J Med Microbiol 52 (2003), 121-125; DOI: 10.1099/jmm.0.05017-0
© 2003 Society for General Microbiology
ISSN 0022-2615
Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil
Lucila O. Fukasawa12,
Maria Cecília O. Gorla3,
Ana Paula S. Lemos3,
Rocilda P. F. Schenkman1,
Maria Cristina C. Brandileone3,
Jay W. Fox4,
Isaias Raw1,
Carl E. Frasch5 and
Martha M. Tanizaki1
1Centro de Biotecnologia, Instituto Butantan, Avenida Vital Brasil 1500, CEP 05504-900, São Paulo, Brazil 2Curso de Pós-Graduação em Biotecnologia, USP-Butantan-IPT, Brazil 3Serviço de Bacteriologia, Instituto Adolfo Lutz, São Paulo, Brazil 4Department of Microbiology, University of Virginia, Charlottesville, VA, USA 5Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD, USA
Correspondence Martha M. Tanizaki tanizaki{at}usp.br
Received 17 July 2002 Accepted 3 October 2002
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Abstract
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A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60 % of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.
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Introduction
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Successful vaccines against Neisseria meningitidis serogroups A, C, Y and W135 offer protection in adults and in children over 2 years of age (Frasch, 1995). However, some recent studies have shown immunological hyporesponsiveness to a second vaccination (Granoff et al., 1998; Richmond et al., 2000). Improved vaccines composed of capsular polysaccharide–protein conjugate antigens have been investigated and these vaccines have been shown to be protective for younger children (Zollinger, 1997). Clinical trials have been performed with capsular polysaccharides from meningococcal serogroups A and C conjugated with the CRM197, a mutant diphtheria toxin (Anderson et al., 1994; Twumasi et al., 1995). Extension of this approach to a group B meningococcal vaccine is not straightforward, since the group B capsular polysaccharide is a poor immunogen in humans (Poolman, 1995). Development of vaccines against serogroup B has therefore focused mostly on the use of outer-membrane vesicles (OMV) containing different proteins (Blake & Gotschlich, 1986). Induction of protective immunity by these OMV vaccines has been studied in several trials and case-control studies. Trials in Chile and Brazil showed poor protection in children less than 4 years of age (Boslego et al., 1995; Milagres et al., 1994). Although efficacy in the range of 50–80 % in children over 4 years old was recorded (Anderson et al., 1994), improvements are clearly needed. Another limitation of OMV vaccines is that the protein antigens that induce bactericidal antibodies show sequence and antigenic variability. The bactericidal antibodies induced by OMV vaccines were found to be directed mainly against two classes of proteins, PorA (Poolman, 1995) and Opa (Rosenqvist et al., 1993). Therefore, interest has increased in finding conserved, surface-exposed proteins in serogroup B meningococcus that are able to induce bactericidal antibodies. NspA (Martin et al., 1997), GNA 33 (Pizza et al., 2000) and NadA (Comanducci et al., 2002), have been described as proteins that satisfy the requirements for broadly distributed surface proteins that induce bactericidal antibodies.
NadA is a novel, surface-exposed protein, which was described recently by Comanducci et al. (2002). It is an oligomeric protein with an apparent molecular mass of 190 kDa in SDS-PAGE. However, it was shown that the recombinant protein obtained by Comanducci et al. (2002) consists of 362 amino acids, including a possible leader peptide of 23 amino acids. The mature protein has a predicted molecular mass of 35 363 Da, and a leucine zipper structure probably maintains its pentameric conformation.
In a previous report, sera of mice immunized with serogroup C strain IMC 2135 were shown to react with a protein of approximately 190 kDa in OMV from the serogroup B strain N44/89, as determined by immunoblotting (Fukasawa et al., 1999). This protein was therefore first studied as a 190 kDa monomer (Fukasawa et al., 2000). Furthermore, antibodies against this protein were found to be abundant in sera of Brazilian patients infected with serogroup B meningococcus (unpublished observations). The object of the present work was to characterize the antigenic properties of this protein further and to confirm its identity as NadA. In view of its potential importance as a component of meningococcal vaccines, the prevalence of this protein in strains isolated from Brazilian patients was also studied.
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METHODS
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Bacterial strains.
The N. meningitidis strains N44/89 (B : 4,7 : P1.19,15 : P5.5,7), IMC 2135 (C : 2a : P1.5,2) and N1002/90 (C : 2b : P1.3 : P5.8) used in these experiments were isolated from Brazilian patients. Strains 3006 (B : 2b : P1.2 : P5.2,8) and M986 (B : 2a : P1.5,2) were from the Center for Biologics Evaluation, FDA, Bethesda, USA, and strain MC58 (B : 15 : P1.7,16b) was provided by Dr Rino Rappuoli.
Immunization of mice.
Male C3H/Hepas mice (Biotério de camundongos isogenicos, Inst. de Ciencias Biomédicas, Universidade de S. Paulo), 35 days old, were injected intraperitoneally with 0.5 ml of an OMV preparation of N. meningitidis N44/89 containing 17 µg protein and 25 µg aluminium hydroxide. A control group was injected with 0.5 ml saline containing 25 µg aluminium hydroxide. Mice were boosted with the same preparations at 14 and 28 days after the first immunization and bled via the retro-orbital plexus at 2 weeks after each injection.
Bactericidal activity.
The bactericidal assay was carried out using Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and serogroup C strains N1002/90 (C : 2b : P1.3 : P5.8), IMC 2135 (C : 2a : P1.5,2) and 3006 (B : 2b : P1.2 : P5.2,8) in 96-well plates, as described by Frasch & Robbins (1978), with modifications. Briefly, the final reaction mixture (50 µl) contained 25 µl of serial twofold dilutions of test serum that had been heat-inactivated at 56 °C for 30 min, 12.5 µl baby rabbit serum as the complement source, screened for absence of anti-meningococcal activity, and 12.5 µl of a suspension of exponential-phase meningococci (60 c.f.u. per well), grown in agar containing 1.0 % (v/v) horse serum. The reaction mixture was incubated at 37 °C for 30 min, and 130 µl tryptic soy agar (TSA), cooled to approximately 45 °C, was added to each well. The mixture was allowed to solidify and the plates were incubated for 18 h at 37 °C in 5 % CO2. Viable counts were done at time 0 (t0), before incubation with complement, in order to estimate the number of c.f.u. for 100 % survival, by plating 12.5 µl bacterial stock suspension onto a 150x150 mm TSA plate. Control wells included on each microtitre plate contained: (i) bacteria, complement and buffer (complement-dependent control) and (ii) bacteria, heat-inactivated complement and buffer (complement-independent control). In addition, a positive control was included in each assay consisting of serial dilutions of a known positive serum. Assays were made in duplicate. Anti-meningococcal serogroup C bactericidal assays were carried out using Brazilian epidemic strains N1002/90 and IMC 2135 and performed as described above, except that the reaction was incubated at 37 °C for 60 min as standardized by Maslanka et al. (1997). The bactericidal titre of the serum was defined as the reciprocal of the serum dilution giving 
50 % killing of the bacteria present at t0.
SDS-PAGE and immunoblot analysis.
SDS-PAGE and immunoblot detection of IgG antibodies against OMV from strain N44/89 were performed as described by Wedege & Froholm (1986) with modifications. OMV proteins (20 µg) were separated by SDS-PAGE on gels containing 12 % acrylamide and transferred electrophoretically to nitrocellulose paper. The transfer was performed at 250 mA for 1 h in a Bio-Rad Electroblotter, as specified by the manufacturer. After transfer, the nitrocellulose was blocked with BSA [3.0 % (w/v) in PBS] for 30 min. The nitrocellulose was then incubated overnight with the serum samples diluted 1 : 50 in PBS-BSA. After washing four times, the strips were incubated with a peroxidase-conjugated goat anti-mouse IgG (Sigma) diluted 1 : 3000 in PBS-BSA. The nitrocellulose was washed three times with PBS and once with 0.05 M sodium acetate, pH 5.0, and incubated for 30 min with hydrogen peroxide, 0.05 % (v/v) (Aldrich), and 3-amino-9-ethylcarbazole, 4 % (w/v) (Sigma), in 0.05 M sodium acetate, pH 5.5. The peroxidase reaction was stopped by washing the strips with water.
Dot-blotting analysis.
Strains were tested by dot-blotting as described previously (Wedege et al., 1990). Briefly, whole-cell suspensions were dotted onto 0.45 µm pore-size nitrocellulose membrane. After drying for 30 min at room temperature, the membrane was blocked for 30 min with 10 ml BSA-PBS. Hybridoma culture supernatant was pipetted directly into blocking buffer at a final dilution of 1 : 10. After overnight incubation at room temperature on a rotator, the membrane was washed four times with PBS and incubated for 3 h with goat anti-mouse IgG conjugated to peroxidase (Sigma), diluted 1 : 3000. The membrane was then washed four times with PBS and developed with 3-amino-9-ethylcarbazole and hydrogen peroxide.
mAbs.
Murine mAbs were generated as described previously (Kearney et al., 1979). Two groups of five BALB/c mice were immunized with three doses, each containing 5 µg OMV protein from strains 3006 or M986, at 2-week intervals. Four days after the last immunization, the spleens were removed and used for the fusion procedure. Positive clones were identified by ELISA using plates coated with the same OMV preparation used to immunize mice. Clones that bound the respective OMVs in ELISA were then examined by immunoblotting. mAb 1079B6 raised against strain 3006 and mAb 4895F9 raised against strain M986 OMV preparations were selected because they bound strongly on the immunoblots to a protein of approximately 170–200 kDa.
Molecular mass determination and peptide sequencing.
`De novo’ sequencing of peptides recovered from in-gel trypsin digestion of protein bands was achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) on a Finnigan LCQ ion-trap mass spectrometer as described elsewhere (Shevchenko et al., 2002).
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RESULTS
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Cross-reactivity among serogroup B and C strains
The mAbs 1079B6, raised against serogroup B strain 3006, and 4895F9, from serogroup B strain M986, were used to study the importance of the 190 kDa protein in the immune response. As shown in Fig. 1(a), mAb 1079B6 recognized a 170–200 kDa protein in OMV preparations from group B strains N44/98, 3006, M986 and MC58 and group C strain IMC 2135 in immunoblot assays. Fig. 1(b) shows the corresponding SDS-PAGE profile. OMV from strain1002/90 did not possess this protein and the amount in OMV from strain MC58 was insufficient to be seen in SDS-PAGE. Complement-mediated bactericidal activity of mAb 1079B6 was tested against strains N44/89, IMC 2135, 3006 and N1002/90. As shown in Table 1, high bactericidal titres were obtained against the three strains that contained the 190 kDa protein in OMV preparations but not against strain N1002/90, which lacked the protein.
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Fig. 1. Analysis of proteins in N. meningitidis OMV preparations. (a) Immunoblot showing binding of mAb 1079B6 to OMV preparations of strain N44/89 (lane 1), IMC 2135 (2), 3006 (3) M986 (4), MC58 (5) and N1002/90 (6). (b) SDS-PAGE profiles, after staining with Coomassie brilliant blue of OMV preparation from strains N44/89 (lane 1), IMC 2135 (2), 1002/90 (3) and MC58 (4). The arrow in (b) indicates a position equivalent to 200 kDa.
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In order to test whether the protein could induce cross-protection when present in the OMV, a group of mice were immunized with three doses of OMV vaccine prepared from strain N44/89. Serum obtained at 15 days after the third dose of this vaccine was used for bactericidal assay against two serogroup B and two serogroup C strains of different serotypes: N44/89 (B : 4,7 : P1.19,15 : P5.5,7), 3006 (B : 2b : P1.2 : P5.2,8), IMC 2135 (C : 2a : P1.5,2) and N1002/90 (C : 2b : P1.3 : P5.8) (Fig. 2). The vaccine induced bactericidal antibodies against the homologous strain and the other serogroup B strain 3006 and also against serogroup C strain IMC 2135, but not against the serogroup C strain N1002/90.
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Fig. 2. Bactericidal activity of pooled mouse antiserum, obtained after immunization with an OMV preparation from N. meningitidis strain N44/89, against meningococcal strains serogroups B (N44/89, 3006) and C (IMC 2135, N1002/90). The bactericidal titres of the antiserum (open bars) and a saline control (filled bars) are shown.
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Molecular mass and partial peptide sequence of the 190 kDa protein
The monoisotopic mass calculated by LC/MS/MS was 37 931 Da. The peptide sequences obtained after trypsin hydrolysis, which covered about 77 % of the total protein, were aligned to the amino acid sequence of NadA from strain MC58 (Comanducci et al., 2002) and, over these regions, the sequences were identical (the amino acid sequence obtained is available as supplementary material in JMM Online at http://jmm.sgmjournals.org/). Thus, the protein investigated in this report is presumed to be NadA.
Presence of the NadA protein in strains isolated from Brazilian patients
mAb 4895F9 was used to test by dot-blotting 100 group B and 100 group C strains isolated from Brazilian patients in 1999. Among the serogroup B strains tested, 36 % were serosubtype 4,7 : P1.19,15, which is the prevalent serosubtype found in Brazilian patients, and 80 % of these isolates bound the mAb in a dot-blot assay (Table 2). Among serotype 4,7 : P1.7,1 isolates, the second most prevalent group, only 40 % of the isolates were positive (Table 2). Therefore, overall, 62 % of the serogroup B strains were positive in the dot-blot assay. Among the serogroup C strains tested, 60 % of the strains were positive. Twenty per cent of serogroup B and C strains that were positive were tested in the immunoblot assay and, in all strains, the mAb reacted with a protein of 170–190 kDa (data not shown).
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DISCUSSION
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A novel protein, NadA, was described by Comanducci et al. (2002) following a reverse vaccinology procedure (Pizza et al., 2000). The NadA monomer has a molecular mass of about 35 kDa and forms very stable oligomers, probably pentamers. It is widely conserved among serogroup B and C strains. The recombinant protein induces bactericidal antibodies (Comanducci et al., 2002). In the present study, native OMV from strains containing NadA were used instead of the recombinant protein.
Pizza et al. (2000) identified a total of 350 candidate vaccine antigens in meningococcus serogroup B through whole-genome sequencing. Most of them are less abundant than the major outer-membrane proteins (Frasch et al., 1985) and that is probably the reason that they have not been studied before. Due to its high apparent molecular mass, NadA is clearly visible in SDS-PAGE. The protein was seen in the OMV preparation of strain N44/89, the most prevalent serogroup B serosubtype in Brazil, and in strain 3006, as well as in the serogroup C strain IMC 2135, which is used in Brazil for production of the serogroup C capsular polysaccharide. However, in strain MC58, from which the recombinant NadA was obtained, the protein seemed to be less abundant, since it was not visible in the protein profile of OMV.
NadA is an immunogenic and conserved protein. The mAb 1079B6, raised against NadA in an OMV preparation from a serogroup B strain, gave a strong bactericidal reaction against homologous and heterologous strains. Furthermore, NadA apparently induces cross-reactivity from the OMV preparations. Bactericidal activity of a pooled serum from mice immunized with OMV from strain N44/89 was found against strains IMC 2135 and 3006, but not against strain N1002/90, which was negative for NadA by immunoblotting. Much of this bactericidal cross-reactivity could be attributed to the 190 kDa protein (NadA), since these vaccine strains have different class 1 (PorA) and class 5 (Opa) proteins. This result is very important, since OMVs seem to be the best way to present antigens of meningococcus B.
About 60 % of serogroup B and C meningococcal isolates from Brazilian patients reacted with the mAb against NadA. This protein was present in 80 % of Brazilian strains of serosubtype B : 4,7 : P1.19,15. This particular serosubtype is the most prevalent in Brazil and accounts for 66 % of all serotyped meningococcal serogroup B isolates (Sacchi et al., 2001). However, it is not clear whether the dot-blot analysis is sufficiently sensitive to detect all NadA-positive strains.
Among the meningococcal B strains isolated in Brazil during 1997–1998, high prevalence of only three subtypes was found: P1.19,15 (66 %), P1.7,1 (11 %) and P1.7,16 (4 %) (Sacchi et al., 2001). NadA was shown to be present in 80 % of P1.19,15 strains, 40 % of P1.7,1 strains and 60 % of P1.7,16 strains (Table 2). The results of the cross-reactivity tests and screening of NadA described here are particularly important for the development of a vaccine for use in Brazil. The high prevalence of P1.19,15 strains and the high frequency of NadA in Brazilian strains may be used to define the serosubtype composition of an OMV vaccine against serogroup B to be used in Brazil. The present study suggests that an appropriate vaccine should contain an OMV preparation from serosubtype P1.19,15 expressing NadA.
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Acknowledgments
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This work was supported by FAPESP grant 00/08464-6 and PADCT/FINEP, Convenio 77.97.1152.00. L. O. F. is the recipient of a scholarship from FAPESP.
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Footnotes
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Abbreviation: OMV, outer-membrane vesicle.
The NadA amino acid sequence obtained is available as supplementary material in JMM Online (http://jmm.sgmjournals.org/).
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Abstract
Introduction
