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Prevalence of genosubtypes (PorA types) of serogroup B invasive meningococcus in the north of Spain from 2000 to 2003

¹Microbiology Department, Hospital Donostia and Basque Country Meningococcal Reference Centre, Paseo Dr Beguiristain s/n, 20014 San Sebastián (Gipuzkoa), Spain ²Microbiology Laboratory, Hospital Txagorritxu, Vitoria, Spain ³Microbiology Laboratory, Hospital Galdácano, Galdácano, Spain ⁴Microbiology Department, Hospital Basurto, Bilbao, Spain

Correspondence Emilio Pérez-Trallero mikrobiol{at}terra.es

Received August 6, 2004
Accepted November 16, 2004

The composition of new vaccines against Neisseria meningitidis serogroup B is based on differences in the variable regions VR1 and VR2 of the class 1 outer-membrane protein (PorA) of meningococci. Genosubtyping of 96 N. meningitidis B isolates from blood or cerebrospinal fluid from 2000 to 2003 in the north of Spain allowed characterization of all the strains. Twenty-six genosubtypes or distinct PorA types were obtained. The most prevalent were P1.5-1, 10-8 (20 strains), P1.19, 15 (14 strains), P1.22, 9 (11 strains) and P1.5, 2 (nine strains), while 17 genosubtypes were represented by only one or two strains. The wide diversity of genosubtypes observed and their differences compared with those found in other regions reveals the difficulty in designing a useful outer-membrane vesicle vaccine applicable to different regions of the world.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Neisseria meningitidis is the main cause of bacterial meningitis in children and young adults throughout the world. Effective polysaccharide vaccines for the prevention of meningococcal disease caused by serogroups A, C, W135 and Y are currently available. Recently, a highly efficacious conjugate vaccine against infections caused by serogroup C has been introduced in several European countries, and protein-based vaccines might be an alternative to prevent epidemics caused by serogroup A meningococci (Norheim et al., 2004). However, an effective vaccine against meningococcus B, the most prevalent serogroup in developed countries, is not currently available. One of the most promising among the alternatives being investigated while waiting for a definitive solution is vaccines based on outer-membrane proteins (OMPs), especially those that use meningococcal class 1 OMP or porin A (PorA) (Frasch et al., 2001). The antigenic variability of the PorA variable regions VR1 and VR2 can be used to identify meningococcal subtypes and forms one of the bases of epidemiological surveillance of meningococcal disease. Moreover, the design of meningococcal outer-membrane vesicle (OMV) vaccines takes into account the meningococcal subtypes most prevalent in the population (Feavers et al., 1996).

The methods most frequently employed to identify subtypes consist of phenotypic techniques using panels of monoclonal antibodies (Abdillahi & Poolman, 1988). Complete serosubtyping of meningococci can rarely be performed and strains that cannot be serosubtyped or only partially serosubtyped are frequently found. This limitation is attributed to the use of incomplete sets of monoclonal antibodies, a lack of PorA expression and the absence of reactivity of the monoclonal antibodies used with some of the numerous VR1 and VR2 variants of the prototype strains (Sacchi et al., 2000; Vogel & Claus, 2003). Genotypic techniques that analyse the VR1 and VR2 sequences of the porA gene overcome these limitations and allow complete identification (Feavers et al., 1992; Sacchi et al., 1998; Diggle & Clarke, 2003). However, information on the distinct genosubtypes or PorA types of meningococci circulating in the various regions of the world is still scarce. In the present study, VR1 and VR2 sequencing was performed to characterize the genosubtypes of serogroup B meningococci isolated from blood or cerebrospinal fluid in the Basque Country in northern Spain over 4 consecutive years.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Epidemiological data.

The Basque Country is situated in the north of Spain and has a population of approximately 2 100 000 inhabitants. According to unpublished data obtained from the Epidemiology Unit of the Department of Health of the Basque Country, there were 279 microbiologically confirmed cases of meningococcal disease in this region from 2000 to 2003, of which 191 corresponded to N. meningitidis serogroup B. The mean annual incidence rate of meningococcal disease, including 97 probable cases of this disease, was 4.5 cases per 100 000 inhabitants and the mean annual incidence of confirmed cases of N. meningitidis B was 2.3 cases per 100 000 inhabitants.

Strains studied.

Ninety-six N. meningitidis B strains isolated in the period 2000–2003 were available (37 strains isolated from cerebrospinal fluid, 47 from blood and 12 from both localizations). Half of the strains were obtained from children aged less than 14 years old.

Serosubtyping.

Serosubtyping was conducted by ELISA following a protocol described previously (Abdillahi & Poolman, 1987) based on 100 µl of a meningococci suspension of approximately 10⁹ c.f.u. ml^–1. Monoclonal antibodies (Rijjksinstituut voor Volksgezondheid) were used to determine 13 serosubtypes. Four serosubtypes were from VR1 (P1.5, P1.6, P1.7 and P1.12) and nine serosubtypes were from VR2 (P1.1, P1.2, P1.4, P1.9, P1.10, P1.13, P1.14, P1.15 and P1.16).

Genosubtyping.

Genosubtype was determined by amplification of two fragments of the porA gene encoding VR1 and VR2, sequencing and subsequent comparison of the deduced amino acid sequence. A suspension containing approximately 10⁶ cells was incubated with 0.1 mg proteinase K ml^–1. Genomic DNA was then extracted using the QIAamp DNA Blood Mini kit (Qiagen). Subsequently, two independent PCRs were performed with specific primers (Tib Molbiol) for both VR1- and VR2-coding regions. For VR1, the U86 forward primer (5'-GCCCTCGTATTGTCCGCACTG-3') and 481 reverse primer (5'-TCGTCGTGGCGTTTGAAAATACCCA-3') were used, and for VR2, the 435 forward primer (5'-GCCATTAATCCTTGGGACAGCAA-3') and 773 reverse primer (5'-GGCATAGTTCCCGGCAAAACCGCCAT-3') were used (Sacchi et al., 1998). The two amplicons obtained were sequenced in an ABI PRISM 3100 Genetic Analyser (Applied Biosystems). To assign the corresponding genosubtype, the deduced amino acid sequences were submitted to the N. meningitidis PorA variable region database (http://neisseria.org/nm/typing/pora/).

The nomenclature recently proposed by Russell et al. (2004) was used.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Using ELISA, nine strains (9.4 %) were completely serosubtypable (VR1 and VR2 serosubtype), 78 strains (81.2 %) were partially serosubtypable (24 strains with VR1 serosubtype and 54 with VR2 serosubtype) and nine strains (9.4 %) were non-serosubtypable (Table 1).

Using genosubtyping, the VR1 or VR2 type of the 96 meningococci studied was identified. Fourteen different VR1 alleles belonging to seven VR1 families and 16 different VR2 alleles belonging to nine VR2 families were found. The 96 strains were grouped into 26 different genosubtypes (Table 1). The most prevalent genosubtypes were P1.5-1, 10-8 (20 strains); P1.19, 15 (14 strains); P1.22, 9 (11 strains); and P1.5, 2 (nine strains); while 17 genosubtypes were represented by only one or two strains. In 42 strains, both VR1 and VR2 sequences were identical to those defining the family prototype sequence (no PorA variant sequences). Genosubtyping identified the VR1 type of 63 strains that could not be identified by serosubtyping (one P1.5-2 strain; 10 P1.7-2 strains; and 52 strains with VR1 types belonging to families not included in the panel of monoclonal antibodies used for serosubtyping). Moreover, genosubtyping identified the VR2 type of 33 strains that could not be identified by serosubtyping (two P1.2 strains; one P1.13-1 strain; three P1.15 strains; two P1.16 strains; and 25 strains with VR2 types from families not included in the panel of monoclonal antibodies) (Table 1).

In only 24 (25 %) strains was the genosubtype (VR1 and VR2) identical to the genosubtype included in one or other of the OMV vaccines used in Norway, Cuba, Brazil and Holland (Bjune et al., 1991; de Moraes et al., 1992; de Kleijn et al., 2000) (Table 2). In 61 strains (63.5 %), either the VR1 type or the VR2 type was identical to one of the VR types included in these vaccines. Twenty-one (21.9 %) N. meningitidis strains did not show even one VR type belonging to one of the VR families included in these vaccines.

The results of the present study, like those of previous studies (Sacchi et al., 2000, 2001; Clarke et al., 2003), demonstrate the advantages of genosubtyping over serosubtyping. Genosubtyping achieves complete identification of both VR1 and VR2 and discriminates the variants among strains (Feavers et al., 1992; McGuinness et al., 1993). The results of the genosubtyping and serosubtyping in this study show greater differences than in previous studies (Sacchi et al., 2000, 2001), partly because the set of monoclonal antibodies used in the present study was small. Moreover, in the present study we observed low or absent reactivity of serosubtype P1.7 monoclonal antibody with the variant type P1.7-2, since, of the 11 strains that presented this variant when genosubtyping was used, only one expressed this serosubtype.

OMV vaccines represent one of the most promising alternatives available to prevent meningococcal B disease. Their efficacy is based on the production of bactericidal antibodies against PorA, and the protection obtained after vaccination is serosubtype and genosubtype specific (Rosenqvist et al., 1995; de Kleijn et al., 2002). When designing OMV vaccines, local knowledge of the prevalence of not only the different serosubtypes but also the genosubtypes of circulating meningococci is essential, since the wide variation found affects the vaccine formula. Countries such as Norway or Brazil, which at one time had a low diversity of serosubtypes or genosubtypes, used monovalent OMV vaccines (P1.7, 16 and P1.19, 15, respectively) to control meningococcal B disease (Bjune et al., 1991; de Moraes et al., 1992). In Holland, a hexavalent vaccine was designed to target a greater diversity of strains (de Kleijn et al., 2000). These vaccines also have a beneficial effect by inducing cross-reactivity against antigenic variants (Vermont et al., 2003), although it has been reported that the sequence variation in meningococcal PorA OMP can reduce the effectiveness of OMV vaccines (Martin et al., 2000).

The results of genosubtyping show wide diversity among the strains analysed in the present study with 26 different genosubtypes. Moreover, in more than 50 % of the strains, the VR1 or VR2 type was a non-prototype sequence. This situation is similar to that described in the USA and in other European regions (Sacchi et al., 2000; Clarke et al., 2003), although the most prevalent genosubtypes are different from those found in the Basque Country.

In our region, with an incidence similar to that of other developed areas, vaccination with one of the formulas developed to date would provide little benefit. Only 25 % of the strains from our environment presented a genosubtype identical to one or other of those included in the monovalent vaccines used in Norway, Cuba and Brazil, or the hexavalent vaccine developed in Holland (Bjune et al., 1991; de Moraes et al., 1992; de Kleijn et al., 2000). Because of the wide diversity of the strains, the design of a vaccine tailored to our environment would be difficult. A vaccine designed for our geographical area with the five most prevalent genosubtypes could target 61 % of the strains and 74 % if it included eight genosubtypes. Any OMV vaccine for this area should undoubtedly include the genosubtype P1.5-1, 10-8, the genosubtype shown by the hypervirulent ET-15 variant of the ST-11/ET-37 complex, which has affected our region since 2000 (Perez-Trallero et al., 2002).

In the present study, genosubtyping was easy to perform and not time consuming, although it was expensive. This technique is able to detect changes that are increasingly emerging in circulating strains.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES