INTRODUCTION

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With the control of Haemophilus influenzae type b disease by vaccination, Neisseria meningitidis has emerged as the most common cause of bacterial meningitis in children and young adults (45, 56). Strains of N. meningitidis can be divided into 12 serogroups based on chemically and antigenically distinctive polysaccharide capsules (14). Five serogroups, designated A, B, C, Y, and W-135, account for virtually all disease-producing isolates (67).

Plain meningococcal polysaccharide vaccines are currently available for the prevention of disease caused by serogroups A, C, Y, and W-135 (18). These vaccines are efficacious in older children and adults but not in infants, the age group at greatest risk of acquiring the disease (18). Second-generation polysaccharide-protein conjugate vaccines are in various stages of clinical development. These vaccines are much more immunogenic in infants than are plain polysaccharide vaccines (16, 28, 32). It is likely, therefore, that highly effective meningococcal conjugate vaccines for the prevention of disease caused by serogroups A, C, Y, and W-135 strains will be licensed for routine use in children in the near future. However, attempts to develop a vaccine for the prevention of group B meningococcal disease have been problematic. Group B strains are a common cause of disease, currently accounting for approximately one-third of disease-producing isolates in the United States (10, 11), half in the United Kingdom (49), and up to 80 to 90% in The Netherlands (55). Therefore, lack of an effective vaccine for the prevention of serogroup B disease will substantially limit the overall effectiveness of a vaccination program for control of meningococcal disease.

To date, experimental meningococcal B vaccines have been designed to elicit serum antibody responses either to the group B capsule or to noncapsular antigens. Capsule-based vaccines are limited by poor immunogenicity, even when the polysaccharide is conjugated to a protein carrier (15, 26). The group B polysaccharide consists of (28)N-acetylneuraminic acid (polysialic acid). The poor immunogenicity of this polysaccharide is attributed to immunologic tolerance as a result of exposure to cross-reacting polysialic acid expressed by a variety of host tissues, particularly the neural cell adhesion molecule (17, 23). In an innovative strategy for overcoming immunologic tolerance to the meningococcal B capsule, Jennings et al. substituted N-propionyl (N-Pr) groups for N-acetyl groups and conjugated the N-Pr meningococcal B polysaccharide derivative to a carrier protein (25). The resulting conjugate vaccine was highly immunogenic in experimental animals, eliciting immunoglobulin G (IgG) antibodies that activated complement-mediated bacteriolysis in vitro and passively protected experimental animals infected with meningococcal B bacteria. However, a subset of the antibodies elicited by this vaccine were also autoantibodies (20, 21). Given the unknown risks of a vaccine that elicits anticapsular antibodies that are also autoantibodies, recent efforts to develop a vaccine for prevention of serogroup B disease have focused on the use of alternative noncapsular antigens as vaccine components.

Non-capsular-antigen-based vaccines include outer membrane vesicles (5, 7, 42, 59, 68), specific outer membrane proteins such as PorA (6, 12, 24, 40, 41, 51, 62, 63), iron-regulated proteins such as transferrin binding protein (3, 31, 43), outer membrane opacity proteins such as Opa and Opc (39, 50), and detoxified lipopolysaccharide (64).

While vaccines based on outer membrane vesicles or recombinant PorA appear to be safe and can induce protective antibody in humans (18, 59), the bactericidal antibody responses are strain specific, especially in infants (18, 59). Therefore, a multivalent PorA approach is required. In initial clinical studies of one multivalent vaccine, an outer membrane vesicle vaccine containing multiple PorA recombinant proteins, only modest serum bactericidal titers were elicited in infants, and there appeared to be antigenic interference between some of the serotype antigens (52). Given the propensity for meningococcal disease during nonepidemic periods to be caused by multiple strains or strain variants (53), together with the frequent temporal shifts in the predominant strains in a community, it will be difficult to develop a universal meningococcal B vaccine with only outer membrane protein components that are highly variable from strain to strain.

Recently, Martin et al. described a membrane protein, designated neisserial surface protein A (NspA), which was reported to be highly conserved across Neisseria strains (36) and elicited serum bactericidal antibody responses in mice to N. meningitidis strains from serogroups A, B, and C (36). Immunized mice also were reported to be protected from a lethal challenge with live meningococcal B organisms (36). Thus, NspA appears to represent a novel and promising vaccine candidate.

To investigate further the vaccine potential of NspA, we cloned the nspA gene, expressed NspA in Escherichia coli, and produced mouse polyclonal anti-recombinant NspA (anti-rNspA) antisera. The resulting antisera were used to evaluate the accessibility of NspA epitopes on the surface of different strains of live encapsulated N. meningitidis group B bacteria and the susceptibility of these strains to antibody-dependent, complement-mediated bacteriolysis. In this report, we confirm that the NspA gene and protein sequences are highly conserved among genetically divergent strains of N. meningitidis group B. However, despite conservation of the protein sequence and expression, we found strain differences in the surface accessibility of NspA epitopes and in the susceptibility of different strains to anti-NspA bactericidal activity.

	MATERIALS AND METHODS

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Bacterial strains. The N. meningitidis strains studied were isolated from patients residing in different countries over a period of more than 30 years (Table 1). The specific strains were selected to be representative of widely divergent "clonal" groups, as defined by multilocus isoenzyme typing and/or multilocus sequence typing (33, 57). Strain M7, which is derived from strain NMB, contains a transposon insertion that blocks capsular polysaccharide biosynthesis (58). All of the other strains were encapsulated.

Cloning, expression, and purification of rNspA and HisTag-NspA. Based on the nucleotide sequence published by Martin et al. (36), the following primers that are complementary to opposing strands at the 5' and 3' ends of the nspA gene, respectively, were synthesized: 5'-ACAGCAGGATCCTTTAACGGATTC-3' and 5'-GTGGATGAAGCTTTGGACATTTC-3'. In addition, the primers contained base substitutions that created cleavage sites for BamHI and HindIII at the 5' and 3' ends, respectively, of the nspA gene. By using the method described for DNA sequencing below, these primers were used in PCR to amplify a 743-bp DNA segment from the genome of N. meningitidis 8047. The fragment, which includes the wild-type promoter region, was subsequently cloned into the multicopy plasmid pSK(+) (Stratagene, San Diego, Calif.). In addition, we obtained as a gift from R. Rappuoli (Chiron Vaccines, Siena, Italy) a plasmid that contains a truncated version of the nspA gene (pTrc.NspA.1). In this construct, the portion of the gene encoding the signal sequence has been replaced with DNA encoding a series of six histidine residues (expression vector pTrcHis [Invitrogen, Carlsbad, Calif.]).

DNA preparation and sequencing. Multiple and single clones of each strain were grown to an absorbance at 620 nm (A₆₂₀) of 1 in 7 ml of Mueller-Hinton broth (Difco, Detroit, Mich.) supplemented with 0.2% glucose. Genomic DNA was prepared with a commercial kit (Qiagen) as specified by the manufacturer for the preparation of genomic DNA from bacteria (47). DNA used for sequencing was obtained by PCR amplification of nspA in the genomic DNA preparation with the primers described above. The Taq polymerase and other reagents used for PCR were from Qiagen. The protocol consisted of a denaturation step (94°C for 5 min) followed by 35 cycles of amplification (94°C for 1 min, 60°C for 2 min, and 72°C for 1.5 min) and 1 cycle of extension (72°C for 5 min). Amplified DNA was purified with a QIAquick PCR purification kit (Qiagen) as specified by the manufacturer (48). DNA sequencing of each clone was performed by McConnel Research (San Diego, Calif.) with the primers given above. For determination of the sequence of the nspA gene of each strain, two or three independent PCR products from single clones were sequenced on both strands. Sequence alignments and translations were performed with MacVector software (Oxford Molecular, Cambridge, Mass.).

Polyclonal anti-rNspA antisera. To prepare antisera against NspA, 50 µg of purified HisTag-NspA or partially purified wild-type rNspA expressed from its own promoter was used to immunize groups of 4- to 6-week old female CD-1 mice (five to eight mice per group). Injections were given intraperitoneally (i.p.). Complete Freund's adjuvant was used for the first dose, and incomplete Freund's adjuvant was used for two subsequent booster doses given at 2- to 3-week intervals. As controls, groups of mice were immunized with the same adjuvants combined with proteins precipitated from culture medium supernatant of E. coli BL21(DE3) cells transformed with the parent plasmid pSK(+), which lacks the nspA gene. In a few experiments, we also immunized mice intramuscularly with 50 µg of rNspA given with 5 µg of purified QuilA (gift of G. Ott, Chiron Corp., Emeryville, Calif.) as the adjuvant, and we also compared the responses of female BALB/c and CD-1 mice. For assessment of serum antibody responses, individual mouse sera obtained 2 to 4 weeks after the third immunization were pooled.

Binding of antisera to the surface of live encapsulated meningococci. The ability of antisera elicited by the recombinant NspA constructs to bind to the surface of pathogenic strains of N. meningitidis group B was determined by flow cytometric detection of indirect fluorescence assay, performed as described previously (21). In brief, bacterial cells were grown to mid-log phase in Mueller-Hinton broth, harvested by centrifugation, and resuspended in blocking buffer (phosphate-buffered saline [PBS] containing 1% [wt/vol] bovine serum albumin and 0.2% [wt/vol] sodium azide) at a density of ~10⁸ cells per ml. Dilutions of test or control antiserum (typically 1:10 to 1:100) were then added and allowed to bind to the cells, which were maintained on ice for 2 h. Following two washes with blocking buffer, the cells were incubated with the fluorescein isothiocyanate-conjugated F(ab')₂ fragment of goat anti-mouse IgG (heavy plus light chains) (Jackson ImmunoResearch, West Grove, Pa.) and fixed with 0.25% formaldehyde in PBS buffer, and the bacterial cells were analyzed by flow cytometry.

Complement-dependent bactericidal antibody activity. The bactericidal assay was adapted from the method previously described from this laboratory (35) with the following modifications. After overnight growth on chocolate agar, several colonies were inoculated into in Mueller-Hinton broth (starting A₆₂₀ of ~0.1) and the test organism was grown for approximately 2 h to an A₆₂₀ of ~0.6. After the bacteria were washed twice in Gey's buffer, approximately 300 to 400 CFU was added to the reaction mixture. The final reaction mixture of 60 µl contained 20% (vol/vol) complement and serial twofold dilutions of test sera or control MAbs in Gey's buffer (instead of barbital buffer as previously described by Mandrell et al. [35]). The complement source was human serum from a healthy adult with no detectable anticapsular antibody to group B polysaccharide when tested by the enzyme-linked immunosorbent assay (ELISA) (22) and no detectable intrinsic bactericidal activity at a final concentration of 20 or 40% against the test strain. In preliminary experiments with a panel of test sera, this complement source gave comparable bactericidal titers to those obtained with agammaglobulinemic serum as the complement source. Serum bactericidal titers were defined as the serum dilution (or antibody concentration) resulting in a 50% decrease in CFU per milliliter after a 60-min incubation of bacteria in the reaction mixture, compared to the control CFU per milliliter at time zero. Typically, bacteria incubated with the negative control antibody and complement showed a 150 to 200% increase in CFU per milliliter during the 60 min of incubation.

Animal protection. The ability of the anti-rNspA antiserum to confer passive protection against N. meningitidis group B bacteremia was tested in infant rats challenged i.p. by a method adapted from that of Saukkonen et al. (54). In brief, 6- to 7-day-old pups from six litters of outbred Wistar rats (Charles River, Hollister, Calif.) were randomly redistributed to the nursing mothers. Groups of five or six animals were challenged i.p. with 100 µl of approximately 5 × 10³ CFU of N. meningitidis group B bacteria. Two strains, M986 and 8047, each of which had been passaged three times in infant rats, were tested. The bacteria isolated from blood cultures after the third pass were grown on chocolate agar overnight and stored frozen at 70°C in vials containing sterile skim milk. On the day of the experiment, the bacteria were grown, washed, and resuspended in Gey's buffer, as described above for the bactericidal assay. Immediately before administration to the animals, the bacterial suspension was mixed with different dilutions of test or control antiserum or MAb. At 18 h after the bacterial challenge, blood specimens were obtained by puncturing the heart with a syringe and needle containing approximately 25 U of heparin without preservative (Fujisawa USA, Deerfield, Ill.). Aliquots of 1, 10, and 100 µl of blood were plated onto chocolate agar. The CFU per milliliter of blood was determined after overnight incubation of the plates at 37°C in 5% CO₂.

Membrane preparations. Single colonies were grown to an A₆₂₀ of 0.7 to 0.9 in 7 ml of Mueller-Hinton broth (Difco) supplemented with 0.2% glucose. The 7-ml culture was then used to innoculate 200 ml of the same medium prewarmed to 37°C. After growth of the culture to an A₆₂₀ of 0.9, the bacteria were collected by centrifugation at 5,000 × g for 15 min. The cell pellets were frozen at 20°C until used for preparation of membrane proteins. Sodium lauroyl sarcosinate-insoluble extracts of bacterial membranes were prepared as described previously for H. influenzae type b (4). Briefly, the cell pellets were resuspended in 10 ml of HEPES buffer (pH 7.4) at 4°C. The cell suspension was then sonicated on ice with several 15-s bursts from a microprobe sonifier (Branson, Danbury, Conn.). Cell debris was removed by centrifugation at 5,000 × g for 20 min, and the membrane vesicles in the supernatant were obtained by ultracentrifugation at 100,000 × g for 1 h at 4°C. The resulting membrane pellet was resuspended in 2 ml of HEPES buffer (pH 7.4) containing 1% (wt/vol) sodium lauroyl sarcosinate (Sigma, St. Louis, Mo.) at ambient temperature. After a 30-min incubation, the sodium lauroyl sarconsinate-insoluble fraction was collected by ultracentrifugation at 100,000 × g for 2 h.

SDS-PAGE and Western blots. NspA protein preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (21% polyacrylamide) as described by Laemmli (27) with a Mini-Protean II electrophoresis apparatus (Bio-Rad, Richmond, Calif.). Samples were suspended in SDS sample buffer (0.06 M Tris-HCl [pH 6.8], 10% [vol/vol] glycerol, 2% [wt/vol] SDS, 5% [vol/vol] 2-mercaptoethanol, 10 µg of bromophenol blue per ml) and either heated to 100°C for 5 min or, in some experiments, left at ambient temperature for 5 min before being loaded directly onto the gel. For Western blots, the gel was equilibrated with buffer (48 mM Tris-HCl, 39 mM glycine [pH 9.0], 20% [vol/vol] methanol) and transferred to a nitrocellulose membrane (Bio-Rad) by using a Trans-Blot (Bio-Rad) semidry electrophoretic transfer cell. The nitrocellulose membranes were blocked with 2% (wt/vol) skim milk in PBS containing 0.2% (wt/vol) sodium azide. Anti-HisTag-NspA antisera were diluted in PBS containing 1% (wt/vol) bovine serum albumin, 1% (wt/vol) Tween 20, and 0.2% (wt/vol) sodium azide. Bound antibody was detected with rabbit anti-mouse IgG-, IgA-, and IgM-alkaline phosphatase conjugate polyclonal antibody (Zymed, South San Francisco, Calif.) and Sigma Fast BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) substrate (Sigma).

Quantitation of capsular polysaccharide antigen. The amount of capsular polysaccharide produced by each strain was determined by an inhibition ELISA. First, extracts of capsular polysaccharide were prepared based on a method described by Corn et al. (13). In this method, individual bacterial clones were grown for 1.5 to 2 h in 7 ml of Mueller-Hinton broth to an A₆₂₀ of 0.5 to 0.7. The bacteria were collected by centrifugation at 5,000 × g for 15 min. The cells were washed once in 0.6 ml of 10 mM HEPES (pH 8.0) and then resuspended in 0.6 ml of the same buffer containing 10 mM EDTA and incubated at 37°C for 1 h. The cells were pelleted at 10,000 × g for 1 min, and the relative amount of meningococcal B polysaccharide antigen released into the supernatant was determined by an inhibition ELISA, performed as described by Azmi et al. (2). The solid-phase antigen in the ELISA was meningococcal B polysaccharide-adipicdihydrazide-biotin absorbed to avidin-coated microtiter plates as previously described (22). The meningococcal B polysaccharide-reactive human paraprotein LIP (2), at concentration of 0.2 µg/ml, was used as the primary antibody. In the absence of inhibitor, this concentration of antibody was sufficient to give an A₄₀₅ of ~0.7 to 1.0 after a 30-min incubation with substrate (2). The titer of polysaccharide released into the supernatant was measured by determining the dilution of supernatant that resulted in 50% inhibition of antibody binding. Controls in this assay included an EDTA extract prepared from strain M7, which does not produce any capsular polysaccharide (58), and purified meningococcal B polysaccharide. To ensure that all of the capsular polysaccharide was released by the EDTA treatment, the same inhibition ELISA was performed with the cell pellet resuspended in the same buffer and volume as the capsule extract. The observable inhibitory activity from the cell pellet was between 0 and 10% of the activity observed in the capsule extracts, with the latter, higher percentage coming from cell pellets of strains that produce the largest amounts of capsule.

	RESULTS

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Figure 1A shows a Coomassie blue-stained SDS-PAGE gel (21% polyacrylamide). The sample in lane 1 contains the HisTag-NspA, purified by Ni-nitrilotriacetic acid Sepharose chromatography of cell lysates solubilized in 6 M guanidine hydrochloride, which resolves as two bands with apparent molecular masses of 20.2 and 24.5 kDa. These bands appear to be different forms of HisTag-NspA rather than different proteins, since both the number and the apparent molecular masses of the bands in the HisTag-NspA sample are dependent on the content of SDS and EDTA in the sample buffer (data not shown).

View larger version (226K): [in this window] [in a new window]   FIG. 1.   (A) Coomassie blue-stained SDS-PAGE gel (21% polyacrylamide). Lanes: 1, HisTag-NspA eluted from the Ni-NTA Sepharose metal affinity column with 8 M urea-0.1 M NaH2PO4-0.01 M Tris-HCl buffer at pH 4.5; 2, ammonium sulfate-precipitated culture medium from E. coli BL21(DE3) transformed with pSK(+) (the parent plasmid without the nspA gene); 3 and 9, ammonium sulfate-precipitated culture medium from E. coli BL21(DE3) transformed with pGMS1.0 [the pSK(+) plasmid containing the wild-type nspA gene without the His tag]; production of rNspA is evident by the appearance of a protein with an apparent molecular mass of 18.6 kDa (arrow); 4 to 8, lauroyl sarcosinate-insoluble outer membrane preparations of meningococcal B strains 8047 (lane 4), CU385 (lane 5), NG6/88 (lane 6), M986 (lane 7), and M136 (lane 8). (B) Binding of anti-HisTag-NspA antiserum as shown by Western blotting. Lanes are identical to those in panel A. Except for HisTag-NspA, which appears as two immunoreactive bands at higher apparent molecular masses (see the text), the presence of NspA in each of the samples is indicated by the arrow at an apparent molecular mass of 18.6 kDa. (C) Coomassie blue-stained SDS-PAGE gel (21% polyacrylamide). Lanes: 1, lauroyl sarcosinate-insoluble membrane proteins from strain 8047 that has not been heated in sample buffer; 2, sample comparable to that in lane 1 that was heated to 100°C for 5 min; 3, ammonium sulfate-precipitated culture medium from E. coli BL21(DE3) transformed with pGMS 1.0 [the pSK(+) plasmid containing the wild-type nspA gene without the His tag] that has not been heated in sample buffer; 4, a sample comparable to that in lane 3 that was heated to 100°C for 5 min. The arrows indicate the shift from the higher apparent molecular weight to the expected molecular weight of NspA, as a result of heating the sample in sample buffer.

According to Martin et al. (36), NspA is associated with the outer membrane. However, recombinant NspA produced in E. coli (without the His tag) is secreted into the culture medium. In their experiments, the "secreted" fraction of the rNspA protein was affinity purified and used as a vaccine. Figure 1A, lane 2, shows the proteins present in ammonium sulfate-precipitated culture medium from E. coli BL21(DE3) transformed with pSK(+), the parent cloning vector without the nspA gene. Lanes 3 and 9 contain duplicate samples of the ammonium sulfate-precipitated culture medium from the same E. coli strain transformed with plasmid pGMS1.0, which contains the wild-type nspA gene. The presence of rNspA is evident by the appearance of a protein with an apparent molecular mass of 18.6 kDa, corresponding to the molecular mass of NspA predicted from the amino acid sequence. This protein is not present in the control (lane 2). Lanes 4 to 8 contain sodium lauroyl sarcosinate-insoluble membrane proteins from strains 8047, CU385, NG6/88, M986, and M136, respectively. All contain a protein having the same mobility (18.6 kDa) as rNspA (lanes 3 and 9).

Confirmation that the 18.6-kDa protein observed in Fig. 1A, lanes 3 to 9, is NspA was obtained by Western blot analysis with anti-HisTag-NspA polyclonal serum as the detecting antibody. The results are shown in Fig. 1B. The 18.6-kDa bands present in Fig. 1B, lanes 3 to 9, are reactive with the anti-HisTag-NspA sera. Note that in lanes 2 and 3 the anti-HisTag NspA antisera also react with higher-molecular-weight proteins. However, these cross-reacting higher-molecular-weight proteins were present in both the control (i.e., without NspA) and rNspA-containing preparations.

When analyzed by SDS-PAGE, NspA is reported to have two forms, a 22-kDa diffuse band and a 18-kDa band (36). However, we found that these two forms resulted because NspA is a heat-modifiable protein. Figure 1C shows an SDS-PAGE gel (21% polyacrylamide) of sodium lauroyl sarcosinate-insoluble outer membrane proteins from strain 8047 and partially purified rNspA. Lane 3 contains rNspA that has not been heated in sample buffer. A diffuse band with an apparent molecular mass of 23 kDa is present. Although not shown, a comparable band was not present in proteins present in ammonium sulfate-precipitated culture medium from E. coli containing the parent cloning vector without the nspA gene. As shown in Fig. 1C, lane 4, when the partially purified rNspA was heated to 100°C in sample buffer, the 23-kDa band disappeared and was replaced by a protein with an apparent molecular mass of 18.6 kDa. A similar shift in mobility was observed in the respective proteins present in the detergent-insoluble outer membrane protein preparation from strain 8047 (compare lanes 1 and 2). In both the rNspA preparation and the outer membrane preparation from strain 8047, the 18.6-kDa band was recognized by the anti-HisTag-NspA antiserum in the Western blot (Fig. 1B). The anti-HisTag NspA antiserum also recognized the respective 23-kDa bands in samples that had not been heated (data not shown).

Binding of anti-rNspA serum to native NspA on the surface of live meningococcal B bacteria. Binding of serum antibodies to the cell surface of live meningococci was measured by flow cytometric detection of indirect immunofluorescence (21). Figure 2 shows the results of a typical experiment examining antibody binding to two test strains: NMB, a fully encapsulated N. meningitidis group B strain, and M7, a transposon-containing capsule-deficient mutant of NMB (58). As expected, the anti-group B polysaccharide MAb (SEAM 3) (21) binds only to the encapsulated strain whereas as the positive control anti-P1.2 (PorA) MAb binds to both the encapsulated strain and the nonencapsulated mutant. The ability of pooled antisera from mice immunized with rNspA to bind to bacteria is also shown. The antisera from mice immunized with wild-type rNspA show an increase in fluorescence intensity with both the encapsulated and nonencapsulated mutant strains when the antisera were tested at dilutions of 1:10 to 1:100. In contrast, polyclonal antisera prepared to proteins precipitated from culture supernatant of the E. coli vector alone (without the nspA gene) showed only low-intensity background fluorescence and were considered negative. Note that in Fig. 2, representative data are shown only for antisera diluted 1:25.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Binding of polyclonal anti-rNspA antisera and control MAbs to encapsulated and nonencapsulated strains of live meningococcal B bacteria as determined by indirect-fluorescence flow cytometry. Strain M7 is a nonencapsulated mutant derived from strain NMB by transposon insertion (58). All antisera were tested at a dilution of 1:25. The control MAbs and antisera include VIG10, a murine MAb with an irrelevant specificity; N. meningitidis subtype MAb anti-P1.2 reagent (Rijksinstituut Voor Volksgezondheid en Mileu, Bilthoven, The Netherlands); and SEAM 3, an anti-capsule-specific murine MAb (21); as well as pooled sera from mice immunized with proteins from the supernatant culture of E. coli BL21(DE3), containing the vector that lacks the nspA gene (anti-E. coli control). The test antisera include anti-rNspA antisera elicited by immunization of mice with ammonium sulfate-precipitated culture medium from E. coli BL21(DE3) that expresses rNspA from plasmid pGMS 1.0 and polyclonal anti-HisTag-NspA antiserum.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   (A) Binding of polyclonal anti-rNspA antiserum and control MAbs to encapsulated meningococcal B strains 8047, CU385, and M986, as determined by indirect-fluorescence flow cytometry. All sera were tested at a dilution of 1:25. The test and control MAbs and antisera are those described in the legend to Fig. 2. (B) Binding of polyclonal anti-rNspA antiserum and control MAbs to encapsulated meningococcal B strains 8047, BZ232, MC58, NG3/88, and NGP165, as determined by indirect-fluorescence flow cytometry. All sera were tested at dilutions of 1:10 and 1:100 as indicated. The irrelevant and anticapsular control MAbs and control anti-E. coli antisera are those described in the legend to Fig. 2.

Strain differences in NspA surface accessibility. The flow cytometric assay was used to assess the ability of pooled IgG anti-rNspA antibodies to bind to the surface of 17 genetically diverse strains of live, encapsulated pathogenic N. meningitidis group B bacteria isolated from patients residing in different countries over a period of more than 30 years. Figure 3A shows examples of antibody binding to the surface of N. meningitidis 8047, the homologous strain from which we cloned the nspA gene, and two heterologous strains, CU385 and M986. All three strains showed no binding with a negative control MAb of irrelevant specificity but bound strongly to two positive control MAbs, an anticapsular antibody and anti-P1.2 (left). In contrast, the anti-rNspA antibody bound only to strains CU385 and 8047 and there was no detectable anti-rNspA antibody binding to strain M986 (right).

Sequence variability in NspA from different strains. One possible explanation for apparent strain differences in reactivity with the anti-rNspA antisera in the flow cytometric assay is polymorphisms in the NspA protein. To test this possibility, we cloned and sequenced the nspA gene from five of the six negative strains (BZ232, NG3/88, NGP165, M136, and M986) and from three of the positive strains (8047, CU385, and NG6/88). The GenBank accession numbers for the nspA gene sequences for these strains are AF175676 to AF175683. For the sixth negative strain, MC58, we obtained the nucleotide sequence of the nspA gene from data generated by The Institute of Genomic Research, as part of their MC58 genome sequencing studies (60). The nspA gene sequences of all 10 strains were highly conserved. In comparison to the DNA sequence published by Martin et al. (36), there were between zero and five nucleotide differences resulting in zero to three amino acid differences. Further, as shown in Fig. 4, with one exception, all of the amino acid variants involved the same respective residues in discrete segments of the protein. These included the signal peptide, which is not present in the mature protein, and two short segments in the carboxyl-terminal 50 amino acids of the protein. Evidently, these differences are inconsequential for recognition of the protein by the anti-rNspA antisera, since there are examples of identical derived amino acid sequences of the mature NspA from strains that were negative for reactivity with anti-rNspA antisera and those that were positive (compare M136 to 8047, NGP165 to NG6/88, and MC58 to CU385).

View larger version (43K): [in this window] [in a new window]   FIG. 4.   Predicted amino acid sequences of NspA from different N. meningitidis serogroup B strains. Sequencing data for N. meningitidis 608B were obtained from Martin et al. (36) (GenBank accession no. U52066), and those for strain MC58 were obtained from The Institute for Genomic Research (49a). Data for N. gonorrhoeae B2 were from GenBank (accession no. U52069), and data for FA1090 were from the University of Oklahoma Neisseria gonorrhoeae Genome Sequencing Project. The numbering corresponds to that of the mature protein. Underlined segments are putative surface-exposed loops (see Fig. 5). NM indicates that the NspA sequence is from N. meningitidis, and NG indicates that it is from N. gonorrhoeae. The superscript + and  indicate strains that were positive or negative, respectively, for anti-rNspA antiserum binding as determined by the flow cytometric assay.

Detergent-insoluble outer membrane fractions prepared from different N. meningitidis group B strains. The indirect-fluorescence cell binding assay provides information on the surface accessibility of NspA epitopes that are likely to be important in interacting with protective antibody. Given the identical or nearly identical nspA genes in the different strains, the failure to detect NspA surface expression in some strains but not others could reflect decreased amounts of NspA in the outer membrane of the former, possibly as a result of strain differences in nspA gene expression.

Production of capsular polysaccharide. The ability of anti-rNspA antibody to bind to the bacterial cell surface could also be influenced by the amount of polysaccharide capsule present. To test this hypothesis, we compared the quantity of capsular polysaccharide produced by each of the strains that were scored as positive or negative for anti-NspA binding by the flow cytometric assay. As described in Materials and Methods, the bacterial cells were suspended in buffer and incubated with EDTA to release capsular polysaccharide. The amount of soluble capsule released was measured by an inhibition ELISA. The data were expressed as the reciprocal dilution of supernatant giving 50% inhibition of binding of an anticapsular MAb to biotinylated meningococcal B polysaccharide adhering to avidin-coated wells. Each strain was tested for capsular polysaccharide production in two to four independent experiments. From these results, a mean value (± standard error) was assigned to each strain. The results for the individual strains are summarized in Table 1. Although there are a few notable outliers (e.g., strains 8047 and NGP165), on average the 6 strains that were negative for reactivity with anti-rNspA antisera by the flow cytometric assay produced threefold more capsular polysaccharide than did the 11 strains that were positive (reciprocal geometric mean dilutions of 676 and 224, respectively [P < 0.05 by the t test]). We also measured the amount of capsular polysaccharide released into the culture medium for representative NspA-positive (CU385 and H44/76) and -negative (M136 and M986) strains by the same inhibition ELISA. In each case, the relative amount of polysaccharide released by each strain into the medium correlated directly with the amount of capsule polysaccharide measured in the bacterial extracts (data not shown). Thus, the smaller amounts of cell-associated capsule in the NspA cell surface positive strains were not a result of greater shedding of capsular material into the broth.

Complement-mediated bactericidal activity of anti-rNspA antiserum. Table 1 summarizes the results of measurement of complement-mediated bactericidal activity of the anti-rNspA antiserum for each of the strains tested. All 17 strains were killed by complement together with similar concentrations of a positive control anti-capsular MAb (SEAM 12; subtype IgG2a [21]). In contrast, the six strains that were negative for anti-rNspA antiserum binding by the flow cytometric assay were resistant to anti-rNspA antibody-induced complement-mediated bactericidal or bacteristatic activity (each showed a 150 to 200% increase in CFU per milliliter compared to the control CFU per milliliter at time zero when assayed at the lowest dilution of the anti-NspA antiserum tested, 1:4).

Passive protection by anti-rNspA antiserum. The ability of the anti-rNspA antiserum to confer passive protection against bacteremia was evaluated in infant rats subjected to i.p. challenge. Two representative strains were selected for this study, i.e., M986, which was negative for NspA surface accessibility by the flow cytometric assay and resistant to anti-NspA-induced bactericidal activity, and 8047, which was positive for NspA surface-accessible epitopes and susceptible to anti-NspA bactericidal activity. The challenge doses used were similar for the two strains (5.5 × 10³ cells of strain M986 and 6.3 × 10³ cells of 8047). To maximize the likelihood of observing protection, the bacteria were resuspended in buffer and different dilutions of test or control antisera immediately before the challenge. Table 2 summarizes the results of quantitative bacterial cultures performed on blood specimens obtained 18 h after challenge. A dose of 2 µg per rat of the positive control anticapsular MAb, SEAM 3, was completely protective against both strains. Two dilutions (1:5 and 1:25) of the anti-rNspA antiserum (the highest dilutions tested) protected against bacteremia caused by strain 8047 (positive for NspA surface epitopes). However, neither dilution conferred protection against strain M986 (negative for NspA surface-accessible epitopes).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In 1997, Martin et al. described NspA as a potential vaccine candidate for the prevention of invasive N. meningitidis disease (36). NspA epitopes appeared to be highly conserved across N. meningitidis based on reactivity with a murine anti-NspA MAb, designated Me-1. This MAb cross-reacted with 248 of 250 meningococcal strains in a colony blot ELISA with whole bacterial antigens. This strain collection included representatives of serogroups A, B, C, 29E, W-135, Y, and Z (including 44 serogroup B organisms). The MAb also was bactericidal against isolates with serogroups A, B, or C. In mice, rNspA administered with QuilA elicited serum bactericidal antibody responses against representative strains of serogroups A, B, and C and protected animals from challenge with live N. meningitidis group B bacteria. Recently, rNspA absorbed to alum also was reported to elicit serum meningococcal bactericidal antibody responses in rabbits and monkeys (37). Based on these data, an rNspA vaccine is being developed for the prevention of meningococcal disease caused by all serogroups. However, serogroup A and C polysaccharide-protein conjugate vaccines are highly immunogenic in human infants (8, 16, 29, 32) and also induce polysaccharide-specific memory B cells (28, 30). Thus, the greatest utility for an rNspA vaccine would be for prevention of serogroup B disease, since, to date, no other approach to vaccination against serogroup B strains has proven to be safe and broadly effective (18). Therefore, in the present study, we focused on further characterization of NspA in a collection of 17 N. meningitidis group B strains that were selected based on ET and/or multilocus sequence typing to be genetically highly diverse (Table 1).

NspA is reported to be accessible at the surface of all intact N. meningitidis strains tested (36, 44). However, we found no detectable anti-rNspA antibody binding to the bacterial surface of 6 (35%) of 17 meningococcal B strains as assessed by indirect-fluorescence flow cytometry. The six strains that were negative for anti-rNspA antiserum binding had nspA genes that were identical or nearly identical to those of representative strains that were positive for anti-rNspA cell surface binding. Furthermore, based on SDS-PAGE and Western blotting, all six strains that were negative for anti-rNspA antisera binding contained NspA in detergent-insoluble outer membrane preparations. Thus, failure of these strains to express NspA epitopes on the bacterial surface is not a result of absence of the gene, lack of expression of the gene, or polymorphisms.

At present it is unclear why there are strain differences in NspA surface epitopes. One possible clue is our finding that the 6 strains that were negative for surface-reactive NspA produced, on average, greater than threefold more capsular polysaccharide than did the 11 strains that were positive for surface-reactive NspA. Conceivably, the presence of larger amounts of capsule interferes with the ability of the anti-rNspA antibody to bind to NspA epitopes, which, in strains with smaller amounts of capsule, are accessible to antibodies. To gain insight into the possible antigenic sites of NspA and into why NspA surface accessibility might be restricted by the presence of increased amounts of capsular polysaccharide, we have developed a tentative model for the structure and the organization of NspA in the bacterial membrane (Fig. 5).

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Secondary-structure prediction for NspA. The boxed areas indicate segments that are >40% identical and >70% similar to encoded amino acid sequences of opacity proteins (Opa) from N. meningitidis, N. gonorrhoeae, N. flavius, N. sicca, and H. influenzae identified in a BLAST sequence alignment search (1) of the Non-Redundant GenBank CDS. The segments having alternating placement of letters are predicted amphiphilic -strands. Vertical segments correspond to putative transmembrane segments. Segments at the top of the figure are predicted to be surface-exposed loops, which are labeled loops 1 to 4.

Based on the pattern of alternating hydrophobic and hydrophilic amino acids, which is characteristic of many -barrel porin structures (66), we predict that NspA contains eight transmembrane -strands and four surface-exposed connecting loops. According to Martin et al., the only significant homology between the deduced amino acid sequence of NspA and those of other proteins are weak homologies to the Neisseria opacity protein (Opa) family in two small segments (~20 amino acids) near the C-terminal end of the protein (36). However, when we compared the N-terminal and C-terminal halves of NspA separately with sequences in the nonredundant GenBank CDS database (1), we found a high degree of homology (>40% identity and >70% similarity) between NspA and Opa proteins from N. meningitidis, N. gonorrhoeae, N. flavius, N. sicca, and H. influenzae (data not shown). The Opa proteins are thought to be integral membrane proteins that have eight transmembrane segments and a -barrel topology in the membrane similar to that of porin (34). The presence of NspA in detergent-insoluble membrane preparations indicates that NspA is located in the outer membrane of meningococci, which would be consistent with the Opa-like membrane topology shown in the model. In addition, the segments of NspA that are most similar to those of the Opa proteins are the putative transmembrane segments and loop 4 indicated by the boxed areas in Fig. 5. Finally, as was found for Opa (46), NspA is a heat-modifiable protein, as shown in Fig. 1C.

The opacity proteins of Neisseria may be virulence factors (65) and, under certain circumstances, can elicit protective antibody (50). However, problems with limited antibody accessibility of the opacity proteins in encapsulated bacteria, variability of amino acid sequences in exposed loop segments, and phase variation of protein expression during clinical infection have limited the ability of Opa to elicit protective antibody consistently (34).

In contrast, there appears to be little or no sequence variation in the putative surface-exposed loops of NspA based on the sequence data produced in this study and by others. However, Plante et al. recently reported that a panel of anti-N. meningitidis NspA MAbs that reacted with all meningococcal strains tested, reacted with only a limited number of N. gonorrhoeae strains, even though the respective amino acid sequences in the two species are 92% identical (44). When the respective NspA sequences of the meningococcal and gonococcal strains were compared (Fig. 4), all of the respective amino acid differences that resulted in changes in hydrophobicity or charge were found to be located in the putative surface-exposed connecting loops (Fig. 5). This finding suggests that the connecting loops in NspA, which are highly conserved in N. meningitidis, may be important epitopes for antibodies that bind to native meningococcal NspA. However, the putative surface loops of NspA are relatively small (10 to 16 amino acids) compared to, for example, the highly immunogenic external VR1 and VR2 loops (loop 1 and loop 4, respectively) of PorA (~10 to 40 amino acids) (61). Although there is some overlap, the NspA loops would appear to be smaller, which might account for lower surface accessibility of NspA epitopes, especially in the presence of abundant capsular polysaccharide.

The full implications of our data with respect to the potential protective efficacy of an rNspA vaccine require additional study. However, our observations suggest that approximately one-third of meningococcal B strains have decreased expression of cell surface NspA epitopes when grown in vitro. Furthermore, strains that are negative for NspA cell surface epitopes are also resistant to anti-NspA-induced complement-mediated bacteriolysis, and for one of the strains tested in the infant-rat model, the anti-NspA antiserum failed to confer protection against bacteremia. In contrast, this antiserum was highly protective against a test strain that was positive for NspA surface-accessible epitopes and that was susceptible to anti-NspA antibody-induced bactericidal activity. Finally, the anti-rNspA-resistant strains also tended to be the same strains that produced the largest amounts of capsular polysaccharide and therefore would be expected to have the greatest virulence. Taken together, these data raise concerns about the ability of a vaccine containing only rNspA to confer broadly protective immunity against meningococcal B disease. An rNspA vaccine, therefore, may need to be supplemented by the inclusion of additional antigens, such as PorA, which is an important target of bactericidal antibody in humans (18). However, before reaching this conclusion, it will be important to investigate the immunogenicity of NspA in humans and to determine whether the addition of other candidate meningococcal antigens to an rNspA vaccine can augment protective immune responses.