An Increase in Antimycobacterial Th1-Cell Responses by Prime-Boost Protocols of Immunization Does Not Enhance Protection against Tuberculosi
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感染与免疫杂志 2006年第4期
Unite de Biologie des Regulations Immunitaires, Inserm, E 352, Institut Pasteur, Paris F-75015, France
Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague, Czech Republic
Unite de Genetique Moleculaire Bacterienne, Institut Pasteur, Paris F-75015, France
Unite deBiochimie des Interactions Macromoleculaires, Institut Pasteur, Paris F-75015, France
ABSTRACT
Bordetella pertussis adenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN--producing CD4+ T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection against Mycobacterium tuberculosis infection. These results question the broadly admitted correlation between the frequency of IFN--producing CD4+ T cells and the level of protection against tuberculosis.
INTRODUCTION
The most feasible strategy to control the increasing number of tuberculosis cases, notably in developing countries, is effective vaccination against Mycobacterium tuberculosis. The only current vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), displays highly variable efficiency (0 to 80%) in preventing adult pulmonary tuberculosis. Several subunit vaccine strategies, including plasmid DNA, bacterial or viral vectors, peptides, or recombinant proteins in adjuvant, have been developed to induce T-cell immunity against mycobacterial antigens. So far, none of these subunit vaccines used alone provides better protection levels than that conferred by BCG. The latter remains the gold standard vaccine model yet is unable to clear infection in mouse model of tuberculosis (9, 26). In the present study, we evaluated the potential of a novel vaccination vector, the adenylate cyclase of Bordetella pertussis CyaA harboring strong M. tuberculosis immunogens, to induce antimycobacterial immunity.
CyaA is a 1,706-amino-acid-long toxin consisting of five functional domains: (i) adenylate cyclase activity (AC) domain (residues 1 to 400), (ii) hydrophobic channel-forming domain (residues 500 to 700), (iii) posttranslationally acylated domain (residues 800 to 1000), (iv) calcium-binding glycine/aspartate-rich repeat domain (residues 1000 to 1600), and (v) C-terminal domain carrying a secretion signal (residues 1600 to 1706) (18). CyaA binds, via its fourth domain, to M2 integrin (CD11b/CD18), expressed on macrophages, neutrophils, natural killer (NK) cells, peritoneal B-1 cells, and notably the major CD8– CD11b+ dendritic cell (DC) population (14). Upon interaction with CD11b, the CyaA AC domain can be endocytosed and degraded for major histocompatibility complex (MHC) II-restricted presentation (20, 34). Several permissive sites have been identified within various domains of CyaA in which it is possible to genetically insert exogenous peptides without interfering with the functions of CyaA (17, 20, 35). T-cell epitopes inserted into these permissive sites are efficiently carried to the MHC-II pathways of antigen-presenting cells (APC) and induce in vivo CD4+ Th1 responses without the need for any adjuvant (7, 34).
Immunization of mice with genetically detoxified CyaA bearing appropriate T-cell epitopes provides full prophylactic protection against lethal lymphocytic choriomeningitis virus challenge (33), efficient therapeutic antitumor immunity (11) and complete therapeutic efficacy against tumor cells expressing an immunogenic oncoprotein of human papillomavirus 16 in a mouse experimental model mimicking human papillomavirus-induced cervical cancer (21, 30).
It is widely admitted that prophylaxis of tuberculosis requires induction of CD4+ Th1 responses. Indeed, induction of high levels of antigen-specific gamma interferon (IFN-) is considered the best antimycobacterial protection correlate (10, 25). Therefore, CyaA appears to be an attractive vector to be evaluated in experimental models of tuberculosis.
In the present study, we investigated the potential in antituberculosis vaccination of fusions of CyaA to the promising immunogen candidates 85A and the 6-kDa early secreted antigenic target (ESAT-6) protein. 85A is a member of the 85 complex consisting of three related secreted proteins (A, B, and C), with various degrees of mycolyl transferase and fibronectin binding activity (2, 38). 85A and 85B are potent targets of antimycobacterial T cells. ESAT-6 is encoded in the ESAT-6 operon within a 10-kb chromosomal region of M. tuberculosis referred to as the region of difference 1 (RD1) that is absent from the BCG genome. ESAT-6 is secreted by a dedicated export system, i.e., the ESX-1 secretion machinery (4). The exact biological function of ESAT-6 has not yet been elucidated but this molecule constitutes a strong immunogen in humans and rodents (4, 27). We constructed recombinant CyaA toxins carrying, in their AC domain, immunodominant epitopes of 85A, restricted in H-2d or H-2b haplotype (16), or the complete sequence of ESAT-6 (1). We first investigated in vitro whether these CyaA constructs were able to deliver to the MHC-II pathway of APC the same epitopes as those generated upon mycobacterial infection. We then studied their immunogenicity in vivo, as subunit immunization vectors.
Boosting vaccination with strong mycobacterial immunogens delivered by a subunit nonreplicative vaccine after priming with live recombinant mycobacterial vaccine has been proposed to improve antimycobacterial immunity. The BCG Pasteur strain stably complemented with the complete ESX-1 system (BCG::RD1) expresses and secretes ESAT-6 (32). This complementation strongly modifies host-mycobacteria interactions as, besides its increased virulence, BCG::RD1 possesses an enhanced capacity to activate cells of the innate or adaptive immune system (22) and to induce strong ESAT-6-specific Th1 CD4+ T-cell responses (32). These characteristics correlate with the enhanced protective capacity of BCG::RD1 against infection with M. tuberculosis in mice and guinea pigs compared to the parental BCG strain (32). We characterized the immunogenicity of CyaA-85A and CyaA-ESAT-6 constructs as boosters after priming with BCG::RD1. Finally, we evaluated the possible protective effect of these CyaA constructs as a subunit vaccine alone or as a booster after priming with BCG::RD1 against aerosol challenge with M. tuberculosis.
MATERIALS AND METHODS
Peptides. The 85A:101-120, 85A:241-260 (16), ESAT-6:1-20 (3), and MalE:100-140 (19) peptides were synthesized by Neosystem (Strasbourg, France).
Recombinant CyaA toxoid construction. Escherichia coli XL1-Blue (Stratagene, Amsterdam, The Netherlands) was used throughout the work for recombinant DNA construction and for expression of mycobacterial antigen/MHC-II epitopes inserted into the AC domain of CyaA. Bacteria transformed with appropriate plasmids derived from pT7CACT1 (29) were grown at 37°C in Luria-Bertani medium supplemented with 150 μg of ampicillin per ml. Construction of the plasmid for production of the CyaA336-ESAT-6 protein was described previously (37).
The CyaA constructs, carrying, between residues 335 and 336, 85A:101-120 (LTSELPGWLQANRHV KPTGS) or 85A:241-260 (QDAYNAGGGHNGVF DFPDSG), were obtained by in-frame insertions of corresponding pairs of annealed synthetic oligonucleotides of the sequences 5'-ACCACTGACCTCTGAACTGCCGGGTTGGCTGCAGGCTAACCGTCACGTGAAACCGACAGGTTCC and 5'-GTACGGAACCGATCGGTTTCACGTGACGGTTAGCCTGCAGCCAACCCGGCAGTTCAGAGGTCAGTG for 85A:101-120 and 5'-GTACCACAGGACGCTTACAACGCTGGTGGTGGTCACAACGGTGTTTTCGACTTCCCGGACTCTGGTTAC and 5'-GTACGTAACCAGAGTCCGGGAAGTCGAAAACACCGTTCTGACCACCACCAGCGTTGTAAGCGTCCTGTG for 85A:241-260. The oligonucleotides were designed (i) to introduce a restriction site for rapid identification of insertion mutants, (ii) to stop CyaA synthesis when inserted in the inverted orientation, and (iii) to destroy the original BsrGI insertion site on ligation.
The orientation and exact sequence of inserted oligonucleotides were verified by DNA sequencing. The recombinant CyaA proteins carrying ESAT-6:1-95, 85A:101-120, or 85A:241-260 were produced in E. coli XL1-Blue, purified from inclusion bodies in 8 M urea, 50 mM Tris-Cl, pH 8, 2 mM EDTA, and characterized as previously described (29). The resulting proteins were free of any detectable adenylate cyclase enzymatic activity.
Insertion of polypeptides into position 336 detoxifies CyaA. To generate CyaA harboring inserts at position 224, we used a derivative of CyaA, CyaA-E5, which is catalytically inactive (but still invasive) as a result of the insertion of the dipeptide Leu-Gln between codons 188 and 189 of CyaA. To construct the plasmid used for production of the detoxified CyaA224-ESAT-6:1-95 protein, the full-length ESAT-6 coding region was PCR amplified with primers ESAT-6N (5'-ACTAGCTAGCATGACAGAGCAGCAGTGGAATTTCGCGGG-3') and ESAT-6K (5'-CGGGGTACCTGCGAACATCCCAGTGACGTTGCCTTCGGT-3'). The 304-bp PCR product was purified, cut by NheI and KpnI, and ligated into plasmid pCACT-OVA-E5 (11) digested by the same enzymes. The nucleotide sequence of the insert was verified by DNA sequencing. The recombinant CyaA224-ESAT-6:1-95 protein was produced in E. coli BLR and purified from inclusion bodies as described previously (30).
Mycobacteria. BCG::RD1 and M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 medium as previously described (31).
Mice and immunization. Female BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from Charles River (Arbresle, France). Six- to 10-week-old mice each received intravenously in the tail vein 50 μg of CyaA harboring mycobacterial immunogens. For immunization with peptides, mice received subcutaneously (s.c.) at the base of the tail 50 μg/mouse of individual peptides emulsified in incomplete Freund's adjuvant. Immunization with BCG::RD1 was performed by subcutaneous injection of 105 CFU/mouse. Mice immunized with BCG::RD1 were placed in isolators in the Pasteur Institute A3 animal facilities. All animal studies were approved by the Pasteur Institute Safety Committee in accordance with French and European guidelines.
Generation of T-cell hybridomas and antigen presentation assays. T-cell hybridomas specific for the 85A:101-120 and 85A:241-260 peptides were generated from BALB/c and C57BL/6 mice, respectively, injected with 50 μg/mouse of the appropriate peptide emulsified in incomplete Freund's adjuvant 10 days postimmunization. Splenocytes were stimulated with 10 μg/ml of the homologous peptide for 4 days. Viable cells were harvested on Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) and were fused with an equal number of BW51-47 thymoma cells by use of polyethylene glycol 1500 (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (23).
BALB/c- and C57BL/6-derived T-cell hybridomas were screened for their capacity to specifically release interleukin-2 (IL-2) upon stimulation with the 85A:101-120 and 85A:241-260 peptides, respectively. To determine their capability to recognize APC infected with mycobacteria, bone marrow-derived DC (BM-DC), generated in antibiotic-free conditions in the presence of 10 ng/ml of granulocyte-macrophage colony-stimulating factor were infected at day 5 of culture with BCG at a multiplicity of infection of 1 for 16 h, washed three times, and incubated with 105 T-cell hybridomas.
The presence of IL-2 in the supernatant of cultures was monitored by standard IL-2-specific enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Proliferation of the CTLL-2 cell line is dependent on IL-2 in a dose-dependent manner. CTLL-2 cells (104 cells/well) were incubated with supernatant of T-cell hybridomas, pulsed 48 h later with 1 μCi of [methyl-3H]thymidine (ICN, Orsay, France)/well for 16 h, and the incorporated cpm were counted in a Wallac Microbeta counter (Perkin-Elmer, Cortaboeuf Cedex, France). L fibroblasts transfected with I-Ad, I-Ed and I-Ab molecules were used as peptide-presenting cells in the same type of antigen presentation assay for determination of the restricting element of the peptide presentation to the T-cell hybridomas.
T-cell assays. To measure T-cell proliferative responses to mycobacterial antigens, splenocytes of immunized mice were cultured (106 cells/well) in 96-well flat-bottom plates in synthetic HL-1 medium (BioWhittaker, Walkersville, MD) as previously described (32) in the presence of various concentrations of appropriate antigens. Cultures were pulsed 72 h later with 1 μCi of [methyl-3H]thymidine/well for 16 h and the incorporated cpm were counted in a Wallac Microbeta counter.
IL-2, IL-4, IL-5, and IFN- production by splenocytes was assessed subsequent to in vitro stimulation with 4 μg/ml of recombinant proteins or 10 μg/ml of synthetic homologous or control peptides. Amounts of IL-2 were determined in culture supernatants after 24 h incubation by a CTLL-2 bioassay. The amounts of IL-4, IL-5, and IFN- were quantified in culture supernatants after 72 h incubation by ELISA as detailed elsewhere (7). IFN- Elispot was performed as previously described (7) by incubation of serial dilutions of splenocytes on anti-IFN- monoclonal antibody (R4-6A2)-coated 96-well Multiscreen filtration plates (Millipore, France). Cells were stimulated with 1 μg/ml of peptide 85A:241-260 or negative control peptide for 36 h without addition of other growth factors. Biotinylated anti-IFN- monoclonal antibody (XMG1.2) and streptavidin-alkaline phosphatase (PharMingen) were used to reveal the spots.
Protection assays against infection with M. tuberculosis. At the indicated time points, unvaccinated and immunized mice (six/group) were challenged by use of a homemade nebulizer via the aerosol route; 2 ml of a suspension containing 106 CFU/ml was aerosolized to obtain an inhaled dose of 100 ± 10 CFU/mouse. Infected mice were placed in isolators. One month postchallenge, the lungs and spleen of the mice were homogenized by use of an MM300 organ homogenizer (QIAGEN, Courtaboeuf, France) and 2.5-mm-diameter glass beads. Serial fivefold dilutions of homogenates were cultured on 7H11 agar supplemented with bovine albumin, dextrose, and catalase (Difco, Becton Dickinson, Sparks, MD). CFU were counted after 15 to 18 days of incubation at 37°C.
RESULTS
CyaA harboring immunodominant epitopes of 85A or complete sequence of ESAT-6. Fragments of 85A containing immunodominant epitopes, i.e., 85A:101-120 (restricted by I-Ed) and 85A:241-260 (restricted by I-Ab) (16), were genetically inserted into the AC domain of CyaA at position 336 (Table 1). In parallel, the complete sequence of ESAT-6 (ESAT-6:1-95) (1) was inserted at positions 224 and 336 of the CyaA AC domain. Indeed, so far, the permissive sites of CyaA have been studied and characterized mainly by insertion of short reporter peptide sequences. Thus, two insertion sites, 224 and 336, were chosen here to evaluate and to compare their potential to carry and deliver the 95-residue-long ESAT-6 sequence.
A CyaA harboring residues 100 to 114 of maltose binding protein of Escherichia coli at position 336 (MalE:100-114), containing an I-Ab/d-restricted epitope (19), was used in all experiments as a negative CyaA control. It was verified that insertion of these amino acid residues, and notably of the long ESAT-6:1-95 polypeptide, did not alter the capacity of the recombinant CyaA constructs to engage its surface receptor CD11b and to compete with a biotinylated intact CyaA tracer for specific binding to CD11b-transfected Chinese hamster ovary (CHO) cells (data not shown).
Incubation with recombinant CyaA and infection with mycobacteria leads to processing and MHC-II-restricted presentation of the same mycobacterial epitopes. Using splenocytes of BALB/c (H-2d) and C57BL/6 (H-2b) mice immunized with the 85A:101-120 and 85A:241-260 synthetic peptides, respectively, we generated panels of T-cell hybridomas specific to these peptides. The 85A:101-120-specific T-cell hybridomas were all restricted by I-Ed and those specific to 85A:241-260 by I-Ab (data not shown). In each genetic background, only a limited number (2 of 15) of the T-cell hybridomas, referred to as AA1 and CG11 for the H-2d haplotype and BF9 and DE10 for the H-2b haplotype cells, were able to recognize histocompatible BM-DC infected in vitro with BCG (Fig. 1A and B). This observation suggests that immunization with a single peptide may lead to generation and presentation of more than one epitope and thereby to stimulation of diverse T-cell receptors.
One can imagine the existence of several fine possibilities of adjustment of a given peptide in the MHC-II presentation groove and the presence of C-terminal or N-terminal sequences of the synthetic peptide which may be present during the presentation of synthetic peptides while the epitope generated during the processing of CyaA and mycobacteria would be much more precise and limited. Of note, all T-cell hybridomas able to recognize APC incubated with CyaA-85A were also able to recognize BCG-infected APC. We only selected T-cell hybridomas specific to the epitopes generated and presented upon mycobacterial infection.
The latter T-cell hybridomas provide a unique tool to examine in vitro whether antigen delivery by CyaA-85A:101-120 and CyaA-85A:241-260 to APC leads to processing and presentation of the same epitopes as those displayed upon infection with mycobacteria. AA1 and CG11 (restricted by I-Ed) and BF9 and DE10 (restricted by I-Ab) hybridomas were indeed able to recognize, in a highly sensitive and specific manner, appropriate histocompatible BM-DC incubated with CyaA-85A:101-120 (Fig. 1C) and CyaA-85A:241-260 (Fig. 1D), respectively. These data demonstrated that in vitro processing of these CyaAs leads to the generation and presentation of immunodominant 85A epitopes that can be recognized by mycobacterium-specific T cells.
To investigate in vitro delivery of ESAT-6 by CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95, we used T splenocytes from BCG::RD1-immunized C57BL/6 mice. These cells (Fig. 2), but not those from BCG-immunized control mice (data not shown), released marked levels of IFN- subsequent to in vitro stimulation with CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95 (Fig. 2). It is noteworthy that ESAT-6:1-20 is the only T-cell epitope on the ESAT-6 sequence, restricted in H-2b haplotype (3) and we have previously shown that T-cell responses from BCG::RD1-immunized C57BL/6 mice to ESAT-6:1-20 were only due to the CD4+ T-cell compartment (32). An in vitro restimulation with the CyaA-MalE:100-114 negative control did not result in IFN- responses, showing the specificity of the antigen presentation. Thus, insertion of ESAT-6 at either position 224 or 336 in CyaA allowed delivery of the ESAT-6 sequence for processing. Moreover, the efficiency of ESAT-6 presentation was markedly improved (100-fold) when it was delivered to APC in the fusion to CyaA, e.g., CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95, compared to presentation of the ESAT-6 recombinant protein alone. This result demonstrates that in vitro processing of CyaA-ESAT-6:1-95 leads to highly efficient generation and presentation of the immunodominant ESAT-6 epitope that is recognized by mycobacterium-specific T cells.
Taken together, these observations strongly suggest that immunization with CyaA carrying mycobacterial antigens may efficiently stimulate T cells capable of recognizing cells infected with mycobacteria in vivo.
In vivo immunogenicity of CyaA harboring mycobacterial immunogens. To investigate the immunogenicity of these CyaAs in vivo, BALB/c and C57BL/6 mice received a single intravenous injection (50 μg/mouse) of purified recombinant CyaA-85A:101-120 and CyaA-85A:241-260, respectively. The proliferative and Th1/Th2 cytokine responses of these mice were evaluated at day 15 postimmunization following in vitro stimulation of their splenocytes with the homologous and control peptides. CyaA-85A:101-120 and CyaA-85A:241-260 induced substantial proliferative, IL-2, and IFN- responses in BALB/c (Fig. 3A and B) and in C57BL/6 (Fig. 3C and D) mice respectively. CyaA224-ESAT-6:1-95 (data not shown) and CyaA336-ESAT-6:1-95 (Fig. 3E and F) were also highly immunogenic in C57BL/6 mice, as shown by strong proliferative and IL-2 responses of their splenocytes stimulated in vitro with the immunodominant ESAT-6:1-20 peptide. However, only low levels of IFN- were obtained in CyaA336-ESAT-6:1-95-immunized mice (Fig. 3F). Immunization with CyaA-MalE:100-114 did not lead to any immune response to 85A and ESAT-6, showing the specificity of the induced T-cell responses. Moreover, no Th2 cytokine (IL-4 and IL-5) responses were detected in these experimental groups (data not shown). Thus, the CyaA constructs bearing the 85A:101-120, 85A:241-260, and ESAT-6:1-95 polypeptides allowed the induction of strong and specific antimycobacterial Th1 immune responses in mice.
Marked boosting effect of CyaA-85A and CyaA-ESAT-6 following priming with BCG::RD1. We next sought to evaluate the efficiency of a heterologous prime-boost vaccination scheme, comprising priming immunization with the BCG::RD1 live attenuated vaccine candidate (32), followed by a booster immunization with CyaA-85A and CyaA-ESAT-6. On day 0, C57BL/6 mice were immunized (s.c.) with 105 CFU/mouse of BCG::RD1. At day 35, groups of mice primed and not with BCG::RD1 received an intravenous (i.v.) injection of 50 μg/mouse of CyaA-85A:241-260, CyaA336-ESAT-6:1-95, and CyaA-MalE:100-114. We have previously demonstrated that C57BL/6 mice immunized with a single dose of BCG::RD1 mounted strong ESAT-6- and 85A-specific proliferative and IFN- responses that were readily detectable 15 to 21 days postimmunization (32).
Fifty days after s.c. immunization of mice with BCG::RD1, the proliferative (Fig. 4A and D) and IFN- (Fig. 4B and E) responses to 85A:241-260 and to ESAT-6 were no longer detectable and near the detection limit. At this time point, significant proliferative responses (Fig. 4A and D) and IFN- (Fig. 4B and E) were detected in mice that were immunized 15 days earlier with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95. Moreover, compared to mice that received only BCG::RD1, their counterparts primed with BCG::RD1 and boosted with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 displayed a consistent increase in both the intensity and the sensitivity of the proliferative response (Fig. 4A and D), as well as a highly increased IFN- response (Fig. 4B and E).
Based on independent but similar experiments, we have observed that a boost with CyaA:MalE:100-114 and with a wild-type CyaA negative control in BCG::RD1-primed mice induced no increase in 85A- and ESAT-6-specific proliferative and 85A-specific IFN- responses (data not shown). The relatively small amounts of anti-ESAT-6 IFN- response in the group of BCG::RD1-primed and CyaA-MalE:100-114-boosted mice can be due to residual ESAT-6-specific response induced by initial BCG::RD1 immunization as it can also be detected in the group of unboosted and BCG::RD1-immunized mice (Fig. 4E). Of note, no anti-85A-specific IFN- response was detected in BCG:: RD1-primed and CyaA-MalE:100-114-boosted mice (data not shown).
At this time point, the frequency of 85A:241-260-specific and IFN--producing T splenocytes in mice primed with BCG::RD1 and boosted with CyaA-85A:241-260 was 50 times higher than those of CyaA-85A:241-260-immunized mice (Fig. 4C). Furthermore, no Th2 cytokine response was detectable in any of the experimental groups (data not shown). These data demonstrated that CyaA harboring mycobacterial immunogens can efficiently boost antimycobacterial Th1 immunity and particularly the IFN- responses induced by BCG::RD1.
Vaccine efficacy of CyaA harboring the mycobacterial immunogens alone and as boosters against infection with M. tuberculosis. We evaluated the potential of the recombinant CyaA to protect mice against M. tuberculosis challenge in groups of C57BL/6 mice (n = 6). Control mice were left unvaccinated or vaccinated with BCG::RD1 at day 0. At day 30, groups of mice received a single i.v. injection of 50 μg/mouse of CyaA-85A:241-260, CyaA336-ESAT-6:1-95, or CyaA-MalE:100-114. At day 60, mice were infected with a small dose of aerosolized M. tuberculosis H37Rv. At 1 month postchallenge, bacterial burden was determined in the lungs and spleen of infected mice.
No protection was observed in CyaA-immunized mice, while the CFU counts in the lungs (Fig. 5A) and spleen (Fig. 5B) of BCG::RD1-immunized mice were 2 logs lower than in unvaccinated mice. Thus, despite induction of powerful T-cell proliferation and of significant Th1 cytokine response to these potent CyaA-delivered immunogens, immunization with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 was not capable of inducing protection against M. tuberculosis challenge in the murine model.
In an independent experiment, C57BL/6 mice were immunized at a 3-week interval by two intraperitoneal injections of CyaA336-ESAT-6:1-95 adjuvanted in alum and again no protection was observed against an H37Rv challenge given 1 week after the last injection (data not shown). It is noteworthy that, subsequent to immunization with CyaA bearing mycobacterial antigen adjuvanted in alum, mice mounted only Th1 but not Th2 responses (our unpublished observation). This fact may be due to the intrinsic capacity of CyaA to orient strongly cell responses to the Th1 type.
We further evaluated the potential of CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 to act as booster vaccines subsequent to priming with BCG::RD1. At day 0, groups of C57BL/6 mice were immunized s.c. with 105 CFU of BCG::RD1 or left unvaccinated. Some groups of BCG::RD1-primed mice were boosted at day 35 with a single injection of 50 μg/mouse of CyaA336-ESAT-6:1-95, CyaA-85A:241-260, or CyaA-MalE:100-114. At day 50, all groups were challenged with a low dose of aerosolized M. tuberculosis H37Rv. It is noteworthy that we have previously determined that, in these experimental conditions and at this time point, BCG::RD1 is no longer detectable in the spleen and lungs of BCG::RD1-immunized hosts (31).
One month postchallenge, M. tuberculosis burden was determined in the lungs and spleen of infected mice. Immunization with BCG::RD1 conferred highly significant protection, i.e., 2 logs at the level of the lungs (Fig. 6A) and >3 logs in the spleen (Fig. 6B). Mice primed with BCG::RD1 and boosted with CyaA336-ESAT-6:1-95 did not exhibit any improved level of protection in the lungs (Fig. 6A) and spleen (Fig. 6B) compared to mice immunized with BCG::RD1 alone. Similarly, mice primed with BCG::RD1 and boosted with CyaA-85A:241-260 did not exhibit any better protection, in terms of lung and spleen CFU, than their counterparts immunized with BCG::RD1 alone. Similar results were obtained when the protection experiment was repeated with mice primed with BCG::RD1 and boosted with CyaA224-ESAT-6:1-95 (data not shown). These results question the rather broadly accepted hypothesis that enhanced protection against tuberculosis correlates with increased IFN- T-cell responses specific to strong M. tuberculosis immunogens (10, 25).
DISCUSSION
In this study, we show that recombinant CyaA toxoids genetically engineered to harbor some promising mycobacterial immunogens can be used to appropriately deliver these immunogens for processing and presentation by APC both in vitro and in vivo and to induce consistent Th1-biased antimycobacterial cell immunity. Indeed, delivery of immunodominant CD4+ T-cell epitopes of antigen 85A and of the entire ESAT-6 polypeptide to APC by recombinant CyaA leads to processing and presentation of the same T-cell epitopes as those generated and presented upon mycobacterial infection. This means that T-cell receptors selected upon immunization with these CyaA constructs were able to recognize cells infected by mycobacteria. Furthermore, on a molar basis, the efficiency of presentation of ESAT-6 was 100-fold higher when this antigen was delivered to APC in fusion to CyaA, compared to presentation of the recombinant ESAT-6 protein.
In line with this result, we have recently shown that CyaA336-ESAT-6:1-95 and also a CyaA construct harboring the complete sequence of the 10-kDa culture filtrate protein (CFP-10) were both able to deliver the mycobacterial antigens to human and bovine APC for specific and highly enhanced ex vivo presentation to T cells obtained from tuberculosis patients and from sensitized donors (39), as well as to T cells from cattle with bovine tuberculosis (37). Thus, these CyaA toxins also have the potential to increase the sensitivity of immunodiagnostic assays.
It is noteworthy that the insertion of the 85A immunodominant regions and the long ESAT-6:1-95 polypeptide did not alter the binding of CyaA to its receptor, CD11b. This has also been checked for CyaA-ESAT-6:1-95 in a human (39) and in a cattle model (37) with the CyaA constructs used in this study. Furthermore, in vivo the CyaA-85A and CyaA-ESAT-6 proteins induced strong antigen-specific lymphoproliferation, IL-2, and IFN- cytokine production in mice in the absence of any IL-4 and IL-5 production. When used as booster vaccines administered following priming with the live attenuated vaccine candidate strain BCG::RD1, the CyaA-85A and CyaA-ESAT-6 constructs were able to strikingly enhance the sensitivity and intensity of induced antigen-specific proliferative and Th1 responses and the frequency of antigen-specific IFN--producing T cells in particular.
It is generally admitted that the best correlate of protection against mycobacterial infection is the level of IFN- responses of CD4+ T cells (10, 25). Our observations, however, challenge this hypothesis, as despite induction of a strong Th1 response to 85A and ESAT-6, prophylactic vaccination with CyaA did not confer on C57BL/6 mice any protection against challenge with a low dose of aerosolized M. tuberculosis H37Rv, as determined by evaluation of the level of bacterial colonization of lungs and spleen. Moreover, boosting with CyaA-85A and CyaA-ESAT-6, subsequent to priming with BCG::RD1, did not improve the protection, despite substantial enhancement of the Th1 responses and notably a consistent increase in antigen-specific IFN- production in mice. This fact could be ascribed to a ceiling of protection in the murine model, which is a limitation of the mouse model of tuberculosis.
In our experimental protocol, mice were immunized with BCG::RD1 at day 0, boosted with CyaA fusion protein at day 30, tested for immunogenicity at day 45, and then challenged with M. tuberculosis at day 60. Our results clearly demonstrated that T cells specific to the immunodominant epitopes of ESAT-6 and 85A are strongly restimulated upon immunization with CyaA harboring these immunogens. One can ask if such T cells stimulated by the CyaA fusions are still present and capable of being restimulated at the time of the challenge, i.e., at day 60. It is important to note that strong Th1 responses were easily detectable in such mice as close as 15 days before the M. tuberculosis challenge. Furthermore, according to our recent observations (24), boosting with the CyaA vector induces long-lasting Th1-polarized responses. Based on these findings, it seems highly unlikely that such strong anti-ESAT-6 and 85A-specific Th1 responses could have totally disappeared at the time of the M. tuberculosis challenge.
We have previously described the enhanced capacity of BCG::RD1, compared to parental BCG, to protect mice and guinea pigs against M. tuberculosis challenge, which directly correlates with the strong anti-ESAT-6 Th1 responses detected in BCG::RD1-vaccinated mice (32). However, the observation that boosting of BCG::RD1-primed mice with CyaA-85A and CyaA-ESAT-6 did not yield any improvement in protection may indicate that the persisting fraction of M. tuberculosis-infected host cells are not sensitive to IFN--producing 85A- and ESAT-6-specific T cells. Therefore, this mycobacterium-colonized cell population seems to escape immune surveillance despite the presence at high frequencies of T cells specific for mycobacterial antigens.
Induction of IFN- T-cell responses appears to be necessary to control the growth of M. tuberculosis in vivo, as suggested by the dramatic exacerbation of tuberculosis in IFN- null mice (6, 12). Here, however, the presence of IFN--producing CD4+ T cells and the increase in their frequency were not sufficient for clearance of M. tuberculosis infection. Indeed, it was also recently reported that the presence of antigen-specific splenocytes producing IFN-, IL-2, and tumor necrosis factor alpha in the spleen and lungs of calves does not correlate with levels of protection induced by a series of M. tuberculosis proteins (15).
The reason for the failure of the CyaA-85A and CyaA-ESAT-6 constructs to induce protection when used as subunit vaccines alone and as a booster upon BCG::RD1 priming remain unclear. It does not appear to be due to the inability of CyaA to induce and/or to enhance the antigen-specific T-cell responses. Indeed, CyaA appears to fulfill the criteria for a useful vaccination vector. CyaA targets DC (14) and can deliver passenger epitopes into the MHC-II presentation pathways (7, 20), with a marked potentiation of the efficiency of MHC-II antigen presentation that depends on CD11b-CyaA interaction (34). Moreover, CyaA is able to initiate T-cell responses in nave mice in the absence of any adjuvant, as also shown here. The present study confirms these previous findings and extends the demonstrations achieved with viral and tumor antigens (11, 30, 33) to bacterial immunogens. Significant protection observed with subunit vaccines based on the ESAT-6, Ag85, and TB10.4 antigens have been achieved by use of dimethyl dioctadecyl ammonium bromide, monophosphoryl lipid A, and trehalose dicorynomycolate as adjuvants (5, 8, 27, 28), all of which possess a strong capacity to stimulate the innate immune system. It is possible that CyaA is not as able as these adjuvants to trigger host innate immunity.
The main antigens currently used in studies on protection against experimental tuberculosis are MTB8.4 (5), Hsp60, Ag85A, Ag85B, and ESAT-6 (27), and ESAT-6-85B (28) and TB10.4-85B (8) fusion proteins. However, it is well established that the majority of M. tuberculosis proteins fail to induce any protective response when used as subunit vaccines and the criteria that define a protective T-cell antigen remain poorly defined. It is admitted that antigens that are unique to the pathogen and are abundantly secreted are of critical importance. Moreover, microarray-based kinetic analyses of gene expression profiles of M. tuberculosis in the lungs of infected mice showed major changes in transcription levels of more than 100 genes around the beginning of the chronic phase of infection (36). Interestingly, a set of these genes are up-regulated only in immunocompetent but not SCID mice. Expression of these genes may reflect adaptation of M. tuberculosis to acquired antimycobacterial immunity, which would support the hypothesis that the growth of M. tuberculosis in immunocompetent mice is influenced by the host immune responses. Interestingly, identification of the genes that are up-regulated upon interaction with host cells has already allowed a successful selection of protective B-cell antigens of Neisseria meningitidis (13). Accordingly, it is tempting to speculate that induction of T-cell responses to the immunogenic proteins that are expressed at enhanced levels when M. tuberculosis bacteria colonize the lungs might provide a promising strategy for containment of persisting M. tuberculosis infection in BCG- and BCG::RD1-immunized mice.
ACKNOWLEDGMENTS
This work was supported by grants from the Institut Pasteur (Programme Transversal de Recherche No. 110) and European Community (Cell Prom Program), Institutional Research Concept No. AVZ50200510, and grant IBS5020311 of the Academy of Sciences of the Czech Republic.
We gratefully acknowledge R. Lo-Man for advice on generation of T-cell hybridomas and Eddie Maranghi for expert animal care in isolators.
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Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Prague, Czech Republic
Unite de Genetique Moleculaire Bacterienne, Institut Pasteur, Paris F-75015, France
Unite deBiochimie des Interactions Macromoleculaires, Institut Pasteur, Paris F-75015, France
ABSTRACT
Bordetella pertussis adenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN--producing CD4+ T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection against Mycobacterium tuberculosis infection. These results question the broadly admitted correlation between the frequency of IFN--producing CD4+ T cells and the level of protection against tuberculosis.
INTRODUCTION
The most feasible strategy to control the increasing number of tuberculosis cases, notably in developing countries, is effective vaccination against Mycobacterium tuberculosis. The only current vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), displays highly variable efficiency (0 to 80%) in preventing adult pulmonary tuberculosis. Several subunit vaccine strategies, including plasmid DNA, bacterial or viral vectors, peptides, or recombinant proteins in adjuvant, have been developed to induce T-cell immunity against mycobacterial antigens. So far, none of these subunit vaccines used alone provides better protection levels than that conferred by BCG. The latter remains the gold standard vaccine model yet is unable to clear infection in mouse model of tuberculosis (9, 26). In the present study, we evaluated the potential of a novel vaccination vector, the adenylate cyclase of Bordetella pertussis CyaA harboring strong M. tuberculosis immunogens, to induce antimycobacterial immunity.
CyaA is a 1,706-amino-acid-long toxin consisting of five functional domains: (i) adenylate cyclase activity (AC) domain (residues 1 to 400), (ii) hydrophobic channel-forming domain (residues 500 to 700), (iii) posttranslationally acylated domain (residues 800 to 1000), (iv) calcium-binding glycine/aspartate-rich repeat domain (residues 1000 to 1600), and (v) C-terminal domain carrying a secretion signal (residues 1600 to 1706) (18). CyaA binds, via its fourth domain, to M2 integrin (CD11b/CD18), expressed on macrophages, neutrophils, natural killer (NK) cells, peritoneal B-1 cells, and notably the major CD8– CD11b+ dendritic cell (DC) population (14). Upon interaction with CD11b, the CyaA AC domain can be endocytosed and degraded for major histocompatibility complex (MHC) II-restricted presentation (20, 34). Several permissive sites have been identified within various domains of CyaA in which it is possible to genetically insert exogenous peptides without interfering with the functions of CyaA (17, 20, 35). T-cell epitopes inserted into these permissive sites are efficiently carried to the MHC-II pathways of antigen-presenting cells (APC) and induce in vivo CD4+ Th1 responses without the need for any adjuvant (7, 34).
Immunization of mice with genetically detoxified CyaA bearing appropriate T-cell epitopes provides full prophylactic protection against lethal lymphocytic choriomeningitis virus challenge (33), efficient therapeutic antitumor immunity (11) and complete therapeutic efficacy against tumor cells expressing an immunogenic oncoprotein of human papillomavirus 16 in a mouse experimental model mimicking human papillomavirus-induced cervical cancer (21, 30).
It is widely admitted that prophylaxis of tuberculosis requires induction of CD4+ Th1 responses. Indeed, induction of high levels of antigen-specific gamma interferon (IFN-) is considered the best antimycobacterial protection correlate (10, 25). Therefore, CyaA appears to be an attractive vector to be evaluated in experimental models of tuberculosis.
In the present study, we investigated the potential in antituberculosis vaccination of fusions of CyaA to the promising immunogen candidates 85A and the 6-kDa early secreted antigenic target (ESAT-6) protein. 85A is a member of the 85 complex consisting of three related secreted proteins (A, B, and C), with various degrees of mycolyl transferase and fibronectin binding activity (2, 38). 85A and 85B are potent targets of antimycobacterial T cells. ESAT-6 is encoded in the ESAT-6 operon within a 10-kb chromosomal region of M. tuberculosis referred to as the region of difference 1 (RD1) that is absent from the BCG genome. ESAT-6 is secreted by a dedicated export system, i.e., the ESX-1 secretion machinery (4). The exact biological function of ESAT-6 has not yet been elucidated but this molecule constitutes a strong immunogen in humans and rodents (4, 27). We constructed recombinant CyaA toxins carrying, in their AC domain, immunodominant epitopes of 85A, restricted in H-2d or H-2b haplotype (16), or the complete sequence of ESAT-6 (1). We first investigated in vitro whether these CyaA constructs were able to deliver to the MHC-II pathway of APC the same epitopes as those generated upon mycobacterial infection. We then studied their immunogenicity in vivo, as subunit immunization vectors.
Boosting vaccination with strong mycobacterial immunogens delivered by a subunit nonreplicative vaccine after priming with live recombinant mycobacterial vaccine has been proposed to improve antimycobacterial immunity. The BCG Pasteur strain stably complemented with the complete ESX-1 system (BCG::RD1) expresses and secretes ESAT-6 (32). This complementation strongly modifies host-mycobacteria interactions as, besides its increased virulence, BCG::RD1 possesses an enhanced capacity to activate cells of the innate or adaptive immune system (22) and to induce strong ESAT-6-specific Th1 CD4+ T-cell responses (32). These characteristics correlate with the enhanced protective capacity of BCG::RD1 against infection with M. tuberculosis in mice and guinea pigs compared to the parental BCG strain (32). We characterized the immunogenicity of CyaA-85A and CyaA-ESAT-6 constructs as boosters after priming with BCG::RD1. Finally, we evaluated the possible protective effect of these CyaA constructs as a subunit vaccine alone or as a booster after priming with BCG::RD1 against aerosol challenge with M. tuberculosis.
MATERIALS AND METHODS
Peptides. The 85A:101-120, 85A:241-260 (16), ESAT-6:1-20 (3), and MalE:100-140 (19) peptides were synthesized by Neosystem (Strasbourg, France).
Recombinant CyaA toxoid construction. Escherichia coli XL1-Blue (Stratagene, Amsterdam, The Netherlands) was used throughout the work for recombinant DNA construction and for expression of mycobacterial antigen/MHC-II epitopes inserted into the AC domain of CyaA. Bacteria transformed with appropriate plasmids derived from pT7CACT1 (29) were grown at 37°C in Luria-Bertani medium supplemented with 150 μg of ampicillin per ml. Construction of the plasmid for production of the CyaA336-ESAT-6 protein was described previously (37).
The CyaA constructs, carrying, between residues 335 and 336, 85A:101-120 (LTSELPGWLQANRHV KPTGS) or 85A:241-260 (QDAYNAGGGHNGVF DFPDSG), were obtained by in-frame insertions of corresponding pairs of annealed synthetic oligonucleotides of the sequences 5'-ACCACTGACCTCTGAACTGCCGGGTTGGCTGCAGGCTAACCGTCACGTGAAACCGACAGGTTCC and 5'-GTACGGAACCGATCGGTTTCACGTGACGGTTAGCCTGCAGCCAACCCGGCAGTTCAGAGGTCAGTG for 85A:101-120 and 5'-GTACCACAGGACGCTTACAACGCTGGTGGTGGTCACAACGGTGTTTTCGACTTCCCGGACTCTGGTTAC and 5'-GTACGTAACCAGAGTCCGGGAAGTCGAAAACACCGTTCTGACCACCACCAGCGTTGTAAGCGTCCTGTG for 85A:241-260. The oligonucleotides were designed (i) to introduce a restriction site for rapid identification of insertion mutants, (ii) to stop CyaA synthesis when inserted in the inverted orientation, and (iii) to destroy the original BsrGI insertion site on ligation.
The orientation and exact sequence of inserted oligonucleotides were verified by DNA sequencing. The recombinant CyaA proteins carrying ESAT-6:1-95, 85A:101-120, or 85A:241-260 were produced in E. coli XL1-Blue, purified from inclusion bodies in 8 M urea, 50 mM Tris-Cl, pH 8, 2 mM EDTA, and characterized as previously described (29). The resulting proteins were free of any detectable adenylate cyclase enzymatic activity.
Insertion of polypeptides into position 336 detoxifies CyaA. To generate CyaA harboring inserts at position 224, we used a derivative of CyaA, CyaA-E5, which is catalytically inactive (but still invasive) as a result of the insertion of the dipeptide Leu-Gln between codons 188 and 189 of CyaA. To construct the plasmid used for production of the detoxified CyaA224-ESAT-6:1-95 protein, the full-length ESAT-6 coding region was PCR amplified with primers ESAT-6N (5'-ACTAGCTAGCATGACAGAGCAGCAGTGGAATTTCGCGGG-3') and ESAT-6K (5'-CGGGGTACCTGCGAACATCCCAGTGACGTTGCCTTCGGT-3'). The 304-bp PCR product was purified, cut by NheI and KpnI, and ligated into plasmid pCACT-OVA-E5 (11) digested by the same enzymes. The nucleotide sequence of the insert was verified by DNA sequencing. The recombinant CyaA224-ESAT-6:1-95 protein was produced in E. coli BLR and purified from inclusion bodies as described previously (30).
Mycobacteria. BCG::RD1 and M. tuberculosis (H37Rv) were grown in Middlebrook 7H9 medium as previously described (31).
Mice and immunization. Female BALB/c (H-2d) and C57BL/6 (H-2b) mice were purchased from Charles River (Arbresle, France). Six- to 10-week-old mice each received intravenously in the tail vein 50 μg of CyaA harboring mycobacterial immunogens. For immunization with peptides, mice received subcutaneously (s.c.) at the base of the tail 50 μg/mouse of individual peptides emulsified in incomplete Freund's adjuvant. Immunization with BCG::RD1 was performed by subcutaneous injection of 105 CFU/mouse. Mice immunized with BCG::RD1 were placed in isolators in the Pasteur Institute A3 animal facilities. All animal studies were approved by the Pasteur Institute Safety Committee in accordance with French and European guidelines.
Generation of T-cell hybridomas and antigen presentation assays. T-cell hybridomas specific for the 85A:101-120 and 85A:241-260 peptides were generated from BALB/c and C57BL/6 mice, respectively, injected with 50 μg/mouse of the appropriate peptide emulsified in incomplete Freund's adjuvant 10 days postimmunization. Splenocytes were stimulated with 10 μg/ml of the homologous peptide for 4 days. Viable cells were harvested on Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) and were fused with an equal number of BW51-47 thymoma cells by use of polyethylene glycol 1500 (Roche Diagnostics GmbH, Mannheim, Germany) as previously described (23).
BALB/c- and C57BL/6-derived T-cell hybridomas were screened for their capacity to specifically release interleukin-2 (IL-2) upon stimulation with the 85A:101-120 and 85A:241-260 peptides, respectively. To determine their capability to recognize APC infected with mycobacteria, bone marrow-derived DC (BM-DC), generated in antibiotic-free conditions in the presence of 10 ng/ml of granulocyte-macrophage colony-stimulating factor were infected at day 5 of culture with BCG at a multiplicity of infection of 1 for 16 h, washed three times, and incubated with 105 T-cell hybridomas.
The presence of IL-2 in the supernatant of cultures was monitored by standard IL-2-specific enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Proliferation of the CTLL-2 cell line is dependent on IL-2 in a dose-dependent manner. CTLL-2 cells (104 cells/well) were incubated with supernatant of T-cell hybridomas, pulsed 48 h later with 1 μCi of [methyl-3H]thymidine (ICN, Orsay, France)/well for 16 h, and the incorporated cpm were counted in a Wallac Microbeta counter (Perkin-Elmer, Cortaboeuf Cedex, France). L fibroblasts transfected with I-Ad, I-Ed and I-Ab molecules were used as peptide-presenting cells in the same type of antigen presentation assay for determination of the restricting element of the peptide presentation to the T-cell hybridomas.
T-cell assays. To measure T-cell proliferative responses to mycobacterial antigens, splenocytes of immunized mice were cultured (106 cells/well) in 96-well flat-bottom plates in synthetic HL-1 medium (BioWhittaker, Walkersville, MD) as previously described (32) in the presence of various concentrations of appropriate antigens. Cultures were pulsed 72 h later with 1 μCi of [methyl-3H]thymidine/well for 16 h and the incorporated cpm were counted in a Wallac Microbeta counter.
IL-2, IL-4, IL-5, and IFN- production by splenocytes was assessed subsequent to in vitro stimulation with 4 μg/ml of recombinant proteins or 10 μg/ml of synthetic homologous or control peptides. Amounts of IL-2 were determined in culture supernatants after 24 h incubation by a CTLL-2 bioassay. The amounts of IL-4, IL-5, and IFN- were quantified in culture supernatants after 72 h incubation by ELISA as detailed elsewhere (7). IFN- Elispot was performed as previously described (7) by incubation of serial dilutions of splenocytes on anti-IFN- monoclonal antibody (R4-6A2)-coated 96-well Multiscreen filtration plates (Millipore, France). Cells were stimulated with 1 μg/ml of peptide 85A:241-260 or negative control peptide for 36 h without addition of other growth factors. Biotinylated anti-IFN- monoclonal antibody (XMG1.2) and streptavidin-alkaline phosphatase (PharMingen) were used to reveal the spots.
Protection assays against infection with M. tuberculosis. At the indicated time points, unvaccinated and immunized mice (six/group) were challenged by use of a homemade nebulizer via the aerosol route; 2 ml of a suspension containing 106 CFU/ml was aerosolized to obtain an inhaled dose of 100 ± 10 CFU/mouse. Infected mice were placed in isolators. One month postchallenge, the lungs and spleen of the mice were homogenized by use of an MM300 organ homogenizer (QIAGEN, Courtaboeuf, France) and 2.5-mm-diameter glass beads. Serial fivefold dilutions of homogenates were cultured on 7H11 agar supplemented with bovine albumin, dextrose, and catalase (Difco, Becton Dickinson, Sparks, MD). CFU were counted after 15 to 18 days of incubation at 37°C.
RESULTS
CyaA harboring immunodominant epitopes of 85A or complete sequence of ESAT-6. Fragments of 85A containing immunodominant epitopes, i.e., 85A:101-120 (restricted by I-Ed) and 85A:241-260 (restricted by I-Ab) (16), were genetically inserted into the AC domain of CyaA at position 336 (Table 1). In parallel, the complete sequence of ESAT-6 (ESAT-6:1-95) (1) was inserted at positions 224 and 336 of the CyaA AC domain. Indeed, so far, the permissive sites of CyaA have been studied and characterized mainly by insertion of short reporter peptide sequences. Thus, two insertion sites, 224 and 336, were chosen here to evaluate and to compare their potential to carry and deliver the 95-residue-long ESAT-6 sequence.
A CyaA harboring residues 100 to 114 of maltose binding protein of Escherichia coli at position 336 (MalE:100-114), containing an I-Ab/d-restricted epitope (19), was used in all experiments as a negative CyaA control. It was verified that insertion of these amino acid residues, and notably of the long ESAT-6:1-95 polypeptide, did not alter the capacity of the recombinant CyaA constructs to engage its surface receptor CD11b and to compete with a biotinylated intact CyaA tracer for specific binding to CD11b-transfected Chinese hamster ovary (CHO) cells (data not shown).
Incubation with recombinant CyaA and infection with mycobacteria leads to processing and MHC-II-restricted presentation of the same mycobacterial epitopes. Using splenocytes of BALB/c (H-2d) and C57BL/6 (H-2b) mice immunized with the 85A:101-120 and 85A:241-260 synthetic peptides, respectively, we generated panels of T-cell hybridomas specific to these peptides. The 85A:101-120-specific T-cell hybridomas were all restricted by I-Ed and those specific to 85A:241-260 by I-Ab (data not shown). In each genetic background, only a limited number (2 of 15) of the T-cell hybridomas, referred to as AA1 and CG11 for the H-2d haplotype and BF9 and DE10 for the H-2b haplotype cells, were able to recognize histocompatible BM-DC infected in vitro with BCG (Fig. 1A and B). This observation suggests that immunization with a single peptide may lead to generation and presentation of more than one epitope and thereby to stimulation of diverse T-cell receptors.
One can imagine the existence of several fine possibilities of adjustment of a given peptide in the MHC-II presentation groove and the presence of C-terminal or N-terminal sequences of the synthetic peptide which may be present during the presentation of synthetic peptides while the epitope generated during the processing of CyaA and mycobacteria would be much more precise and limited. Of note, all T-cell hybridomas able to recognize APC incubated with CyaA-85A were also able to recognize BCG-infected APC. We only selected T-cell hybridomas specific to the epitopes generated and presented upon mycobacterial infection.
The latter T-cell hybridomas provide a unique tool to examine in vitro whether antigen delivery by CyaA-85A:101-120 and CyaA-85A:241-260 to APC leads to processing and presentation of the same epitopes as those displayed upon infection with mycobacteria. AA1 and CG11 (restricted by I-Ed) and BF9 and DE10 (restricted by I-Ab) hybridomas were indeed able to recognize, in a highly sensitive and specific manner, appropriate histocompatible BM-DC incubated with CyaA-85A:101-120 (Fig. 1C) and CyaA-85A:241-260 (Fig. 1D), respectively. These data demonstrated that in vitro processing of these CyaAs leads to the generation and presentation of immunodominant 85A epitopes that can be recognized by mycobacterium-specific T cells.
To investigate in vitro delivery of ESAT-6 by CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95, we used T splenocytes from BCG::RD1-immunized C57BL/6 mice. These cells (Fig. 2), but not those from BCG-immunized control mice (data not shown), released marked levels of IFN- subsequent to in vitro stimulation with CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95 (Fig. 2). It is noteworthy that ESAT-6:1-20 is the only T-cell epitope on the ESAT-6 sequence, restricted in H-2b haplotype (3) and we have previously shown that T-cell responses from BCG::RD1-immunized C57BL/6 mice to ESAT-6:1-20 were only due to the CD4+ T-cell compartment (32). An in vitro restimulation with the CyaA-MalE:100-114 negative control did not result in IFN- responses, showing the specificity of the antigen presentation. Thus, insertion of ESAT-6 at either position 224 or 336 in CyaA allowed delivery of the ESAT-6 sequence for processing. Moreover, the efficiency of ESAT-6 presentation was markedly improved (100-fold) when it was delivered to APC in the fusion to CyaA, e.g., CyaA224-ESAT-6:1-95 and CyaA336-ESAT-6:1-95, compared to presentation of the ESAT-6 recombinant protein alone. This result demonstrates that in vitro processing of CyaA-ESAT-6:1-95 leads to highly efficient generation and presentation of the immunodominant ESAT-6 epitope that is recognized by mycobacterium-specific T cells.
Taken together, these observations strongly suggest that immunization with CyaA carrying mycobacterial antigens may efficiently stimulate T cells capable of recognizing cells infected with mycobacteria in vivo.
In vivo immunogenicity of CyaA harboring mycobacterial immunogens. To investigate the immunogenicity of these CyaAs in vivo, BALB/c and C57BL/6 mice received a single intravenous injection (50 μg/mouse) of purified recombinant CyaA-85A:101-120 and CyaA-85A:241-260, respectively. The proliferative and Th1/Th2 cytokine responses of these mice were evaluated at day 15 postimmunization following in vitro stimulation of their splenocytes with the homologous and control peptides. CyaA-85A:101-120 and CyaA-85A:241-260 induced substantial proliferative, IL-2, and IFN- responses in BALB/c (Fig. 3A and B) and in C57BL/6 (Fig. 3C and D) mice respectively. CyaA224-ESAT-6:1-95 (data not shown) and CyaA336-ESAT-6:1-95 (Fig. 3E and F) were also highly immunogenic in C57BL/6 mice, as shown by strong proliferative and IL-2 responses of their splenocytes stimulated in vitro with the immunodominant ESAT-6:1-20 peptide. However, only low levels of IFN- were obtained in CyaA336-ESAT-6:1-95-immunized mice (Fig. 3F). Immunization with CyaA-MalE:100-114 did not lead to any immune response to 85A and ESAT-6, showing the specificity of the induced T-cell responses. Moreover, no Th2 cytokine (IL-4 and IL-5) responses were detected in these experimental groups (data not shown). Thus, the CyaA constructs bearing the 85A:101-120, 85A:241-260, and ESAT-6:1-95 polypeptides allowed the induction of strong and specific antimycobacterial Th1 immune responses in mice.
Marked boosting effect of CyaA-85A and CyaA-ESAT-6 following priming with BCG::RD1. We next sought to evaluate the efficiency of a heterologous prime-boost vaccination scheme, comprising priming immunization with the BCG::RD1 live attenuated vaccine candidate (32), followed by a booster immunization with CyaA-85A and CyaA-ESAT-6. On day 0, C57BL/6 mice were immunized (s.c.) with 105 CFU/mouse of BCG::RD1. At day 35, groups of mice primed and not with BCG::RD1 received an intravenous (i.v.) injection of 50 μg/mouse of CyaA-85A:241-260, CyaA336-ESAT-6:1-95, and CyaA-MalE:100-114. We have previously demonstrated that C57BL/6 mice immunized with a single dose of BCG::RD1 mounted strong ESAT-6- and 85A-specific proliferative and IFN- responses that were readily detectable 15 to 21 days postimmunization (32).
Fifty days after s.c. immunization of mice with BCG::RD1, the proliferative (Fig. 4A and D) and IFN- (Fig. 4B and E) responses to 85A:241-260 and to ESAT-6 were no longer detectable and near the detection limit. At this time point, significant proliferative responses (Fig. 4A and D) and IFN- (Fig. 4B and E) were detected in mice that were immunized 15 days earlier with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95. Moreover, compared to mice that received only BCG::RD1, their counterparts primed with BCG::RD1 and boosted with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 displayed a consistent increase in both the intensity and the sensitivity of the proliferative response (Fig. 4A and D), as well as a highly increased IFN- response (Fig. 4B and E).
Based on independent but similar experiments, we have observed that a boost with CyaA:MalE:100-114 and with a wild-type CyaA negative control in BCG::RD1-primed mice induced no increase in 85A- and ESAT-6-specific proliferative and 85A-specific IFN- responses (data not shown). The relatively small amounts of anti-ESAT-6 IFN- response in the group of BCG::RD1-primed and CyaA-MalE:100-114-boosted mice can be due to residual ESAT-6-specific response induced by initial BCG::RD1 immunization as it can also be detected in the group of unboosted and BCG::RD1-immunized mice (Fig. 4E). Of note, no anti-85A-specific IFN- response was detected in BCG:: RD1-primed and CyaA-MalE:100-114-boosted mice (data not shown).
At this time point, the frequency of 85A:241-260-specific and IFN--producing T splenocytes in mice primed with BCG::RD1 and boosted with CyaA-85A:241-260 was 50 times higher than those of CyaA-85A:241-260-immunized mice (Fig. 4C). Furthermore, no Th2 cytokine response was detectable in any of the experimental groups (data not shown). These data demonstrated that CyaA harboring mycobacterial immunogens can efficiently boost antimycobacterial Th1 immunity and particularly the IFN- responses induced by BCG::RD1.
Vaccine efficacy of CyaA harboring the mycobacterial immunogens alone and as boosters against infection with M. tuberculosis. We evaluated the potential of the recombinant CyaA to protect mice against M. tuberculosis challenge in groups of C57BL/6 mice (n = 6). Control mice were left unvaccinated or vaccinated with BCG::RD1 at day 0. At day 30, groups of mice received a single i.v. injection of 50 μg/mouse of CyaA-85A:241-260, CyaA336-ESAT-6:1-95, or CyaA-MalE:100-114. At day 60, mice were infected with a small dose of aerosolized M. tuberculosis H37Rv. At 1 month postchallenge, bacterial burden was determined in the lungs and spleen of infected mice.
No protection was observed in CyaA-immunized mice, while the CFU counts in the lungs (Fig. 5A) and spleen (Fig. 5B) of BCG::RD1-immunized mice were 2 logs lower than in unvaccinated mice. Thus, despite induction of powerful T-cell proliferation and of significant Th1 cytokine response to these potent CyaA-delivered immunogens, immunization with CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 was not capable of inducing protection against M. tuberculosis challenge in the murine model.
In an independent experiment, C57BL/6 mice were immunized at a 3-week interval by two intraperitoneal injections of CyaA336-ESAT-6:1-95 adjuvanted in alum and again no protection was observed against an H37Rv challenge given 1 week after the last injection (data not shown). It is noteworthy that, subsequent to immunization with CyaA bearing mycobacterial antigen adjuvanted in alum, mice mounted only Th1 but not Th2 responses (our unpublished observation). This fact may be due to the intrinsic capacity of CyaA to orient strongly cell responses to the Th1 type.
We further evaluated the potential of CyaA-85A:241-260 and CyaA336-ESAT-6:1-95 to act as booster vaccines subsequent to priming with BCG::RD1. At day 0, groups of C57BL/6 mice were immunized s.c. with 105 CFU of BCG::RD1 or left unvaccinated. Some groups of BCG::RD1-primed mice were boosted at day 35 with a single injection of 50 μg/mouse of CyaA336-ESAT-6:1-95, CyaA-85A:241-260, or CyaA-MalE:100-114. At day 50, all groups were challenged with a low dose of aerosolized M. tuberculosis H37Rv. It is noteworthy that we have previously determined that, in these experimental conditions and at this time point, BCG::RD1 is no longer detectable in the spleen and lungs of BCG::RD1-immunized hosts (31).
One month postchallenge, M. tuberculosis burden was determined in the lungs and spleen of infected mice. Immunization with BCG::RD1 conferred highly significant protection, i.e., 2 logs at the level of the lungs (Fig. 6A) and >3 logs in the spleen (Fig. 6B). Mice primed with BCG::RD1 and boosted with CyaA336-ESAT-6:1-95 did not exhibit any improved level of protection in the lungs (Fig. 6A) and spleen (Fig. 6B) compared to mice immunized with BCG::RD1 alone. Similarly, mice primed with BCG::RD1 and boosted with CyaA-85A:241-260 did not exhibit any better protection, in terms of lung and spleen CFU, than their counterparts immunized with BCG::RD1 alone. Similar results were obtained when the protection experiment was repeated with mice primed with BCG::RD1 and boosted with CyaA224-ESAT-6:1-95 (data not shown). These results question the rather broadly accepted hypothesis that enhanced protection against tuberculosis correlates with increased IFN- T-cell responses specific to strong M. tuberculosis immunogens (10, 25).
DISCUSSION
In this study, we show that recombinant CyaA toxoids genetically engineered to harbor some promising mycobacterial immunogens can be used to appropriately deliver these immunogens for processing and presentation by APC both in vitro and in vivo and to induce consistent Th1-biased antimycobacterial cell immunity. Indeed, delivery of immunodominant CD4+ T-cell epitopes of antigen 85A and of the entire ESAT-6 polypeptide to APC by recombinant CyaA leads to processing and presentation of the same T-cell epitopes as those generated and presented upon mycobacterial infection. This means that T-cell receptors selected upon immunization with these CyaA constructs were able to recognize cells infected by mycobacteria. Furthermore, on a molar basis, the efficiency of presentation of ESAT-6 was 100-fold higher when this antigen was delivered to APC in fusion to CyaA, compared to presentation of the recombinant ESAT-6 protein.
In line with this result, we have recently shown that CyaA336-ESAT-6:1-95 and also a CyaA construct harboring the complete sequence of the 10-kDa culture filtrate protein (CFP-10) were both able to deliver the mycobacterial antigens to human and bovine APC for specific and highly enhanced ex vivo presentation to T cells obtained from tuberculosis patients and from sensitized donors (39), as well as to T cells from cattle with bovine tuberculosis (37). Thus, these CyaA toxins also have the potential to increase the sensitivity of immunodiagnostic assays.
It is noteworthy that the insertion of the 85A immunodominant regions and the long ESAT-6:1-95 polypeptide did not alter the binding of CyaA to its receptor, CD11b. This has also been checked for CyaA-ESAT-6:1-95 in a human (39) and in a cattle model (37) with the CyaA constructs used in this study. Furthermore, in vivo the CyaA-85A and CyaA-ESAT-6 proteins induced strong antigen-specific lymphoproliferation, IL-2, and IFN- cytokine production in mice in the absence of any IL-4 and IL-5 production. When used as booster vaccines administered following priming with the live attenuated vaccine candidate strain BCG::RD1, the CyaA-85A and CyaA-ESAT-6 constructs were able to strikingly enhance the sensitivity and intensity of induced antigen-specific proliferative and Th1 responses and the frequency of antigen-specific IFN--producing T cells in particular.
It is generally admitted that the best correlate of protection against mycobacterial infection is the level of IFN- responses of CD4+ T cells (10, 25). Our observations, however, challenge this hypothesis, as despite induction of a strong Th1 response to 85A and ESAT-6, prophylactic vaccination with CyaA did not confer on C57BL/6 mice any protection against challenge with a low dose of aerosolized M. tuberculosis H37Rv, as determined by evaluation of the level of bacterial colonization of lungs and spleen. Moreover, boosting with CyaA-85A and CyaA-ESAT-6, subsequent to priming with BCG::RD1, did not improve the protection, despite substantial enhancement of the Th1 responses and notably a consistent increase in antigen-specific IFN- production in mice. This fact could be ascribed to a ceiling of protection in the murine model, which is a limitation of the mouse model of tuberculosis.
In our experimental protocol, mice were immunized with BCG::RD1 at day 0, boosted with CyaA fusion protein at day 30, tested for immunogenicity at day 45, and then challenged with M. tuberculosis at day 60. Our results clearly demonstrated that T cells specific to the immunodominant epitopes of ESAT-6 and 85A are strongly restimulated upon immunization with CyaA harboring these immunogens. One can ask if such T cells stimulated by the CyaA fusions are still present and capable of being restimulated at the time of the challenge, i.e., at day 60. It is important to note that strong Th1 responses were easily detectable in such mice as close as 15 days before the M. tuberculosis challenge. Furthermore, according to our recent observations (24), boosting with the CyaA vector induces long-lasting Th1-polarized responses. Based on these findings, it seems highly unlikely that such strong anti-ESAT-6 and 85A-specific Th1 responses could have totally disappeared at the time of the M. tuberculosis challenge.
We have previously described the enhanced capacity of BCG::RD1, compared to parental BCG, to protect mice and guinea pigs against M. tuberculosis challenge, which directly correlates with the strong anti-ESAT-6 Th1 responses detected in BCG::RD1-vaccinated mice (32). However, the observation that boosting of BCG::RD1-primed mice with CyaA-85A and CyaA-ESAT-6 did not yield any improvement in protection may indicate that the persisting fraction of M. tuberculosis-infected host cells are not sensitive to IFN--producing 85A- and ESAT-6-specific T cells. Therefore, this mycobacterium-colonized cell population seems to escape immune surveillance despite the presence at high frequencies of T cells specific for mycobacterial antigens.
Induction of IFN- T-cell responses appears to be necessary to control the growth of M. tuberculosis in vivo, as suggested by the dramatic exacerbation of tuberculosis in IFN- null mice (6, 12). Here, however, the presence of IFN--producing CD4+ T cells and the increase in their frequency were not sufficient for clearance of M. tuberculosis infection. Indeed, it was also recently reported that the presence of antigen-specific splenocytes producing IFN-, IL-2, and tumor necrosis factor alpha in the spleen and lungs of calves does not correlate with levels of protection induced by a series of M. tuberculosis proteins (15).
The reason for the failure of the CyaA-85A and CyaA-ESAT-6 constructs to induce protection when used as subunit vaccines alone and as a booster upon BCG::RD1 priming remain unclear. It does not appear to be due to the inability of CyaA to induce and/or to enhance the antigen-specific T-cell responses. Indeed, CyaA appears to fulfill the criteria for a useful vaccination vector. CyaA targets DC (14) and can deliver passenger epitopes into the MHC-II presentation pathways (7, 20), with a marked potentiation of the efficiency of MHC-II antigen presentation that depends on CD11b-CyaA interaction (34). Moreover, CyaA is able to initiate T-cell responses in nave mice in the absence of any adjuvant, as also shown here. The present study confirms these previous findings and extends the demonstrations achieved with viral and tumor antigens (11, 30, 33) to bacterial immunogens. Significant protection observed with subunit vaccines based on the ESAT-6, Ag85, and TB10.4 antigens have been achieved by use of dimethyl dioctadecyl ammonium bromide, monophosphoryl lipid A, and trehalose dicorynomycolate as adjuvants (5, 8, 27, 28), all of which possess a strong capacity to stimulate the innate immune system. It is possible that CyaA is not as able as these adjuvants to trigger host innate immunity.
The main antigens currently used in studies on protection against experimental tuberculosis are MTB8.4 (5), Hsp60, Ag85A, Ag85B, and ESAT-6 (27), and ESAT-6-85B (28) and TB10.4-85B (8) fusion proteins. However, it is well established that the majority of M. tuberculosis proteins fail to induce any protective response when used as subunit vaccines and the criteria that define a protective T-cell antigen remain poorly defined. It is admitted that antigens that are unique to the pathogen and are abundantly secreted are of critical importance. Moreover, microarray-based kinetic analyses of gene expression profiles of M. tuberculosis in the lungs of infected mice showed major changes in transcription levels of more than 100 genes around the beginning of the chronic phase of infection (36). Interestingly, a set of these genes are up-regulated only in immunocompetent but not SCID mice. Expression of these genes may reflect adaptation of M. tuberculosis to acquired antimycobacterial immunity, which would support the hypothesis that the growth of M. tuberculosis in immunocompetent mice is influenced by the host immune responses. Interestingly, identification of the genes that are up-regulated upon interaction with host cells has already allowed a successful selection of protective B-cell antigens of Neisseria meningitidis (13). Accordingly, it is tempting to speculate that induction of T-cell responses to the immunogenic proteins that are expressed at enhanced levels when M. tuberculosis bacteria colonize the lungs might provide a promising strategy for containment of persisting M. tuberculosis infection in BCG- and BCG::RD1-immunized mice.
ACKNOWLEDGMENTS
This work was supported by grants from the Institut Pasteur (Programme Transversal de Recherche No. 110) and European Community (Cell Prom Program), Institutional Research Concept No. AVZ50200510, and grant IBS5020311 of the Academy of Sciences of the Czech Republic.
We gratefully acknowledge R. Lo-Man for advice on generation of T-cell hybridomas and Eddie Maranghi for expert animal care in isolators.
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