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Journal of Clinical Microbiology, June 2004, p. 2718-2723, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2718-2723.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

DNA Sequence Analysis of the PorB Protein of Nonserotypeable Serogroup C ET-15 Meningococci Suggests a Potential Mutational Hot Spot on Their Serotype Antigens

Dennis K. S. Law,* Averil M. Henderson, and Raymond S. W. Tsang

CNS Infection and Vaccine Preventable Bacterial Diseases Division, National Microbiology Laboratory, Population and Public Health Branch, Health Canada, Winnipeg, Manitoba, Canada

Received 18 November 2003/ Returned for modification 9 January 2004/ Accepted 2 March 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nucleotide sequences of the PorB proteins from 28 nonserotypeable serogroup C ET-15 meningococci recovered from invasive meningococcal disease cases were determined. PCR amplification of the porB genes responsible for encoding the serotype antigen was used for DNA sequence determination and identification of the nature of the serotype antigen. DNA sequencing revealed that three strains were of serotype 2a, and of the remaining 25 strains, 20 were found to have an identical single point mutation in the region of the VR3 gene, which encodes surface-exposed loop VI, where the serotype 2a epitope resides. This nonsynonymous mutation was confirmed by synthetic peptide immunochemical analysis to confer new serospecificity to these serotype 2a mutants. This finding of a potential novel mutational hot spot on the PorB proteins of meningococci may have implications for pathogenesis and vaccine development.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Invasive meningococcal disease (IMD) presenting as meningitis and/or septicemia is a serious disease with an overall case fatality rate of about 10%, even in developed countries, such as Canada and the United States (14, 18). Most epidemiological studies of Neisseria meningitidis strains associated with both disease cases and carriers involve the detection and characterization of specific antigens, such as the capsule, outer membrane proteins (OMPs), and lipooligosaccharide, that are present on the bacterial envelope surface. Based on the immunological specificities of its capsular polysaccharides, N. meningitidis is divided into 13 serogroups (27). Of these, five serogroups (A, B, C, Y, and W135) are responsible for most of the disease worldwide (11). Meningococci can be further subdivided into serotypes and subserotypes by the detection of serologically distinct epitopes present on their class 2 or class 3 (PorB) and class 1 (PorA) OMPs. Differences in lipooligosaccharide epitopes allow strains to be divided into one or more of at least 12 different immunotypes. Therefore, a strain of N. meningitidis can be characterized serologically by its serogroup, serotype, subserotype, and immunotype (for example, C:2a:P1.5,2:L3,7,9) (7).

The serotype antigen of N. meningitidis resides on its PorB OMPs, which are transmembrane proteins with eight predicted surface-exposed loops (I to VIII) that are variable in terms of their lengths and amino acid sequences. These surface-exposed loops are interspaced and anchored by nine membrane-spanning regions that are relatively conserved (25). The two classes of PorB proteins are encoded by either one of their respective porB genes, which are mutually exclusive, so that a strain will produce either a class 2 or a class 3 PorB OMP. Sequence analysis of PorB proteins from different serotypes of meningococci identified four regions with a high level of amino acid sequence variability, termed VR1 to VR4, that correspond to surface-exposed loops I, V, VI, and VII, respectively (5, 33). In ET-37 meningococci, the serotype 2a epitope resides in surface-exposed loop VI, which corresponds to VR3 of their class 2 PorB OMPs. The eight-amino-acid sequence that is believed to correspond to the VR3-2a epitope is NNEVGSTK (15, 25).

The population biology of meningococci has been studied by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (10). Despite the genetic diversity of the organisms, a few electrophoretic types, such as subgroup I, subgroup III, ET-5, ET-37, lineage III, and cluster A4, are repeatedly identified from invasive disease cases (3). One of these hypervirulent clones (the ET-37 clonal complex) has a worldwide distribution and is associated with both endemic and epidemic diseases (29). Beginning in the early 1990s, a new genetic variant of the ET-37 clone emerged and caused a significant increase in meningococcal disease in Canada (2). This new variant, designated ET-15, expresses C:2a:P1.5,2 surface antigens and has a unique genetic marker that distinguishes it from the ET-37 clone. ET-15 has the same profile of housekeeping enzymes as ET-37, with the exception that it has a distinct fumarase enzyme allele, allele 2, instead of the allele (allele 1) possessed by the ET-37 clone. The difference, at the genetic level, between fumarase locus alleles 1 and 2 is due to only one base pair change, from G to A, at position 640 of the fumC gene (28).

Since it was first identified in Canada in 1986, this ET-15 clone of serogroup C meningococci has caused two waves of increased meningococcal disease activity and localized outbreaks, first in 1989 to 1993 (31) and again in 2000 to 2001 (12). In analyzing the serogroup C strains responsible for clusters of IMD cases in 2001, Tsang et al. reported the occurrence of antigenic variants of endemic C:2a:P1.5,2 ET-15 meningococci (20, 21, 22). One such variant, which had all of the characteristics of the endemic ET-15 clone in its serogroup and subserotype antigens but which was nonserotypeable with a panel of commonly used serotyping monoclonal antibodies, was linked to an outbreak of IMD in a community of men who have sex with men in Toronto, Ontario, Canada (20). In order to understand the nature of the serotype antigen in this cluster of nonserotypeable outbreak strains, we sequenced their porB genes and found that a single nucleotide substitution had occurred at surface-exposed loop VI of the PorB protein, where the serotype epitope resides, resulting in a change in one amino acid in hypervariable region 3 (VR3). In this study, we analyzed all nonserotypeable serogroup C strains obtained from IMD cases in Canada in 2001 and 2002 by DNA sequencing of their porB genes, which encode their PorB proteins. Our finding of an identical single point mutation in the PorB proteins of serogroup C, serotype 2a, ET-37 or ET-15 meningococci, which may have implications for pathogenesis and vaccine development, is discussed.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Sources and identities of meningococcal isolates. Isolates of N. meningitidis from IMD cases were submitted to the Health Canada National Microbiology Laboratory (NML) by provincial and territorial public health laboratories across Canada for the national surveillance of IMD (19). Their identities were confirmed by standard biochemical tests. A total of 244 serogroup C meningococci obtained from individual IMD cases in 2001 and 2002 were included in this study. A detailed analysis of the nature of the serotype antigens of 28 nonserotypeable strains was done by DNA sequencing of their porB genes.

Serological characterization of meningococci. Serogrouping was done by bacterial agglutination with rabbit antisera produced to the different serogroups. Serotyping and subserotyping of meningococci was done by an indirect whole-cell enzyme-linked immunosorbent assay (ELISA) (1) with monoclonal antibodies to the following serotype and subserotype antigens: 1, 2a, 2b, 4, 14, 15, P1.1, P1.2, P1.4, P1.5, P1.6, P1.7, P1.9, P1.10, P1.12, P1.13, P1.14, P1.15, and P1.16 (Rijksinstituut voor Volksgezondheid en Milieu, National Institute of Public Health, Bilthoven, The Netherlands).

Genetic characterization of meningococci. MLEE was performed by using a previously described method (16). Partial sequences of the porB genes, which encode segments VR1 to VR4, including the serotype antigen or epitope, were determined by methods described by Sacchi et al. (15).

Synthetic peptides, OMP antigens, and indirect ELISA. 9-mer synthetic peptides representing the serotype 2a and 2a mutant serotype epitopes (corresponding to VR3 of the PorB protein) as well as control peptides containing sequences corresponding to the carboxyl-terminal end of the anthrax protective antigen (see Table 3 for sequences of the various peptides) were purchased from United Biochemical Research, Inc., Seattle, Wash.


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  		TABLE 3. Serological specificity of the serotyping antigen of the 2a mutant strain studied by indirect ELISA with synthetic peptides and rabbit antisera developed against whole cells of the 2a mutant strain

 
For use as antigens in the indirect ELISA, these peptides were conjugated to bovine serum albumin (BSA) via cysteine residues incorporated into their amino-terminal ends by use of an Imject maleimide-activated immunogen conjugation kit (Pierce Biotechnology, Rockford, Ill.). The peptide-protein conjugate was diluted to 5 µg/ml in sterile phosphate-buffered saline (PBS) for antigen coating of Nunc Maxisorp immunoplates (Nalge Nunc International, Rochester, N.Y.) at 4°C overnight. After three washes with 0.85% saline-0.05% Tween 20 (saline-Tween), wells were blocked with 2% BSA in PBS at 37°C for 90 min. Rabbit antisera to the serotype 2a mutant strain were serially diluted and added to both antigen-coated wells and non-antigen-coated control wells for incubation at 4°C overnight. After four washes with saline-Tween, rabbit antibodies bound to the plates were detected with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) diluted 1:2,000 in 2% BSA in PBS. After a final four washes with saline-Tween, 2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) substrate (Roche Diagnostics, Laval, Quebec, Canada) was added for color development at room temperature. All reagents were added in a volume of 100 µl per microtiter well, with the exception of the blocking and washing steps, where 300 µl per well was used.

When OMP antigens were tested against a mouse monoclonal antibody to serotype 2a, OMPs at 10 µg/ml in PBS were used to coat plates at 4°C overnight. Mouse antibody binding to antigens was then detected with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch Laboratories) diluted 1:5,000 in 2% BSA in PBS.

Production of rabbit antisera to the serotype 2a mutant antigen. New Zealand White rabbits were immunized by multiple intravenous injections of increasing doses of either inactivated or live whole cells of the serotype 2a mutant strain (6). The immunization schedule included three injections of formalin-inactivated cells given on Mondays, Wednesdays, and Fridays for each of the first three weeks, beginning with a dose of 0.5 x 108 cells in the first week, increasing to a dose of 1 x 108 cells in the second week, and finally ending with a dose of 2 x 108 cells in the third week. A final single intravenous injection of approximately 2 x 108 live cells was given in the fourth week. Sera were collected from the rabbits in the fifth week.

Absorption of the immune serum with a typical C:2a:P1.5,2 strain (strain 2241C) was carried out to remove common cross-reacting antibodies. The absorbing strain was grown on four large plates of Columbia blood agar at 36°C in 5% CO2 for 18 to 20 h, harvested with 0.5% formal saline, and washed in sterile PBS. Absorption was carried out with packed cells for 1 h at 37°C, followed by overnight incubation at 4°C, and the absorbed serum was clarified by centrifugation.

Nucleotide sequence accession numbers. The partial sequences of the porB genes of strains 2001-031, 2001-202, 2001-203, 2001-351, and 2001-400 were deposited in GenBank (National Center for Biotechnology Information) with the assigned accession numbers AY394653, AY394650, AY394651, AY394652, and AY394654, respectively. The partial porB gene sequences of some of the strains showing the potential mutational hot spot were assigned GenBank accession numbers AY234206 to AY234211.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of serogroup C meningococci causing IMD in Canada. In 2001, 289 meningococcal isolates were submitted to the NML from individual IMD cases across Canada, and of these 289 isolates, 173 (59.8%) were serogroup C meningococci. In 2002, there were 184 individual IMD cases, and 71 (38.6%) of them were due to serogroup C meningococci. Out of the 244 serogroup C meningococci isolated from IMD cases in 2001 and 2002, 240 belonged to the ET-37 clonal complex (170 isolates in 2001 and 70 in 2002), which can be subdivided into ET-15 and the ET-15 complex (166 isolates in 2001 and 69 in 2002) and ET-37 (but non-ET-15; 4 isolates in 2001 and 1 isolate in 2002) (Table 1).


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  		TABLE 1. MLEE profiles for 244 serogroup C meningococci collected from IMD cases in Canada during 2001 and 2002

 
Most of the serogroup C isolates associated with IMD cases in Canada were serotype 2a (149 out of a total of 173 isolates [86%] in 2001 and 65 out of 71 isolates [91.5%] in 2002), and 28 of the isolates were nonserotypeable (23 isolates [13%] in 2001 and 5 isolates [7%] in 2002).

porB gene sequences of nonserotypeable serogroup C strains causing IMD in Canada. Of the 28 nonserotypeable group C isolates, 27 were found by MLEE to belong to the ET-37 clonal complex (25 isolates were ET-15 and 2 were ET-37 but non-ET-15), and 1 isolate was not found to belong to the ET-37 clonal complex.

Three of the 27 ET-37 clonal complex isolates were sequenced as serotype 2a: 2 isolates had typical serotype 2a nucleotide sequences in all four VR segments of their porB genes (VR1-C, VR2-Eb, VR3-2a, and VR4-C), and in 1 isolate, a nonsynonymous single base pair substitution was found in its porB VR4 segment. Therefore, 24 serogroup C ET-37 or ET-15 clonal complex isolates were not typical serotype 2a. Twenty of these 24 isolates (83%) were found to have PorB VR sequences identified as VR1-C, VR2-Eb, VR3-2a(a), and VR4-C by the scheme developed by Sacchi et al. (15). Their PorB VR1, VR2, and VR4 segments were identical to those of typical serotype 2a PorB. However, their VR3 segments were found to contain an identical nonsynonymous single nucleotide substitution at position 700 of loop VI. Hence, these 20 isolates were regarded as serotype 2a variants. This single base change from guanine to adenine in the 2a mutants led to a change in the encoded amino acid from the negatively charged glutamic acid, which has an acidic side chain, to the positively charged lysine, which has a basic side chain. In all 20 of these isolates, the nonsynonymous single point mutation led to the formation of the 2a mutant peptide epitope, with the sequence NNKVGSTK.

The 20 identical 2a mutants came from the following provinces: British Columbia (3 isolates—2 in 2001 and 1 in 2002), Alberta (1 isolate in 2002), Ontario (12 isolates—11 in 2001 and 1 in 2002), and Quebec (4 isolates in 2001). The 11 2a mutants isolated from Ontario in 2001 included 6 isolates that caused an outbreak in the city of Toronto among a community of men who have sex with men (20).

The remaining five nonserotypeable isolates (four belonging to the ET-37 clonal complex and one belonging to the non-ET-37 clonal complex) were not related to the serotype 2a mutants described above. The porB VR gene sequences of these five isolates are summarized in Table 2. The three isolates from Alberta had identical nucleotide sequences in segments VR1 to VR4 of their porB genes. Segments VR1 and VR2 of these three isolates had nucleotide sequences identical to those identified for strains M990 (B:6:P1.6; accession number X67936) and B16B6 (B:2a:P1.5,2; accession number X67937). Their porB gene sequences encoding segments VR3 and VR4 each had differences of 3 bp from that of strain B16B6 (accession number X67937).


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  		TABLE 2. porB gene sequences of five IMD-related serogroup C meningococci that were not related to the most common serotype 2a mutants found in Canada

 
The isolate which was typed as C:NT:P1.7,1 was found to be unrelated to serotype 2a based on its unique porB gene sequence. The PorB VR types of this isolate were found to be VR1-C, VR2-F, VR3-B(a), and VR4-E for segments VR1 to VR4, respectively. The single serogroup C isolate which was neither ET-37 nor ET-15 was typed as C:NT:P1.6. Its PorB VR types were found to be C, Eb, 2a(c), and C(c).

Immunochemical analysis of the serotype 2a mutant antigen. Both typical 2a peptides and 2a mutant peptides were tested against rabbit antisera prepared against the serotype 2a mutant antigen by immunization with whole cells of 2a mutant bacteria. Table 3 compares the reactivities of the different peptides (2a, 2a mutant, and irrelevant peptides) with the rabbit antisera prepared against the serotype 2a mutant epitope. Only the 2a mutant peptide showed strong reactions with the antibodies present in the antisera produced against whole bacterial cells of the 2a mutant strain. Table 4 compares the reactions of the anti-2a monoclonal antibody and rabbit antisera produced to both typical 2a strains and 2a mutant strains with OMPs prepared from both typical 2a strains and 2a mutant strains and their respective peptides. The anti-2a monoclonal antibody did not react with any of the peptides or OMPs prepared from the 2a mutant strain, a result which confirmed the nonserotypeable nature of the mutant strain.


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  		TABLE 4. Reactions of typical 2a and 2a mutant OMP antigens and synthetic peptides against antisera developed to typical 2a and 2a mutant bacterial whole cells

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most serogroup C meningococci recovered from disease cases in North America, Europe, and Africa belong to the ET-37 clonal complex, which is known to cause both endemic and epidemic diseases in different parts of the world (29). All ET-37 N. meningitidis strains express a class 2 but not a class 3 porin OMP, where the serotype antigen resides. Ninety-one percent of 228 ET-37 meningococcal strains studied by Wang et al. (29) expressed the serotype 2a antigen, and the rest were either serotype 2b (6 isolates) or nonserotypeable (14 isolates). In our study of 240 ET-37 meningococci (including 235 ET-15 and ET-15 complex strains) collected from IMD cases in Canada in 2001 and 2002, 213 (88.75%) were typed as 2a. Twenty-seven isolates were nonserotypeable. Of these 27 isolates, 2 were found to have typical serotype 2a porB gene sequences in all four VR segments, while 1 had VR sequences identical to those of a typical serotype 2a strain in the VR1, VR2, and VR3 segments but with a nonsynonymous single point mutation in the VR4 segment. Twenty isolates were found to have VR gene sequences typical of serotype 2a, with the exception of a single point mutation in the VR3 segment, which encodes the serotype 2a epitope. The remaining four isolates may have had multiple mutations in their VR genes, suggesting possible recombination events.

Although it is possible that a single mutation might have occurred in the endemic C:2a:P1.5,2 strain, with subsequent multiplication and spread to cause these 20 IMD cases, circumstantial evidence does not support this hypothesis. On the contrary, the same mutation might have occurred in different isolates of the endemic clone. The evidence supporting this hypothesis include the following. (i) These 20 isolates were determined by an ELISA to have different subserotype antigens, although sequencing of their porA genes was not done to confirm the ELISA subserotype results (5 isolates were typed as C:NT:P1.5,2; 9 isolates were typed as C:NT:P1.2; and 6 isolates were typed as C:NT:P1.– [nonsubserotypeable]). (ii) Besides the Toronto outbreak isolates, which had identical fingerprints (20), preliminary pulsed-field gel electrophoresis analysis done with two different restriction enzyme-digested genomic DNAs from 12 other 2a mutant isolates showed that most of them had different pulsed-field gel electrophoresis patterns, with the exception of two pairs of isolates (one pair isolated from Ontario and one pair isolated from Quebec) (NML, unpublished data). (iii) These 20 isolates represented three MLEE types; 18 were ET-15 and 2 were ET-15 complex (1 with a unique aconitase allele and 1 with a unique glutamate dehydrogenase allele). (iv) These 20 isolates were collected from four different provinces (British Columbia, Alberta, Ontario, and Quebec) mostly within the short period of 1 year. This situation is in contrast to our recent experience with another unique antigenic variant (C:2a:P1.7,1) of endemic serogroup C ET-15 meningococci, which were isolated almost exclusively in one province only (Quebec), without any substantial spread to other provinces within 2 years of its initial detection in 2001 (22; NML, unpublished). If a single successful mutation had occurred and spread to result in these 20 isolates, they would have been similar to the 6 C:NT:P1.2 isolates found in a cluster of IMD cases in Toronto, Ontario; all isolates would have had identical subserotype antigen P1.2 and identical MLEE patterns for all 13 housekeeping enzyme alleles (20).

Another explanation for the occurrence of these 20 2a mutant isolates might be recombination: different serogroup C strains might have recombined with a strain that successfully acquired this single point mutation. Recombination has been reported to occur frequently in Neisseria species (4, 26), and this event may explain the finding over a short period of time of 20 different isolates showing the same mutation in their PorB proteins in individual IMD cases. Although many single nucleotide polymorphisms are clearly evident from the Neisseria PorB typing website (http://neisseria.org/nm/typing/porb), it is not clear from this database or from the literature how often isolates that are encountered in clinical specimens show any one particular type of mutation at any specific site in their porB genes. Also, strains showing the novel mutation described in this article have not been reported in the literature or in the GenBank and Neisseria PorB typing databases (last accessed on January 2004).

In order to test the hypothesis that a mutational hot spot exists in the VR3 segment of the PorB protein of N. meningitidis, it may be necessary to examine a larger number of isolates collected over a longer period of time and with additional markers to establish the relationships among isolates collected independently from individual cases. Recently, phylogenetic techniques were used to investigate the effect of evolutionary selection on the PorB protein. These data indicated that the VR3 segment is highly variable and is subject to strong positive evolutionary selection, possibly driven by the human immune response to this antigen (23).

When rabbit antisera were generated to the serotype 2a mutant antigen by immunization with formalin-inactivated 2a mutant bacterial cells, the antibodies in this antisera reacted much more strongly to the synthetic 2a mutant peptide (NNKVGSTK) than to the typical 2a peptide (NNEVGSTK). These serological data obtained from multiple experiments (Table 3) provided confirmation that the single nucleic acid substitution indeed caused a change in the serological specificity of the 2a serotype antigen. Furthermore, our serological data also repeatedly showed that the anti-2a monoclonal antibody reacted with neither typical 2a peptides nor synthetic 2a mutant peptides (Table 4); these results also suggested that the monoclonal antibody to the 2a antigen recognizes a conformational epitope on the 2a PorB protein. This suggesion was further confirmed by testing OMPs prepared from serotype 2a strains dissolved in either PBS or sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer. Only native 2a OMP antigens in PBS and not denatured 2a OMP antigens in sample buffer showed a reaction with the anti-2a monoclonal antibody (data not shown).

Nucleotide sequence data from the mid-1990s suggested that protein I of Neisseria gonorrhoeae but not the class 2 or class 3 PorB of N. meningitidis exhibited evidence of positive Darwinian selection based on the pattern of synonymous versus nonsynonymous substitutions in the porB genes (17). However, more recent and comprehensive sequence data presented in 2002 suggested that strong positive selection is exerted on the meningococcal PorB protein, presumably due to the host immune response developed during natural infection (23). In this study of serogroup C meningococci isolated from IMD cases in Canada, we have shown that mutations in surface-exposed loop VI of the meningococcal PorB protein occur at a fairly high frequency; 20 out of 24 nonserotypeable strains (83%) or 20 out of 240 serogroup C strains, regardless of their serotype nature (8.3%), had the same mutation. These data suggest that the mutation site is a potential hot spot in the class 2 PorB protein of ET-37 complex (including ET-15 complex) meningococci and that this site is prone to change possibly because of evolutionary selection (or immune-driven adaptation).

Since first recognized in the mid-1980s as causing a significant percentage of IMD cases in Canada, serogroup C meningococci have continued to cause not only an increase in the proportion of endemic IMD but also an increase in the number of IMD cases, including localized outbreaks in schools and other settings. The proportion of IMD cases due to serogroup C meningococci in Canada has increased overall from 25% in 1985 to 64% in 1992 (18, 19). With the prevalence of these organisms in the Canadian population, it can be expected that serum antibodies to the different surface components of the meningococci, including the serotype 2a antigen, are developing in the population. Infection with meningococci is usually characterized by serum antibody responses to their porin proteins, including the serotype epitopes (13, 30). Also, antibodies to the serotype antigen have bactericidal and opsonic properties (8, 9, 32). Considering the endemic nature of serogroup C meningococci (mostly serotype 2a) in Canada, it is possible that serum antibodies to the serotype 2a antigen are developing in the population and are exerting immune selection on the meningococci. A number of codon sites in surface-exposed loop VI of the class 2 PorB protein have been reported to be under strong immune selection for amino acid change (23). Hence, it is possible that changes in the PorB OMP of serotype 2a meningococci may allow the organisms to evade host defense. These new variants may have an increased ability to cause disease, as they are able to escape natural immunity against the more widespread PorB serotype 2a antigen. Therefore, surveillance should include the detection of such mutant strains.

The finding of a potential mutational hot spot on the PorB protein may have an adverse effect on the development of vaccines against meningococci based on their PorB proteins. Similar immune selection has been reported for the subserotype antigen on the PorA protein (24). Therefore, meningococcal vaccines based on OMPs such as PorA or PorB may hasten the selection process for mutations in potential vaccine targets and result in mutant strains being selected.


   ACKNOWLEDGMENTS
 
We thank the directors and staff of the provincial and territorial public health laboratories for supplying the meningococcal strains, the NML DNA Core Facility for providing the DNA sequencing service, and Jan Stoltz for performing MLEE.

We thank the Health Canada Genomics R&D Fund for financial assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: CNS Infection and Vaccine Preventable Bacterial Diseases Division, National Microbiology Laboratory, 1015 Arlington St., Winnipeg, Manitoba R3E 3R2, Canada. Phone: (204) 789-6017. Fax: (204) 789-2018. E-mail: dennis_law{at}hc-sc.gc.ca. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, June 2004, p. 2718-2723, Vol. 42, No. 6
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.6.2718-2723.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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