JAC Advance Access originally published online on March 2, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):924-930; doi:10.1093/jac/dkl066
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Immunogenicity of meningococcal PBP2 during natural infection and protective activity of anti-PBP2 antibodies against meningococcal bacteraemia in mice
Neisseria Unit, National Reference Center for Meningococci, Department of Molecular Medicine, Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex 15, France
* Corresponding author. Tel: +33-1-45-68-89-58; Fax: +33-1-40-61-30-34; E-mail: lzaranto{at}pasteur.fr
Received 4 November 2005; returned 15 December 2005; revised 2 January 2006; accepted 14 February 2006
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Abstract |
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Objective: To evaluate the immunogenicity of the meningococcal penicillin-binding protein 2 (PBP2) and its potential as a vaccine candidate.
Methods: The immunogenicity of meningococcal PBP2 was investigated using acute and convalescent sera from patients who recovered from meningococcal disease. Sera were tested against purified recombinant PBP2s corresponding to meningococcal isolates of different genetic lineages, of different serogroups and with various susceptibility levels to penicillin G. Mice were vaccinated with recombinant PBP2 and challenged with Neisseria meningitidis. A purified anti-PBP2 rabbit IgG was also used for passive protection experiments in mice.
Results: Convalescent patients' sera recognized PBP2s from different strains, showing that this protein is immunogenic in meningococcal disease. Vaccination with purified recombinant PBP2 and purified anti-PBP2 rabbit IgG antibody conferred protection against experimental meningococcaemia in mice.
Conclusion: These data argue for considering meningococcal PBP2 as a vaccine candidate.
Keywords: Neisseria meningitidis , vaccine , penicillin-binding proteins , animal model
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Introduction |
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Management of meningococcal disease requires immediate antibiotic therapy for the patient as well as chemoprophylaxis and vaccination (when applicable) of household contacts to prevent secondary cases and epidemics. Penicillin G is the antibiotic of choice against meningococcal disease,1 but meningococcal strains with diminished susceptibility are being increasingly recorded.2,3 Current vaccines against Neisseria meningitidis are based on capsular polysaccharide and are available only against strains belonging to serogroups A, C, Y and W135, not against strains of serogroup B. Protein candidates such as the major outer membrane porin (PorA) and the transferrin-binding protein B (TbpB) have been explored to develop vaccines against strains of serogroup B.4,5 Outer membrane vesicle-based vaccines have also been developed.6–8 However, these strategies are hindered by the narrow specificity of the vaccine and by the high degree of variability of N. meningitidis owing to horizontal DNA exchanges between strains, resulting in alterations of genetic loci and emergence of escape variants.9
Penicillin-binding proteins (PBPs) are conserved proteins that play a major role in peptidoglycan biosynthesis.10 Their possible role as an immunogenic protein and a vaccine candidate has not been analysed in N. meningitidis. Three PBPs (PBP1, PBP2 and PBP3) have been reported in N. meningitidis,11 but others were identified in the genome sequences.12,13 PBP2, encoded by the penA gene, is a 60 kDa protein which is homologous to high-molecular-weight class B PBPs and most likely catalyses a transpeptidation reaction that is necessary for the cross-linking of peptidoglycan in the meningococcal cell wall. Highly related penAS and different penAI alleles have been described in susceptible strains (PenS) and strains with reduced susceptibility to penicillin G (PenI), respectively.14 While the 5' half of penA is highly conserved, alterations in the 3' half of the gene are responsible for reduced susceptibility to penicillin G and occur through DNA transformation.15 The active serine residue SXXK, the SXN and the KTG motifs, which are all located in the C-terminal transpeptidase region, are usually conserved, but a high degree of polymorphism is observed in the surrounding sequences.14,16
The aim of this work was to test the meningococcal PBP2 as an immunogen and a vaccine candidate against N. meningitidis.
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Materials and methods |
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Bacterial strains and media
Meningococcal strains used in this study and their characteristics are listed in Table 1.
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Phenotypic and genotypic characterizations were performed as described previously.17 Bacteria were grown at 37°C under 5% CO2 on GCB medium (Difco, Detroit, MI, USA) with Kellogg's supplements.18 Penicillin G susceptibility testing was performed using Etest (Solna, Sweden) on Mueller–Hinton agar supplemented with sheep blood as described previously.19 Escherichia coli strains DH5 and BL21(DE3) pLysS were used to prepare recombinant plasmids and for overproduction of recombinant PBP2, respectively. When required, kanamycin and chloramphenicol were added to the L medium (Difco, Detroit, MI, USA) at concentrations of 40 and 15 mg/L, respectively.
Cloning and expression of recombinant PBP2 proteins
penA genes, lacking the sequence encoding the 41 residues of the N-terminal transmembrane domain (41 amino acid residues) of PBP2, from several meningococcal PenS and PenI isolates were cloned into the pET28b expression vector (Novagen, Nottingham, UK). The E. coli BL21(DE3) pLysS strain was then transformed with these recombinant plasmids and His6-tag-recombinant proteins PBP2CS and PBP2CI (from meningococcal PenS and PenI isolates, respectively) were overexpressed under the control of the T7 bacteriophage promoter. Purification of the recombinant proteins was performed using a nickel nitrilotriacetic acid-agarose column (Qiagen, Düren-Germany), as reported previously.16 After the SDS–PAGE analysis, the fractions containing purified PBP2 were pooled, dialysed against PBS, adjusted to 20% glycerol (v/v) and stored at –20°C until use.
Immunodetection of recombinant PBP2 proteins
Six paired sera were obtained from six patients (age range 5–16 years) who recovered from invasive meningococcal infection. They consisted, for each patient, of an acute serum obtained at admission to hospital and a convalescent serum obtained 7–20 days later. Control sera were obtained from an 11-year-old patient who was admitted to hospital for an infection not caused by N. meningitidis. Immunodetection of purified recombinant PBP2CS and PBP2CI proteins corresponding to meningococcal isolates belonging to different serogroups and genetic lineages was performed using dot blot. For each protein, two concentrations (300 and 150 ng) were spotted, except for PBP2CS from strain LNP8013, for which 100 and 50 ng were used. Four different dilutions of sera were tested (1 : 500, 1 : 1000, 1 : 2000 and 1 : 5000) and the IgG response was revealed using a goat anti-human IgG (CALTAG laboratories, Burlingame, CA, USA). Purified meningococcal pilin (a major surface protein of N. meningitidis) was used as a positive control.20 A negative control test was performed with all sera using purified maltose-binding protein of E. coli. ELISA against N. meningitidis whole cells was performed as described previously.21
Vaccination experiments in mice
Five 6-week-old female BALB/c mice were immunized using 12 µg of the purified PBP2CS. Four control mice were injected with PBS. Injections were performed subcutaneously (sc) on days 0, 7 and 14, using complete Freund's adjuvant (CFA; Difco, Detroit, MI, USA) for the first dose and incomplete Freund's adjuvant (IFA) for the second one. On day 28, mice were bled to test their immune response using ELISA, using 96-well PBP2CS-coated plates with 1 µg of the purified PBP2CS. ELISA was performed as described previously.21 Vaccinated and control mice were challenged intraperitoneally (ip) with 5 x 105 cfu of strain LNP8013 (PenS). Blood cfu counts were performed on GCB plates, 3 h after the bacterial challenge that corresponded to the highest level of bacteraemia in this model.
Polyclonal rabbit anti-PBP2 IgG
Two 3 kg female rabbits were injected sc with 3 µg of the purified PBP2CS with CFA on day 0 and with IFA on days 7 and 21. Immune sera were collected on day 28. Pre-immune and immune sera (1 : 5000 diluted) were tested against several purified PBP2CS and PBP2CI recombinant proteins corresponding to strains from different serogroups and genetic lineages (strains LNP8013, LNP17723, LNP16454, LNP18425 and LNP17041 in Table 1) using western blot as described previously.22
For anti-PBP2 IgG purification, total IgGs from rabbit immune sera were first precipitated using 33% (NH4)2SO4 and dialysed against PBS 17.5 mM, pH 6.3. This fraction was adsorbed on a cyanogen bromide-activated Sepharose 4B column (Pharmacia, Sweden) previously coated with a bacterial extract of BL21(DE3) pLysS E. coli harbouring the pET28b expression vector. The protein concentration in the eluted fraction was determined by measuring the absorbance at 280 nm.
Serum bactericidal activity (SBA) assays were performed as described previously.23 SBA titre was expressed as the reciprocal of the dilution that allowed 50% bacterial killing.
Passive protection in mice
All meningococcal strains tested were passaged in mice to maintain virulence and stored at –70°C in GC broth with 30% glycerol prior to infective challenge. They were grown overnight at 37°C in 5% CO2 on GCB agar and then subcultured in BHI broth for 4 h to reach the exponential phase and adjusted to 106 cfu/mL. Five-week-old female BALB/c mice (Janvier, France) were injected intravenously at time 0 with 30, 3 or 0.3 µg of purified rabbit anti-PBP2 IgG. These doses were calculated to achieve in mice a concentration (0.3–30 µg/mL) of specific IgGs similar to that expected in humans (
0.3–0.5% of total IgGs). For each experiment, control mice were injected with PBS or with a purified rabbit polyclonal IgG antibody against the meningococcal cytoplasmic regulatory protein, CrgA.24 Two hours later, mice were challenged ip with 5 x 105 cfu of the bacterial suspension in 0.5 mL of PBS. Blood cfu counts from three mice were performed 1, 2, 3 and 4 h after the bacterial challenge in two independent experiments.
To test the specificity of the rabbit anti-PBP2 IgG, 5-week-old female BALB/c mice were injected at time 0 with a solution containing 30 µg of purified rabbit anti-PBP2 IgG alone or with 20 µg of purified recombinant PBP2CS. Two hours later, mice were challenged ip with 3 x 106 cfu of the bacterial suspension in 0.5 mL of PBS. Blood cfu counts from four mice were performed 4 h after the bacterial challenge in two independent experiments.
The paired and two-tailed Student's t-tests were performed to determine the statistical significance of results (P < 0.05). The paired t-test assumes that the differences are sampled from a Gaussian distribution. This assumption was tested with GraphPad using the method of Kolmogorov and Smirnov. All data passed the normality test.
Approval of experimental protocols
Sera from consenting patients, either with meningococcal disease or not, were studied at our National Reference Centre for Meningococci, according to our current procedures, approved by the Ministry of Health, for assessing the aetiological diagnosis of suspected meningococcal infections. All animal experimental designs and protocols were approved by the Institut Pasteur Review Board.
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Results |
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PBP2 is immunogenic in natural meningococcal disease
We first analysed the immunogenicity of PBP2 during natural meningococcal disease. Purified recombinant PBP2CS and PBP2CI (Figure 1a), corresponding to meningococcal isolates belonging to different genetic lineages, different serogroups (B and C) and with various susceptibility levels to penicillin G (Table 1), were spotted on a nitrocellulose membrane and immunodetected using a pair of sera (C1) from a patient with confirmed meningococcal infection (positive culture for a serogroup C N. meningitidis). Only an IgG signal was detected using convalescent serum against all PBP2CS and PBP2CI proteins (Figure 1c). Similar results were obtained with five other pairs of sera obtained from several patients with confirmed meningococcal infection due to isolates of several serogroups (data not shown). No signal was detected with both sera against the unrelated maltose-binding protein, MalE of E. coli, while a positive signal was observed against a major meningococcal immunogenic protein, the pilin, with the convalescent serum (Figure 1c). This increase in anti-PBP2 immunoblotting paralleled the immune response of patients' sera (dilution 1 :1000) tested using ELISA against N. meningitidis whole cells (Figure 1b). No signal was observed with a pair of control sera (C3) obtained from a patient with no meningococcal infection (Figure 1b). These data indicate that meningococcal PBP2 induces a specific immune response during natural meningococcal infection.
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Vaccination with the recombinant PBP2 protein
In order to evaluate the possible protection conferred by PBP2 immunization, we vaccinated five BALB/c mice with the purified recombinant PBP2CS corresponding to the penicillin-susceptible strain LNP8013 (Figure 1a). High anti-PBP2 antibody titres were observed using ELISA as compared with control mice, with no difference in antibody titres among vaccinated mice (data not shown). Mice were challenged ip with the strain LNP8013. Vaccinated mice showed significantly lower cfu counts in blood when compared with control mice (P = 0.0304) (Figure 2). These results suggest that PBP2CS is immunogenic and is able to induce a protective response against N. meningitidis. However, we were not able to show a complement-dependent bactericidal activity using sera from vaccinated mice (SBA titres < 4).
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Passive protection by rabbit anti-PBP2 IgG
Anti-PBP2 sera were obtained after immunization of rabbits with the purified recombinant PBP2CS lacking the N-terminal transmembrane domain (41 amino acid residues). Pre-immune sera from immunized rabbits did not recognize purified recombinant PBP2CS and PBP2CI proteins corresponding to meningococci belonging to different serogroups and genetic lineages (Table 1). In contrast, immune sera showed strong signals with all recombinant proteins tested regardless the phenotype of the corresponding strain (Figure 3 and data not shown). Similar results were also obtained when membrane extracts from these strains were tested (data not shown). These results confirm that the purified PBP2 protein is also immunogenic in rabbit and that anti-PBP2 antibodies recognize PBP2s from several different meningococcal strains.
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Purified specific anti-PBP2CS IgGs from one immunized rabbit were prepared as described in the Materials and methods section. They showed similar recognition profiles to the original immune serum (data not shown). No complement-dependent bactericidal activity against strain LNP8013 was detected (SBA titres <4). Purified IgGs were then used to confer passive protection against N. meningitidis bacteraemia in ip challenged mice. Bacterial challenge was performed 2 h after intravenous injection of purified IgG. We first performed passive protection experiments against the serogroup C N. meningitidis strain LNP8013, a penicillin-susceptible strain harbouring a wild-type penAS allele. There was no significant difference in blood cfu counts 1 and 2 h after bacterial challenge among control and anti-PBP2 IgG-injected mice (Figure 4). However, rabbit anti-PBP2 IgG-injected mice showed a reduction in bacteraemia after 3 and 4 h when compared with controls, and this reduction was dose dependent for the three doses of purified IgG (30, 3 and 0.3 µg) (data not shown). No protection was observed in control mice injected with PBS or with purified rabbit IgG against the meningococcal cytoplasmic regulatory protein, CrgA (Figure 4).24
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0.05.We next analysed the ability of the anti-PBP2 IgG to protect passively against ip challenges with three LNP8013 isogenic mutants with reduced susceptibility to penicillin G that were obtained by transformation and allelic replacement of the penAS allele with an altered penAI allele into the strain LNP8013. All these mutants have alterations within the 3' transpeptidase-encoding region of the penA gene. The TR-TH41, TR16454 and TR16504 strains were obtained previously by the transformation of LNP8013 with the PCR amplified penA allele from three different meningococcal strains (TH-41, LNP16454 and LNP16504, respectively).16 As shown in Figure 4, the anti-PBP2 IgG reduced bacteraemia after challenge with all three penA mutants, regardless of the alterations in the PBP2 protein they express. The passive protection was analysed against a higher bacterial dose also (108 cfu per mouse). Even at this high dose, mortality is low in this model and bacteria are cleared from mice within 24 h (data not shown). Two groups of seven mice were injected iv with purified IgG (30 µg) or PBS (control) and were then challenged 2 h later by ip injection of 108 cfu per mouse with the strain LNP8013. Mortality was higher (2/7) in the control group than in the group treated with the purified specific anti-PBP2CS IgG (0/7).
To confirm the specificity of the passive protection observed using the purified anti-PBP2CS IgGs further, we performed a set of experiments where the bacterial challenge was performed 2 h after injection of purified anti-PBP2CS IgG alone or with purified recombinant PBP2CS. A significantly higher level (P = 0.00012) of bacteraemia was observed in mice treated with both purified anti-PBP2CS IgG and purified PBP2CS recombinant protein than in mice treated with purified IgG alone. Anti-PBP2CS IgG recognized only one band in the purified preparation of the recombinant PBP2CS (Figure 5). Altogether, these results strongly suggest that the passive protection conferred by the anti-PBP2CS IgG is due to the specific recognition of, and binding to, the meningococcal PBP2.
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Discussion |
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Protein-based vaccines against N. meningitidis are hindered by the polymorphism and variability of the vaccine candidates. PBP2 is a variable protein among different clinical isolates with different phenotypes of susceptibility to penicillin G. However, PBP2 shows a highly conserved N-terminal part as well as highly conserved catalytic motifs in its C-terminal part.16 Protection against experimental meningococcaemia in mice is highly relevant to the pathophysiology of meningococcal infection in humans, as invasive meningococcal infection starts when bacteria invade the bloodstream.25
The results obtained in this work using convalescent sera demonstrate the immunogenicity of meningococcal PBP2. PBP2 is associated with the membrane fraction in N. meningitidis and it is also accessible in these fractions for binding of radiolabelled penicillin G.16 Moreover, whole cell ELISA using anti-PBP2 IgG clearly showed a dose-dependent binding of anti-PBP2 antibodies to intact bacteria (data not shown). These data cast light on the immunogenic/antigenic properties of meningococcal PBP2.
Two pieces of evidence eliminate the possibility that the protective effects observed with anti-PBP2 antibodies could be due to the presence of contaminating antigens/antibodies: (i) purified recombinant PBP2C protein was able to neutralize the protection conferred by anti-PBP2 IgG and (ii) the purified anti-PBP2 IgG recognized only one band corresponding to the recombinant PBP2CS.
The protection was not correlated with complement-mediated bactericidal activity of anti-PBP2 IgG. Despite the absence of bactericidal activity we showed that anti-PBP2 IgG conferred passive protection in a mouse model of meningococcal bacteraemia after ip challenge with three isogenic mutants expressing different PBP2s. Such an absence of complement-mediated bactericidal activity was observed previously for GNA2132, a novel meningococcal vaccine candidate. Similarly, anti-GNA2132 antibodies were shown to confer protection to infant rats against meningococcal bacteraemia.23 Therefore, the in vivo protection observed during experimental meningococcal infection may be explained by opsonophagocytic activity.
Two reports have shown that the whole sequence or a fragment of the PBP2a coding region, administered by DNA vaccination, can elicit an immune response against methicillin resistant Staphylococcus aureus.26,27 Anti-PBP2 antibodies seem to protect against experimental meningococcaemia in mice using strains harbouring homologous as well as heterologous PBP2, suggesting that conserved regions/structures on PBP2 may be targeted. Eight amino acids within the C-terminal transpeptidase domain of PBP2 were reported to be modified in strains with reduced susceptibility to penicillin G. These altered positions are located around the conserved KTG motif.16 Antibodies may be directed against the conserved surrounding sequences and may therefore be responsible for the protective effect in mice. These sequences may also be necessary for the structure that forms the active site that is targeted by the antibodies.28 Alternatively, antibodies may be directed against the conserved amino-terminal part of PBP2. A DNA vaccine containing an internal fragment of PBP2a coding region comprising the serine protease domain as antigen was able to induce a protective response against methicillin-resistant S. aureus. This protection was correlated with enhanced phagocytosis mediated by anti-PBP2a antibodies.27 This result may be in favour of the protection conferred by antibodies directed against the conserved sequences surrounding the catalytic site of PBP2.
Protection against experimental meningococcaemia was more pronounced in mice passively immunized with a purified rabbit anti-PBP2 IgG than in mice vaccinated with purified recombinant PBP2. This may be due to differences in antibody levels, their subclass distribution, and affinity and avidity. Indeed, Sabhnani et al.29 demonstrated that F1, a highly immunogenic antigen of Yersinia pestis, was able to induce high titre antibodies with similar IgG subclass distribution in different strains of mice but with different affinity/avidity. Furthermore, rabbit anti-F1 antibodies showed better avidity/affinity than mouse antibodies, suggesting that antibodies elicited in different hosts might have different protective effects, correlating with different affinity/avidity properties.
The ideal vaccine candidate against meningococcal infections should be present in all strains of N. meningitidis and have conserved regions that can be targeted by protective antibodies. Our data show that PBP2 seems to fulfil these criteria, allowing us to propose PBP2 as a promising vaccine candidate. The results from the present study, using isogenic strains expressing different PBP2s, strongly suggest that PBP2 antibodies are directly responsible for the protection observed in mice against a meningococcal challenge. One original aspect is that vaccination with PBP2 would induce protection against a protein that is involved in chromosome-mediated resistance to the antibiotic of choice in meningococcal disease.
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None to declare.
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Footnotes |
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Present address. Laboratory of Microbiology, The Rockefeller University, New York, New York 10021, USA 
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Acknowledgements |
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We thank Dario Giorgini and René Pirés for their help in immunization experiments. We are grateful to Bruno Dupuy for the generous gift of MalE purified protein. This work was supported by the Institut Pasteur. M. L. is supported by a grant from CAPES (process no. BEX 1407/01-5).
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References |
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