Meningococcal molecular mimicry and the search for an ideal vaccine

32 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICALMEDICINE.AND Hxrem

Meningococcal

molecular

mimicry

(1991) 85,

SUPPLEMENT

1, 32-36

and the search for an ideal vaccine*

J. McLeod GrifEss, Ryohei Yamasaki, Michele Estabrook and Janice J. Kim Centerfm Zmmunochemistry and Departments of Laboratory Medicine, Medicine and Pediatrics, University of California, San Francisco, USA; Veterans Administration Medical Center, 4150 Clement Street (I 13A), San Francisco, CA 94121, USA The carbohydrates expressed on the surface of meningococcal strains of groups B and C mimic those commonly found on human cells and thus are not functionally antigenic in infancy. In order to develop an effective vaccine, it will be necessaryto find ways of circumventing this molecular mimicry. Three possible ways of achieving this are discussed. (i) The surface polysaccharides can theoretically present conformationally different epitopes, someof which might be recognized as antigemc by the host. Experimeutal evidence is nresented that such differences do indeed exist; what is needed is to determine which of these conformations are unique to the organism and hence potentially antigenic. (ii) Precursors of the surface lipooligosaccharides may be unable to mimic human antigens, and so may be potential candidates for vaccine development. (iii) Natural immunity to some strains of meningococci -develops in young children who are colonized with strains of Neisseria lactamica, and it is possible that its development could be enhanced by widespread intentional colonization by N. lactamica strains that are particularly efficient inducers of broad immunity. Introduction The pathogenetic potential of the meningococcus is based on the organism’s ability to express on its surface carbohydate organelles- that exactly mimic those on the surface of human cells. The ar2-8 polysialyl (NeuNAc) capsule of group B strains is commonly found on human cell surface glycoconjugates during foetal life and continues to be expressed, albeit at lower densities, throughout life (FINNE et al., 1983). As a result, this capsular polysaccharide is not immunogenic in its purified form (WYLE et al., 1972) and, even when presented on the surface of the oraanism durina disseminated disease, only briefly b&&s toleranc&and induces a short-lived imnumoglobulin (Ig) M response (GRIFFISS et al., 1984). The 1~2-9polysialyl capsule of group C organisms is also expressedon human glycoconjugates during foetal life but is lost by 2 years of age (FUKUDA et al., 1985). As a result, children are able to respond immunologically to this polysaccharide at about 2 years of age, whereas they are tolerant to it earlier in life (PELTOLA et al., 1985). The NeuNAc ~2-6 Gal repeating unit of the group W polysaccharide is found at the terminus of most oligosaccharides that adorn human glycoconjugates; the NeuNAc ar2-6 Gal repeating unit of group Y polysaccharides is not known to be present on human cells, and organisms of this serogroup cause disease primarily in individuals with deficiencies of the terminal complement components. ‘This is report number 48 from the Center for Immunochemistry.

Meninaococcal outer membranes contain short triantenn-uy glycolipids, termed lipooligosaccharides or LOS (GRIFFISS et al., 1988; JENNINGS et al., 1983), that resemble the structures of glycosphingolipids (GSL), the analogous glycolipids in the membranesof human cells (MANDRELL et al., 1988, 1990; JOHNet al., 1991). Most pathogenic meningococcal strains terminate the longest chain of their LOS with lacto-N-neotetraose (Figure) (JENNINGS et al., 1983; GRIFFISS et al., 1988; JOHN et al., 1991). The neotetraose structure is also found in GSL of the paragloboside series (MANDRELL et al., 1988a, 1988b) and is the building block of human ABO blood groups. The N-neotetraose is polysialylated in the membranes of human cells; those meningococcal strains that can make sialic acid (serogroups B, C, W and Y) also sialylate the lacto-N-neotetraose substituent of their LOS (MANDRELLet al., 1991). Group A meningococci which cannot make sialic acid (FROSCH et al., 1989) do not endogenously sialylate LOS, but they and the gonococcus exogenously sialylate their LOS with use of a sialyl transferase that can ‘borrow’ sialic acid of human origin (CMP-NANA) and attach it to their LOS (MANDRELL et al., 1988a, 1988b, 1991). Sialylation of membrane glycoconjugates aborts the assemblage of the complement membrane attack complex composed of U-9 that would otherwise result in lysis of the cells (FEARON& AUSTEN,1980). Defence of the blood stream against meningococcal dissemination depends upon complement-mediated immune lysis (GRIFFISS, 1982). Sialylation of the meningococcal surface by polysialyl capsules and/or sialyation of LOS therefore serves 2 pathogenetic purposes; it aborts complement-mediated immune lysis (JARVIS81VEDROS,1987) and uptake and killing by polymorphonuclear leucocytes (ESTABROOK et al:, 1991) and, by providing structures that exactly minuc those on human cell surfaces, it prevents recognition and the generation of antibodies that could tilt the balance in favour of complement-mediated clearance (GRIFFISS et al., 1984). In order to make a vaccine that will prevent meningococcal disease during infancy, we must find ways of circumventing the organisms’ molecular mimicry. In this report, we discuss 3 such approaches and summarize our data in support of each of them. Polysialyl capsules Polysaccharides have less structural rigidity than proteins, which assumerigid OLhelixes or l3 sheetsdue to hydrogen bonding between amino acid residues. The group B and C meningococcal polysaccharides are ar2, 8- and o-2, 9-linked polysialic acids, respectively, and there are 2.or 3 extra carbons, respectively, between the glycosidically linked residues compared with common glycoses, in which anomeric carbons

33 TERMINAL SEOUENCE

SUBTERMINAL LACTOSAMINE

I

INTERNAL IACTOSES

LACTO-N-NEOTETFJAOSE

BASAL OLGOSACCHAFIIDE

LIFOIDAL MOLN

0

T OLK;OSACCHARDE

3

Figure. Structure of the 4.8 kDa lipooiigosaccluuide made by N. meningihdti, N. lactmniu and N. gcmmhma (Gwmss et al., 1988; Kw CI al., 1983)and gonococci (Grumssn ol., 1988; MANDRELLII 1989). The struc~re is drawn from published structures for meohgococ ci(J~tw~~~~efaI., al., 1988b, 1990; Pst~~ups.ef al., 1990; JOHNII al., 1991; GIBSONet al., 1989~; Y,uus.ucnr er al., 1991). The terminal L.acNAc and internnl Lac form the hcto-wncotetraosc SubstiNent. Monoclonal entibodia 3Fll and 6FS4 find their cpitopes in the terminal LacNAc (ZOLLINGER et al., 1979; CI al., 1988b, 1990; JOHN et al., 1991; YAMASAKI cf ol., 1991). The tcrmhl Gal of Gtumss cf ol., 1988; DUDAS & A~rrr~m. 1988; ~~NDRELL lam-rwxoteumse is the preferred site for sialyhtion (~~MDRELL et al., 1990). The 3.6 kDa ncisscrial lipooligosacchuide (SCHNEIDERer ol., 1985) terminates .st the internal hc (DUDAS & ~LPICELLA.1988; JOHN ef aI., 1991). This struc~re binds mottoclonnl antibodies D6A and 2-1-B (DUDAS & 1988; EST.UROOK et ol., 1990; JOHN et al., 1991). The terminal Gal of the 3.6 kJh lipooligospccharide may provide an alternative site for APKELLA, sidylaion.

are glycosidically linked to the rigid pyranose carbons. Groups A, W and Y meningococcal polysaccharides are also glycosidically linked between pyranose residues, but from one pyranose ring to the extra ring C6 carbon of an adjacent hexose residue. Because of this, conformations of B and C polysaccharides as well a8 the other 3 ‘pathogenic’ capsular polysaccharides, may be more diverse than the common glycoses (YA~~ASAKI, 1988).

As antibcdies recognize shapes, each polysaccharide can theoretically assume several epitopic shapes. The conformation of polysaccharide capsulesis somewhat constrained by other elements in the cell membrane. This may be why uriiied polysaccharides are often less immunogenic Jan the same structures on the surface of the mtact organisms. It could also explain why the same structure may present different epitopes in a meningococcal membrane from those in et al., 1986). a human cell membrane (MANDRELL Other glycoconjugates (mammalian GSL and glycoproteins and bacterial proteins) in the respective membranes would be expected to interact with polysialyl polymers and distort their conformations. It is theoretically possible, then, that the group B and C polysaccharides can exist in conformations that are suffficiently distinct from those on the surface of human cells to render them both immunogenic and unable to abort the complement membrane attack complex. In order to understand expression of epitopes

within the B and C polysaccharides, it is essential for us to understand their three-dimensional (3D) structures. For the 3D structural determination of the ar-2,8-linked polysialic acid, we performed twodimensional (2D) nuclear Overhauser effect (NOE) nuclear magnetic resonance and molecular modelling of the B lysaccharide in solution (YAMASAKI, 1988). The appl?cation of NOE to conformational analysis is based on the following principle: NOE intensities can be observed from any pair of protons that are less than 5 A apart. The NOE intensities become larger the shorter are the distances between protons. Therefore, once complete assignments of their spectra have been made we can use the distance constraints obtained by the NOE data to determine 3D stmctur~ of molecules of interest. Pure absorption 2D NOE experiments provide the most quantitative data, and comparative analysis of experimental and theoretical NOE data becomesa powerful tool for determining accurate conformations. Models for theoretical NOE experiments are selected from modelling analyses based on the experimental NOE data. We found that group B polysaccharide exists in t are in equilibrium in helical conformations solution. One pitch (%ll 9 ) consistsof between 3 and 4 sialic acid residues, depending on the ‘tightness’ of the coil, with + (C7-C8-08-C2) and (OS-CZ-OS-C8) angles in the ranges of: $115-1Y 5”, ~-30-O” or +55-l lS”, ~90-120”. The orientation of carboxy and NHAc groups alternates on opposite sides of the

34 helical coil. These angles may not be identical to those assumedby the polymer on the bacterial surface, but certain mouse monoclonal antibodies (Mabs) bind both purified polysaccharide and the bacteria, indicating that at least some epitope shapesare shared. The helical structure of the B polymer explains why its antibodies do not bind to short ar-2,8-linked sialic acid oligomers that cannot form such secondary structures. Further, since the polymer adopts different shapes within the Q and II, angles, expression of epitopes within the B polymer can be complex. Immunological data have shown that humans recoanize at least two enitooes within each of the group B and C polysialyl-capsules, but that only one induces antibodies that are bactericidal: i.e.. that restore the balance in favour of effective assemblageof the membrane attack complex (GRIFFISS et a!., 1982). The goal, then, is to sort these conformations and determine those that are unique to the organism and potentially able to induce protective antibodies. Interestinalv, -_. one of the enitones conformed on the 02-8 capsule of group B meningococci is also conformed bv the ar2-9 cansule of PTOUD C oreanisms (urmublished observations). Tl& e&tone % both immunogenic in adults and induces bactericidal antibodv. Groun B diseasehas been virtuallv absent from m&ary recruits in the United Statesof America since the advent in 1971 of routine immunization against group C disease; could this be due to cross-protection by the shared polysialyl epitope? LOS mecursots Metiingococcal outer membrane LOS mimic one of the maior classesof human GSL, the naragloboside series. IQrther, like paraglobosidePitself;thestructure is either endogenously sialylated by the organism’s own sialic acid, depending unon the strain’s serogroup, or exogenously sialjlaied by human-derived siahc acid. or both (MANDRELL et al.. 1991). On the face of it, these molecules do not seemto be l&omising vaccine candidates, but carbohydrate structures, unlike proteins, are not synthesized at the ribosomal level and moved intact into the membrane. Rather, each necessary glycosyl transferase is co-ordinately synthesied and acts sequentially to attach each glycosyl residue. As glycosyl transferasesresponsible for terminal sequences cannot act until their substrates have been created by precursor enzymes, each transferase must wait until a biosynthetic precursor has been made by a different transferase that acts at an earlier step before it can catalyse attachment of the next sugar residue. Thus there is an orderly process through which each sequential biosynthetic step must proceed. We have focused on one LOS precursor that migrates in sodium dodecyl sulphate-polyacrylamide electrophoresis gels as a 3.6 kDa molecule (Figure) (SCHNEIDER et al., 1985). The o-chain of this LOS is the internal lactose (Lac) of lacto-N-neotetraose (DUDAS & APICELLA,‘ 1988; JOHN et al., 1991). Because it lacks the terminal Gal of the LacNAc substituent, which is the favoured sialic acid acceptor on lacto-N-neotetraose, this LOS may not be sialylated on the organisms’ surfaces. However, the terminal Gal of the internal Lac substituent may act as an alternative sialic acid acceptor. This possibility is currently being investigated. We have mapped the

epitopes expressed by this LOS molecule and found that one epitope, identified by the Mab D6A, is highly conserved among group A strains and cryptically expressed by strains of other serogroups (KIM et al., 1988; ESTABROOK et al., 1990). Children who are infected with meningococcal strains that do not overtly express the epitope on their surface nonetheless make antibodies of the same specificity as Mab D6A during disseminated disease.This observation is consistent with the belief that the structure that conforms the epitope is a necessary precursor in the biosynthesis of complete meningoccoccal LOS, but that- its expression is occluded-by the addition of subsequent sugars and/or bv sialvlation. Mab D6A is bactericidal fo; most meningococci that express the epitope and infants as young as 3 weeks of age have newly acquired bactericidal activity in their sera during convalescence in the absence of capsular polysaccharide antibody. There are both theoretical reasons and supportive data for believing that a precursor LOS molecule could induce protective antibodv during infancv. and we are currently working with coll
35 tericidal for the strain infecting them (GRIFFISS et al., 1984). N. lactamica, not N. meningitidis, is the neisserial species that most commonly colonizes the oropharynges of infants and young children (GOLDet al., 1978;

Hu, 1986). Colonization by N. lactamica can induce

antibodies that are bactericidal for meninaococcal strains of different serogroups and serotypes OLD et al.. 1978X N. &tarn& strains from south China. whkre m&iugococcal disease is rare, often make LOS

that bind Mab D6A, whereas strams from northern Chinese Drovinces. where eDidemic meninaococcal diseased&s occur; do not (I&i et al., 1989).-Surface exuression bv N. lacrumiea strains of LOS that bear th; Mab DhA-detined epitope suggests a way by which antibody that binds this epitope could be induced during the early years of life. Conclusion Meninaococcal caDsular Dolvsaccharide vaccines were developed for ;se in &lit&y recruits (ARTENSTEIN et al.. 19711. Children do not resnond to the polysaccharides c&aining sialic acid (Goups C, W and Y) before 2 years of age? and the immune response to the group A capsule 1snot maintained by young children (F%LTOLA et al., 1985). These vaccines are useful for containing small outbreaks in civilian populations (COUNTSet al., 19841, but they have not-b&n used to prevent endenhicd&&e and db not maintain control of eDidemics for lona (Hu. 1986; .1977): REINC~LD et al., 1985; WAHDAN et-il., Conjugation to protein carriers to improve immunogenicity should not be tried for group C or, particularly, group B polysaccharides until their eDitows have been maDDed and structurallv

under-

&&, as they are stru&ually identical to oligosaccharides that nlvcosvlate human cells durine foetal life and early i&&y (I&NE et al., 1983; FUK~DA et al., 1985) and share some epitope conformations with the

human-derived polymers. Once non-shared epitopes have been identified and means of assuring maiutenance of non-self conformations

found, conjugation

could be tried. In fact, conjugation might ensure epitopic maintenance. However, conjugate vaccines are too expensive for use in developing countries. A simple, cheap, vaccine is needed that prevents endemic disease throughout childhood and provides protracted control of epidemic disease. The immune response to outer membrane proteins is relatively short-lived (ZOLLINGER et al., 1982); their greater usefulness is as carrier proteins for conjugated carbohydrate antigens. The search for such a vaccine has frustrated us for 2 decades, as we were unprepared for the redundant systems of mimicry that the meningococcus uses to evade host recognition and immune clearance. Our emerging understanding of this complexity, however, combined with the availability of new techniques of carbohydrate

chemistry,

have given us several possi-

bilities for circumventing this problem and hope that an effective vaccine will be made in the near future. Acknowledgement This work was supported by the US Public Health Service through grants AI 21620, AI 21171, AI 28871, AI 22998; the Thrasher Award no. 2802-o; the World Health Organization Programme on Vaccine Development; and the Research

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