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1 The Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3SY, UK
2 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Hertfordshire EN6 3QG, UK
Correspondence
Martin C. J. Maiden
maiden{at}zoo.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank accession numbers for the sequences reported in this paper are AF439155–AF439260.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pollard & Maiden, 2001). Vaccines directed against the serogrouping antigen, the capsular polysaccharide, are effective against some meningococci (Gotschlich et al., 1969; Jennings & Lugowski, 1981), but poor immunological reactivity and immunological similarity to host antigens has hampered the development of such vaccines against serogroup B meningococci, which are responsible for most disease in Europe, North and South America and Australasia (Finne et al., 1983; Reingold et al., 1985; Wyle et al., 1972). Outer-membrane proteins (OMPs) are major candidates in the search for alternative vaccine components either in purified form (Boslego et al., 1995) or as part of complex outer-membrane vesicle (OMV) formulations (Bjune et al., 1991; Claassen et al., 1996).
Proteins expressed in response to iron-limitation, many of which function as receptors in iron acquisition pathways, are attractive potential vaccine components as their expression is essential for bacterial growth in vivo and their metabolic function requires that they are surface-exposed (Schryvers & Stojiljkovic, 1999). One iron-repressible OMP which has generated interest as a vaccine candidate for both the meningococcus and gonococcus is FetA, previously known as FrpB (Ala'Aldeen et al., 1994; Beucher & Sparling, 1995; Gotschlich et al., 1991). Immunological support for the candidacy of this protein was provided by the observations that FetA antibodies are present in sera obtained from patients convalescent for meningococcal disease (Black et al., 1986) and murine mAbs raised against FetA were bactericidal and specific to the isolate against which they were raised (Pettersson et al., 1990). The FetA protein is a component of some meningococcal vaccines which have undergone phase III trials and appears to elicit an immune response in vaccinees (Wedege et al., 1998). The FetA protein typically represents between 1 and 10 % of the protein content of meningococcal OMV vaccines (Frasch et al., 2001).
FetA has an apparent Mr of approximately 70 000 and interacts with at least one other component of the outer membrane, the RmpM protein (Prinz & Tommassen, 2000). It is a member of the TonB-dependent class of OMPs of Gram-negative bacteria (Beucher & Sparling, 1995) and the change in nomenclature from FrpB to FetA was proposed as, in gonococci, this protein is a receptor for the Escherichia coli siderophore ferric enterobactin (Carson et al., 1999). A preliminary topology model for FetA envisaged 26 membrane-spanning 
-sheet structures and 13 surface-exposed loop structures (Pettersson et al., 1995). The longest of these, loop 7, corresponded to a region of variable amino acid sequence which included the epitopes for several anti-FetA mouse mAbs. Other, shorter polymorphic regions were also located within the surface-exposed loops of the proposed structure (van der Ley et al., 1996).
Here, the extent of the antigenic diversity of FetA was investigated by nucleotide sequence determination of the fetA genes present in a collection of 107 meningococcal isolates. This collection was assembled in 1997 to be representative of meningococci isolated globally between 1940 and 1996, and its members had been characterized at various genetic loci. The collection included 73 members of major meningococcal invasive lineages and 34 additional isolates (Maiden et al., 1998). Direct nucleotide sequence determination of PCR products identified 60 fetA alleles, encoding 56 unique amino acid sequence variants. Comparisons of the alleles confirmed the existence of a variable region (VR) which was presumed to contain immunodominant epitopes, an idea that was supported by the serological reactivities of polyclonal mouse sera raised against FetA variants. Immunological analyses also indicated that sera raised against particular variants were poorly cross-reactive with other variants in mice. This genetic and immunological diversity has implications for the vaccine coverage that would be attainable by vaccines that included FetA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This collection consisted principally of invasive isolates from diverse geographical locations representing all known invasive genotypes and serogroups. Inoculum cultures of meningococci were grown on Mueller–Hinton (MH) agar (Oxoid) at 37 °C in an atmosphere of 5 % CO2, resuspended into MH broth. Iron-starved meningococci were grown by inoculation to an OD600 of 0·1–0·3 in MH broth supplemented with 30 µM iron-chelator ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDHA). These cultures were incubated for 5 h at 37 °C in a shaking incubator. Meningococcal DNA was prepared from cell suspensions using an IsoQuick nucleic acid extraction kit (Orca Research) according to the manufacturer's instructions.
Nucleotide sequence determination.
The nucleotide sequences of meningococcal fetA genes were determined directly from PCR-amplified DNA fragments. The genes were amplified from chromosomal DNA preparations with primers 4 and 12 (van der Ley et al., 1996). In some cases an additional primer, A11 (5'-TTGCGGCAGGTTTTGCCCACGC-3'), was used in combination with primer 12. Amplification reactions were as follows: reaction buffer (10 mM Tris/HCl, pH 8·3, 50 mM KCl, 1·5 mM MgCl2, 0·001 %, w/v, gelatin); 200 µM each of dATP, dCTP, dGTP, dTTP; 1 µM each primer; 0·5 µl Taq polymerase per 100 µl (AmpliTaq; Perkin Elmer); 1 µl template DNA per 100 µl (approx. 50 ng µl-1). Reaction conditions were 30 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2·5 min, followed by incubation at 72 °C for a further 5 min. The PCR products were precipitated by incubation at room temperature for 15 min with 20 % (w/v) PEG8000, 2·5 M NaCl. After centrifugation for 10 min at 12 000 g, the precipitates were washed in 70 % (v/v) ethanol, dried and resuspended in 10–20 µl sterile distilled water (Embley, 1991). The nucleotide sequences of the amplified fetA genes were determined at least once on each DNA strand by cycle sequencing with BigDye Ready Reaction Mix (Applied Biosystems), used in accordance with the manufacturer's instructions. Extension reactions were performed with the amplification primers and the following sequencing primers: S1, 5'-CGGCGCAAGCGTATTCGG-3'; S2, 5'-CCGAATACGCTTGCGCCG-3'; S3, 5'-CCATCAGGAAATCAAACCGC-3'; S4, 5'-GCGGTTTGATTTCCTGATGG-3'; S5, 5'-CCATCAAAGACGCGCTTGCC-3'; S8, 5'-CGCGCCCAATTCGTAACCGTG-3'; S9, 5'-CCGAATACGCTGGCACCG-3'; S13, 5'-TACGCAGGCAATGTAAAAGGC-3' and S15, 5'-TTGCAGCGCGTCRTACAGGCG-3'. Unincorporated dye terminators were removed by precipitation of the termination products with 95 % (v/v) ethanol and the labelled extension products separated and detected with an ABI Prism 377 automated DNA sequencer (Applied Biosystems). Sequences were assembled from the resultant chromatograms with the STADEN suite of computer programs (Staden, 1996).
Phylogenetic analysis.
The nucleotide sequences were aligned and translated using the SeqLab alignment program in the GCG suite of programs (Genetics Computer Group, Madison, WI, USA) (Womble, 2000). The sequence data were exported and further analysed using the Molecular Evolutionary Genetics Analysis (MEGA) software package versions 1.02 and 2.0 (Kumar et al., 1994). The relationships among the alleles, generated from pairwise allele comparisons using Hamming distance matrices (equivalent to p-distances), were visualized by split decomposition (Bandelt & Dress, 1992) with the program SPLITSTREE (Huson, 1998). The ‘fit’ parameter indicated how well the graph represented the distance matrix. The distribution of nucleotide changes within the fetA alleles was analysed with the Synonymous Nonsynonymous Analysis Program (SNAP; available at http://hiv.lanl.gov/). This program calculated pairwise synonymous and non-synonymous distances (Nei & Gojobori, 1986) for a sequence alignment. The output from SNAP was imported into Microsoft Excel and manipulated to generate a graph showing the levels of synonymous and non-synonymous substitution along the gene.
Meningococcal membrane preparations.
These were made using a modification of a spheroplast lysis method (Witholt et al., 1976). Meningococcal cultures (25 ml) were grown under iron-restricted conditions, heat-killed at 55 °C for 30–60 min and the cells were harvested by centrifugation at 7800 g for 10 min. Cells were resuspended in 200 mM Tris/HCl (pH 8), 1 mM EDTA (5 ml). Subsequently, 200 mM Tris/HCl (pH 8), 1 mM EDTA, 1 M sucrose (5 ml) was added. At 90 s after this addition 1·25 mg lysozyme was added, followed at 2 min 15 s by 10 ml water. Samples were incubated with shaking at room temperature for 30 min and sedimented by centrifugation at 2800 g for 20 min to obtain spheroplasts. These were homogenized in 10 ml ice-cold water and centrifuged at 2800 g for 20 min to collect a membrane fraction which was resuspended in 0·5 ml 0·06 M Tris/HCl, pH 8·0. The protein concentration of the preparation was measured using the Bio-Rad protein assay kit using bovine serum albumin as standard.
Construction of fetA mutant strains.
DNA manipulations used standard published protocols (Sambrook et al., 1989). The fetA gene from isolate Z1318 was cloned into the plasmid vector pTrcHis2 (Invitrogen) to create plasmid pEAT1, which was digested with restriction endonuclease EcoO109I and ligated to a marker for kanamycin resistance derived by restriction endonuclease digestion of Tn5 from E. coli, to yield plasmid pEAT2. The fetA : : KanR construct present in pEAT2 was amplified by PCR using fetA amplification primers, one of which was modified to contain a meningococcal uptake sequence on the 5' end. The construct was digested with NsiI and the digestion product was used to transform N. meningitidis isolates using a modification of the previously published method of van der Ley & Poolman (1992). Meningococci were cultured overnight on MH agar and resuspended to an OD600 of 0·2–0·3 in MH broth (5 ml) supplemented with MgCl2 (10 mM). The restriction endonuclease digest was added, the mixture incubated for 3 h at 37 °C and transformants were selected by plating the mixture onto MH agar supplemented with kanamycin (25 µg ml-1) and incubation at 37 °C. The identity of transformants was confirmed by PCR amplification with primers 4 and 12 (van der Ley et al., 1996).
Western blot analysis.
Membrane protein samples (approx. 75 µg total protein) were boiled in SDS sample buffer for 5 min, the proteins were separated on 7·5 % polyacrylamide gels (Laemmli, 1970) and then electrophoretically transferred to nitrocellulose (Hybond-C super; Amersham) (Towbin et al., 1979). The membranes were rinsed in several changes of Tris-buffered saline (TBS: 200 mM NaCl, 10 mM Tris/HCl, pH 7·6) and blocked for 1 h at room temperature with TBS with powdered milk (5 %, w/v, TBS-milk). The primary antibody (a FetA-specific rabbit serum, supplied by Dr J. Holst, National Institute for Public Health, Oslo, Norway, and used at a final dilution of 1 : 500) was added and the membranes were incubated overnight with gentle agitation. The membranes were rinsed several times with TBS and the secondary antibody conjugate (goat anti-rabbit horseradish peroxidase conjugate; Sigma) was added at a dilution of 1 : 2000 in TBS-milk and incubated at room temperature with gentle agitation for 1 h. Following further washes with TBS the bound antibody was detected by incubation with 3,3'-diaminobenzidine peroxidase (Sigma) and photographed.
Production of polyclonal antibodies.
For each isolate, groups of 10 BALB/c mice were immunized intraperitoneally with approximately 106 live, iron-restricted meningococcal cells. A further 104 heat-killed iron-restricted cells were administered 21 days after the initial immunization and the mice were terminally bled on day 35 after the first immunization. For each isolate the sera from all 10 mice were pooled, diluted 1 : 100 in PBS and adsorbed with a cell suspension of heat-killed iron-restricted meningococci for 60 min with gentle agitation at room temperature. The heat-killed suspensions were prepared from a 50 ml 5 h culture of meningococci in iron-restricted MH broth. These were heat-killed at 55 °C, 3 ml samples were removed and the meningococci were collected by sedimentation by centrifugation. Each of the resultant pellets was washed three times in PBS, resuspended in 0·5 ml PBS and stored overnight at 4 °C. The cells were collected by sedimentation and resuspended in 0·5 ml of the serum to be adsorbed. The sera were subjected to repeated rounds of adsorption until they no longer reacted with the cell preparations from the corresponding fetA mutant in ELISAs. Serum 3 was adsorbed once against isolate H44/76 fetA, while serum 1, serum 2 and serum 4 were adsorbed three times against isolate Z4673 fetA, isolate Z4699 fetA and isolate Z4662 fetA, respectively.
ELISA.
ELISA plates were coated with approximately 106 heat-killed iron-restricted whole cells per well for each isolate and dried at 37 °C overnight. The plates were washed three times in washing buffer (1x PBS, 0·03 %, w/v, Brij-35) and 200 µl dilution buffer (1x PBS, 0·03 %, w/v, Brij-35, 5 % newborn bovine serum) was added to each well and the plates were incubated for 1 h at room temperature. The dilution buffer was discarded and dilution buffer (100 µl per well) was added to the control wells. The four FetA-specific sera were diluted to working concentration, 100 µl of serum was added to each well and the plates were incubated overnight at 4 °C. The plates were washed three times and incubated for 1 h 30 min at room temperature with 100 µl goat anti-mouse IgG-HRP conjugate at a dilution of 1 : 2000 per well. Following three washes the plates were incubated with 100 µl TMblue (Intergen) per well for 20 min. The reaction was stopped with 100 µl 1 M sulphuric acid per well. Results were expressed as A450 values. All ELISA experiments were carried out in triplicate using multiple cell preparations and the results presented as mean values. Prior to studying the panel of 104 isolates, the specificity and variability of the whole-cell ELISA was confirmed using wild-type and mutant strains. The standard deviations were typically about 10 % of the mean A450 (data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Over the entire sequence the mean uncorrected p-distance among alleles was 5·42 % and ranged from 0·05 % (corresponding to a single nucleotide difference) to 10·11 % (199 nt differences). Representation of fetA allelic diversity by split decomposition produced a star-like phylogeny with a low fit parameter of 24·2 % (data not shown), indicating that the inter-allele relationships did not conform to a tree-like phylogeny and were poorly resolved. Repeating the analysis on a subset of these data, comprising the 12 fetA alleles identified in the 33 representatives of the major invasive serogroup A meningococci included in the isolate collection, revealed a phylogenetic network with improved resolution (a fit parameter of 73 %, Fig. 1). Inspection of the aligned variable sites in this subset showed that allelic differences comprised both mosaic gene sequences and single nucleotide substitutions (Fig. 2). Similar patterns of variation were found in other allele subsets (data not shown).
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). The highest level of dS was recorded at codon 265 and the highest level of dN at codon 360 of the alignment. Of the 42 codons present in the aligned VR region, 36 contained non-synonymous substitutions, 28 % of those present in the whole sequence. A total of 32 of these codons contained both synonymous and non-synonymous substitutions with synonymous substitutions predominating in 21 of these codons. Translation of the VR nucleotide sequences resulted in 24 unique peptides which were classified into five VR families on the basis of peptide sequence similarity, with each family identified by a number preceded by the letter F. Variants within each family were indicated by the addition of a supplementary number (e.g. F1-1, F1-2 and F2-1; Table 1). A total of 13 of the variants were represented by a single allele and 12 of these alleles were present in only one isolate. Families F2 and F4 were each represented by a single variant in this isolate collection.
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). Four FetA-variant-specific polyclonal sera were produced by the immunization of mice with whole-cell preparations of the parental isolates, followed by cross-adsorption with the appropriate mutant. The sera were named as follows: serum 1, produced with isolate Z4673 (fetA-02, F1-5); serum 2, produced with isolate Z4699 (fetA-01, F3-3); serum 3, produced with isolate H44/76 (fetA-01, F3-3); and serum 4, produced with isolate Z4662 (fetA-04, F3-9). In ELISA experiments, which compared the reactivity of each of the adsorbed sera with the mutant and wild-type isolate used in its production, working dilutions for the sera were identified at a dilution of 1 in 128 for serum 3 and 1 in 32 for the remaining sera, although serum 1 showed a higher level of non-specific binding in this experiment (data not shown). These dilutions were used in subsequent ELISA experiments.
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a–d). In these experiments most isolates showed no better reactivity with a given serum than the fetA-negative mutants. However, those isolates that contained a fetA gene encoding the FetA VR against which the serum was raised generally reacted well although there was little evidence for cross-reactivity between distinct variants belonging to the same FetA variant family. The significance of the differences between the reactivity of each serum with the isolates expressing FetA with the VR against which the serum was raised and their reactivity with the other isolates in the collection was assessed using a Wilcoxon Rank Sum test. For each serum, the reactivity of isolates with the homologous VR was significantly greater than with the other isolates in the collection (serum 1, P<0·0001; serum 2, P=0·0005; serum 3, P=0·0007; serum 4, P<0·0001). The least sensitive and specific serum was serum 1 (Fig. 5a). In this case a number of isolates shown to contain a gene encoding an F1-5 variant did not react with the serum. This probably reflected the variable level of expression of fetA among these isolates; for example, RT-PCR analysis of mRNA from the least reactive F1-5 isolate, Z6425, indicated that in this case the fetA gene was not transcribed. Serum 2 and serum 3, which were raised against different isolates with the same fetA allele, were similar in their patterns of reactivity, indicating that much of the variation among isolates could be explained by differences in the whole-cell preparations used. Neither of these sera reacted with isolate Z4239. Both RT-PCR and Western blot analyses showed that this isolate did not express FetA at detectable levels (data not shown). This appears to be the result of the presence of additional cytosine residues, between the -35 and the -10 boxes of the promoter, increasing the distance from 17 bp, in a well expressed gene, to 21 bp in isolate Z4239 (K. Barski, unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These include antigenic variation, the expression of alternative surface antigen variants encoded in the genome, and antigenic diversity, which is a consequence of the short-term evolution of mutable parts of surface components (Robertson & Meyer, 1992). These adaptations present problems for vaccine development (Maskell et al., 1993) which can be addressed by the identification of conserved antigens or the development of vaccines containing cocktails of multiple variants of antigens. To this end the extent of variation of one meningococcal vaccine component, FetA, was systematically assessed. The nucleotide sequences of the 107 fetA genes were highly diverse, with 60 alleles encoding 56 FetA protein variants. As the isolate collection employed in this analysis was chosen to be representative of the meningococci recovered from cases of invasive disease, it is probable that there is even greater diversity of this protein among asymptomatically carried populations of meningococci. The sequence variation present in the fetA alleles did not conform to a tree-like phylogeny and the mosaic gene structures observed, which are common in a range of meningococcal genes, were consistent with the generation of allelic variation by intragenic recombination events, although there was also evidence for single point mutations generating new alleles. The difficulties in resolving the phylogenetic relationships among large numbers of fetA alleles was likely to be a consequence of multiple recombination and point mutation events occurring during their evolution.
The three-dimensional structure of the E. coli FepA protein assisted the interpretation of the uneven distribution of nucleotide changes within the fetA gene. FepA and FetA are highly divergent in terms of protein sequence. However, they are functional homologues and they do share a region of sequence homology (Carson et al., 1999). This region was highly conserved in FetA, with 62 of the 79 amino acids in this region identical among the 56 variants, and the highest value for dS was recorded in this region (Fig. 2
). The N-terminal portion of the E. coli FepA protein forms a globular domain which acts as a plug in the 22-stranded membrane spanning -barrel pore formed by the remainder of the protein (Buchanan et al., 1999; Newton et al., 1999; Scott et al., 2001). The patterns of nucleotide sequence variation present among the fetA alleles were consistent with the FetA protein conforming to a similar structure to FepA, with a conserved N-terminal domain and a less conserved C-terminal domain.
Many of the sequence changes, and especially non-synonymous substitutions, were localized in a region equivalent to the region of variable sequence identified previously from three full-length and five partial meningococcal fetA gene sequences (van der Ley et al., 1996), although the greater number of sequences presented here enabled a more precise definition of the extent of this region. This region corresponded to loop 7 of the FetA structural model and was previously shown to contain the epitopes for several mouse mAbs (van der Ley et al., 1996). The localization of most variation in an area corresponding to a putative surface-exposed loop on the -barrel structure of an OMP was redolent of the two major VRs found in the meningococcal PorA porin (Maiden et al., 1991; McGuinness et al., 1990, 1993).
In common with the PorA VRs, the sequences of the FetA VR were highly divergent, but the peptide sequence could be resolved into distinct families. The sequence changes within and among sequences, which were likely to be a consequence of positive, diversifying selection imposed by host immune responses, were extensive and it was not possible to reconstruct the evolutionary history of these variants. The nomenclature system proposed for these variants was therefore a practical one and was not intended to imply definitive phylogenetic relationships among VRs.
The evidence for diversifying selection acting on the VR, together with the presence of epitopes for bactericidal mouse mAbs in this region, suggested that the VR was an immunodominant part of the FetA protein. This was supported by the immunological reactivities of the polyclonal mouse sera generated with the fetA mutant meningococci. Most of the reactivity of the four adsorbed sera could be ascribed to the FetA protein, specifically to the VR, and the results of these experiments were similar to those previously obtained for the PorA VRs, which contain epitopes recognized by the immune systems of both animal models and humans (Rosenqvist et al., 1995; Saukkonen et al., 1987).
In addition to the sequence diversity of FetA, variable expression in both meningococci and gonococci (Carson et al., 2000) may also reduce the effectiveness of this protein as a vaccine component. This provides a further parallel with the PorA protein which is also phase-variable (van der Ende et al., 2000). Perhaps the most likely role for the FetA protein is as a component of OMV vaccines, along with other antigens such as PorA. These vaccines elicit bactericidal responses in people, including infants, but the immunity induced is strain-specific, probably as a consequence of antigenic diversity of their components (Martin et al., 2000). If FetA is to be used as a vaccine component consideration will have to be given to the conditions used for meningococcal growth. In common with most similar vaccines (Frasch et al., 2001), the meningococci used to produce OMV vaccines that have been evaluated to date have not been grown under iron limitation and the human immune reactions that have been observed against FetA (Rosenqvist et al., 1995) in vaccinees may be a consequence of the apparently high levels of FetA expressed by isolate H44/76 (Fig. 4), which is the isolate used for the production of the Norwegian OMV vaccine (Fredriksen et al., 1991).
In practical terms OMV vaccines could be used to achieve partial protection and could have a major impact on disease levels if OMVs derived from each invasive genotype predominant in a particular area were included in vaccine formulations. This strategy, however, is dependent on meningococcal populations exhibiting sufficient stability with respect to antigenic diversity and may be impractical in countries such as the UK where many diverse disease-associated lineages are present. OMV vaccines may have a role in reducing the burden of meningococcal disease caused by a single lineage, for example the outbreak experienced by New Zealand from the late 1990s onwards (Bremner et al., 1999). However, the data presented here highlight the challenges of developing comprehensive meningococcal vaccines from the major OMPs of this highly diverse bacterium.
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ACKNOWLEDGEMENTS |
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Received 19 November 2002;
revised 10 March 2003;
accepted 3 April 2003.
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