57BL/6, TLR2−/−, TRIF-deficient (TRIFdef) mice (A), or C3H/HeOuJ and C3H/HeJ mice (B) immunized with PBS or whole cell pertussis vaccine were incubated for 4 days with 500 ng/ml FHA Cytokines IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (whole cell pertussis vaccine), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.

https://doi.org/10.1371/journal.pone.0015692.g006

Discussion

In the present study, we demonstrate that TLR4 signalling by LPS contributes to generation of adaptive immune responses after immunization with N. meningitidis OMVs or whole cell pertussis vaccine, while TLR2 activation was not required. Instead, TLR2−/− mice overall showed higher rather than lower responses compared to C57BL/6 mice after immunization with the N. meningitidis and B. pertussis vaccines. For example, IL-6 serum levels just after vaccination tended to be higher in the

TLR2−/− mice. At the antibody level, IgG1 and IgG3 tended to be higher in N. meningitidis OMVs immunized TLR2−/− mice, whereas IgG3 and IgE tended to be higher in TLR2−/− mice immunized with whole cell pertussis vaccine. Finally, spleen cells of TLR2−/− mice immunized with N. meningitidis OMVs restimulated with P1.5-1,2-2 PorA peptides produced higher levels of several cytokines, including significantly higher levels of IL-13. Together these results indicate that TLR2 activation inhibited immune responses, especially Th2 responses, after vaccination. Similarly, TLR2-deficient mice were shown to have greatly enhanced Th1 and Tc1 responses after vaccination with yellow fever vaccine YF-17D [13]. Moreover, it has been demonstrated recently that activation of TLR2 by the yeast cell wall derivative zymosan induces a Treg response [42], [43]. On the other hand, TLR2 ligands have been shown to have adjuvant activity in several studies [10], [11], [27], [44]. In addition to induction of a Treg response, TLR2 activation has also been shown to induce a Th1 reponse [45], or Th2 response [46]–[48]. How TLR2 activation can lead to all these different responses is currently not clear, but it might depend for example on which ligand is used, whether the ligand is recognized by TLR2/TLR1 or TLR2/TLR6, or which cell type is targeted. In any case, our results show that TLR2 activation is not required for induction of adaptive immunity when TLR4 is also activated. However, even TLR4-deficient mice still develop adaptive immune responses after vaccination. This residual response might be due to TLR2 activation. To address this issue, also mice deficient in both receptors should be tested. Moreover, the vaccines might contain ligands of other TLRs or PRRs, which also contribute to the immunogenicity.

TRIF-deficient mice clearly showed altered immune responses after immunization with N. meningitidis OMVs and whole cell pertussis vaccine. Antigen- specific IgG2b and IgG2c levels were significantly reduced for both vaccines, which resulted in less bactericidal antibodies in the case of N. meningitidis OMVs. In addition, spleen cells of TRIF-deficient mice immunized with N. meningitidis OMVs restimulated with PorA peptides produced significantly higher amounts of IL-4, IL-5, and IL-13, while IFN-γ production was reduced. Together these data show that activation of the TRIF pathway leads to a Th1 response after immunization with N. meningitidis OMVs or whole cell pertussis vaccine. Therefore, targeting of the TRIF pathway is important, because for both vaccines optimal responses after vaccination should include Th1-responses. Induction of the TRIF pathway eventually leads to the induction of type I interferon, which is not induced by the MyD88 pathway downstream of TLR2 or TLR4 [3], [32], [49]. We have shown previously that ligands of TLR3, TLR4, TLR7, and TLR9 enhance the immunogenicity of a N. meningitidis outer membrane vaccine by promoting Th1-dependent antibody production, whereas ligands of other TLRs did not show adjuvant activity [25]. Importantly, activation of TLR3, TLR4, TLR7, and TLR9 leads to the induction of type I interferon, but activation of the other TLRs does not [50]. Possibly, TLR-induced type I interferon drives Th1 development and contributes to the adjuvant effect of TLR ligands. Indeed, the adjuvant effect of poly IC by inducing CD4⁺ Th1 immunity and humoral immunity depends on type I interferon [51], [52]. Interestingly, the vaccine adjuvant Monophosphoryl lipid A, a low-toxicity LPS derivative, has been shown to preferentially activate the TRIF pathway [53].

We also show that TLR4-deficient mice have impaired immune responses after immunization with N. meningitidis OMVs and whole cell pertussis vaccine. After whole cell pertussis immunization of the TLR4-deficient C3H/HeJ mice, IL-6 and IL-12p70 were almost undetectable in the serum. Moreover, IgG, IgG1, and IgG2a levels were significantly lower in these mice. In contrast, others have found that antibody levels are not decreased in TLR4-deficient mice after immunization with whole cell pertussis vaccine [54], [55]. However, in these studies alum was added to the vaccine as an adjuvant, which mainly enhances humoral immunity. Presumably, the adjuvant effect of LPS is more redundant in the presence of another potent adjuvant like alum.

Surprisingly, TLR4-deficient C3H/HeJ mice did not have significantly decreased OMV-directed antibody titers compared to wild-type C3H/HeOuJ mice. By contrast, we previously found that LPS-deficient N. meningitidis outer membrane complexes (OMCs) induced more than 1000-fold lower antibody titers in TLR4-proficient mice than wild type OMCs [25]. This suggests that LPS improves the immune response not only by activating TLR4, but also by other mechanisms. Possibly, LPS could target the vaccine to antigen presenting cells by binding to other receptors, for example CD14 or C-type lectins.

In contrast to antibody levels, cytokine production after restimulation with PorA peptides was severely impaired in spleen cells from TLR4-deficient C3H/HeJ mice. Similarly, it has been demonstrated that whole cell pertussis vaccine immunization led to comparable antibody titers in TLR4-deficient and wild type mice, but IFN-γ and IL-17 production of T cells after antigen restimulation was much lower for TLR4-deficient mice [55]. These results suggest that TLR4 activation is more important for the induction of effector T cells than for antibody production. Remarkably, the TLR4-deficient spleen cells restimulated with PorA peptides or FHA proliferated equally well as spleen cells from the wild-type mice. This suggests that in TLR4-deficient mice, antigen-specific CD4⁺ T cells were generated without effector function. Interestingly, it has been shown that DCs that had a mature phenotype but did not produce IL-12 and other pro-inflammatory cytokines, promoted expansion of CD4+ T cell populations lacking effector function [56]. Possibly, this type of DCs was induced in the TLR4-deficient mice. After whole cell pertussis vaccine immunization, serum levels of IL-6 and IL-12p70 were very low in C3H/HeJ mice. In addition, it has been demonstrated that serum IL-12p70 production in vivo after administration of a TLR9 ligand depended entirely on DCs [57]. Unfortunately, in our experiments N. meningitidis OMVs were not potent enough to enhance serum IL-12p70 levels even in wild-type animals, but in a recent report where a 10-fold higher dose of a comparable preparation of native N. meningitidis OMVs was used, IL-12p70 was elevated in serum of wild-type mice but not in TLR4-deficient mice. Serum levels of MCP-1, TNF-α, IL-6, and IL-10 were also much lower in the TLR4-deficient mice [58].

In conclusion, our results show that two successful LPS-containing vaccines against the Gram-negative bacterial pathogens N. meningitidis and B. pertussis require TLR4 signaling for optimal immunogenicity and induction of Th1 responses in mice. On the other hand, TLR2 activation was not required. These findings confirm that TLR4 and the TRIF pathway are attractive targets for adjuvants in vaccines.

Materials and Methods

Ethics statement

Animal experiments were approved by the Netherlands Vaccine Institute's Committee on Animal Experiments (DEC), project review nrs. 200800223 and 200800224.

Animals

Specific-pathogen-free C57BL/6J, C3H/HeOuJ (TLR4-proficient) and C3H/HeJ (TLR4-deficient) mice were purchased from Charles River Laboratories. TLR2−/− mice [31] and TRIF-deficient mice [32], both from a C57BL/6 background, were kindly provided respectively by Dr. Shizuo Akira (Osaka University, Japan) and Dr. Bruce Beutler (Scripps Research Institute, La Jolla, CA) via Dr. Tom van der Poll (Academic Medical Center, Amsterdam, the Netherlands). For experiments, female mice were used, aged 10–14 weeks.

Vaccine preparation

The OMV vaccine was obtained from the PorB/RmpM-negative N. meningitidis strain TR52 [P1.5-1,2-2] [59] by extraction of bacteria with 0.5% deoxycholate in 0.1 M Tris-HCl-10 mM EDTA (pH 8.6) and were purified by differential centrifugation [60]. OMVs were stored at 4°C and diluted prior to immunization to 20 µg/ml in PBS. For preparation of the whole cell pertussis vaccine, B. pertussis strains 134 and 509 were grown in defined synthetic medium [61]. After 18–22 hours of culture, bacteria were heat inactivated for 10 min at 56°C in the presence of 16 mM formaldehyde. Next cells were centrifuged for 10 min at 16,100×g and resuspended in PBS to 200 international opacity units (IOU)/ml. The suspensions were stored at 4°C. Prior to immunization, both strains were mixed 1∶1 and diluted to a final concentration of 16 IOU/ml in PBS.

Immunizations

Mice were immunized subcutaneously on days 0 and 21 with 250 µl PBS or 5 µg of N. meningitidis P1.5-1,2-2 OMVs in 250 µl PBS. Whole cell pertussis vaccine (4 IOU in 250 µl PBS) was administered subcutaneously on days 0 and 28. Groups that received PBS contained 3 mice per group and groups that received OMVs or whole cell pertussis vaccine contained 6 animals per group. A blood sample of all mice was taken 2 and 4 hours after immunization. From the mice that received PBS or N. meningitidis P1.5-1,2-2 OMVs blood and spleen were taken on day 28. From the mice that received whole cell pertussis vaccine blood and spleen were taken on day 42. Sera were collected and stored at −20°C. Single-cell suspensions of spleen cells were produced by mechanical dissociation of organs through 70-µm-pore-size nylon filters. Spleen cells were used either freshly or after freezing and storage at −135°C.

Antigen-specific antibodies

Antigen-specific IgG, IgG1, IgG2a/c, IgG2b, and IgG3 antibodies were determined by enzyme-linked immunosorbent assay (ELISA) as described previously [25] with slight modifications. Briefly, flat-bottom 96-well microtiter plates (Immulon 2; Nunc) were coated overnight at room temperature with 100 µl/well 5 µg/ml N. meningitidis P1.5-1,2-2 OMVs (4 µg/ml PorA) in PBS, or 2 IOU/ml of B. pertussis strain 509 (inactivated as described above) in PBS. Plates were incubated with serial dilutions of the sera of the immunized mice (100 µl/well) at 37°C for 1 h. Antibody isotypes were detected with anti-mouse antibodies conjugated to horseradish peroxidase (Southern Biotechnology Associates, Inc.). Next, peroxidase substrate was added to the wells and the reaction was stopped by the addition of 2 M H₂SO₄. A four-parameter curve fit was made for optical density values of the serial dilutions, and the antibody titer was calculated in reciprocal dilutions that gave 50% of the maximum absorbance. The results are expressed as log₁₀ titers.

Total IgE

Total IgE levels in the sera were measured by ELISA as described previously [25]. Briefly, flat-bottom 96-well microtiter plates were coated with rat anti-mouse IgE monoclonal antibody (BD Biosciences) and incubated overnight at 4°C. Next, the plates were incubated for 1 h at room temperature with the sera of the immunized mice and purified mouse IgE clone C48-2 (BD Biosciences) as a standard. For detection of IgE, biotinylated anti-mouse IgE (BD Biosciences) was used followed by streptavidin-HRP (Sanquin). Peroxidase substrate was used as a substrate, and the reaction was stopped with 2 M H₂SO₄. The absorbance was determined at 450 nm.

Serum bactericidal assay

The sera of immunized mice were diluted 1∶5 in Gey's balanced salt solution plus 0.5% bovine serum albumin and then heat inactivated for 30 min at 56°C. Next, twofold serial dilutions of the sera, together with N. meningitidis P1.5-1,2-2 (10⁴ CFU/ml), were incubated in 96-well plates at room temperature for 15 min. After the addition of baby rabbit complement (20% of total volume) plates were incubated at 37°C for 1 h. Bacteria were plated on GC medium base (Difco Laboratories) supplemented with IsoVitaleX (Becton Dickinson) and grown overnight at 37°C in 5% CO₂ in a humid atmosphere. The serum bactericidal titer was determined as the reciprocal serum dilution that gave more than 90% killing of the number of bacteria used. The results are expressed as log₁₀ titers.

Peptide synthesis

Overlapping 18-mer peptides covering the entire B. pertussis P.69 Prn protein or N. meningitidis P1.5-1,2-2 PorA protein were prepared by solid-phase synthesis using N^α-(9-fluorenyl)methoxycarbonyl (FMOC)-protected amino acids and a Syro II simultaneous multiple-peptide synthesizer (MultiSyntech GmbH, Witten, Germany). The purity and identity of the synthesized peptides were assessed by reverse-phase high-performance liquid chromatography and mass spectometry.

Spleen cell proliferation

Spleen cells from immunized mice were cultured at 1.5×10⁵ in 150 µl IMDM (Gibco BRL) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 300 µg/ml L-glutamine (Gibco BRL), 10% heat-inactivated fetal calf serum (FCS) (Gibco BRL), and 50 µM beta-mercaptoethanol in 96-well round-bottom plates (Greiner). Spleen cells were co-cultured with either medium, 1 µM of P1.5-1,2-2 PorA peptides, or 500 ng/ml purified FHA (Kaketsuken). On day 4, 100 µl of supernatant was removed and stored at −20°C. Next, 0.5 µCi (18.5 kBq) [³H]thymidine (Amersham) was added to the wells, and cells were cultured for another 18 h. Cells were harvested, and [³H]thymidine incorporation was determined as counts per minute using a Wallac 1205 Betaplate liquid scintillation counter. Results are expressed as stimulation indices from triplicate wells, calculated as (counts per minute of cultures in the presence of antigen)/(counts per minute of cultures in the presence of medium only).

Luminex

A 6-plex Bio-Plex assay (Bio-Rad) containing beads for mouse IL-1β, IL-6, IL-10, IL-12p70, RANTES, and TNF-α was used to measure levels of these cytokines in the sera of the mice taken 2 and 4 hours after immunization. A 7-plex Bio-Plex assay (Bio-Rad) containing beads for mouse IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ was used to determine amounts of these cytokines in the supernatants of spleen cells restimulated with antigen or peptide. Cytokine concentrations were determined with a Bio-Plex system (Bio-Rad).

Statistics

One-way analysis of variance (ANOVA) was performed, followed by the post-hoc Dunnett t test, to analyze differences in means between the experimental groups of C57BL/6 mice, TLR2−/−, and TRIF-deficient mice (GraphPad Prism 4). The unpaired two-tailed Students' t test was used to analyze differences in means between the experimental groups of C3H/HeOuJ and C3H/HeJ mice. Variances were compared with the F-test. In case of significantly different variances (p<0.05), the Welch's correction was included (GraphPad Prism 4). In case the SD of one of the means was 0, the One-sample T test was used instead (SPSS Statistics 17.0). P values of <0.05 were considered significant.

Acknowledgments

We thank Shizuo Akira, Bruce Beutler and Tom van der Poll for generously providing mouse strains, Peter Hoogerhout, Humphrey Brugghe and Hans Timmermans for peptide synthesis, and Arno van der Ark for providing the whole cell pertussis vaccine.

Author Contributions

Conceived and designed the experiments: FF CB JvP CvE PvdL. Performed the experiments: FF RS MP HvD BK. Analyzed the data: FF MP CvE PvdL. Contributed reagents/materials/analysis tools: CvE PvdL. Wrote the paper: FF CB JvP CvE PvdL.

Citation: Fransen F, Stenger RM, Poelen MCM, van Dijken HH, Kuipers B, Boog CJP, et al. (2010) Differential Effect of TLR2 and TLR4 on the Immune Response after Immunization with a Vaccine against Neisseria meningitidis or Bordetella pertussis. PLoS ONE 5(12): e15692. https://doi.org/10.1371/journal.pone.0015692

References

1. Manicassamy S, Pulendran B (2009) Modulation of adaptive immunity with Toll-like receptors. Semin Immunol 21: 185–193.S. ManicassamyB. Pulendran2009Modulation of adaptive immunity with Toll-like receptors.Semin Immunol21185193

2. Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124: 783–801.S. AkiraS. UematsuO. Takeuchi2006Pathogen recognition and innate immunity.Cell124783801

3. O'Neill LA, Bowie AG (2007) The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7: 353–364.LA O'NeillAG Bowie2007The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling.Nat Rev Immunol7353364

4. Palsson-McDermott EM, O'Neill LA (2004) Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 113: 153–162.EM Palsson-McDermottLA O'Neill2004Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4.Immunology113153162

5. van Duin D, Medzhitov R, Shaw AC (2006) Triggering TLR signaling in vaccination. Trends Immunol 27: 49–55.D. van DuinR. MedzhitovAC Shaw2006Triggering TLR signaling in vaccination.Trends Immunol274955

6. Iwasaki A, Medzhitov R (2004) Toll-like receptor control of the adaptive immune responses. Nat Immunol 5: 987–995.A. IwasakiR. Medzhitov2004Toll-like receptor control of the adaptive immune responses.Nat Immunol5987995

7. Sousa Reise (2006) Dendritic cells in a mature age. Nat Rev Immunol 6: 476–483.Reise Sousa2006Dendritic cells in a mature age.Nat Rev Immunol6476483

8. Pulendran B (2004) Modulating vaccine responses with dendritic cells and Toll-like receptors. Immunol Rev 199: 227–250.B. Pulendran2004Modulating vaccine responses with dendritic cells and Toll-like receptors.Immunol Rev199227250

9. Gavin AL, Hoebe K, Duong B, Ota T, Martin C, et al. (2006) Adjuvant-enhanced antibody responses in the absence of Toll-like receptor signaling. Science 314: 1936–1938.AL GavinK. HoebeB. DuongT. OtaC. Martin2006Adjuvant-enhanced antibody responses in the absence of Toll-like receptor signaling.Science31419361938

10. Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J, et al. (2002) Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 8: 878–884.L. AlexopoulouV. ThomasM. SchnareY. LobetJ. Anguita2002Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice.Nat Med8878884

11. Latz E, Franko J, Golenbock DT, Schreiber JR (2004) Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity. J Immunol 172: 2431–2438.E. LatzJ. FrankoDT GolenbockJR Schreiber2004Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity.J Immunol17224312438

12. Geeraedts F, Goutagny N, Hornung V, Severa M, de Haan A, et al. (2008) Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling. PLoS Pathog 4: e1000138.F. GeeraedtsN. GoutagnyV. HornungM. SeveraA. de Haan2008Superior immunogenicity of inactivated whole virus H5N1 influenza vaccine is primarily controlled by Toll-like receptor signalling.PLoS Pathog4e1000138

13. Querec T, Bennouna S, Alkan S, Laouar Y, Gorden K, et al. (2006) Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity. J Exp Med 203: 413–424.T. QuerecS. BennounaS. AlkanY. LaouarK. Gorden2006Yellow fever vaccine YF-17D activates multiple dendritic cell subsets via TLR2, 7, 8, and 9 to stimulate polyvalent immunity.J Exp Med203413424

14. Kimman TG, Vandebriel RJ, Hoebee B (2007) Genetic variation in the response to vaccination. Community Genet 10: 201–217.TG KimmanRJ VandebrielB. Hoebee2007Genetic variation in the response to vaccination.Community Genet10201217

15. Stephens DS, Greenwood B, Brandtzaeg P (2007) Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 369: 2196–2210.DS StephensB. GreenwoodP. Brandtzaeg2007Epidemic meningitis, meningococcaemia, and Neisseria meningitidis.Lancet36921962210

16. Holst J, Martin D, Arnold R, Huergo CC, Oster P, et al. (2009) Properties and clinical performance of vaccines containing outer membrane vesicles from Neisseria meningitidis. Vaccine 27: Suppl 2B3–12.J. HolstD. MartinR. ArnoldCC HuergoP. Oster2009Properties and clinical performance of vaccines containing outer membrane vesicles from Neisseria meningitidis.Vaccine27Suppl 2B312

17. Greco D, Salmaso S, Mastrantonio P, Giuliano M, Tozzi AE, et al. (1996) A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. Progetto Pertosse Working Group. N Engl J Med 334: 341–348.D. GrecoS. SalmasoP. MastrantonioM. GiulianoAE Tozzi1996A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. Progetto Pertosse Working Group.N Engl J Med334341348

18. Gustafsson L, Hallander HO, Olin P, Reizenstein E, Storsaeter J (1996) A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine. N Engl J Med 334: 349–355.L. GustafssonHO HallanderP. OlinE. ReizensteinJ. Storsaeter1996A controlled trial of a two-component acellular, a five-component acellular, and a whole-cell pertussis vaccine.N Engl J Med334349355

19. Mann PB, Wolfe D, Latz E, Golenbock D, Preston A, et al. (2005) Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection. Infect Immun 73: 8144–8152.PB MannD. WolfeE. LatzD. GolenbockA. Preston2005Comparative toll-like receptor 4-mediated innate host defense to Bordetella infection.Infect Immun7381448152

20. Zughaier SM, Tzeng YL, Zimmer SM, Datta A, Carlson RW, et al. (2004) Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway. Infect Immun 72: 371–380.SM ZughaierYL TzengSM ZimmerA. DattaRW Carlson2004Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway.Infect Immun72371380

21. Ingalls RR, Lien E, Golenbock DT (2000) Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis. J Endotoxin Res 6: 411–415.RR IngallsE. LienDT Golenbock2000Differential roles of TLR2 and TLR4 in the host response to Gram-negative bacteria: lessons from a lipopolysaccharide-deficient mutant of Neisseria meningitidis.J Endotoxin Res6411415

22. Ingalls RR, Lien E, Golenbock DT (2001) Membrane-associated proteins of a lipopolysaccharide-deficient mutant of Neisseria meningitidis activate the inflammatory response through toll-like receptor 2. Infect Immun 69: 2230–2236.RR IngallsE. LienDT Golenbock2001Membrane-associated proteins of a lipopolysaccharide-deficient mutant of Neisseria meningitidis activate the inflammatory response through toll-like receptor 2.Infect Immun6922302236

23. Pridmore AC, Wyllie DH, Abdillahi F, Steeghs L, van der Ley P, et al. (2001) A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2. J Infect Dis 183: 89–96.AC PridmoreDH WyllieF. AbdillahiL. SteeghsP. van der Ley2001A lipopolysaccharide-deficient mutant of Neisseria meningitidis elicits attenuated cytokine release by human macrophages and signals via toll-like receptor (TLR) 2 but not via TLR4/MD2.J Infect Dis1838996

24. Steeghs L, Kuipers B, Hamstra HJ, Kersten G, van Alphen L, et al. (1999) Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response. Infect Immun 67: 4988–4993.L. SteeghsB. KuipersHJ HamstraG. KerstenL. van Alphen1999Immunogenicity of outer membrane proteins in a lipopolysaccharide-deficient mutant of Neisseria meningitidis: influence of adjuvants on the immune response.Infect Immun6749884993

25. Fransen F, Boog CJ, van Putten JP, van der Ley P (2007) Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup B. Infect Immun 75: 5939–5946.F. FransenCJ BoogJP van PuttenP. van der Ley2007Agonists of Toll-like receptors 3, 4, 7, and 9 are candidates for use as adjuvants in an outer membrane vaccine against Neisseria meningitidis serogroup B.Infect Immun7559395946

26. Zughaier S, Steeghs L, van der Ley P, Stephens DS (2007) TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A. Vaccine 25: 4401–4409.S. ZughaierL. SteeghsP. van der LeyDS Stephens2007TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A.Vaccine2544014409

27. Jackson DC, Lau YF, Le T, Suhrbier A, Deliyannis G, et al. (2004) A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Proc Natl Acad Sci U S A 101: 15440–15445.DC JacksonYF LauT. LeA. SuhrbierG. Deliyannis2004A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses.Proc Natl Acad Sci U S A1011544015445

28. Cartwright K, Morris R, Rumke H, Fox A, Borrow R, et al. (1999) Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins. Vaccine 17: 2612–2619.K. CartwrightR. MorrisH. RumkeA. FoxR. Borrow1999Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins.Vaccine1726122619

29. de Kleijn ED, de Groot R, Labadie J, Lafeber AB, van den Dobbelsteen G, et al. (2000) Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7–8 years of age. Vaccine 18: 1456–1466.ED de KleijnR. de GrootJ. LabadieAB LafeberG. van den Dobbelsteen2000Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7–8 years of age.Vaccine1814561466

30. Luijkx TA, van Dijken H, Hamstra HJ, Kuipers B, van der Ley P, et al. (2003) Relative immunogenicity of PorA subtypes in a multivalent Neisseria meningitidis vaccine is not dependent on presentation form. Infect Immun 71: 6367–6371.TA LuijkxH. van DijkenHJ HamstraB. KuipersP. van der Ley2003Relative immunogenicity of PorA subtypes in a multivalent Neisseria meningitidis vaccine is not dependent on presentation form.Infect Immun7163676371

31. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, et al. (1999) Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11: 443–451.O. TakeuchiK. HoshinoT. KawaiH. SanjoH. Takada1999Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components.Immunity11443451

32. Hoebe K, Du X, Georgel P, Janssen E, Tabeta K, et al. (2003) Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424: 743–748.K. HoebeX. DuP. GeorgelE. JanssenK. Tabeta2003Identification of Lps2 as a key transducer of MyD88-independent TIR signalling.Nature424743748

33. Poltorak A, He X, Smirnova I, Liu MY, Van HC, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085–2088.A. PoltorakX. HeI. SmirnovaMY LiuHC Van1998Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.Science28220852088

34. Finkelman FD, Holmes J, Katona IM, Urban JF Jr, Beckmann MP, et al. (1990) Lymphokine control of in vivo immunoglobulin isotype selection. Annu Rev Immunol 8: 303–333.FD FinkelmanJ. HolmesIM KatonaJF Urban JrMP Beckmann1990Lymphokine control of in vivo immunoglobulin isotype selection.Annu Rev Immunol8303333

35. Coffman RL, Lebman DA, Rothman P (1993) Mechanism and regulation of immunoglobulin isotype switching. Adv Immunol 54: 229–270.RL CoffmanDA LebmanP. Rothman1993Mechanism and regulation of immunoglobulin isotype switching.Adv Immunol54229270

36. Holdsworth SR, Kitching AR, Tipping PG (1999) Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis. Kidney Int 55: 1198–1216.SR HoldsworthAR KitchingPG Tipping1999Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis.Kidney Int5511981216

37. Nicholson A, Lepow IH (1979) Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity. Science 205: 298–299.A. NicholsonIH Lepow1979Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity.Science205298299

38. Goldschneider I, Gotschlich EC, Artenstein MS (1969) Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med 129: 1307–1326.I. GoldschneiderEC GotschlichMS Artenstein1969Human immunity to the meningococcus. I. The role of humoral antibodies.J Exp Med12913071326

39. Neuberger MS, Rajewsky K (1981) Activation of mouse complement by monoclonal mouse antibodies. Eur J Immunol 11: 1012–1016.MS NeubergerK. Rajewsky1981Activation of mouse complement by monoclonal mouse antibodies.Eur J Immunol1110121016

40. Zhou L, Chong MM, Littman DR (2009) Plasticity of CD4+ T cell lineage differentiation. Immunity 30: 646–655.L. ZhouMM ChongDR Littman2009Plasticity of CD4+ T cell lineage differentiation.Immunity30646655

41. Maynard CL, Weaver CT (2008) Diversity in the contribution of interleukin-10 to T-cell-mediated immune regulation. Immunol Rev 226: 219–233.CL MaynardCT Weaver2008Diversity in the contribution of interleukin-10 to T-cell-mediated immune regulation.Immunol Rev226219233

42. Dillon S, Agrawal S, Banerjee K, Letterio J, Denning TL, et al. (2006) Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest 116: 916–928.S. DillonS. AgrawalK. BanerjeeJ. LetterioTL Denning2006Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance.J Clin Invest116916928

43. Manicassamy S, Ravindran R, Deng J, Oluoch H, Denning TL, et al. (2009) Toll-like receptor 2-dependent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity. Nat Med 15: 401–409.S. ManicassamyR. RavindranJ. DengH. OluochTL Denning2009Toll-like receptor 2-dependent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity.Nat Med15401409

44. Cervantes-Barragan L, Gil-Cruz C, Pastelin-Palacios R, Lang KS, Isibasi A, et al. (2009) TLR2 and TLR4 signaling shapes specific antibody responses to Salmonella typhi antigens. Eur J Immunol 39: 126–135.L. Cervantes-BarraganC. Gil-CruzR. Pastelin-PalaciosKS LangA. Isibasi2009TLR2 and TLR4 signaling shapes specific antibody responses to Salmonella typhi antigens.Eur J Immunol39126135

45. Sieling PA, Chung W, Duong BT, Godowski PJ, Modlin RL (2003) Toll-like receptor 2 ligands as adjuvants for human Th1 responses. J Immunol 170: 194–200.PA SielingW. ChungBT DuongPJ GodowskiRL Modlin2003Toll-like receptor 2 ligands as adjuvants for human Th1 responses.J Immunol170194200

46. Agrawal S, Agrawal A, Doughty B, Gerwitz A, Blenis J, et al. (2003) Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J Immunol 171: 4984–4989.S. AgrawalA. AgrawalB. DoughtyA. GerwitzJ. Blenis2003Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos.J Immunol17149844989

47. Dillon S, Agrawal A, Van Dyke T, Landreth G, McCauley L, et al. (2004) A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells. J Immunol 172: 4733–4743.S. DillonA. AgrawalT. Van DykeG. LandrethL. McCauley2004A Toll-like receptor 2 ligand stimulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mitogen-activated protein kinase and c-Fos in dendritic cells.J Immunol17247334743

48. Redecke V, Hacker H, Datta SK, Fermin A, Pitha PM, et al. (2004) Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J Immunol 172: 2739–2743.V. RedeckeH. HackerSK DattaA. FerminPM Pitha2004Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma.J Immunol17227392743

49. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, et al. (2003) Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301: 640–643.M. YamamotoS. SatoH. HemmiK. HoshinoT. Kaisho2003Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science301640643

50. Smith PL, Lombardi G, Foster GR (2005) Type I interferons and the innate immune response—more than just antiviral cytokines. Mol Immunol 42: 869–877.PL SmithG. LombardiGR Foster2005Type I interferons and the innate immune response—more than just antiviral cytokines.Mol Immunol42869877

51. Longhi MP, Trumpfheller C, Idoyaga J, Caskey M, Matos I, et al. (2009) Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant. J Exp Med 206: 1589–1602.MP LonghiC. TrumpfhellerJ. IdoyagaM. CaskeyI. Matos2009Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant.J Exp Med20615891602

52. Le Bon A, Schiavoni G, D'Agostino G, Gresser I, Belardelli F, et al. (2001) Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14: 461–470.A. Le BonG. SchiavoniG. D'AgostinoI. GresserF. Belardelli2001Type I interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo.Immunity14461470

53. Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, et al. (2007) The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science 316: 1628–1632.V. Mata-HaroC. CekicM. MartinPM ChiltonCR Casella2007The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4.Science31616281632

54. Banus S, Stenger RM, Gremmer ER, Dormans JA, Mooi FR, et al. (2008) The role of Toll-like receptor-4 in pertussis vaccine-induced immunity. BMC Immunol 9: 21.S. BanusRM StengerER GremmerJA DormansFR Mooi2008The role of Toll-like receptor-4 in pertussis vaccine-induced immunity.BMC Immunol921

55. Higgins SC, Jarnicki AG, Lavelle EC, Mills KH (2006) TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J Immunol 177: 7980–7989.SC HigginsAG JarnickiEC LavelleKH Mills2006TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells.J Immunol17779807989

56. Sporri R, Reis e Sousa C (2005) Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function. Nat Immunol 6: 163–170.R. SporriC. Reis e Sousa2005Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function.Nat Immunol6163170

57. Hou B, Reizis B, DeFranco AL (2008) Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms. Immunity 29: 272–282.B. HouB. ReizisAL DeFranco2008Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms.Immunity29272282

58. Durand V, Mackenzie J, de Leon J, Mesa C, Quesniaux V, et al. (2009) Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles. Vaccine 27: 1912–1922.V. DurandJ. MackenzieJ. de LeonC. MesaV. Quesniaux2009Role of lipopolysaccharide in the induction of type I interferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles.Vaccine2719121922

59. Peeters CC, Rumke HC, Sundermann LC, Rouppe van der Voort EM, Meulenbelt J, et al. (1996) Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 14: 1009–1015.CC PeetersHC RumkeLC SundermannEM Rouppe van der VoortJ. Meulenbelt1996Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine.Vaccine1410091015

60. Fredriksen JH, Rosenqvist E, Wedege E, Bryn K, Bjune G, et al. (1991) Production, characterization and control of MenB-vaccine “Folkehelsa”: an outer membrane vesicle vaccine against group B meningococcal disease. NIPH Ann 14: 67–79.JH FredriksenE. RosenqvistE. WedegeK. BrynG. Bjune1991Production, characterization and control of MenB-vaccine “Folkehelsa”: an outer membrane vesicle vaccine against group B meningococcal disease.NIPH Ann146779

61. Thalen M, van den IJsel J, Jiskoot W, Zomer B, Roholl P, et al. (1999) Rational medium design for Bordetella pertussis: basic metabolism. J Biotechnol 75: 147–159.M. ThalenJ. van den IJselW. JiskootB. ZomerP. Roholl1999Rational medium design for Bordetella pertussis: basic metabolism.J Biotechnol75147159