Clonal properties of meningococci from epidemic meningitis

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TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1991) 85, hWJS,,ENT 1, 24-31

Clonal properties of meningococci from epidemic meningitis

Mark Achtman Ma+anck Institut fiir Molekulare Genetik, Ihnestrasse 73, D-1000 Berlin 33, Germany

Abstract Methods from the field of population genetics now

enable the classification of eoidemic strains of Neisser- ia meningitidis and have r&olved the relationships between apparently distinct epidemics. The diversity of serogroup A bacteria seems quite limited and only a few strains have been responsible for the epidemics of recent decades. Meningococci express both constant and highly variable antigens, the variability of which is determined by the clonal background of the epidemic strain. The development of an improved vaccine is being pursued but still faces technical problems.

Introduction Bacterial meningitis and septicaemia, sometimes

caused by Neisseria nzeningitidis of serogroups B and C, are rare childhood diseases today. Before the second world war, however, epidemics of these diseases occurred world-wide at irregular intervals; these epidemics were usually caused by N. meningitidis of serogroup A, affected civilians of all age groups as well as military personnel, and attained annual morbidity rates of up to 1% of the population. Since that period, serogroup A epidemics have occurred only sporadically in Europe and the Americas (PELTO- LA, 1983), while intermediate levels of serogroup B and C disease have been recorded in England and Norway (POOLMAN et al., 1986). However, major serogroup A epidemics lasting 2-3 years, with a mortality rate of at least lo%, have continued to return approximately every 10 years to the Sahel region of sub-Saharan Africa (Fig. l), called the ‘meningitis belt’ (LAPEYSSONNIE, 1963), and to China (Hu, 1987). 600 000 cases of disease occurred in the meningitis belt between 1939 and 1962 (LAPEYSSON- NIE, 1963); epidemic meningitis occurs predominant- ly during the hot summer and disappears at the beginning of the rainy season (GREENWOOD, 1987; SALIH et al., 1990a). Several million cases of disease were registered in China in 1966 and the predominant epidemic season is during the cold winter.

Not every person exposed to pathogenic meningococci develops clinical symptoms. Instead, healthy individuals can carry meningococci in the nasopharynx and, even during an epidemic, the healthy carriage rate is much higher than the chsease rate (MILLER et al., 1944; AYCOCK & MUELLER, 1950; BLAKEBROUGH et al., 1982; CAUGANT et al., 1988; HA&SAN-KING et al., 1988). Because the menmgococcal carriage rate in West Africa is very low in inter-epidemic periods, irrespective of serogroup (HASSAN-KING et al., 1988), an epidemic may be regarded as a flare in the meningococcal carriage rate which results in disease in some carriers due to unknown secondary factors. This conclusion does not necessarily apply to meningococci which are not serogroup A. High meningococcal carriage rates in the absence of disease have been recorded (DUDLEY &

Fig. 1. The African meningitis belt (modified from LAPEYSSONNIE, 1%3). The shaded region corresponds approximately to the Sahel region, where dry savannah predominates and where meningitis epidemics occur at fairly regular 10 year intervals.

BRENNAN, 1934) and serogroup B and C strains have been isolated from healthy carriers which are rarely or never isolated from cases of disease (CAUGANT et al., 1986a, 1988). However, the genetic complexity of serogroup A is limited (see below) and it is possible that all serogroup A meningococci which are efficient at nasopharyngeal colonization are potentially capable of causing epidemic disease.

Clonal analysis During the 1980s the electrophoretic variation of

cytoplasmic isoenzymes (and in some cases of major outer membrane proteins) has been used to subdivide several Gram-negative species into distinct electrophoretic types (ETs) (SELANDER et at., 1986; ACHTMAN & PLUSCHKE, 1986). Some ETs differ only marginally from each other and have been grouped into ‘clones’, while other ETs are highly distinct and defme clones with only a single representative. Each clone is postulated to consist of bacteria which have descended from a single ancestral cell; its members are uniform for numerous properties in addition to isoenzymes, although less stable properties may differ between isolates due to genetic recombination, selec- tion pressures or random evolutionary drift. Certain clones have been isolated repeatedly over many decades and from different continents while others have been isolated only rarely and from discrete locations. The species N. meningitidis contains hun-

dreds of distinct ETs (CAUGANT et al., 1986a, 1986b, 1987a, 1987b), but only 21 clones comprising 34 ETs were discerned among 423 serogroup A bacteria from 23 different epidemics (OLYHOEK et al., 1987) (Fig. 2). Related clones were assigned to one of four subgroups (Fig. 2). The subgroups were designated A-I to A-IV and the clones were designated I-l to IV-4. Very recent analysis has identified the additional subgroups V and VI (D. A. Caugant, personal communication). A different terminology has been used for serogroups B and C, where groups of ETs are designated as a ‘complex’ named after the most common ET (ET5 complex, ET37 complex, etc.) (CAUGANT et al., 1986b, 1987a, 1987b) and clones have not been defined. Probably complex and subgroup are equivalent designations.

GENETIC DISTANCE .60 A0 20 0

I I I I I 1: I ' CLONES

SUBGROUPS

I

y~!:t:

I I I I I I: I I .60 .40 .20 0

Fig. 2. Clonal analysis of serogroup A bacteria. From ACHTMAN (1990), based on data from OLYHOEK ef al. (1987). Each line corresponds to one of 34 eiectrophoretic types (ETs) which differed from each other in at least one of 7 cytoplasmic isoenzym es or one of two major outer membrane proteins. The number of differences between any two ETs is reflected by the genetic distance at which their two lines diverge. ETs which diverge to the right of the dotted line are assigned to common clones, of which the three indicated are discussed in detail in the text. The clones are grouped in four so-called subgroups.

Epidemic spread Most or all of the serogroup A bacteria isolated

from any one epidemic belonged to a single clone although a few epidemics yielded some unrelated isolates (OLYHOEK et al., 1987). Concurrent epidemics in neighbouring countries were always associated with the same clone and the last 3 epidemic waves in the African meningitis belt were caused by the wave-specific clones I-l, IV-l and III-l, respectively (Fig. 3). The last 3 epidemic waves in China were each associated with one subgroup with one predominant clone, namely subgroup III and clone III-l, subgroup V and clone V-l, and again subgroup

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III and clone III-1 (Fig. 3). The epidemiological pattern of disease during these epidemic waves differed with the individual clone.

c1o?le I-l Clone I-l bacteria were first isolated during an

inter-epidemic period in Algeria (1962). Epidemics caused by clone I- 1 broke out in Morocco and Senegal in 1967 and spread in 1968 to Algeria, Greece, Iran, Sudan, Niger, and Mali. Most other North African countries and countries in the African meningitis belt were visited by epidemics between 1969 and 1974 and almost all bacteria isolated from those epidemics were assigned to this clone (OLYHOEK et al., 1987). Clone I-l also caused outbreaks in Canada (1971-1973) and the United States of America (1975-1976) as well as epidemics in nothem Nigeria and Rwanda (1977- 1979) (OLYHOEK er al., 1987) and in New Zealand (1986) (P. Moore, personal communication). Since the early 197Os, these bacteria have been isolated from endemic disease in numerous other countries and we concluded that their spread (Fig. 4) was best considered to represent that of a meningococcal pandemic (OLYHOEK et al., 1987).

Clone IV-I Most bacteria isolated from cases of inter-epidemic

disease in West Africa between 1963 and 1990 belonged to clone IV-l (OLYHOEK et al., 1987; SALIH et al., 1990b; WANG et al., 1991). In 2 African countries, these bacteria were a minor component of epidemics otherwise associated with clones II-3 and I-l, respectively. In addition, all bacteria tested from West African epidemics in the early 1980s were clone IV-l, including over 300 strains isolated in The Gambia between 1982 and 1988 (OLYHOEK et al., 1987; CROWE et al., 1989). Somewhat surprisingly, none of the serogroup A bacteria isolated since the second world war outside Africa belonged to clone IV-l.

Clone III-1 and subgroup ZZZ Recent information has revealed that clone III-1 has

been involved in 2 pandemics since the early 1960s (Fig. 5). In the first pandemic, most of the bacteria were of clone III-1 but a small fraction of bacteria belonging to clones 111-2, III-3 and III-4 were also isolated, indicating that this should properly be considered a subgroup III pandemic. The earliest documented epidemic caused by subgroup III (LI et al., 1991) began in China in 1966 with 3 000 000 registered cases of meningococcal meningitis (Hu, 1987. The next III-1 epidemic (ACHTMAN~~U~., 1992) began in 1969 in Russia (KUZEMENSKA 61 KRIZ, 1987). Subgroup III also caused an outbreak in Norway, beginning in 1969 (HJETLAND et al., 1990), followed by an epidemic in Finland which began in 1973 (OLYHOEK et al., 1987; PELTOLA, 1987). Although Sweden did not suffer an epidemic, most serogroup A bacteria isolated there between 1973 and the mid-1980s were clone III-1 (SALIH et al., 1990b). The last epidemic of this pandemic wave occurred in Brazil in the late 1970s (OLYHOEK et al., 1987; JACOBSON et al., 1976; BRYAN et al., 1990). A second pandemic began in the early 1980s in Asia, with an epidemic in Nepal (COCHI et al., 1987) and a rise in morbidity in China (LI et al., 1991), both caused by

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Epidemics and Clones in Different African Countries within the “Meningitis Belt” Year: 1905-07 1920-22 1939-40 1943-4s 1949-51 1960-62 1969-72 1981-84 1988-89

Counts I

Gambia Senegal Mali Ivory Coast

EthioF lia

Epidemics and Clones in China

Fig. 3. Epidemic WBWS in Africa and China. From ACHTMAN (1990). Dark boxes represent epidemics which occurred concurrently in several African countries or Chinese provinces; other isolated epidemics have been ignored. Those epidemics from which bacteria were isolated and characterized are indicated by the clonal designation of the bacteria, whereas others from which no bacteria have been tested are indicated by a + sign.

Fig. 4. Pandemic spread of clone I-l. From OLYHOEK (1987), as modified by ACHTMAN (1990). Dots indicate countries from which these bacteria have been isolated.

Fig. 5. Pandemic spread of clone III-I. From ACHTMAN (1990), modified to include the epidemic in Kenya which began in 1989. The dates indicate the beginnings of epidemics caused by clone III-1 in the indicated countries. The north-west MOW from Saudi Arabia (1987) terminated with a bar is intended to symbolize spread of clone III-1 by pilgrims returning from the 1987 Haj pilgrimage, which did not result in epidemics.

clone III-1 and other bacteria of subgroup III. This should be designated as a second pandemic because the major Chinese epidemic in the mid-1970s was caused by subgroup V bacteria (LI et al., 1991). No bacteria have yet been tested from a major epidemic in northern India which occurred in 1985 (MOORE et al., 1988) and the next documented III-1 outbreak was during the annual pilgrimage (Haj) to Mecca of 1987 (MOORE et al., 1988, 1989). All post-Mecca isolates described below were uniformly clone III-l. Numer- ous pilgrims carried clone III-1 serogroup A meningococci back to their countries of origin, including the USA (MOORE et al., 1989) and the UK, where 33 cases were noted among pilgrims and their contacts (JONES & SUTCLIFFE, 1990), but the importation of these bacteria has not resulted in an epidemic outside East Africa. In 1988, epidemics caused by clone III-1 bacteria began in Chad (MOORE et al., 1989), Sudan (SALIH et al., 1990a), and Ethiopia (HAIMANOT et al., 1990), and epidemics, the causative bacteria of which have not yet been tested, began in 1989 in Kenya and Uganda. Before 1988, clone III-1 had never been isolated from Africa (OLYHOEK et al., 1987).

Serological properties Stable antigens

In addition to the capsular polysaccharide, meningococci express large amounts of other anti- genie cell surface components (OLYHOEK et al., 1987; FRASCH et al., 1985), namely lipopolysaccharide (LPS), pili, and the major outer membrane proteins termed H.8, class 1, class 2/3, class 4, class 5 and class 6. Some of these components are members of antigen families which can differ in molecular weight and antigenicity between different meningococci while

others seem to be uniform. The class 4 protein is electrophoretically invariant among different meningococci (OLYHOEK et al., 1987; FRASCH er al., 1985) and all strains tested to date react with a monoclonal antibody (Mab) directed against the equivalent gonococcal protein called P.111. Similarly, although molecular weight variants of the H.8 protein have been observed (ACHTMAN et al., 1992; BLACK et al., 1985), that protein always reacts with a Mab directed against the gonococcal H.8 protein. Extensive com- parisons of electrophoretic migration with clonal assignments have not been performed but most clone IV-1 strains expressed one electrophoretic variant of H.8 and most subgroup III strains expressed a different variant (ACHTMAN et al., 1992). Some serogroup A clones express a class 6 protein, whereas others do not (OLYHOEK et al., 1987); no electrophoretic difference was observed between the class 6 proteins from different clones and no serological reagent is yet available.

In recent years, serological typing schemes based on monoclonal reagents have been developed for serogroup B and C strains based on either the ‘serotype antigen’, the primary antigenic component of which is the outer membrane class 2 or 3 protein, the outer membrane class 1 protein (‘serosubtype’), or the LPS (‘immunotype’ (FRASCH et al., 1985; ABDIL- LAHI & POOLMAN, 1988). Some of these reagents also react with serogroup A bacteria and a few serogroup A-specific reagents have also been produced. Almost all serogroup A meningococci express a class 3 protein which reacts with serotype 4 and 21 reagents (CROWE et al., 1989; FFCASCH et al., 1985; AJJDILLAHI er al., 1988) and none expresses a class 2 protein (OLYHOEK et al., 1987). Seven electrophoretic variants of the

28

class 1 protein were associated with distinct clones of seroaroun A strains (OLYHOEK et al.. 1987) and these 7 el&trhphoretic variants fell into 5 skrosubtype specificities (ACHTMAN, 1990; CROWE et al., 1988). 98% of the bacteria of clone IV-l are of the P1.7 serosubtype; the remaining 2% do not express a class 1 protein and all African P1.7 isolates tested were clone IV-l. Subgroup III is associated with both the P1.9 and P1.x serosubtvnes. 98% of subzroun III is P1.9,~ and, except for 6;le strain which-was-P1.5,9 with a class 1 protein differing in electrophoretic migration, the other exceptions did not express a class 1 protein. Whereas P1.9 is also found within serogroup B and C strains, no strain outside subgroup III has vet been found which reacts with the P1.x reagent. Thus, the use of P1.7, P1.9 and P1.x reagents allows ready distinction m Africa between clones IV-l and III-l. The dual serosubtype of subgroup III probably reflects the fact that the class 1 protein possesses 2 independent variable regions (MCGUINNESS et al., 1990) and theoretically all serogroup A strains should express a dual serosubtype. It is conceivable that a dual serosubtype scheme would enable the reliable recognition of individual clones or subgrouns. However, currentlv a dual serosubtype ha; be&i determined only for subgroup III. and recent results with Chinese isolates have shown that subgroup V expresses the reactivities P1.7 and P1.10 (LI et al., 1991), which are otherwise associated with subgroup I and clone IV- 1, respective- .ly (CROWE et al., 1988). Thus, the currently available reagents do not allow reliable recognition of serogroup A clones and subgroups except for subgroup III. Variable antigens

LPS immunotypes L4, L6, L7, L8, L9, LlO, Lll and L12 have been described in various combinations within serogroup A bacteria (ZOLLINGER & MAN- DRELL. 1980: KIM er al.. 1988). There was no unique correlation between LPS immunotype and clonal analysis (OLYHOEK et al., 1987). Among the highly related clone IV-l bacteria isolated in The Gambia, most isolates expressed L9 LPS but 9/105 expressed LPS with different serological reactivities (CROWE et al., 1989). In contrast, in&unotypes L8 to‘L13 were found within subgroup III even when isolates from one city taken within a few weeks were tested (ACHTMAN et al., 1992; KIM et al., 1988). Because the expression of LPS has been shown to vary in the laboratory for some gonococcal strains (SCHNEIDER et al., 1988), this meningococcal variability may reflect genetic instability in certain clones.

Meningococci can alter the expression at high frequency of other antigens, namely pili and the outer membrane class 5 proteins, both in the human host and in the laboratory (MEYER & VAN PUTTEN, 1989). The term ‘phase variation’ designates the reversible change from expression to lack of expression, and ‘antigenic variation’ designates the alternative ex- nression of different antigens by the same strain. The variability of these antigens has been compared for clone IV-l (CROWE et al., 1989) and subgroup III (ACHTMAN et al., 1992). Mab SMl recognizes an epitope located in an invariable N-terminal domain of pilin from gonococci and many meningococci (VIRJI et al., 1989). Six electrophoretic pilin variants were distinguished by western blots with Mab SMl within

clone IV-l bacteria (CROWE et al:, 1989). Each strain expressed between 0 and 2 distmct pilin bands and variation from expression to non-expression or in electrophoretic migration was common (CROWE et al., 1989). Whereas only 69% of Gambian clone IV-l strains and 57% of other clone IV-l strains were piliated, 97% of subgroup III strains reacted in Western blots with SMl (ACHTMAN et al., 1992). Furthermore, the subgroup III pilin also reacted (ACHTMAN et al., 1991b) with a new monoclonal reaeent UlOl. which is snecilic for so-called class 2a pili-(WANG er al., 1991j. Thus, for at least these clones, the expression of conservative pilin epitopes is clonally associated despite continuous changes in primary sequence of the pilin gene due to recombination events.

Gambian clone IV-l bacteria expressed a median of 2 different class 5 proteins (range O-4) (ACHTMAN et al.. 1991). but onlv 5 different oroteins 5a, 5b. 5c, 5d’ and 5e were found among over 300 isolates, whether the bacteria were isolated during the 1982-1983 enidemic or thereafter, from the nasopharvnx, blood or cerebrospinal fluid,-from healthy carriers or from diseased oatients KROWE et al.. 1989). Examination of clone IV-1 bacteria isolated between 1963 and 1985 from other West African countries revealed expression of only 3 additional proteins, Sf, 5g and 5h (ACHTMAN er al., 1988). Both rabbit hyperimmune sera and mouse Mabs showed that the proteins 5b, 5d, 5e and 5g were strongly cross-reactive while the proteins 5a, 5c, 5f and 5h were serologically distinct (ACHTMAN et al.. 1988 & 1991). Current information indicates that meningococci possess 4 independent opa genes, each encoding a class 5 protein (AHO et al., 1991). In aonococci, where 11 independent opa genes exist; each gene consists of 3 strongly c&&rved regions interspersed with 2 hyper-variable regions called HVl and HV2 (CONNELL et al., 1990). HVl and HV2 are probably responsible for the serological specificity of the proteins synthesized and cross- reaction between 2 proteins can be explained by antibodies being directed against an epitope in such an HVl or HV2 sequence which is present in more than one protein. Because of the conserved sequences, recombination events can occur, generating new combinations of HVl and HV2 (CONNELL et al., 1988).

Investigations are under way to determine whether the 8 class 5 proteins in clone IV-l arose by such mechanisms of recombination. However, proteins indistinguishable from 5a, 5c, 5f or 5h were also among the class 5 proteins found when 200 subgroup III strains isolated between 1966 and 1989 from both pandemic waves were tested, and only a single new protein, 5i, was found among these strains (ACHTMAN et al., 1992). Because the geographical sources of clone IV-l and subgroup III were distinct until 1988, any recombinational events which might have led to the evolution of these opa genes must have occurred a very long time ago. Within this context of genetic stability, it is noteworthy that, within subgroup III, 5h was expressed only by strains isolated before the Mecca epidemic of 1987, and all strains expressing 5i were epidemiologically related to that epidemic, consistent with the gene for 5h having been replaced by a gene for 5i.

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polysaccharide (CROWE et al., 1989). Their isolation suggests that herd immunity in The Gambia was directed against the class 1 protein and LPS. More importantly, their existence indicates that effective immunization with a new vaccine might stimulate the development of strains resistant to the antibodies induced by immunization.

Protective antibodies Meningococcal disease apparently results in perma-

nent immunity in normal individuals because, of the rare individuals who suffer from more than one episode, most are immunologically deficient and many lack one of the terminal complement components (Ross & DENSEN, 1984). Epidemics of meningococcal disease can be interrupted by vaccination with A/C polysaccharide vaccine (FRASCH, 1989) but protection seems to last only 3-5 years (REING- OLD et al., 1985) and very young children respond poorly to vaccination. Efforts are under way to develop a new conjugate vaccine containing polysaccharide conjugated to a protein carrier, in order to stimulate long-lasting immunity after immunization of young children within existing vaccine program- mes. Although choosing a protein carrier which stimulated protective antibodies in its own right would increase the efficacy of such a vaccine, it is currently unclear which protein would be the most suitable.

Vaccine development necessitates using in vitro assays for measuring potency which correlate well with human protection. Because disease in USA army recruits was correlated strongly with both carriage df meningococci and the absence of bactericidal killing in sera from those recruits (GOLDSCHNEIDER et al., 1969a. 1969b). the bactericidal test is often used as an indicaior of p;btective activity. Indeed, the frequency of sera with detectable bactericidal activity rises with ap;e whereas meningococcal meningitis is most common in those age gropus with low leiels of bactericidal activitv (GOLDSCHNEIDER et al.. 1969bX However. during the Gambian epidemic df 1983,’ bactericidal activity was lacking in both acute phase sera and sera from individuals who later became healthy carriers (ACHTMAN et al., 1992) and the primary difference between the 2 groups was that healthy carriers developed bactericidal antibodies more often than did reconvalescent patients. The specificity of bactericidal antibodies remains unknown but they often seem able to distinguish between related bacteria (ACHTMAN et al., 1992; GOLDSCHNEIDER et al., 1969a, 1%9b) and the specificity is probably not directed against the capsular polysaccharide. This type-specificity was one of the reasons that the serotyping, subtyping and immunotyping schemes were developed. Indeed some murine Mabs directed against the class 1 protein or LPS, and occasionally against the serotype antigen, are bactericidal and can protect newborn rats against meningococcal sepsis (SAUKKONEN et a&, 1987). The class 1, class 2 and class 5 proteins are nnmunogenic both in human volunteers vaccinated with an ex- perimental vaccine and after disease (MANDRELL & ZOLLINGER, 19891, but the concentration of antibodies directed against these proteins did not correlate well with the degree of bactericidal activitv. Thus, at the moment,the bactericidal test rema& one of the primary Indicators of potential human protection but further research on the nature of protective human antibodies is needed. Furthermore, after the end of the Gambian epidemic, a few meningococci were isolated which were resistant to bactericidal killing. These bacteria synthesized dimi- nished levels of class 1 protein, expressed LPS which differed serologically from that of previously isolated strains, and synthesized elevated levels of capsular

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Clonal properties of meningococci from epidemic meningitis