Vaccine 32 (2014) 6240–6250

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HBHA vaccination may require both Th1 and Th17 immune responses to protect mice against tuberculosis Claudie Verwaerde a,b,c,d,∗ , Anne-Sophie Debrie a,b,c,d , Christophe Dombu d , Damien Legrand c , Dominique Raze a,b,c,d , Sophie Lecher a,b,c,d , Didier Betbeder d , Camille Locht a,b,c,d a

Inserm U1019, Lille, France CNRS UMR8204, Lille, France c Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France d Univ Lille Nord de France, Lille, France b

a r t i c l e

i n f o

Article history: Received 14 November 2013 Received in revised form 12 August 2014 Accepted 8 September 2014 Available online 22 September 2014 Keywords: HBHA Tuberculosis Th17 response Nanoparticle CpG

a b s t r a c t Almost one century after the discovery of the BCG vaccine, tuberculosis remains a major cause of global mortality and morbidity, emphasizing the urgent need to design more efficient vaccines. The heparinbinding haemagglutinin (HBHA) appears to be a promising vaccine candidate, as it was shown to afford protection to mice against a challenge infection with Mycobacterium tuberculosis when combined with the strong adjuvant DDA/MPL (dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A), a TLR4 ligand. In this study, we investigated the immunological response and protection of mice immunized with HBHA formulated in lipid-containing nanoparticles and adjuvanted with CpG, a TLR9 ligand. Subcutaneous immunization with this HBHA formulation led to a marked Th1 response, characterized by high IFN-␥ levels, but no significant IL-17 production, both in spleen and lung, in contrast to DDA/MPL MPL-formulated HBHA, which induced both IFN-␥ and IL-17. This cytokine profile was also observed in BCG-primed mice and persisted after M. tuberculosis infection. No significant protection was obtained against challenge infection after vaccination with the nanoparticle-CpG formulation, and this was associated with a failure to mount a memory immune response. These results suggest the importance of both Th1 and Th17 immune responses for vaccine-induced immunity. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Tuberculosis (TB) remains a major cause of global mortality and morbidity despite large vaccination coverage [1] with the Bacille Calmette-Guérin (BCG), the only vaccine available against this disease. Although BCG protects reasonably well infants against severe and deadly forms of TB [2], it provides insufficient protection against the pulmonary form of the disease in adults [3]. In the two last decades, many efforts have been devoted to the development of new vaccines against TB [4–6]. Some vaccine candidates aim at replacing BCG, such as recombinant forms of BCG or attenuated forms of Mycobacterium tuberculosis. Others are being designed to strengthen BCG-induced immunity. Only a handful of

∗ Corresponding author at: Center for Infection and Immunity of Lille, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019 Lille Cedex, France. Tel.: +33 3 20 87 11 54; fax: +33 3 20 87 11 58. E-mail address: [email protected] (C. Verwaerde). http://dx.doi.org/10.1016/j.vaccine.2014.09.024 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

antigens have been tested so far as potential booster vaccines; one of them is HBHA, a protein antigen involved in extrapulmonary dissemination of M. tuberculosis [7–9]. It is strongly recognized by T cells from latently infected individuals in contrast to patients with active TB, and a decline in HBHA-specific T cell responses in latently infected subjects was shown to be associated with risk of progression to TB disease [9–11]. In mouse models, HBHA has been shown to provide protection against challenge with M. tuberculosis [12,13]. However, the protective effect of HBHA in mice strongly depends on the use of the Th1 polarizing adjuvant dimethyldioctadecyl-ammonium bromide/monophosphoryl lipid A (DDA/MPL). As this adjuvant is not suitable for human use, we explored alternative ways to simulate HBHA-mediated protective immune responses. As an attractive antigen delivery system, we tested here phospholipid-rich nanoparticles to vectorize HBHA. The use of nanoparticles has a number of advantages, including the protection of the antigen against degradation, potential cell targeting and cellular antigen delivery, and the possibility to combine

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Fig. 1. Epithelial cell delivery of HBHA using nanoparticles. Human bronchial epithelial cells were incubated for 30 min at 37 ◦ C with 10 ␮g FITC-labelled HBHA either alone (a) or formulated with 30 ␮g of PGLA particle (b), NP particle (c) or NPL particle (d). HBHA delivery into cells was observed using a fluorescence microscope after washing and fixation (×63).

several antigens and immunostimulartory molecules. Furthermore, phospholipid-rich nanoparticles have already undergone clinical investigations in humans [14]. We found that HBHA formulated in phospholipid-rich nanoparticles induced humoral and cellular anti-HBHA responses in mice, which could be strongly enhanced by the addition of CpG ODN. Nevertheless, this formulation provided no protection against challenge with M. tuberculosis, in contrast to HBHA formulated with DDA/MPL. This lack of protection in spite of the strong Th1 response was accompanied by a lack of Th17 cytokine induction by the nanoparticle-CpG formulation. In contrast, the formulation with DDA/MPL induced both Th1- and Th17-type responses, suggesting that the induction of Th17 cells may play a role in protection against tuberculosis by HBHA.

HBHA was purified from BCG cultures by chromatography on heparin-Sepharose CL-6B, followed by HPLC, as described [7,10]. For FITC labelling, HBHA was isolated from a recombinant Mycobacterium smegmatis overproducing the BCG HBHA (unpublished data). Purified protein derivative (PPD) was purchased from Statens Serum Institute (Copenhagen, Denmark). 2.3. FITC labelling of HBHA One milligram of HBHA was labelled with 100 ␮g FITC by overnight incubation in 0.1 M carbonate/bicarbonate buffer (pH 9.5) at room temperature. FITC-labelled HBHA was separated from free FITC by two passages on a Sephadex G10 column.

2. Materials and methods 2.4. Nanoparticles and antigen formulation 2.1. Animals and cells All experiments were performed with 6-week-old female C57Bl/6 mice (Charles River Laboratory, L’Arbresle, France). For challenge infection with pathogens, mice were transferred to a biosafety level 3 laboratory and housed in isolators. Human bronchial epithelial SV40-transformed cells (16HBE) were maintained in DMEM/F12 medium with 10% FCS (Invitrogen).

PGLA nanoparticles were prepared as previously described [15]. Polysaccharide cationic nanoparticles (NP) were prepared from maltodextrin; NPL are NP with anionic phospholipids incorporated in their core [16,17]. HBHA (5 ␮g) was incorporated into nanoparticles by incubation for 15 min with nanoparticles (15 ␮g) in a final volume of 30 ␮l. 2.5. Immunization, prime-boost protocol and challenge infection

2.2. Bacteria and antigen preparation M. tuberculosis H37Rv and the BCG Pasteur strain (isolate 1173P2, WHO, Stockholm, Sweden) were grown in liquid Sauton medium or on solid Middlebrook 7H11 medium (Difco, Detroit, USA). Stock solutions were titrated and stored at −80 ◦ C until use.

Animals were immunized three times at 2-week intervals with 5 ␮g HBHA encapsulated into 15 ␮g nanoparticles with or without 10 ␮g of CpG (ODN 1826, Invivogen). Alternatively, mice were immunized with 5 ␮g HBHA emulsified in 200 ␮l with DDA/MPL (150 ␮g DDA and 25 ␮g MPL/injection; Sigma).

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Fig. 2. Antibody and cytokine immune responses after s.c. immunization of mice with HBHA formulated in NPL particles. (A) C57Bl/6 mice (n = 6–7/group) were immunized 3 times with 5 ␮g of HBHA formulated with NPL particles (nano-HBHA) or DDA/MPL (HBHA-DDA/MPL) and their antibody (sera diluted 1/400) and cellular (IFN-␥ and IL-5) anti-HBHA responses were determined 1 week after the last immunization. The results shown are representative of 3 independent experiments. Horizontal lines represent the means. (B) Mice were immunized as in A and challenged 4 weeks later with 100 CFU Mtb by aerosol. The bacterial load were analysed 8 weeks later in spleen and lung.

In prime-boost experiments, mice were first primed subcutaneously (s.c.) with 5 × 105 colony forming units (CFU) BCG and 8 months later boosted three times at a 2-week interval with different preparations of HBHA. Animals were challenged at the indicated time points by aerosol with ∼100 CFU of M. tuberculosis H37Rv. Eight weeks later, their spleens were divided in two equal parts, one for CFU counting and the other for splenocyte culture. The right lung was used for CFU counting and the left lung for cell preparations. The bacterial loads were determined by plating serial dilutions of organ homogenates onto Middlebrook 7H11 medium agar.

(Southern Biotech, AL, USA), were added, and the plates were incubated for 1 h. After washing, the presence of antibodies was revealed with the TMB substrate (BD). 2.8. Statistical analysis The results were analysed using the Mann–Whitney U test (GraphPad Prism program). A value of P ≤ 0.05 was considered to be significant. 3. Results

2.6. Lymphocyte preparations and cytokine production 3.1. Selection of the optimal nanoparticle to deliver HBHA Spleen and lung cells were prepared as previously described [18,19]. Cells were added at 7.5 × 105 cells per well in 96-well plates (BD Falcon) and stimulated with medium, 10 ␮g/ml HBHA or PPD or 1 ␮g/ml of Concanavalin A (Sigma). Cell supernatants were harvested 48 h later, and cytokine levels were determined by ELISA using the BD OptEIATM kit (for IFN-␥, IL-5, IL-10) or a pair of specific monoclonal antibodies (for IL-17; BD Pharmingen) accordingly to manufacturer’ protocols. 2.7. Antibody determination Microtiter plates were coated overnight at 4 ◦ C with 100 ␮l of HBHA solution (1 ␮g/ml in carbonate buffer, pH 9.5) and saturated with 3% BSA in PBS for 2 h. Serum samples were added onto the wells and kept overnight at 4 ◦ C. After washing, different goat anti-mouse Ig isotype-horseradish peroxidase conjugates

Three types of nanoparticles were tested in order to deliver HBHA: an anionic nanoparticle composed of PGLA, and two positively charged nanoparticles composed of maltodextrin, one of which containing a lipid core composed of 70% phospholipids (termed, respectively, NP and NPL). The optimal formulation was determined in an in vitro cell assay using human bronchial epithelial cells incubated with the different FITC-labelled HBHA-containing nanoparticles. As shown in Fig. 1, the best cell delivery was observed with HBHA incorporated into NPL. This formulation was thus used in the following study. 3.2. Induction of immune responses with HBHA-NPL HBHA encapsulated in NPL particles (nano-HBHA) was injected s.c. three times in mice, and both humoral and cellular immune

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Fig. 3. Effect of addition of CpG to HBHA preparation. (A) C57Bl/6 mice (n = 5/group) were immunized 3 times with 5 ␮g of HBHA formulated with NPL particles (nano-HBHA), with NPL particles and co-incorporated CpG (nano-HBHA-CpG), with NPL particles and CpG given at a different site (nano-HBHA/CpG) or with DDA/MPL (HBHA-DDA/MPL), and their anti-HBHA IgG and IgG1, IgG2a, (sera diluted 1/200 for IgG, 1/500 for IgG1, 1/100 for IgG2a,) and cellular (IFN-␥, IL-5 and IL-17) anti-HBHA response were determined 1 week after the last immunization. Negative controls included mice immunized with NPL particles without HBHA and CpG (nano) and mice immunized with NPL without HBHA but with incorporated CpG (nano-CpG). The results shown are representative of two independent experiments. Horizontal lines represent the means. (B) C57Bl/6 mice (n = 5/group) were immunized three times with 5 ␮g of HBHA formulated with DDA or NPL particles plus 25 ␮g MPL (DDA-HBHA/MPL or nano-HBHA/MPL) or plus 15 ␮g CpG (DDA-HBHA/CpG or nano-HBHA/CpG). IFN-␥ and IL-17 production were determined 1 week after the last immunization after in vitro stimulation of splenocytes with HBHA (10 ␮g/ml).Results are expressed in the form of ratios which are obtained by dividing the cytokine levels found in CpG-containing formulation by the values obtained with the corresponding MPL-containing formulation. They are the mean of values obtained with 2 and 3 pooled spleen cell preparations for each group.

responses were analysed one week later. As a comparator, HBHA formulated with DDA/MPL (HBHA-DDA/MPL) was used. The negative control consisted of nanoparticles without HBHA. S.c. immunization with nano-HBHA led to the production of anti-HBHA antibodies (Fig. 2A), although at a level inferior to

that obtained with HBHA-DDA/MPL. Upon in vitro stimulation with HBHA, splenocytes from mice immunized with nano-HBHA produced lower IFN-␥, but higher IL-5 levels than splenocytes from mice immunized with HBHA-DDA/MPL (Fig. 2A). When mice were challenged by aerosol with M. tuberculosis, significant

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Fig. 4. Cytokine profile induced by nano-HBHA-CpG in BCG-primed mice (A) and after mycobacterial challenge (B). C57Bl/6 mice (n = 5/group) were s.c injected with BCG and 10 weeks later immunized twice with 5 ␮g of HBHA formulated with NPL particles (nano-HBHA), with NPL particles and co-incorporated CpG (nano-HBHA-CpG) or with DDA/MPL (HBHA-DDA/MPL). (A) The cellular anti-HBHA response (IFN-␥, IL-5 and IL-17) was determined in the spleen 1 week after the last immunization. NPL particles without HBHA and CpG (nano) served as a negative control. (B) Four weeks after the last immunization, animals were i.n. challenged with 2.5 × 105 BCG bacteria. Four weeks later, both splenocytes and lung cells were in vitro stimulated with HBHA or PPD, and the IFN-␥ and IL-17 production determined in the supernatants collected 48 h later. NPL particles without HBHA and CpG (nano) served as a negative control.

protection was only seen in HBHA-DDA/MPL vaccinated mice (Fig. 2B). 3.3. Addition of CpG into NPL In order to enhance nano-HBHA immunogenicity, we added the TLR9 ligand CpG, an already approved adjuvant for clinical application, to nano-HBHA. CpG ODN1826 was selected because it is a very potent Th1 adjuvant in murine models both in vitro and in vivo, [20], which was previously used with success in vaccine strategies against intracellular pathogens [21,22]. Ten microgram GpG was either co-incorporated with HBHA into NPL (nano-HBHACpG) or administered at approximately 1 cm from the nano-HBHA injection site (nano-HBHA/CpG). As shown in Fig. 3A, the addition of CpG induced an increase in total anti-HBHA IgG production, approaching the level obtained with HBHA-DDA/MPL. In particular for the Th1-related IgG2a, a marked increase was observed upon the

addition of CpG. This effect was only observed for nano-HBHACpG, but not for nano-HBHA/CpG. Upon in vitro stimulation of splenocytes with HBHA, the IFN-␥ response of nano-HBHA-CpGimmunized mice was comparable to that of HBHA-DDA/MPLimmunized mice, whereas only a weak IFN-␥ response was seen in the nano-HBHA and nano-HBHA/CpG groups (Fig. 3A). The addition of CpG, either within the nanoparticle or given at a different site, completely abolished the IL-5 production seen in the nano-HBHA group. Finally, in contrast to HBHA-DDA/MPL-immunized mice, no significant amount of IL-17 was produced in any other group. Thus, a highly polarized and Th1-type restricted immune response was induced in mice immunized with nano-HBHA-CpG. In order to determine whether the low IL-17 production observed in the nano-HBHA-CpG group was due to the immunostimulant CpG, or to the carrier (i.e. nanoparticles), we performed a back to back comparison of the two carriers combined with the two immunostim ulants and examined the relative IL-17 and

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Fig. 5. Lack of protection in s.c. nano-HBHA-CpG immunized, BCG-primed mice against aerosol challenge with M. tuberculosis. C57Bl/6 mice (n = 5–7/group) were s.c. primed with PBS or 5 × 105 CFU BCG and 8 months later immunized 3 times with 5 ␮g HBHA formulated with NPL particles and co-incorporated CpG (nano-HBHA-CpG) or with DDA/MPL (HBHA-DDA/MPL). Mice immunized with NPL particles without HBHA and CpG (nano) or with NPL without HBHA but with incorporated CpG (nano-CpG) served as a negative control. The mice were challenged 8 weeks later by aerosol with ∼100 CFU M. tuberculosis, and the bacterial burden was determined in the spleens and lungs 8 weeks later. Horizontal lines represent the means.

IFN-␥ production depending on the immunostimulant added. The results, expressed as fold changes of cytokine levels measured after vaccination with CpG- compared to MPL-containing formulations, indicated that, when compared to MPL, the addition of CpG with either the DDA or the NPL carriers led to a drop in IL-17 synthesis (Fig. 3B). In contrast, the addition of CpG in either carrier formulation led to an increase in IFN-␥ production. These results indicate that the immunostimulant CpG was the major driver of the low IL-17 and high IFN-␥ production, rather than the carrier NPL.

3.4. Nano-HBHA-CpG-induced cytokine profile is maintained in the context of BCG priming or after challenge We have previously shown that HBHA administrations given after BCG priming strongly increases BCG-mediated protection against M. tuberculosis challenge [12]. Here, we tested whether BCG priming influences the cytokine profile induced by nano-HBHACpG immunization. Mice received s.c. BCG, followed 10 weeks later by two s.c. nano-HBHA, nano-HBHA-CpG or HBHA-DDA/MPL injections, given at a 2-week interval. One week later, the splenocytes were restimulated with HBHA, and the secreted cytokines were measured. After BCG priming, we observed significant IFN-␥ but weak IL-5 secretion in the nano-HBHA-CpG and HBHA-DDA/MPL immunized groups, and only barely IL-17 in the nano-HBHA-CpG group (Fig. 4A), indicating that BCG priming did not modify the cytokine profile induced by nano-HBHA-CpG immunization. To investigate whether this polarized immune response is maintained following pulmonary infection, mice were BCG-primed, boosted twice with nano-HBHA-CpG and then intra-nasally challenged with BCG. Four weeks later the cytokine responses were measured in spleen and lung cells upon stimulation with PPD or HBHA. Whereas no marked difference in the IFN-␥ production was seen between groups, immunization with nano-HBHA-CpG resulted in a general reduction of IL-17 synthesis, both in lung and spleen (Fig. 4B). This reduction was seen after both HBHA and PPD stimulation, indicating a general inhibition of the IL-17 production. Even the basal production of IL-17 in unstimulated lung cells was abolished in animals receiving nano-HBHA-CpG. This absence of IL-17 production was also noticed in the nano-HBHA group, but

affected essentially the specific anti-HBHA response, and not the anti-PPD response. 3.5. Lack of protection after nano-HBHA-CpG immunization In order to investigate the potential protective effect of nano-HBHA-CpG against M. tuberculosis challenge in BCG-primed mice, mice were immunized three times with different formulations of HBHA 8 months after BCG priming and challenge d by aerosol with M. tuberculosis 8 weeks later, according to the protocol defined previously [12]. As shown in Fig. 5, only animals immunized with HBHA-DDA/MPL were protected against aerosol challenge in the lungs (0.7 log reduction). A trend (0.3 log reduction) was observed after immunization with nano-HBHA-CpG, although this did not reach statistical significance. This could to be due in part to the presence of CpG, since a slight reduction was also observed with nano-CpG in the absence of HBHA (BCG/nano vs BCG/nanoCpG; 0.2 log reduction). However, this CpG effect was only found in BCG-primed animals, but not in control mice (i.e. PBS/nano-CpG vs PBS/nano), suggesting a booster effect of CpG on BCG priming as previously reported [23]. No significant protection was observed in the spleen with or without BCG vaccination, although there was a trend in the reduction of the bacterial burden was seen 8 months after BCG administration, (respectively, 0.43 and 0.87 log reduction between PBS-nano vs BCG-nano and PBS/nano-CpG vs BCG/nanoCpG primed animals). These results are somewhat in contrast to earlier work [12]. However, in the previous study a different mouse strain was used (Balb/c), as well as another route of infection (intraveneous), which may explain the differences in these results. 3.6. Lack of memory response in BCG-primed, nano-HBHA-CpG immunized mice after challenge In an attempt to understand why nano-HBHA-CpG immunization conferred only weak protection in spite of the strong Th1 response, we analysed the immune response developed in mice after infection. As indicated in Fig. 6A whereas the anti-HBHA antibody response was maintained after challenge of HBHA-DDA/MPL

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Fig. 6. Lack of anti-HBHA memory induced by nano-HBHA-CpG immunization. C57Bl/6 mice (n = 5–7/group) were s.c. primed with PBS or 5 × 105 CFU BCG and 8 months later immunized 3 times with 5 ␮g HBHA formulated with NPL particles and co-incorporated CpG (nano-HBHA-CpG) or with DDA/MPL (HBHA-DDA/MPL). Mice immunized with NPL particles without HBHA and CpG (nano) or with NPL without HBHA but with incorporated CpG (nano-CpG) served as a negative control. The mice were challenged 8 weeks later by aerosol with ∼100 CFU M. tuberculosis and sacrificed 8 weeks after challenge. (A) The anti-HBHA IgG, IgG1 and IgG2a levels were measured in the sera either 1 week after the last immunization or at the time of sacrifice (sera diluted 1/400 for IgG, 1/200 for IgG1 and IgG2a). Individual mice were analysed, and each symbol represents a different mouse. Horizontal lines represent the means. (B) IFN-␥, IL-5 and IL-17 levels were analysed on spleen and lung cell culture supernatants after in vitro stimulation with HBHA.

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Fig. 7. Lack of protection after i.n. immunization with nano-HBHA-CpG. C57Bl/6 mice (n = 7/group) were either i.n. or s.c. immunized three times with 5 ␮g of HBHA formulated with NPL particles and co-incorporated CpG (nano-HBHA-CpG) or with DDA/MPL (HBHA-DDA/MPL). Mice immunized with NPL particles without HBHA but with incorporated CpG (nano-CpG) served as a negative control. Animals were aerosol challenged 4 weeks later with ∼100 CFU M. tuberculosis and sacrificed 8 weeks later. (A) IFN-␥, IL-5 and IL-17 levels were analysed on spleen and lung cell culture supernatants after in vitro stimulation with HBHA for 48 h and (B) the bacterial loads were determined in the spleen and lungs. Horizontal lines represent the means.

immunized mice, it decreased in the nano-HBHA-CpG group. Likewise, while spleen cells from HBHA-DDA/MPL immunized animals secreted diverse cytokines upon HBHA stimulation, only background cytokine secretion levels were observed in nanoHBHA-CpG group (Fig. 6B). Weaker responses to HBHA were also

found in the lungs of the nano-HBHA-CpG group, compared to the HBHA-DDA/MPL group. Thus, despite the strong immunogenicity of HBHA achieved after s.c. delivery with nanoparticles plus CpG, this antigen formulation appeared unable to maintain a robust T cell immunity. Several

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studies have suggested that the generation of vaccine-induced IL17 may be prerequisite for long-lasting memory immunity against tuberculosis [24–26]. The absence of production of this cytokine in our vaccination protocol may thus account for the lack of memory response observed. 3.7. Intranasal immunization elicits IL-17 synthesis but is not sufficient for protection Since the intranasal (i.n.) vaccination has been reported to be the preferred route for promoting Th17 responses [27–29], nano-HBHA-CpG was administered three times i.n. to mice, which were then challenged by aerosol with M. tuberculosis 4 weeks later. Control groups consisted of animals s.c. immunized with nano-HBHA-CpG or HBHA-DDA/MPL. In the i.n. nano-HBHA-CpGimmunized mice, we observed measurable, but low IL-17 synthesis after in vitro HBHA stimulation both in the spleens and the lungs, in contrast to s.c. nano-HBHA-CpG-immunized mice (Fig. 7A). This IL-17 production remained, however, far below that obtained by HBHA-DDA/MPL immunization, which was also the case for the production of IFN-␥. Intranasal vaccination with nano-HBHA-CpG also did not result in significant protection against M. tuberculosis challenge (Fig. 7B). 4. Discussion In this study we evaluated the capacity of nanoparticles to deliver HBHA, a potential subunit vaccine candidate against TB. The best delivery of HBHA into cells was achieved with a cationic NPL, which is consistent with the high content of HBHA in basic amino acids [30]. These NPL are of small size (60 nm), facilitating their migration to the draining lymph node and consequently the contact with antigen-presenting cells and the initiation of immune responses [31–33]. S.c. injection of HBHA encapsulated in NPL induced anti-HBHA immune responses. The addition of a low dose of CpG strongly enhanced this response, to a level comparable to that obtained with the strong adjuvant formulation HBHA-DDA/MPL. This enhanced response was only observed when CpG was co-entrapped with HBHA into nanoparticles, allowing for concomitant delivery to the same antigen-presenting cells, and confirming previous reports [34–37]. CpG directed the anti-HBHA immune response mainly towards a Th1 type profile, characterized by IFN-␥ production and antibodies of the IgG2a isotype. It suppressed the Th2related IL-5 cytokine production, consistent with previous reports [38–41]. No or only very low levels of IL-17 were detected after s.c. immunization with nano-HBHA-CpG, in contrast to HBHA-DDA/MPL. Even in BCG-primed animals boosted with nano-HBHA-CpG, immunization did not induce IL-17, and no IL-17 was induced in nano-HBHA-CpG-immunized mice after M. tuberculosis challenge. Neither in the spleen nor in the lungs was IL-17 detected after stimulation with HBHA or with other mycobacterial antigens, such as PPD, indicating a generalized suppression of the Th17 response. Various cytokines, such as IL-4, IL-10 or IL-27, have been described to inhibit IL-17 synthesis [42]. However, we did not detect significant differences in the production of these cytokines at the time of sacrifice between animals immunized with nano-HBHA-CpG vs HBHA-DDA/MPL (data not shown). The link between CpG and IL-17 production is complex, and conflicting reports are found in the literature, showing either a positive or a negative association [43–49]. However, these studies used different protocols of administration, which may have impacted on the nature of the different antigenpresenting cell populations present at the site of immunization [50].

It has now increasingly become clear that the production of IFN-␥ is important but not sufficient for protection against TB [51–53], and the results reported here support this idea. Although nano-HBHA-CpG administration induced a strong IFN-␥ response, this did not result in significant protection against M. tuberculosis challenge infection, whereas HBHA-DDA/MPL provided significant protection, without superior IFN-␥ production. The main difference between these two formulations resided in the differential production of IL-17. This cytokine was recently shown to play a crucial role in the formation of granulomas during M. tuberculosis infection [54] and in the establishment of a strong memory T cell response [24]. Studies performed with IL-17KO or IL-17RA-/mice indicated that IL-17 is not essential to control mycobacterial growth, but its lack resulted in reduced survival late in the course of infection [24,25,55]. Importantly, IL-17 was shown to be involved in vaccine-induced immunity [24–26], and the results presented here also suggest the role of IL-17 in maintaining vaccine-induced memory, even if the definitive proof of its role in HBHA-conferred protection will imply its depletion by specific anti-IL-17 antibodies. Challenge infection failed to induce a recall of both humoral and cellular responses in mice s.c. immunized with nano-HBHA-CpG, in contrast to animals immunized with HBHA-DDA/MPL, which induced a marked IL-17 synthesis and showed persistent immune responses after challenge. One hypothesis is that this general immunosuppression may be due to the generation of regulatory T and B cells following CpG administration, and possibly amplified by M. tuberculosis challenge. There is indeed a fine balance between the generation of Th17 and Treg lineages. Although originating from a same precursor, these two CD4+ T cell subsets diverge early, depending on their cellular environment, into distinct phenotypes with opposite activities [56]. Illustrating this complexicity is the opposite effect of CpG on the generation or suppression of Treg activity described in the literature [57–59]. Whether in our model, CpG indeed generates Treg cells awaits further investigation, especially through kinetic studies on the cytokine profile and the phenotype of the cells that expand in the lungs after immunization and challenge infection. In addition to the choice of the adequate combination of adjuvants, the site of antigen administration and the nature of the different immune cell types present at this site, are also important to optimize immune responses [60]. The mucosal tissue represents an attractive site to induce protective immunity, particularly against M. tuberculosis, for which it is the natural portal of entry. It has been shown that pulmonary immunization with Ag85B associated with CpG in nanoparticles elicits both IFN-␥ and IL-17 synthesis, whereas intradermal injection with the same preparation failed to induce IL-17 [51]. In that study, only immunization via the pulmonary route resulted in some protection against a challenge infection. By contrast, in another study, pulmonary administration of Ag85A with CpG was shown to induce high levels of IFN-␥ but no IL-17 and failed to protect mice against challenge infection [52]. In this study, we found that i.n. immunization with nano-HBHA-CpG induced measurable but low levels of IL-17 in addition to IFN-␥, confirming the importance of the immunization site to orient the immune response. However, both these IL-17 and IFN-␥ levels were low and not sufficient to protect animals against M. tuberculosis challenge. This weak immunogenicity is rather surprising since we have previously shown that NPL is able to induce a marked immune response to the encapsulated antigen by the i.n. route [36]. HBHA has the capacity to interact with heparin sulphate present at the surface of epithelial cells, which may therefore potentially have impacted on the transit of the nano-particles through the epithelium. We are now investigating new formulations of encapsulated HBHA, hoping to obtain improved mucosal immunity and protection.

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