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An integrated molecular and immunological approach towards a meningococcal group B vaccine
An integrated molecular and immunological approach towards a meningococcal group B vaccine M.R. Lifely*, C. Moreno’ and J.C. Linden* There has been a notable lack of success in producing an effective vaccine against Neisseria meningitidis group B infections, despite such prophylaxis being available for group A and C disease. The reasons for this are reviewed and evidence presented that a vaccine based on the group B capsular polysaccharide should be pursued. To be effective, a clear understanding of, and improvement in the poor immunogenic&y of the polysaccharide is required. Consequently, the nature of the antigenic structure involved in immune recognition has been evaluated at the molecular level and reasons for the poor immunogenicity of the B polysaccharide are presented. Methods of increasing the immunogenic@ are proposed with the intention of undertaking human volunteer trials. Keywords:
Bacteria;
meningitis;
Neisseria
meningikfis
Neisseria meningitidis is a Gram-negative diplococcus which causes meningitis in humans. Virulent strains (i.e. those isolated from the bloodstream or cerebrospinal fluid) are invariably associated with a capsule which surrounds and helps the organism to evade the host defence systems. The capsules consist of a high molecular weight anionic polysaccharide, and form the basis for serological classification of the meningococci into distinct serogroups (A, B, C, 29-E, H, I, K, L, W135, X, Y, Z)‘. Within each serogroup there are many antigenically different outer membrane proteins (OMP) and lipopolysaccharides which define a serotyping system used to further subdivide the organism’%-. Acquisition of meningococcal meningitis is through airborne droplet spread, followed by colonization of the nasopharynx. Carriage of the organism in the throat, however, is relatively common (5-30% depending upon the season) and only rarely does carriage progress to cause symptoms of disease. The most common form of disease occurs through invasion of the meningococci into the bloodstream, and penetration of the bloodbrain barrier (the meninges, and hence the name of the disease). The organisms are then able to multiply in the Departments of Experimental Immunobiology* and Physical Chemistry*, Wellcome Research Laboratories, Langley Court, Beckenhv, Kent, BR3 3BS UK, and ‘MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Rd, London, W12 OHS 026&410X’87/05001 0
1987 Butterworth
l-l
cerebrospinal fluid causing inflammation of the brain, often leading to death or mental retardation. Onset of disease may also be rapid, and the sequence from noticeable symptoms or diagnosis to death often occurs within 24 hours. Meningococcal meningitis is a disease occurring worldwide in both endemic and epidemic forms. Endemic disease has a rate of between 1 and 5 per 100 000 per annum, whereas epidemics of up to 500 per 100 000 population have occurred. Serogroups A, B and C have been shown to constitute greater than 90% of the isolates from patients, with group B alone accounting for 5@70% of cases. In the late 196Os, vaccines were developed against serogroup A and C meningococci4; these vaccines consisted of the purified high molecular weight capsular polysaccharide derived from the respective organism, and their safety and efficacy has been demonstrated in a number of controlled field trials and epidemic situations”,6. Unfortunately, however, the serogroup C vaccine is ineffective in children under two years old’ (that age group most susceptible to disease) and the serogroup A vaccine in children below 6 month?. More seriously, there has been a spectacular lack of success in producing an effective vaccine against group B disease. In early trials, the high molecular weight group B capsular polysaccharide proved to be very poorly immunogenic in humans”. The group B polysaccharide is a (2-8)~u-linked homopolymer of N-acetylneuraminic acid (Figure I), which is interesting in two respects. First, this structure is identical to that found in
1 co3-
.oH2C9+~oH
3
AcHN
OH 5 cx -
NeuNAc
9 polyracchsride - (2 -
9),-
C polysaccharide - (2 -
9bR
K92 E Coli
-
[l2-88)-(2-9B)1~
Figure 1 The structure of N-acetylneuraminic acid and the repeating units of the N. meningitidis group B and C, and the E. co/i K92 capsular polysaccharides
6 $03.00
& Co. (Publishers)
Ltd
Vaccine, Vol. 5, March 1987
11
Meningococcal group B vaccine: M. R. Life/y et al.
the capsular polysaccharide of Escherichia coli Kl (colominic acid), the major cause of neonatal meningitis, suggesting a common mechanism by which these polymers evade the host defences, and second, group C polysaccharide, a homopolymer of (2-+9)-a-linked Nacetylneuraminic acid (Figure I), is structurally very similar to the B polysaccharide, which contrasts with the difference between the polymers in immunogenicity in adults.
Poor immunogenicity of the B polysaccharide A prerequisite for an effective meningococcal group B vaccine would seem to be a basic understanding of the poor immune response to the purified B polysaccharide. Although hypotheses have been postulated to explain the poor immunogenicity of the B ,{O’Ysaccharide, namely, sensitivity to neuraminidases , crossreactivity with ‘self’ antigens”, and intrinsic ‘floppiness’ of the purified B polysaccharide’*, clear-cut experimental evidence has been scarce. Consequently, a clear and rational programme of group B meningococcal vaccine development has been difficult to achieve. With this in mind, our interpretation of the poor immunogenicity of the B polysaccharide is described in detail in this section.
Conformational
determinants
Chemical and immunological evidence. Polymeric carbohydrate antigens of bacterial origin often show structural similarities to cell-surface components of the host. In this situation it would be advantageous if the immune system of the animal were to respond to produce antibodies directed against conformational determinants present on the secondary or tertiary structure of the polymer, thus providing immunity but preventing autoimmune cross-reactivity. This may occur, for instance, for type III group B streptococcal polysaccharide”, where sialic acid seems to direct the antigenit conformation; this same situation may also apply to meningococcal group B polysaccharide. Present on cell surfaces of the host are sialogangliosides and sialoproteins containing short chains (generally 2-4 residues) of (2-8)~a-linked sialic acid, identical to the repeating unit of the B polysaccharide. It is likely therefore that the animal is tolerant to this particular ‘self’ epitope, since if not, and antibodies were to recognise a straightforward (2+8)-a-linked sequential or continuous determinant, it would initiate an autoimmune response against gangliosides and glycoproteins bearing this linkage. Consequently, the host forms antibodies directed to one or more conformational or discontinuous determinants defined by the three-dimensional structure of the B polysaccharide. Evidence in support of this hypothesis has come from a number of workers in the field. Egan et al.” showed that the capsular polysaccharide from E. co/i K92 is a heteropolymer containing alternate (2+8)-aand (2--+9)-a-linked sialic acid residues (Figure I), which cross-reacts immunologically with meningococcal group C polysaccharide but not with group B polysaccharide. This suggests that antibodies against the C polysaccharide recognize a linear (i.e. structural) determinant, whereas antibodies against the B polysaccharide recognize a conformational determinant. 12
Vaccine,
Vol. 5, March
1987
The effect of reduction of carboxyl groups in the B polysaccharide (i.e. -C02H + -CH,OH) on its antigenicity was studied more recently’“. Correlation between the degree of carboxyl reduction and the antigenicity of the modified polysaccharide was obtained by a solid phase radioimmune inhibition assay (Table I) which showed that a modest degree of carboxy1 reduction (<20%) resulted in a substantial loss of antigenicity of the polymer, far greater than would be expected for an antibody which recognized a linear or continuous determinant. Thus a low degree of carboxyl reduction is likely to cause considerable disruption of the three-dimensional structure of the B polysaccharide with a concomitant reduction of antigenicity. Low molecular weight colominic acid (E. cofi Kl capsule; M r = 10’) also had a poor affinity for anti-B antibody and modification of only 2% of the residues caused a further loss of antigenicity. In contrast, the C polysaccharide showed only a modest decrease in antigenicity following carboxyl reduction, thus suggesting the presence of a structural determinant on the molecule. Not only are the -C02H groups important for maintaining antigenicity of the B polysaccharide. however. since N-deacetylation of the polymer caused almost complete loss of antigenic activity, shown by its failure to a lutinate with a group B monoclonal latex reagent ?! . Moreover, although following N-reacetylation the polysaccharide recovered (-NH_ 7 + -NHC0.CH3) its ability to agglutinate with the latex reagent, this was not observed when the N-deacetylated polymer was Npropionylated (-NH, -+ -NHCO.CH,CH,). Thus. there is a specific requirement for N-acetyl groups in the B polysaccharide in order to preserve antigenicitv. Perhaps the most compelling evidence obtained m support of the difference in determinant specificity of B and C polysaccharides has been obtained by Jennings and co-workers” who purified a series of oligosaccharides from both polymers and used a radioimmunoassay inhibition technique to determine the nature of the epitopes. Polyclonal rabbit antisera were raised against the group C polysaccharide and experiments to inhibit the precipitation of the polysaccharide with the antiserum were performed with the a-methyl ketoside of sialic acid and the series of (2+9)-a-linked oligomers from the di- to the hexasaccharide. No inhibition was observed for the monomer. but the di, tri- and tetramer showed increasing inhibitory properties. No further increase in inhibition was observed with the pentamer and hexamer. consistent with the estimate of the maxi-
Table 1 Correlation between degree of carboxyl antigenicity of B and (0-Ac’)C polysaccharides
reduction
and
Incubation conditions
Carboxyl reduction (%)
Relative concentration giving 50% inhibition (pg ml-‘)
_ 15 min 1h 4h
0 2.4 5.9 13.1
1 .o 9.5 37 135
EDC
20 min 1h 4h
4.5 19.4 21.7
17.3 36.7 36.7
EDC
20 min 1h 4h
0 9.2 19.7 21.6
Polysaccharide B pH 3.4 B
(0-Ac+)C
1 .o 0.06 0.5
1.o
Meningococcal group B vaccine: M. R. Life/y et
mum size of an antibody binding site. In complete contrast, experiments to inhibit the precipitation of the B polysaccharide with a horse anti-B serum, using a series of (2-+8)-a-linked oligomers to the heptasaccharide, failed to show any inhibition and the use of larger oligosaccharides up to 14 residues long resulted in only poor inhibition. A procedure using a direct binding assay has also shown that a horse anti-B serum has low affinity for (2-S)-a-linked oligosaccharides up to 10 residues long. We have recently extended these results through the determination of oligosaccharide chain length’s by (a) calorimetric measurement of formaldehyde released from the non-reducing end residue after periodate oxidation, (b) radiolabelling of the reducing end residue by reduction with borotritiide, and (c) determination of the ratio of the non-reducing end and internal residues by gas-liquid chromatography. (2-+8)-aLinked oligosaccharides up to 28 residues long very poorly inhibited binding of an anti-B murine monoclonal antibody to B polysaccharide in a solid phase radioimmune inhibition assay, and also only weakly agglutinated a monoclonal B latex reagent’(j. Thus, the determinants present on the B polysaccharide are inadequately expressed on oligosaccharides up to 28 residues long. The estimate of the linear hexasaccharide or heptasaccharide as the upper limit for the size of an antibody bindin site as demonstrated by the classical studies of Kabat has been displayed for the interaction of the meningococcal group C polysaccharide with a rabbit anti-C serum. This has manifestly been demonstrated not to be the simple case for the B polysaccharide, in which the antigenic determinant has been adequately expressed only in the high molecular weight polymer. This situation is analogous to that found for protein antigenic sites2” which may occur on a continuous or linear region of the surface of the polypeptide chain and have thus been termed ‘continuous’, ‘sequential’ or ‘primary’ antigenic sites. Alternatively, a protein may incorporate surface residues from different parts of the polypeptide chain which are brought into close proximity due to the folding constraints on the molecule and have been termed ‘discontinuous’ antigenic sites. Accordingly, we propose that meningococcal group C polysaccharide has continuous and group B polysaccharide discontinuous determinants, but in order to determine the nature of the epitopes present on the surface of the B polysaccharide a greater understanding of the molecular architecture of the molecule has been required. In this context, Kabat etaL21 recently demonstrated that a human monoclonal antibody with specificity for the B polysaccharide cross-reacted with polynucieotides and denatured DNA but had only minimal cross-reactivity with native DNA. It is tempting to speculate on the nature of this cross-reaction, given that B polysaccharide has conformational determinants, and Kabat et al. favour the hypothesis22,23 that the spatial arrangement of charged groups on molecules may constitute antigenic determinants which are cross-reactive with seemingly unrelated substances. The authors stress, however, that the presence of these antibodies in serum at a concentration of 23 mg ml-’ has no& caused any signs or symptoms of disease. N.m.r. spectroscopy has been our principal tool in probing the molecular architecture and for discovering
al.
distinguishing molecular features between group B, group C and E. coli K92 polysaccharides, although confirmatory studies using molecular mechanics and computer graphics have also been employed. N.m.r. studies of flexibility. The relative mobility and flexibility of the polysaccharides was probed by measuring the ‘%Z n m.r. spin-lattice relaxation times (NT,) of the CH and CH2 carbons where N is the number of attached hydrogens. This parameter for a particular 13C nucleus can be related to a molecular correlation time TR which is a measure of the speed of motion of that part of the molecule in solution. Hence, if all the NT, values are equal it is possible to deduce that the molecular fragment is tumbling in solution as a rigid body. Extension of the theory is possible to include the detection of additional internal degrees of flexibility (segmental motion), and measurement at two n.m.r. field strengths is necessary to remove ambiguity in the results24. Measurement of the NT, values at two magnetic field strengths showed that there is a field dependence, and this was used to confirm the correlation times. The results for the B, (0-AC-)C and K92 polysaccharides are listed in Table 22”,2h. The constancy of the NT, values for C-4, C-6, C-7 and C-8 of the B polysaccharide repeating unit indicates that there is no segmental motion for the backbone of this species. At a field strength of 5.9 T, C-9 of the B polysaccharide has a NT, longer than that of the rigid backbone, but at 9.4 T this becomes shorter than those of the carbons in the rigid part. This demonstrates that C-9 has some extra degree of motion over and above the overall molecular tumbling. For the (0-AC-)C polysaccharide, increased T1 values are observed for C-7, C-8 and C-9, something not observable for the (0-Ac+)C polysaccharide, which consists of a mixture of non-O-, 7-O-, 8-O-, and 7,8-di0-acetylated residues. These longer NT, values demonstrate extra segmental motion for C-7, C-8 and C-9, all involved in the chain backbone. The NT, values for C-3 are generally longer than those for the rigid parts of the molecule and this may be a result of increased molecular flexibility due to ringpuckering motions. If it is assumed that the molecule is tumbling isotropically, and this is likely to be true for a coiled polymer, then there is a single rotational correlation time, sR. Using the NT, values for the rigid parts of the polysaccharide one obtains for the B polysaccharide TR = 6.7 X lo-‘s at 5.9 T and zR = 7.1 X lo-' s at 9.4 T. The model developed by Doddrell et a1.24 has been applied to the internal rotation (q;) of the pendant -CH20H (C-9) of the serogroup B polysaccharide and applying this specifically to the B polysaccharide Table 2
‘% n.m.r. spin-lattice relaxation times (NT,), seconds
Carbon atom
B(5.9 T)
B(9.4 T)
(O-AC-)C (5.9 T)
K92 (8.8 T) (2+8)_u_ (2+g)_a_
c3 c4 c5 C8 c7 C8 c9
0.16 0.16 0.17 0.17 0.15 0.16 0.19
0.56 0.44 0.37 0.43 0.42 0.43 0.35
0.17 0.14= 0.16 0.14 0.19= 0.17 0.20
0.27 0.21 0.21 0.22 0.21 0.22 0.33
0.25 0.21 a 0.22 0.22 0.24a 0.23 0.28
aAssignments may be reversed
Vaccine,
Vol. 5, March
1987
13
Meningococcal group B vaccine: M. R. Life/y et al
gives, at 5.9 T, zo =1.3 x 10p”‘s; and at 9.4 T, r. = 0.7 x lo-” s. The consistency of these correlation times is obvious from the constancy obtained for the B polysaccharide at the two field strengths. The agreement for r. is clearly not so good as for ra, but the results at both field strengths indicate that the -CH20H group has an internal rotational correlation time 50-100 times faster than the overall molecular tumbling. The molecules under consideration are homopolymers of sialic acid and. hence the NT, values measured represent an average for all the monosaccharide units throughout the chain, including end groups. Consequently, increased flexibility at the ends of the polymer chain could invalidate the motional model used. This situation is unlikely, because of the high molecular weight which effectively gives the end groups little relative weight. Also, if a particular carbon in different sialic acid residues along the chain possessed a variety of NT, values, it is unlikely that the relaxation data for that carbon would fit the observed, single exponential decay. An important conclusion of this study is the relative flexibility of the B and C polysaccharides in solution. The lack of segmental motion for the former (except for C-9) is compatible with some form of three-dimensional structure, whereas the C polysaccharide gives indications of greater flexibility in solution. Immunologically, these results suggest that the B polysaccharide bears conformational epitopes and that group C determinants are sequential. Similar experiments on the molecular dynamics of the B and C polysaccharides have been reported by Egan et ~1.‘~. ‘, and their results for the molecular correlation times, ra, are in the same range as those derived by us. An exact comparison cannot be drawn because of the different experimental conditions used. For example, their solution concentration was four times higher than in our study, with a related increase in viscosity. However, by measuring spectra at 37°C compared to our 27”C, this may have been somewhat counterbalanced. In addition, their experiments were performed at magnetic field strengths of 2.4 T and 6.4 T and are thus probing a slightly different time-scale. The main difference between the two sets of results is that we observe differences in the 13C relaxation times for the side-chain carbons of the (0-AC-)C polysaccharide. It appears that the previous study used the (0-Ac+)C polysaccharide, which is a mixture of partially acetylated species, giving rise to a loss of peak resolution and chemical shift changes on 0-acetylation that lead to assignment difficulties. This may account for their inability to differentiate NT, values for the side-chain and hence derive relative flexibility information on the two polymers. It should be remembered that n.m.r. NT, measurements only probe fluctuations that occur on the timescale of the n.m.r. observation frequency (2-4 X 10” ss’), and that slower segmental motion may not affect NT,. Indeed, by using the Stokes-Einstein-Debye equation, the absolute magnitudes of the experimental NT, values, ours2s.2h and those of Egan et uI.“.~*, indicate that all the polysaccharides possess considerable internal flexibility at other frequencies. Like the B polysaccharide, the (2+8)-a-linked (B) residues of the K92 polysaccharide showed a constancy 14 Vaccine, Vol. 5, March 1987
of ‘% relaxation-times for C-4/8, which indicates that there is no segmental motion for the backbone of these residues. However, C-9(B), the pendant -CH20H group, had a longer NT, than the rigid backbone, demonstrating some extra degree of motion besides the overall molecular tumbling. The (2-+9)-a-linked (C) residues of the K92 polysaccharide also showed a constancy of NT, values for C-4/8, and only C-9 showed any significant additional flexibility. These results differ from those obtained for the meningococcal (0-AC-)C polysaccharide where the NT, values indicated flexibility along the whole of the C-7, 8, 9 side-chain. Use of the model of isotropic reorientation leads to a correlation time, ra. of 4.0 x lo-” s, and an internal rotation correlation time for the C-9(B) -CH*OH group, ro(B), of 0.6 x lO_"'s. Furthermore, since it appears that only C-9 of the (2+9)-a-linked (C) residues showed any significant additional flexibility, it is possible to use the model of Doddrell et d2’, allowing one degree of internal motion, on the C-9(C) group, giving Q;(C) = 0.8 x lo-“’ s. Thus, the C-9 groups of both (2+8)-u-linked (B) and (2+9)-a-linked (C) residues have an internal rotational correlation time -SO times faster than the overall molecular tumbling. The K92 polysaccharide is characterized by internal or segmental motion of only the C-9(B) and C-9(C) parts of the molecule, which rotate -50 times faster than the overall molecular tumbling. Since C-9(C) is involved in the ketosidic linkage, however, this suggests that the polysaccharide chains will have a considerable degree of freedom of motion, similar to that predicted for the (0-AC-)C polysaccharide, but different to the relative rigidity envisaged for the B polysaccharide. N.m.r. studies ofpolysaccharide conformation. More definitive structural information on the differences between B and C polysaccharides comes from ‘H n.m.r. studies of the carbohydrate conformations. Initially the monomer NeuNAc was studied as the data for this compound were in the literature although it is important to remember that this relates to the more stable p anomer in aqueous solution2”. Since then the conformations of both anomers of NeuNAc have recently been shown to be identica13”. Chemical shifts (6) and spin coupling constants (.I) were obtained for N-acetylneuraminic acid (NeuNAc) by first-order analyses in our work2s. These are listed in Table 3 and are in close agreement with those published. The assignments are as previously given and were confirmed by double resonance and two-dimensional chemical shift correlation experiments. The ring coupling constants, .13a,l. J 1.5., and J5,(, are typical of axial-axial hydrogen interactions and confirm the assignment of the ring conformation as ‘C.5. The coupling constants observed for the C-7-C-9 side-chain are averages over internal rotation that is fast on the n.m.r. timescale, although the barriers to rotation are such that the side-chain exists in staggered conformations. Haasnoot et al.“, have formulated predictive rules for vicinal H-H coupling constants in carbohydrate systems based on the gauche or antiperiplanar nature of the coupled hydrogens and the relative disposition of the oxygen substituents. These predictions explain the magnitudes of the observed coupling constants given in Table 3 giving detailed conformational information
Meningococcal group B vaccine: M. R. Life/y et TRANS
GAUCHE
C7-C8
;l*lI)OH
C-8-C-7
GAUCHE
(y$
al.
C-7-C-8
y$;
H8
“‘1
/“”
1 I
/
‘;fj$:c
HOHZC,,, ;;@:c
,;;;‘&:Ac
H8l I I I ’ ac OH (H)OH2C / N-AcNeu Figure 2
OH
OH
H8
P B polysaccharide
and C polysaccharide
The conformations of the sialic acid sidechains for (2+8)-u- and (2-9)u-linked
Table 3 ‘H n.m.r. data for 8-N-acetylneuraminic acid, saccharide (0-AC-)C polysaccharide and K92 polysaccharide Hydrogen
NeuNAc
B
(0-AC-)C
Chemical shifts (S)= 3a
3e 4 5 6 7 8 9 9’
1.89 2.32 4.08 3.95 4.07 3.57 3.77 3.63 3.86
Coupling constants (Hz) J 3a.3e 13.06 J 3a.4 J 3e.4
11.62
J4.5 J5.6
10.21 10.37
J 6.7
J78 J 8.9
Js.9, J 9.9’
4.97
1.31
9.12 6.30 2.63 11.76
1.72 2.62 b b b 3.87 4.11 4.05 b
1.72 2.74 b b 3.82 3.62 3.98 3.87 3.75
12.1 11.8 4.5 b
12.3 11.5 4.5 b
b
10.1 I .2 9.0 6.2 2.3 10.3
residues as deduced from the n.m.r. spectroscopic studies
B polyK92
“6
248
2-9
1.79 2.70 3.73 3.85 3.87 3.85 4.18 3.75 4.13
1.82 2.88 3.84 3.86 3.64 3.67 4.00 3.92 3.77
<3 8.4 5.2 3.1
aFrom Me&I, taking the N-acetyl resonance as 6 2.07. %esolution insufficient to allow determination of the n.m.r. parameters
the important deduction being that H-7 and H-S are antiperiplanar. The total confirmation is shown in
with
Figure 2.
The ‘H n.m.r. spectrum of the (0-AC-)C polysaccharide is shown in Figure 3, and the n.m.r. parameters, where they can be resolved, are also given in Table 3. Because of the viscous nature of the solution, the relaxation times (T2) of the nuclei are shorter and this results in broader lines. This effect was lessened by measuring the spectrum at 70°C. Computer resolution enhance’ment3* was necessary and under these conditions, by analysis of the coupling patterns and
“9
nn
hR
“7
Figure 3 360 MHz’H n.m.r. spectrum of (O-AC-)C polysaccharide, excluding H3a and H3e
through the use of spin decoupling, the bands due to H-7 and H-S are assigned. The signal for H-8 is easily assigned because, apart from H-4, it is the only nucleus coupled to three other protons. Thus, irradiating the signal at 6 3.98 caused no effect at either of the H-3 resonances but removed the 9.0 Hz coupling on the band at 6 3.62, thereby confirming the assignments of H-7 and, H-S. The coupling constant is typical of the antiperiplanar arrangement of H-7 and H-8 as in NeuNAc, and is similar to the monosaccharide value, indicating the same total conformation. The B polysaccharide was examined under the same conditions as for the (0-AC-)C polysaccharide, and the ‘H n.m.r. spectrum is shown in Figure 4. The assignments of the resonances again follow from application of the techniques used on the other polysaccharide. The H-3 resonances show coupling constants similar to those of the other substances and confirm that the ring conformation is unchanged. The other protons give rise to three separate bands with considerable overlap. At highest frequency is a two-proton band, followed by a signal from a single hydrogen, and a four-proton complex band at lowest frequency. On resolution enhanceVaccine,
Vol. 5, March
1987
15
Meningococcal group B vaccine: M. R. Life/y et al. b.“‘s,b,“~
and this provides additional support for the strict threedimensional requirements for an immunological response with the B polysaccharide. The determination of the conformations of the units in the K92 polysaccharide required the assignment of the ‘H n.m.r. resonances in a very complicated spectrum with much peak overlap. This was achieved only through the use of a two-dimensional “C/‘H correlation n.m.r. experiment, taking advantage of the fact that the ‘%Z spectrum is easily assignable. In this experiment the results are plotted as a contour map. A peak appears where the ‘-‘C chemical shift of the signal of a CH,, fragment intersects with its ‘H chemical shifts’“. An expansion of the region containing C-4 to C-9 for both (2+8)-aand (2-9)~u-linked residues is shown in Figure 5. Having assigned the ‘H shifts it was then possible to use computer resolution enhancement to obtain the ‘H coupling constants to yield the conformational information’h. In the following argument H-4(B) and C-4(B) will, for example, represent H-4 and C-4. respectively, of the (2-G)-a-linked residues in the K92 polysaccharide. The N-acetyl methyl and C-3 methylene groups are trivially assigned and the expanded contour plot (Figure 5) enables the assignment of the other ‘H chemical shifts. The digital resolution in the projection of the ‘H spectrum precludes further analysis. but recourse to a resolution-enhanced one-dimensional spectrum (Figure 6) allows the relevant coupling constants to be extracted. In addition, the assignments and, particularly, the ‘H shifts of the signals of the hydrogens attached to C-4(B), C-4(C), and C-7(C) which have very similar “C shifts, were confirmed by doubleresonance difference experiments. Figlue 6 shows that the resonances at d 4.18 and 6 4.00, assigned to H-g(B) and H-g(C). respectively, each have three coupling constants, as expected. The resonance due to C-O(B) at F 4.13 showed a large coupling constant (-12 Hz) consistent with the expected geminal H-9( B)-H-9’( B) coupling. and 21
H7
“8
HO
wt.4
Figure 4 and H3e
360 MHz n.m.r. spectrum of B polysaccharide, excluding H3a
ment, the signal at 6 4.11 can be seen to possess three couplings, indicating that it must be due to H-4 or H-8. The couplings do not match those for H-3a and, thus, the band cannot be due to H-4. The band assigned to H-8 shows a quartet structure, indicating three moderate couplings of similar magnitude (3-4 Hz). The next band at 6 4.05 shows two couplings, one of = 12 Hz typical of a geminal interaction, and is therefore assigned to one of the non-equivalent protons in the C-9 methylene group (the two protons are expected to have different chemical shifts because of the chiral nature of the molecule). The single proton resonance at 6 3.87 has only one coupling constant of 3.5 Hz and, as this is repeated in the band due to H-8, it is assigned to H-7, with J7.x 3.5 Hz. The coupling constant Jh,, is not resolved and, as in previous molecules, must be
6B 6C
6C 78 48
9B
58 5C
3.67(7C) 3.75
3.67
Figure 5 Two-dimensional contour plot of the correlation between ‘% and ‘H chemical shifts of the K92 polysaccharide, with assignments as marked, excluding C3(H2) and the N-AC methyl signals
16 Vaccine,
Vol. 5, March
1987
Meningococcal group B vaccine: M. R. Life/yet al.
larger than the monosaccharide polysaccharide2h
repeating
unit of the
Stability of the three-dimensional structure Since there are no a determinants in the B polysaccharide with a poor immune response, we have assumed that the three-dimensional structure is unstable, thus resulting in poor immunogenicity. A potential source of antigenic instability has been suggested for the B polysaccharide through the formation of internal esters or lactones under mild conditions, resulting in a considerable loss in antigenicityX4. This internal esterification in the highly water soluble B polysaccharide was observed because reaction of the polymer with a carbodiimide or with 48% HF resulted in a water insoluble product. Infrared spectra of the modified polymers showed a major band near 1750 cm-‘, consistent with ester formation (Figure 7b); this band virtually disappeared after mild alkali treatment (Figure 7a). More relevantly, esterification was shown to occur by incubating the B polysaccharide below pH 6 and infrared spectra showed that the degree of esterification increased as the pH was lowered. “C n.m.r. spectroscopy of the fully esterified polymer provided conclusive evidence for cross-linking between the carboxy1 group of one residue and HO-9 of an adjoining residue (Figure 8) to form a six-membered intramolecular ester or lactone ringZ4. Following the development of a gas-liquid chromatographic procedure” to give more sensitive and precise measurement of the degree of esterification of B polysaccharide, it was shown’” that the salt form of the polymer underwent no ester formation whereas about 80% esterification occurred in the free acid form. In contrast, the C polysaccharide, whether 0-acetylated or not, underwent no ester formation either in the salt or free acid form. Under more forcing conditions of activation of carboxyl groups with a carbodiimide, and in the absence of 0-acetyl groups, the C polysaccharide underwent partial internal esterification (about 50%) in contrast to the full esterification of the B polymer. “C n.m.r. spectroscopy showed that the activated carboxyl groups condense with an adjacent HO-8 group (Figure 8) to form a six-membered Intramolecular esterzjkation.
priori reasons to equate conformational
4.20
4.15
,.,o
4.05
3.95
4.00
3.90
3.85
3.80
3.75
PPM
Figure 6 360 MHz ‘H n.rn.r. spectrum of the K92 polysaccharide, excluding H3a and H3e from both residues of the repeating unit
smaller coupling, with second-order effects, to the resonance assigned to H-8(B). Analysis of the H-8(B) multiplet showed three couplings, viz J7.x (B), Jx.<)(B), and Jx,u8 (B), each of =4 Hz. In a decoupling experiment, irradiation of the H-8(B) resonance collapsed the adjacent H-9(B) resonance to a geminally coupled doublet, and perturbed the spectrum at 6 3.85 and 6 3.75. Using double-resonance difference spectroscopy, the resonance at 6 3.75 showed the geminal coupling constant, thus confirming it as that of H-9’(B). It follows that the resonance at 6 3.85 is due to H-7(B), which, in the decoupled spectrum, appears as a broad singlet, showing that J6,, (B) is
a
8
b
n
z, 8 9
Figure 7 Infrared spectrum of B polysaccharide before (a) and after (b) carbodiimide treatment
Vaccine,
Vol. 5, March
1987
17
Meningococcal group B vaccine: M. R. Life/y et
Figure 8
al
The mechanism of internal esterification of B polysaccharide (left) and (0-AC-)C polysaccharide (right)
internal ester ring analogous to that formed in the B polysaccharide. A good correlation was observed between the degree of esterification and the antigenicity of the B polysaccharide by countercurrent immunoelectrophoresis (CIE)“4. At pH 5.5, the Na+ salt of the polymer underwent 2-3% esterification and little reduction in antigenicity, whereas at pH 5 and -9% esterification the antigenicity was dramatically lowered (Table 4). These results emphasize the ease with which esterification can occur in the B polysaccharide, thus causing destabilization of the three-dimensional structure of the polymer and resulting in loss of antigenicity. The n.m.r. approach to molecular conformation has been coupled with theoretical molecular mechanics calculations in order to probe the conformations about chemical bonds for which no coupling constants are available and in order to explain the propensity of the (2-8)~a-linkages to undergo internal esterification2”. NeuNAc models were joined together to form disaccharides linked either (2-+8)-u- or (2&9)-a- using the graphics facilities of the Wellcome Molecular Modelling system. Atom centred partial atomic charges, calculated by the CNDO method were included and the geometry was fixed at the known crystal structure with the ring and side chain conformational information from n.m.r. as an additional constraint. The molecular mechanics energy calculations are based on variations in bond length, bond angle, torsion angle, van de Waals contacts and coulombic interactions and the minimum energy conformation obtained. The only unknowns remaining are the two torsional angles at the anomeric linkage and these were optimized to give minimum energy forms by sequential bond rotations. These final conformations are shown in Figure 9 for the B and C type linkages, respectively. Examination of these models shows that the (2-+8)-a-linkages impart a geometry in which the carboxyl group in the free acid form is perfectly arranged to undergo internal esterification
Table 4 Correlation between degree of esterification and the antigenicity of B polysaccharide
PH
(“/)
Minimal concentration giving positive band by C.I.E. (pg ml-‘)
3.0 4.0 5.0 5.5 6.0 Salt form
53 30 8.6 2.5
>I00 >I00 >I00 0.4 0.4 0.4
Incubation
18
Degree of esterification
Vaccine, Vol. 5, March 1987
Minimal concentration giving full inhibition by C;I.E. (pg ml-‘) ND ND 400 80 40 20
0
O‘i
\
0
0
Figure 9 Calculated conformations about the glycosidic linkage for NeuNAc disaccharides with a (2+8)-a-(left) and a (2+9)-u-(right) linkage. Derivation of the rest of the structures is described in the text
with the adjacent HO-9 group. Conversely, it can be seen that the relative disposition of the carboxyl group and the HO-8 in the (2-+9)-a-linkages is most unfavourable for internal esterification. A second source of antiTemperature dependence. genie instability of the three-dimensional structure of B polysaccharide has been s;Egested by the experiments of Mandrel1 and Zollinger- who observed a considerable decrease in avidity of group B specific antibodies with an increase in temperature from 4 to 37°C. whereas that of anti-C antibodies decreased only marginally. We interpret these results in terms of a loss of the three-dimensional structure and therefore of the specific determinants of the B polysaccharide with the increase in temperature, whereas the continuous determinants of the C polysaccharide remain unaffected. However, it must be stressed that there is no n.m.r. evidence for a temperature-dependent conformational change of the B polysaccharide. Neuraminidase sensitivity. The sensitivity of the B polysaccharide to neuraminidases may contribute to decrease immunogenicity further”‘. although there is admittedly little experimental evidence to prove or disprove this point. Unpublished experiments in our laboratory have indicated that the B polysaccharide (or colominic acid) is rapidly eliminated from liver and spleen after intravenous injection, in comparison with the elimination of B512 dextran of a similar molecular weight. Reluctance to accept the relevance of neurami-
Meningococcal group B vaccine: M. R. Life/y et al.
nidase degradation of B polysaccharide in decreasing irnmunogenicity has been due to the argument that (O-AC-)C polysaccharide, which is a good immunogen in adults, is also sensitive to neuraminidases and should therefore be degraded as rapidly as the B polysaccharide in viva. However, the difference in the antigenit stabilities of the two polysaccharides caused by their contrasting determinant specificities makes it probable that the epitopes on the B polysaccharide will be destroyed far more rapidly than those on the (O-AC-)C polysaccharide. Cross-reactive brain determinants Although there is a consensus of opinion that tolerance to the B polysaccharide in the host is due to crossreactive tissue components, there seems to be some confusion over which of these components are responsible. We, and others, believe that the immune response is suppressed most effectively when directed against the ubiquitous cell surface sialogangliosides and sialoproteins, containing only short chains (about 24 residues) of (2+8)-a-linked sialic acid residues identical to the repeating unit of the B polysaccharide; hence, the immune response is directed with some success against conformational determinants on the polysaccharide. Finne and co-workers37%38, however, have shown that a polysialoglycoprotein present in brain tissue, and referred to as N-CAM (neural cell adhesion molecule) protein”‘, contains long chains of (2-43)~a-linked sialic acid residues. This polysialoglycoprotein, found in developing fetal brain tissue, but declining rapidly after birth, was originally reported to cross-react with a horse anti-B serum”, a result which has been confirmed by us using mouse anti-B monoclonal antibodies4’. They have suggested that immunological tolerance to the B polysaccharide may be due to this cross-reactive brain antigen, and have urged caution in efforts to develop an effective vaccine containing the B polysaccharide component. They have argued the breakdown of natural tolerance caused by an ‘artificial’ vaccine might initiate an autoimmune process and might have adverse effects due to interference in the normal physiological function of the polysialoglycoproteins in brain tissue. They have also voiced their concern over immunotherapeutic approaches involving passive administration of antibodies against group B meningococci or E. coli Kl. This is a view which we (and others) do not share. Zollinger et al.“‘, have already stated their opposition to the likelihood of cross-reaction in vivo between anti-B polysaccharide antibodies and brain polysialoproteins; the vaccines they administered (with no ill-effects) were not artificial but were in a ‘natural’ hydrophobic complex with OMPs. Moreover, vaccine-induced antibodies to B polysaccharide or antibodies from convalescent patients, which were predominantly of the immunoglobulin M(IgM) class and of low avidity, appeared to be equivalent to the naturally occurring anti-B antibodies present in 80-90% of adults. Animals hyperimmunized with group B meningococci or with a B polysaccharideOMP complex showed no ill-effects, despite high circulating antibody levels. In addition, we have found4” that monoclonal antibodies against not only the B polysaccharide but also against the polysaccharide from another pathogenic serogroup (W135) of N. meningitidis bind to human fetal brain tissue. Yet the W13.5
polysaccharide is one component of a commercially available tetravalent vaccine which has proved to be safe and effective in children42 and adults4”. It has not been adequately explained how tolerance to cross-reactive brain polysialo roteins would operate in the adult. It has been argued’ Pthat the lack of an IgG anti-B response in experimental animals and humans is because the polysialoproteins would be readily accessible to IgG (but not IgM) antibodies with B specificity. Since, however, as Finne et al. 1’,37338report, the polysialoproteins decline rapidly after birth, then, unless similar molecules are present in other organs, the maturing immune system would not be in contact with such an antigen. Although IgG responses have been described as more susceptible to tolerance than their IgM counterparts, such a state would not be maintained without the persistence of the tolerogen. The immunological cross-reaction observed between B polysaccharide and the brain polysialoprotein does not necessarily imply any untoward pathological consequences, especially if the antigen is embryonic and/or separated from the immune system by the blood-brain barrier. Although molecular mimicry caused by sharing of antigens between parasite and host has been detected before, and potential immunological implications for the host described, there is very little experimental evidence that cross-reactive antibodies are harmfu121,4L47, and it is certainly within the bounds of reason to expect, with the increasingly sophisticated and sensitive techniques available, that many further examples of cross-reactions between the extremely complex and continually altering brain tissue components and foreign antigens will arise without adverse consequences for the host. However, by stating this we do not imply that such cross-reactivities should be ignored. On the contrary, it is our opinion that they should be the subject of much more research and careful monitoring.
Enhancement of immunogenicity of the B polysaccharide Introduction We interpret the poor immunogenicity of B polysaccharide as due to suppression of the immune response when directed against the ubiquitous short chains of (2+8)-u-linked sialic acid determinants, such as those present in sialogangliosides, and the response is therefore directed against conformational or discontinuous determinants present on the B polysaccharide. The polysaccharide adopts a preferred three-dimensional structure in solution which facilitates internal esterification, thereby radically altering the antigenic structure of the native molecule, and the sensitivity of the polymer to neuraminidases contributes to decrease immunogenicity further. As a consequence, purified B polysaccharide does not induce specific antibodies’, and the response against whole bacteria, or the polysaccharide in a complexed form, is relatively short lived and consists almost exclusively of IgM antibodies4s.4y. These factors may also explain the failure of previous attempts to raise an immune response with (2+8)-alinked sialic acid oligosaccharides covalently coupled to a tetanus toxoid Carrie?‘. The stabilization of the three-dimensional structure, held together in a rather Vaccine, Vol. 5, March 1987
19
Meningococcal group B vaccine: M. 17.Life/y
et al.
precarious way, seems to be a basic requirement therefore for immunogenicity of the B polysaccharide and this has been at the core of our efforts to manufacture an effective vaccine against group B disease. B polysaccharide-outer membrane protein (OMP) complex Noncovalent B polysaccharide complexes with outer membrane proteins (OMPs) from N. meningitidis have been prepared in several laboratories. Zollinger et al.4X studied the effect in a small group of human volunteers of two vaccine preparations both containing B polysaccharide complexed to type 2 OMPs. One vaccine was a partially purified B polysaccharide, which had a sialic acid:protein ratio of 1.6. The second vaccine preparation was a combination of a partially purified B polysaccharide mixed with an outer membrane complex of protein and lipopolysaccharide (LPS); the mixture was subsequently processed to remove LPS and the final vaccine had a sialic acid:protein ratio of 1.1. Both vaccine lots gave encouraging results in that an enhancement of immunogenicity of the B polysaccharide component was observed, even though the antibodies were restricted mostly to the IgM class and were relatively short lived. Raised antibody titres to the type 2 OMP were also observed4s. The results support the view that the three-dimensional structure of the B polysaccharide can, to some extent, be stabilized by maintaining the polysaccharide in a complex with OMPs. Frasch and co-workers”’ prepared outer membrane complexes of serotype 2 proteins and LPS, which were subsequently treated with Brij-96, a nonionic detergent, to selectively remove the LPS. The resulting serotype 2 protein vaccine was soluble and relatively more immunogenic in mice” than previously prepared particulate OMP vaccines”. The immunogenicity of the serotype 2 protein vaccine could be further improved by mixing with an e ual weight of the purified B or C polysaccharides2~’ 7. The disadvantages of such a vaccine are threefold: (i) the LPS content was relatively high (=10%)5’.5s; (ii) most of the B polysaccharide (~80%) did not complex with the protein”‘, and it was not surprising, therefore, that anti-B responses in mice were very low”‘; and (iii) there are many antigenically distinct OMPs within group B meningococci2, whereas the B polysaccharide is always present. A vaccine based on OMPs would require multiple serotype roteins for complete protection. More recently 49 , we developed a methodology for preparing the B polysaccharide complexed to type 2 or
type 6 OMPs, which had several advantages: (i) they were naturally formed complexes prepared in high yield directly from culture supernatants by a mild method; (ii) they were low in nucleic acid (<2%) and lipopolysaccharide (<0.5%) content; and (iii) the B polysaccharide and protein components were exclusively associated in a high molecular weight (>2 x 10’) complex held together by hydrophobic interactions, which probably occur through binding of hydrophobic regions on the protein with a phospholipid moietysh covalently attached to the reducing end residue of the B polysaccharide chains. Using the methods of chain length analysis’s discussed earlier in this review, this lipid moiety was shown to be present on all of the B polysaccharide chains, which were estimated to have an average length of ~200 residues. Immunization of mice with a B polysaccharide-type 6 OMP complex resulted in a mean 3 to 4-fold rise in anti-B titres4” which were mostly of the IgM class (Figure 10). In most experiments, however, the anti-B response declined rapidly after the first week and was barely detectable by day 21. Immunized mice were, however, protected against challenge with group B N. meningitidis strains of the same (type 6) or of a different serotype (type 2a) from that used for immunization (Table 5) when the immunizing dose was 1000 ng per
days 7
21 10wlmoure
7
14
21
,
14
7
1vglmoure
21
O.l~glmoura
Figure 10 Anti-B responses (solid-phase radioimmunoassay) of CBA mice immunized with 10, 1, or 0.1 pg of group B polysaccharide-type 6 OMP complex given intraperitoneally (0) or subcutaneously (E4). The values obtained with non-immunized mice are indicated to the left of the graph. Results are expressed as the geometric mean of five mice + standard errors
Table 5 Protection of mice from challenge with N. meningitidis after primary and secondary immunization with a B polysaccharide-type and purified B polysaccharide Death/total after challenge’with Primary immunizationa
Secondary immunizatior?
7619
1000 ng complex 100 ng complex 100 ng complex 1000 ng B polysaccharide 100 ng B polysaccharide None
None None 1 .ng complex None None None
118d 518e 118d 818 818 819
Ww
B, twe2)
6 OMP complex strain 7622 kvoupB,twe 6) 118’ 418e ND 818 719 719
aPrimary immunization was given intraperitoneally on day 0. bSecondary immunization was given intraperitoneally on day 10. ‘Challenge with IO4 CFU of the corresponding strain, in combination with iron dextran. Data are recorded as deaths per total 72 h after challenge and 21 days after immunization. “x2 shows significance at p < 0.01. eNot significant: p > 0.1
20
Vaccine, Vol. 5, March 1987
Meningococcal group B vaccine: M. R. Life/y et
mouse, although no significant protection was afforded with an immunizing dose of 100 ng per mouse. However, primary immunization with 100 ng followed by secondary immunization with as little as 1 ng complex was sufficient to give almost complete protection (Table 5). No protection was observed following immunization with the purified B polysaccharide. The results suggest, firstly, that the anti-B antibodies are responsible for prevention of death in mice, since challenge with either the homologous or a heterologous strain of group B meningococci resulted in clear protection, and, secondly, priming with the complex leads to the buildup of memory cells although the secondary anti-B response, similar to the primary response, contains mostly IgM and very little IgG antibody. As mentioned above, immunization of mice with the purified B polysaccharide resulted in a lack of protection. Although the classification of the polysaccharide as a poor immunogen is well documented, this designation must be qualified by defining the term immunogen. Immunogenicity is most commonly defined only as implying the induction of circulating antibodies; however, we have shown that the purified B polysaccharide is immunogenic if the term encompasses the induction of immune memory. We found that neither primary nor secondary immunization of mice with the purified B polysaccharide resulted in a significant antibody response. However, when primary immunization with B polysaccharide was followed one month later by a second immunization with 10s formalin-fixed group B meningococci, a clear dose-dependent secondary response was obtained with an optimum at 0.1 pg of B polysaccharide per mouse 4y. Therefore, although B polysaccharide is non-immunogenic in the sense that it does not induce humoral responses, it is perfectly capable of inducing immune memory. Stabilization with aluminium ions One of the factors suggested in this review to be responsible for the poor immunogenicity of the B polysaccharide is the intrinsic instability of the polymer due to its tendency to undergo internal esterification under mildly acidic conditions”4, thus resulting in the loss of conformational or discontinuous antigenic determinants that are characteristic of the native polysaccharide. Neuraminidase sensitivity has also been implicated in the oor immunogenicity of the polymer12. We have shown P’ that the Ca2+ salt of B polysaccharide undergoes internal esterification at a slower rate than the Naf salt, a fact that has been ascribed to coordination of Ca2+ ions to the HO-9 of sialic acid residues in the polymer. Because intramolecular esterification occurs in B polysaccharide through condensation of -CO?H and HO-9 groups of adjacent residues”4, cations, binding principally to the -C02H group, but
Table 6
1:0.2 1:0.3
also by coordination of -OH groups, were therefore of potential use in inhibiting the rate of ester formation in the polymer. This rationale was used to select cations to examine: (i) their ability to complex with the B polysaccharide; (ii) their effect on esterification and neuraminidase susceptibility of the B polysaccharide; (iii) their effect on stabilization of the main epitope(s) of the B polysaccharide; and (iv) the resultant immunogenicity of the polysaccharide. Evidently, each of these possibilities is meaningful only when the previous ones have been answered in the affirmative. Binding occurred spontaneously between B polysaccharide and A13+ ions”, either by equilibrium dialysis or by direct incubation. It seemed clear that sequestration of Al”+ by the polysaccharide occurred in preference to Ca*+ binding and that this complex was dissociated by neither ethanol precipitation nor gel filtration. The nature of the binding is still unclear, but nuclear magnetic resonance data, although preliminary, suggest that, apart from the obvious interaction with the carboxylic groups, additional interactions take place involving -OH groups at C-95x. Binding between A13+ and B polysaccharide resulted in a clear resistance to neuraminidase digestion (Figure 21). Moreover, at acid pH, Al”+ forms of B polysaccharide, as compared with the Ca2+ and Na+ salts, showed markedly reduced rates of esterification (Table 6). This, in itself, resulted in a persistent antigenicity of B polysaccharide measured both by immunoprecipitation (counterimmunoelectrophoresis) and by radioimmunoassay”‘. Since the antigenic stability was enhanced through complexation with Al 3+ ions, the effect of Al”+ complexes of the B polysaccharide on the immune response in mice was tested. The results were disappointing with no significant increase in the anti-B titres when compared with the preimmunization levels or with antibody levels in mice immunized with the Ca2+ salt of the B polysaccharide. As already mentioned in this section,
60
60 TIME
100
120
140
OF INCUBATION
160
160
200
220
(hl
Figure 11 Rate of release of sialic acid by neuraminidase digestion of B polysaccharide complexed to Al ‘+ ions; the polymers had molar ratios of sialic acid/A13’ of I:0 (U), 1:0.14 (0) and 1:0.31(A)
Effect of A13+ binding on esterification in B polysaccharide at low pH
Molar ratio of sialic acid/A13+ in B polysaccharide 1 :o 1 :o.i
al.
Esterification (%) after incubation for 24 h with molar ratio of sialic acid/H+ of 1:0.2
1:0.4
1:o.a
12.9 3.6 <0.5 <0.5
27.1 15.0 7.4 2.3
48.9 41.0 27.7 15.6
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21
Meningococcal group B vaccine: M. R. Life/y et al.
the lack of antibody production does not necessarily reflect a lack of effective interaction with the immune system, since immunization with purified B polysaccharide leads to immune memory. The interaction of native B polysaccharide with the immune system was apparently transient and of an abortive nature, and complexing with Al”+ did not alter this situation. However, since B polysaccharide is known to be a far better immunogen when associated with meningococcal outer membrane proteins, the effect of binding of Al”+ ions to a B polysaccharide-type 6 OMP complex on the immune response in mice was tested”‘. Animals were immunized intraperitoneally with 10 ug doses of the group B-type 6 protein complex given with either 10-s M Alz(SO&, with 100 ug AI(O or with no addition of Al’+ ions. The anti-B response examined seven days later (Table 7) clearly showed a 2 to 3-fold increase when A12(S0 4)_Twas added, without alteration of the anti-OMP titres, and almost a lo-fold increase with Al(OH)3, both results being highly significant statistically. This potentiation was not apparent when the same experiment was performed with a meningococcal group C polysaccharide-OMP complex administered in identical fashion to the group B complex. These results were interpreted in terms of stabilization of B polysaccharide in its native form rather than an adjuvant effect, based on the following observations: (i) administration of protein-B polysaccharide complexes in alhydrogel (as an adjuvant) boosted both anti-B and antiprotein responses, whereas Al’+ as a salt stimulated the production of anti-B antibodies alone; and (ii) Al”+ forms of protein-C polysaccharide complexes did not show this enhancing effect. Why is it, then, that purified B polysaccharide, as the Al”+ salt, did not seem to stimulate antibody production? The answer to this question probably lies in the way phagocytic cells handle, process, and present antigens to B cells. The rapid clearance of the B polysaccharide from liver and spleen following intravenous injection suggests that this relatively short-lived antigen can stimulate the production of memory cells but not antibody, which requires B polysaccharide stabilization to preserve the conformational determinants and, possibly, B and T cell proliferation factors together with T helper function. Probably none of these activities are stimulated by polysaccharide alone, but protein-polysaccharide complexes do so. Al”+ is not the only cation capable of enhancing the anti-B response in protein-B polysaccharide complexes. Results obtained in our laboratory with ruthenium red as a complexing agent have indicated similar chemical stability and antigenic stimulation. It cannot be established at the moment whether reduction of Table 7
Immunization of CBA mice with a B polysaccharid&ype
internal esterification or increased resistance to neuraminidases is more important in promoting the immunogenicity of B polysaccharide, and, although we favour the former as being the most significant, it is possible that both phenomena play a role. Whatever the explanation may be, it is important that these findings suggest the interesting possibility of using Al”+ to enhance the immunogenicity of N. meningitidis group B vaccines for immunization of humans.
Immunobiology of the B polysaccharide response Generally, the immune response to purified polysaccharides is thymus-independent with production of IgM antibodies, and no enhancement of the response is observed upon subsequent immunizations. There are exceptions to these rules, however, since a booster response can be induced in young children to the meningococcal A polysaccharide”“, and high levels of IgG antibodies can be induced in mice with the group C polysaccharide6’. We must bear in mind, however, that unlike many thymus-independent antigens that persist for a very long time in the lymphoid organs, A polysaccharide (and B polysaccharide as well) is unstable and degrades relatively fast in viva. The induction of an immune response to polysaccharides can be enhanced by injecting the polysaccharide (or oligosaccharide thereof) coupled, either covalently or non-covalently, to protein carrier@‘. This usually results in a shift from a thymus-independent to a thymus-dependent antigen, a more robust response than that of the polysaccharide alone, and the induction of IgG antibodies and memory cells, as demonstrated, amongst others. for the group C polysaccharide”‘,“2,6’. The unique immunological characteristics of B polysaccharide, however, make unsafe any prediction of its behaviour deduced from the properties of other similar polysaccharides. In fact, coupling (2-S)u-linked sialic acid oligosaccharides (from B polysaccharide) to protein carriers proved unsuccessful”“. For this reason, we have studied in detail the immunobiology of the B polysaccharide response in mice. Athymic mice were shown@ to be capable of producing IgM antibodies to the B polysaccharide when immunized with the polysaccharide complexed with type 6 OMPs, whereas no immunogenicity could be demonstrated with the purified polysaccharide. When athymic mice were given T cells obtained from the spleen of normal mice, the primary, anti-B immune response to the B polysaccharide-type 6 OMP complex in Al(OH)3 (given five days after transfer) was low and comparable to a control group of athymic mice receiving no cells. However, when the animals were reimmunized in the same way 15 days later, the anti-B
6 OMP complex administered in combiration with Al&SO&
Group
Antigen at day Oa
Additive to antigen
Anti-B response at day 7b
1 2 3 4 5 6
+ -
None None 1O-3 M AIP(SO& 1O-3 M AIZ(SO& 100 pg AI 100 pg AI(
1.6(1.38) 0.49 (1.04) 3.92 (1.16) 0.65 (1.20) 14.7 (1.83) 0.46 (1.04)
+ + -
or AI(
p valueC
<0.025
Ten trg polysaccharide-type 6 OMP complex given intraperitoneally in 5% lactoseO.01 M sodium phosphate buffer pH 7.3 (0.5 ml). Control mice (groups 2,4 and 6) received lactose-phosphate buffer pH 7.3 (0.5 ml). ‘Geometric average of five mice per group expressed in pg lgiml serum, with standard error in parentheses. %alculated by the student’s 1test when comparing groups 3 and 5 with group 1
22
Vaccine,
Vol. 5, March
1987
Meningococcal group B vaccine: M. R. Life/y Table 8 Effect of T-cell transfer upon the anti-B response of BALB/c nu/nu mice immunized with a B polysaccharide-type 6 OMP complex Anti B titrea Day
Action
Group 1
Group 2
-5
Cell transfer 2.3 x10* Balblc T cells
+
-
0.1 (1.01)
0.13 (1.04)
d.46 (1.19) 0.32 (1.09)
d.,, (1.15) 0.35 (1.27)
8f.03 (1.64) 1.23 (1.58)
0+.56(1.52) 0.55 (1.53)
-1 0 +7 +14 +15 +22 +29
Primary vaccination*
Secondary vaccination b
aGeometric average of five mice per group expressed in pg Ig/ml serum, with standard error in parenthesis. ‘7en Kg B polysaccharide-type 6 OMP complex + 100 ttg AI given intraperitoneally in PBS (0.2 ml)
titre at day 22 was 14 times higher than in control mice (Table 8). These results demonstrate clearly that a primary immune response to the B polysaccharidetype 6 OMP complex is thymus-independent; this in itself is rather surprising, since the presence of large polysaccharide chains presented in combination with protein carriers should be able to stimulate both thymus-independent and thymus-dependent responses. Secondary responses are quite different, however, and here there is a substantial shift towards a thymusdependent component. In order to clarify whether the secondary response depended on the accumulation of T and/or B memory cells, lethally irradiated mice were injected with either immune or non-immune T-cells or B-cells. Both donors and recipients were immunized with the B polysaccharide-type 6 OMP complex given with a purified meningococcal group Y polysaccharide (control, thymus-independent antigen) and Al(O The response to B, Y and type 6 OMP was measured 7 days after immunization. The results can be summarized as follows: (i) Transfer of immune T cells (but not immune B cells) resulted in an enhancement of the response for both B polysaccharide and type 6 OMP. (ii) Transfer of immune as compared to non-immune cells resulted in a slight reduction in the response to Y polysaccharide. These experiments demonstrate the presence of memory T cells for both B polysaccharide and type 6 OMP, but no memory B cells could be found. The truly thymus-independent antigen, Y polysaccharide, performed as expected with no build-up of memory. This series of experiments also demonstrated the failure of T-suppressor cells to explain the poor immune response to the purified B polysaccharide since: (i) athymic mice respond poorly to the purified polysaccharide; (ii) addition of normal spleen cells to athymic mice does not decrease the response to the polysaccharide-OMP complex; and (iii) cyclophosphamide treatment of normal mice does not result in an increased anti-B response. Bearing in mind that the epitope on B polysaccharide is most probably conformational and very likely to be chemically unstable, one would tend to regard the response to B polysaccharide as ‘abortive’, in the sense that it is triggered by the antigen only temporarily without reaching the period when it becomes antigen
et al.
independent, i.e. a plasma cell. This could be the reason why the B lymphocytes (specific for this antigen) undergo antigen-driven mitogenic cycles and only partial differentiation, without ever reaching full maturation to produce IgG antibodies or to maintain a sustained production of IgM. Therefore, modifications that stabilize the antigen, like aluminium salts of the polysaccharide, may improve its immunogenicity without changing completely its ‘abortive’ nature. Also it could explain why anti-B antibodies are of low affinity. This is probably the reason why the secondary response of mice, although substantially more robust than the primary one, does not result in a shift towards the production of IgG antibodies specific for B polysaccharide. It is unlikely, however, that a situation such as restricted IgM production is going to apply rigidly to every other strain of mice or other animal species, since it has been reportedh5 that NZB mice produce IgG antiB antibodies and other murine strains, as well as New Zealand albino rabbits, have been found to produce IgG antibodies when immunized with the B polysaccharide-type 6 OMP complex in our laboratories. Two injections of the same vaccine given two to three weeks apart resulted in a substantial increase of the anti-B and anti-OMP antibodies, and to a lesser extent this was also observed when a different serotype was used for the second immunization”4. These facts ought to be considered if planning immunization strategies using similar vaccines in human volunteers. Conclusions In this article, we have attempted to show that an effective vaccine requires stabilization of the threedimensional structure of the B polysaccharide, which our proposed vaccine seems to be able to do to an extent, partly through association with OMP proteins49, and partly through interactions with Al”+ ions5’. The poor immunogenicity of the B polysaccharide has been studied at a molecular level through the use of n.m.r. spectroscopy25; with the aid of molecular modelling using computer graphics, this has led to a detailed understanding of one of the underlying mechanisms involved in destabilization of the three-dimensional structure of the polysaccharide, namely, esterification’5,‘4. Although studies of the secondary and tertiary structure of proteins have been conducted*’ and the relationship to immunogenicity delineated, this is the first report, to our knowledge, of such an undertaking with a polysaccharide. It must be emphasized, however, that this study is, as yet, incomplete, since there are questions which remain unanswered. What, for instance, is the complete three-dimensional structure of B polysaccharide? Where is the location and what is the nature of the conformational or discontinuous determinant(s) on the polymer? Can the determinant(s) be stabilized sufficiently to give a longer lasting immune response, and can a shift from IgM to IgG antibodies be induced? Evidently, these questions are interrelated and, in attempts to give answers to them, results using one approach will inevitably provide information which may be used to confirm or modify results from another. It is also evident that in answering these, further questions are posed: Do conformational/ discontinuous epitopes occur on other polysaccharides? Can these epitopes be stabilized to allow an immune Vaccine,
Vol. 5, March
1987
23
Meningococcal group B vaccine: M. R. Life/y et al.
response to be mounted? Perhaps mimicking of unstable epitopes by designing synthetic compounds of carbohydrate or non-carbohydrate origin may be the basis of future vaccines. An obvious deterrent to the study of polysaccharide three-dimensional structures has been the need to develop suitable techniques. The breathtaking speed with which n.m.r. spectroscopy has advanced in recent years in structural and conformational studies of carbohydrates, however, has made this technique invaluable for future studies of carbohydrate conformation in relation to immunogenicity. Continuous determinants have been successfully delineated on a number of polysaccharides’7,‘“.66,67 through the use of monoclonal or polyclonal antibodies and specific carbohydrate inhibitors, which mimic the primary structure and are generally smaller than the hexasaccharide. However, this approach cannot be applied to discontinuous determinants on the polymer with any confidence, precisely because the carbohydrate inhibitors cannot be readily predicted. The determination of polysaccharide secondary and tertiary structures through the use of n.m.r. and other spectroscopies, X-ray diffraction and theoretical predictions will undoubtably aid in this respect. At present, it is difficult to speculate on the methods which may be used to stabilize particular discontinuous determinants, although these should become more evident as information on the determinants is generated. In this article, for example, the meningococcal group B polysaccharide has been shown to have discontinuous determinants which are destabilized by internal esterification involving -CO?H and HO-9 groups of adjoining residues (see Figure 8). It was logical to predict that blocking of either or both of these groups would inhibit ester formation and therefore stabilize the determinants on the polymers. It is hoped that in the next few years, we will see more examples of discontinuous determinants on oligoand polysaccharides. This, in turn, will give impetus to the question of the relationship between discontinuous determinants and their immunology and, finally, to the stabilization or synthesis of these determinants as potential immunogens. The schematic diagram in Figure 12 illustrates many of the factors involved in the immune response to the meningococcal group B polysaccharide. The polymer may be degraded by neuraminidases or antigenically modified by esterification under mild conditions. Continuous determinants, i.e. short chains of (2+8)-w-linked sialic acids, within the polysaccharide do not appear to be recognized as ‘foreign’ by the immune system due to the ubiquitous presence of sialogangliosides and sialoproteins on cell surfaces of the host, bearing identical sialic acid structures. Therefore, discontinuous determinants, held together in a precarious way, on the three-dimensional structure of the polysaccharide are important for immunogenicity and must be stabilized in order for the B polysaccharide to interact effectively with the immune system; this achieved, an anti-B response is mounted. It has been speculated that anti-B antibodies, which cross-react with polysialoglycoproteins (N-CAM proteins) found in fetal and neonatal brain tissue, may initiate an autoimmune process’ ‘. It is highly unlikely, however, that interaction of N-CAM proteins with the immune system occurs (discussed earlier in this review), and we do not regard this as a
24
Vaccine,
Vol. 5, March
1987
Figure 12 A schematic diagram of factors involved in the immune response to B polysaccharide, as explained in the text
serious drawback in efforts to manufacture an effective vaccine containing the group B polysaccharide. However, we must stress that careful monitoring of any new vaccine is essential, and to ignore this cross-reactivity would be irresponsible. We believe that the potential cross-reactivity of foreign antigens with brain components, particularly at the early developmental stage. should be the subject of further close examination and research. Immunization of mice with a meningococcal group B polysaccharide-type 6 OMP complex given with Al(OH)3 resulted in an increased anti-B response5’. Memory T-cells were stimulated and. on secondary immunization with the same vaccine, a boosted response to both B polysaccharide and type 6 OMP components of the vaccine was observed’“. This secondary response did not require OMP serotypes to be the same for primary and secondary immunization, suggesting that the potential protective value of T-helper cells induced could be maintained even when the vaccine and field strains do not share the same serotype. Moreover, a secondary anti-type 6 OMP response could be induced when mice. primed with the B polysaccharide-type 6 OMP complex. were challenged with a B polysaccharide-type 2a OMP complexh4. Although small (16%) in comparison with the homologous challenge, it was highly significant 0, < 0.0005 for the student’s f-test) with a 2 to 3-fold antibody increase over non-immunized controls. Our chemical, molecular and immunological studies have led us to propose reasons for the poor immunogenicity of B polysaccharide and from this has emerged a candidate vaccine for use in humans. Volunteer studies are now in progress.
Acknowledgements Many of our colleagues at the Wellcome Research Laboratories have made significant contributions to this endeavour and we gratefully acknowledge their invaluable help. In particular we thank Mrs J. Esdaile, Miss U.T. Nowicka (immunology), Mr D.C. Brown (bacteriology), Dr A.S. Gilbert (IR). Dr R.C. Glen, Dr
Meningococcal group 8 vaccine: M. R. Life/y et
J.G. Vinter (molecular modelling) and Mrs J.M. Williams (n.m.r.). We also thank Dr A.J. Beale for his encouragement and support. 22
References
4
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Gotschlich, EC. Meningococcal meningitis. In: Bacteria/ Vaccines (Ed. Germanier, Ft.) Academic Press, Inc., 1984, Ch. 8, p. 237-255 Frasch, C.E. Noncapsular surface antigens of Neisseria meningitidis. Semin. Infect. Dis. 1979, 2, 304 Tsai, C-M., Frasch, C.E. and Mecca, L.F. Five structural classes of major outer membrane proteins in Neisseria meningitidis. J. Bacferiol. 1981, 146, 69 Gotschlich, EC., Goldschneider, I. and Artenstein, MS. Human immunity to the meningococcus IV. lmmunogencity of group A and group C meningococcal polysaccharides in human volunteers. J. Exp. Med. 1969, 129, 1367 Artenstein, M.S., Gold, R., Zimmerly, J.G., Wyle, F.A., Schneider, H. and Harkins, C. Prevention of meningococcal disease by group C polysaccharide vaccine. N. Engl. J. Med. 1970, 282, 417 Erwa, H.H., Haseeb, M.A., Idris, A.A., Lapeyssonnie, L., Sanborn, W.R. and Sippel, J.E. A serogroup A meningococcal polysaccharide vaccine. Studies in the Sudan to combat cerebrospinal meningitis caused by Neisseria meningitidis group A. Bull. WHO 1973, 49, 301 de Taunay, A., Galvao, P.A., de Morais, J.S., Gotschlich, EC. and Feldman, R.A. Disease prevention by meningococcal serogroup C polysaccharide vaccine in pre-school children: results after 11 months in Sao Paula. Pediatr. Res. 1974, 8, 429 Peltola, H., Makela, P.H., Kayhty, H.M., Jousimies, H., Herva, E., Hallstrom, K. et al. Clinical efficacy of meningococcal group A vaccine in children three months to five years of age. fV. Engl. J. Med. 1977, 297, 686 Wyle, F.A., Artenstein, MS., Brandt, B.L., Tramont, EC., Kasper, D.L., Altieri, P.L., Berman, S.L. and Loventhal, J.P. Immunologic response of man to group B meningococcal polysaccharide vaccines J. Infect. Dis. 1972, 126, 514 Gotschlich, E.C., Liu, T.-Y. and Artenstein, MS. Human immunity to meningococcus. Ill. Preparation and immunochemical properties of the group A, group B, and group C meningococcal polysaccharides. J. Exp. Med. 1969, 129, 1349 Finne, J., Leinonen, M. and Make@ P.H. Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenosis. Lancer 1983, 2,355 I Griffiss, J. McL. immunological Recognition and Effector Mechanisms in infectious Diseases (Eds. Torrigiani, G. and Bell, R.) Schwabe, Basel, 1981, p. 137-152 Jennings, H.J., Lugowski, C. and Kasper, D.L. Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide. Biochemistry 1981, 20, 4511 Egan, W., Liu, T.-Y., Dorow, D., Cohen, J.S., Robbins, J.D., Gotschlich, E.C. and Robbins, J.B. Structural studies on the sialic acid polysaccharide antigen of fscherichia co/i strain BOS-12. Biochemistry 1977, 16, 3687 Lifely, M.R., Gilbert, A.S. and Moreno. C. Rate, mechanism, and immunochemical studies of lactonisation in serogroup B and C polysaccharides of Neisseria meningitidis. Carbohydr. Res. 1984, 134,229 Lifely, M.R., Nowicka, U.T., Krambovitis, E. and Esdaile, J. Antigenicity of meningococcal group B oligo- and polysaccharides of defined chain length. Fiffh lntemafional Pafhogenic Neisseria Conference, Amsterdam, 1986 Jennings, H.J., Roy, R. and Michon, F. Determinant specificities of the group B and C polysaccharides of Neisseria meningifidis. J. immunol. 1985, 134, 2651 Lifely, M.R., Nowicka, U.T. and Moreno, G. Analysis of the chain length of oligomers and polymers of sialic acid isolated from Neisseria meningifidis group B and C and Escherichia co/i Kl and K92. Carbohydr. Res. 1986, 156, 123 Kabat, E.A. Structural Concepts in Immunology and lmmunochemistry, 2nd Edition, Holt, Rinehart and Winston, New York, 1976, Ch. 6 Atassi, M.Z. Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites. Eur. J. Biochem. 1984, 145, 1 Kabat, E.A., Nickerson, K.G., Liao, J., Grossbard, L., Osserman, E.F., Glickman, E. et al. A human monoclonal macroglobulin with
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specificity for a(2+8)-linked poly-hl-acetyl neuraminic acid, the capsular polysaccharide of group B meningococci and Escherichia co/i Kl, which cross-reacts with polynucleotides and with denatured DNA. J. Exp. Med. 1986,184,642 Lafer, E.M.. Rauch, J., Andrzejewski, C., Mudd, D., Furie, B., Schwarz, R.S. and Stellar, B.D. Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids. J. Exp. Med. 1981, 153, 897 Heidelberger, M. Precipitating cross-reactions among pneumococCal types. Infect Immun. 1983, 41, 1234 Doddrell, D., Glushko, V. and Allerhand, A. Theory of Nuclear Overhauser Enhancement and 13C-‘H dipolar relaxation in protondecoupled carbon-13 n.m.r. spectra of macromolecules. J. Chem. Phys. 1972, 56, 3683 Lindon, J.C., Vinter, J.G., Lifely, M.R. and Moreno, C. Conformational and dynamics differences between N. meningitidis serogroup B and C polysaccharides, using n.m.r. spectroscopy and molecular mechanics calculations. Carbohydr. Res. 1984, 133, 59 Lifely, M.R., Lindon, J.C.. Williams, J.M. and Moreno, C. Structural and conformational features of the Escherichia co/i K92 capsular polysaccharide. Carbohydr. Res. 1985, 143, 191 Egan, W., Ferretti, J.A. and Marshall, G.R. Relaxation parameters and motional properties in biological macromolecules. Bull. Magn. Reson. 1981, 2, 15 Egan, W. Magnetic Resonance in Siology (Ed. Cohen, J.S.) Wiley, New York, 1980, 1, 197-258 Brown, E.B., Brey, W.S. and Weltner. W. Cell-surface carbohydrates and their interactions. I. N.m.r. of N-acetyl neuraminic acid. Biochim. Biophys. Acta. 1975, 399, 124 Dabrowski, U., Friebolin, H., Brossmer, R. and Supp, M. ‘H-n.m.r. studies at N-acetyl-o-neuraminic acid ketosides for the determination of the anomeric configuration, II. Tetrahedron Lett. 1979, 48, 4637 Haasnoot, C.A.G., De Leeuw, F.A.A.M. and Altona, C. Prediction of anti and gauche vicinal proton-proton coupling constants for hexapyranose rings using a generalised Karplus equation. Bull. Sot. Chem. Be/g. 1980, 89, 125 Lindon, J.C. and Ferrige, A.G. Digitisation and data processing in Fourier transform n.m.r. Prog. Nucl. Magn. Reson. Spectrosc. 1980, 14,27 Bodenhausen, G. and Freeman, R. Correlation of Proton and Carbon-13 n.m.r. spectra by heteronuclear two-dimensional spectroscopy. J. Magn. Reson. 1977, 28, 471 Lifely, M.R., Gilbert, AS. and Moreno, C. Sialic acid polysaccharide antigens of Neisseria meningitidis and Escherichia co/i: Esterification between adjacent residues. Carbohydr. Res. 1981, 94, 193 Lifely, M.R. and Cottee, F.H. Formation and identification of two novel anhydro compounds obtained by methanolysis of N-acetylneuraminic acid and carboxyl-reduced, meningococcal B polysaccharide. Carbohydr. Res. 1982, 107, 187 Mandrell, R.E. and Zollinger, W.D. Measurement of antibodies to meningococcal group B polysaccharide: Low avidity binding and equilibrium binding constants, J. Immunol. 1982, 129, 2172 Finne, J. Occurrence of unique polysialosyl carbohydrate units in glycoproteins of developing brain. J. Biol. Chem. 1982, 257, 11966 Finne, J., Finne, U., Deagostini-Bazin, H. and Goridis, C. Occurrence of a(2+8)-linked polysialosyl units in a neural cell adhesion molecule. Biochem. Biophys. Res. Commun. 1983, 112, 482 Hoffman, S., Sorkin, B.C., White, PC., Brackenbury, R., Mailhammer, R., Rutishauser, U., Cunningham, B.A. and Edelman, G.M. Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes. J. Biol. Chem. 1982, 257, 7720 Gregson, N.A., Moreno. C. and Lifely. M.R. Monoclonal antibodies against meningococcal polysaccharide with cross-reactivity against brain antigens. Biochem. Sot. Trans. 1985, 13, 462 Zollinger, W.D., Boslego, J.E., Frasch. C.E. and Froholm, L.O. Safety of vaccines containing meningococcal group B polysaccharide. Lancet 1984, ii, 166 Cadoz, M., Armand, J., Arminjon, F., Gire, R. and Lafaiz, C. Tetravalent (A&Y, W135) meningococcal vaccine in children: Immunogenicity and safety. Vaccine 1985, 3, 340 Griffiss. J. McL., Brandt, B.L., Altieri, P.L., Pier, G.B. and Berman, S.L. Safety and immunogenicity of group Y and group WI35 meningococcal capsular polysaccharide vaccines in adults. Infect. Immun. 1981, 34, 725 Kaplan, M.H. Immunologic relation of streptococcal and tissue antigens I. Properties of an antigen in certain strains of group A Streptococci exhibiting immunologic cross-reactions with human heart tissue. J. Immunol. 1963, 90, 595
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Zabriskie, J.B., Hsu, KC. and Seegal, B.C. Heart reactive antibody associated with rheumatic fever: Characterization and diagnostic significance. C/in. Exp. /mmuno/. 1970, 7, 147 Fischetti, V.A. and Manjula, B.N. Biologic and immunologic implications of the structural relationship between streptococcal M protein and mammalian tropomysin. In: Seminars in infectious Disease Vol IV: Bacterial Vaccines (Eds. Robbins, J.B., Hill, J.C. and Sadoff, J.C.) Thieme-Stratton, New York, 1982, Ch. 58, p. 41 I-418 Jann, K. and Westphal, 0. Microbial polysaccharides. In: The Antigens, Vol Ill (Ed. Sela, M.) Academic Press, New York, 1975, p. I-125 Zollinger, W.D., Mandrell, R.E., Griffiss, J.M., Altieri, P. and Berman, S. Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man. J. C/in. Invest. 1979, 63,836 Moreno, C., Lifely, M.R. and Esdaile, J. Immunity and protection of mice against Neisseria meningifidis group B by vaccination, using polysaccharide complexed with outer membrane proteins: A comparison with purified B polysaccharide. Infect. Immun. 1985, 47, 527 Jennings, H.J. and Lugowski, C. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J. Immunol. 1981, 127, 1011 Frasch, C.E. and Peppler, M.S. Protection against group B Neisseria meningitidis disease: Preparation of soluble protein and proteinpolysaccharide immunogens. Infect. Immun. 1982, 37, 271 Peppler, MS. and Frasch, C.E. Protection against group B Neisseria meningitidis disease: Effect of serogroup B polysaccharide and polymixin B on immunogenicity of serotype protein preparations. Infect. Immun. 1982, 37, 264 Craven, D.E. and Frasch, C.E. Protection against group B meningococcal disease: Evaluation of serotype 2 protein vaccines in a mouse bacteremia model. infect. Immun. 1979, 26, 110 Wang, L.Y. and Frasch, C.E. Development of a Neissena meningitidis group B serotype 2b protein vaccine and evaluation in a mouse model. Infect. Immun. 1984, 46, 408 Frasch, C.E., Coetzee, G., Zahradnik, J.M., Feldman, H.A. and Koornhof, H.F. Development and evaluation of group B serotype 2 protein vaccines: Report of a group B field trial. Med. Trap. 1983, 43, 177 Gotschlich, E.C., Fraser, B.A., Nlshimura, O., Robbins. J.B. and LIU. T.-Y. Lipid on capsular polysacchandes of Gram-negative bacteria. J. Biol. Chem. 1981, 256, 8915
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Moreno, C., Lifely, M.R. and Esdaile, J. Effect of aluminium ions on chemical and immunological properties of meningococcal group B polysaccharide. Infect. Immun. 1985, 49, 587 58 Lindon, J.C., Moreno, C. and Lifely, M.R. Unpublished observations 59 Gotschlich, E.C., At ,trian, R., Cvjetanovic, B. and Robbins, J.B. Prospects for the pri>vention of bacterial meningitis with polysaccharide vaccines. 61 1. WHO 1978, 56, 509 60 Moreno, C. and Esdaile, J. lmmunoglobulin isotype In the murine response to polysaccharide antigens. Eur. J. Immunol. 1983, 13, 262 61 Avery, O.T. and Goebel, W.F. Chemo-immunological studies on conjugated carbohydrate-proteins. V. The immunological specificity of an antigen prepared by combining the capsular polysaccharide III pneumococcus with foreign protein. J. Exp. Med. 1931, 54, 437 62 Beuvery, E.C., Miedema, F., van Delft, R.W., Haverkamp, J., Leussink, A.B., te Pas, B.J., Teppema, K.S. and Tiesjema, R.H. Preparation and physiochemical and immunological characterization of polysaccharid+outer membrane protein complexes of Neissena meningifidis. Infect. Immun. 1983, 40, 369 63 Beuvery, E.C., Miedema, F., van Delft, R. and Haverkamp, J. Preparation and immunochemical characterization of meningococcal group C polysaccharide-tetanus toxoid conjugates as a new generation of vaccines. Infect. Immun. 1983, 40, 39 64 Moreno, C., Esdaile, J. and Lifely, M.R. Thymic-dependence and immune memory in mice vaccinated with meningococcal polysaccharide group 6 complexed to outer membrane protein. immunology 1986,57, 425 65 Frosch, M., Giirgen, I., Boulnois, G.J., Timmls. K.N. and BitterSuermann, D. NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: Isolation of an IgG antibody to the polysaccharide capsules of Escherichia co/i Kl and group B meningococci. Proc. Nat/ Acad. Sci. USA 1985, 82, 1194 66 Kasper, D.L., Baker, C.J., Galdes, B., Katzenellenbogen, E. and Jennings, H.J. lmmunochemical analysis and immunogenicity of the type II group B streptococcal capsular polysaccharide. J. C/in. Invest. 1983, 72, 260 67 Heidelberger, M., Jann, K. and Jann, B. Cross-reactions of fschenchia co/i K and 0 polysaccharides in antipneumococcal and antlSalmonella sera. J. Exp. Med. 1985, 162, 1350 57
Bacteria;
meningitis;
Neisseria
meningikfis
Neisseria meningitidis is a Gram-negative diplococcus which causes meningitis in humans. Virulent strains (i.e. those isolated from the bloodstream or cerebrospinal fluid) are invariably associated with a capsule which surrounds and helps the organism to evade the host defence systems. The capsules consist of a high molecular weight anionic polysaccharide, and form the basis for serological classification of the meningococci into distinct serogroups (A, B, C, 29-E, H, I, K, L, W135, X, Y, Z)‘. Within each serogroup there are many antigenically different outer membrane proteins (OMP) and lipopolysaccharides which define a serotyping system used to further subdivide the organism’%-. Acquisition of meningococcal meningitis is through airborne droplet spread, followed by colonization of the nasopharynx. Carriage of the organism in the throat, however, is relatively common (5-30% depending upon the season) and only rarely does carriage progress to cause symptoms of disease. The most common form of disease occurs through invasion of the meningococci into the bloodstream, and penetration of the bloodbrain barrier (the meninges, and hence the name of the disease). The organisms are then able to multiply in the Departments of Experimental Immunobiology* and Physical Chemistry*, Wellcome Research Laboratories, Langley Court, Beckenhv, Kent, BR3 3BS UK, and ‘MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, Ducane Rd, London, W12 OHS 026&410X’87/05001 0
1987 Butterworth
l-l
cerebrospinal fluid causing inflammation of the brain, often leading to death or mental retardation. Onset of disease may also be rapid, and the sequence from noticeable symptoms or diagnosis to death often occurs within 24 hours. Meningococcal meningitis is a disease occurring worldwide in both endemic and epidemic forms. Endemic disease has a rate of between 1 and 5 per 100 000 per annum, whereas epidemics of up to 500 per 100 000 population have occurred. Serogroups A, B and C have been shown to constitute greater than 90% of the isolates from patients, with group B alone accounting for 5@70% of cases. In the late 196Os, vaccines were developed against serogroup A and C meningococci4; these vaccines consisted of the purified high molecular weight capsular polysaccharide derived from the respective organism, and their safety and efficacy has been demonstrated in a number of controlled field trials and epidemic situations”,6. Unfortunately, however, the serogroup C vaccine is ineffective in children under two years old’ (that age group most susceptible to disease) and the serogroup A vaccine in children below 6 month?. More seriously, there has been a spectacular lack of success in producing an effective vaccine against group B disease. In early trials, the high molecular weight group B capsular polysaccharide proved to be very poorly immunogenic in humans”. The group B polysaccharide is a (2-8)~u-linked homopolymer of N-acetylneuraminic acid (Figure I), which is interesting in two respects. First, this structure is identical to that found in
1 co3-
.oH2C9+~oH
3
AcHN
OH 5 cx -
NeuNAc
9 polyracchsride - (2 -
9),-
C polysaccharide - (2 -
9bR
K92 E Coli
-
[l2-88)-(2-9B)1~
Figure 1 The structure of N-acetylneuraminic acid and the repeating units of the N. meningitidis group B and C, and the E. co/i K92 capsular polysaccharides
6 $03.00
& Co. (Publishers)
Ltd
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11
Meningococcal group B vaccine: M. R. Life/y et al.
the capsular polysaccharide of Escherichia coli Kl (colominic acid), the major cause of neonatal meningitis, suggesting a common mechanism by which these polymers evade the host defences, and second, group C polysaccharide, a homopolymer of (2-+9)-a-linked Nacetylneuraminic acid (Figure I), is structurally very similar to the B polysaccharide, which contrasts with the difference between the polymers in immunogenicity in adults.
Poor immunogenicity of the B polysaccharide A prerequisite for an effective meningococcal group B vaccine would seem to be a basic understanding of the poor immune response to the purified B polysaccharide. Although hypotheses have been postulated to explain the poor immunogenicity of the B ,{O’Ysaccharide, namely, sensitivity to neuraminidases , crossreactivity with ‘self’ antigens”, and intrinsic ‘floppiness’ of the purified B polysaccharide’*, clear-cut experimental evidence has been scarce. Consequently, a clear and rational programme of group B meningococcal vaccine development has been difficult to achieve. With this in mind, our interpretation of the poor immunogenicity of the B polysaccharide is described in detail in this section.
Conformational
determinants
Chemical and immunological evidence. Polymeric carbohydrate antigens of bacterial origin often show structural similarities to cell-surface components of the host. In this situation it would be advantageous if the immune system of the animal were to respond to produce antibodies directed against conformational determinants present on the secondary or tertiary structure of the polymer, thus providing immunity but preventing autoimmune cross-reactivity. This may occur, for instance, for type III group B streptococcal polysaccharide”, where sialic acid seems to direct the antigenit conformation; this same situation may also apply to meningococcal group B polysaccharide. Present on cell surfaces of the host are sialogangliosides and sialoproteins containing short chains (generally 2-4 residues) of (2-8)~a-linked sialic acid, identical to the repeating unit of the B polysaccharide. It is likely therefore that the animal is tolerant to this particular ‘self’ epitope, since if not, and antibodies were to recognise a straightforward (2+8)-a-linked sequential or continuous determinant, it would initiate an autoimmune response against gangliosides and glycoproteins bearing this linkage. Consequently, the host forms antibodies directed to one or more conformational or discontinuous determinants defined by the three-dimensional structure of the B polysaccharide. Evidence in support of this hypothesis has come from a number of workers in the field. Egan et al.” showed that the capsular polysaccharide from E. co/i K92 is a heteropolymer containing alternate (2+8)-aand (2--+9)-a-linked sialic acid residues (Figure I), which cross-reacts immunologically with meningococcal group C polysaccharide but not with group B polysaccharide. This suggests that antibodies against the C polysaccharide recognize a linear (i.e. structural) determinant, whereas antibodies against the B polysaccharide recognize a conformational determinant. 12
Vaccine,
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The effect of reduction of carboxyl groups in the B polysaccharide (i.e. -C02H + -CH,OH) on its antigenicity was studied more recently’“. Correlation between the degree of carboxyl reduction and the antigenicity of the modified polysaccharide was obtained by a solid phase radioimmune inhibition assay (Table I) which showed that a modest degree of carboxy1 reduction (<20%) resulted in a substantial loss of antigenicity of the polymer, far greater than would be expected for an antibody which recognized a linear or continuous determinant. Thus a low degree of carboxyl reduction is likely to cause considerable disruption of the three-dimensional structure of the B polysaccharide with a concomitant reduction of antigenicity. Low molecular weight colominic acid (E. cofi Kl capsule; M r = 10’) also had a poor affinity for anti-B antibody and modification of only 2% of the residues caused a further loss of antigenicity. In contrast, the C polysaccharide showed only a modest decrease in antigenicity following carboxyl reduction, thus suggesting the presence of a structural determinant on the molecule. Not only are the -C02H groups important for maintaining antigenicity of the B polysaccharide. however. since N-deacetylation of the polymer caused almost complete loss of antigenic activity, shown by its failure to a lutinate with a group B monoclonal latex reagent ?! . Moreover, although following N-reacetylation the polysaccharide recovered (-NH_ 7 + -NHC0.CH3) its ability to agglutinate with the latex reagent, this was not observed when the N-deacetylated polymer was Npropionylated (-NH, -+ -NHCO.CH,CH,). Thus. there is a specific requirement for N-acetyl groups in the B polysaccharide in order to preserve antigenicitv. Perhaps the most compelling evidence obtained m support of the difference in determinant specificity of B and C polysaccharides has been obtained by Jennings and co-workers” who purified a series of oligosaccharides from both polymers and used a radioimmunoassay inhibition technique to determine the nature of the epitopes. Polyclonal rabbit antisera were raised against the group C polysaccharide and experiments to inhibit the precipitation of the polysaccharide with the antiserum were performed with the a-methyl ketoside of sialic acid and the series of (2+9)-a-linked oligomers from the di- to the hexasaccharide. No inhibition was observed for the monomer. but the di, tri- and tetramer showed increasing inhibitory properties. No further increase in inhibition was observed with the pentamer and hexamer. consistent with the estimate of the maxi-
Table 1 Correlation between degree of carboxyl antigenicity of B and (0-Ac’)C polysaccharides
reduction
and
Incubation conditions
Carboxyl reduction (%)
Relative concentration giving 50% inhibition (pg ml-‘)
_ 15 min 1h 4h
0 2.4 5.9 13.1
1 .o 9.5 37 135
EDC
20 min 1h 4h
4.5 19.4 21.7
17.3 36.7 36.7
EDC
20 min 1h 4h
0 9.2 19.7 21.6
Polysaccharide B pH 3.4 B
(0-Ac+)C
1 .o 0.06 0.5
1.o
Meningococcal group B vaccine: M. R. Life/y et
mum size of an antibody binding site. In complete contrast, experiments to inhibit the precipitation of the B polysaccharide with a horse anti-B serum, using a series of (2-+8)-a-linked oligomers to the heptasaccharide, failed to show any inhibition and the use of larger oligosaccharides up to 14 residues long resulted in only poor inhibition. A procedure using a direct binding assay has also shown that a horse anti-B serum has low affinity for (2-S)-a-linked oligosaccharides up to 10 residues long. We have recently extended these results through the determination of oligosaccharide chain length’s by (a) calorimetric measurement of formaldehyde released from the non-reducing end residue after periodate oxidation, (b) radiolabelling of the reducing end residue by reduction with borotritiide, and (c) determination of the ratio of the non-reducing end and internal residues by gas-liquid chromatography. (2-+8)-aLinked oligosaccharides up to 28 residues long very poorly inhibited binding of an anti-B murine monoclonal antibody to B polysaccharide in a solid phase radioimmune inhibition assay, and also only weakly agglutinated a monoclonal B latex reagent’(j. Thus, the determinants present on the B polysaccharide are inadequately expressed on oligosaccharides up to 28 residues long. The estimate of the linear hexasaccharide or heptasaccharide as the upper limit for the size of an antibody bindin site as demonstrated by the classical studies of Kabat has been displayed for the interaction of the meningococcal group C polysaccharide with a rabbit anti-C serum. This has manifestly been demonstrated not to be the simple case for the B polysaccharide, in which the antigenic determinant has been adequately expressed only in the high molecular weight polymer. This situation is analogous to that found for protein antigenic sites2” which may occur on a continuous or linear region of the surface of the polypeptide chain and have thus been termed ‘continuous’, ‘sequential’ or ‘primary’ antigenic sites. Alternatively, a protein may incorporate surface residues from different parts of the polypeptide chain which are brought into close proximity due to the folding constraints on the molecule and have been termed ‘discontinuous’ antigenic sites. Accordingly, we propose that meningococcal group C polysaccharide has continuous and group B polysaccharide discontinuous determinants, but in order to determine the nature of the epitopes present on the surface of the B polysaccharide a greater understanding of the molecular architecture of the molecule has been required. In this context, Kabat etaL21 recently demonstrated that a human monoclonal antibody with specificity for the B polysaccharide cross-reacted with polynucieotides and denatured DNA but had only minimal cross-reactivity with native DNA. It is tempting to speculate on the nature of this cross-reaction, given that B polysaccharide has conformational determinants, and Kabat et al. favour the hypothesis22,23 that the spatial arrangement of charged groups on molecules may constitute antigenic determinants which are cross-reactive with seemingly unrelated substances. The authors stress, however, that the presence of these antibodies in serum at a concentration of 23 mg ml-’ has no& caused any signs or symptoms of disease. N.m.r. spectroscopy has been our principal tool in probing the molecular architecture and for discovering
al.
distinguishing molecular features between group B, group C and E. coli K92 polysaccharides, although confirmatory studies using molecular mechanics and computer graphics have also been employed. N.m.r. studies of flexibility. The relative mobility and flexibility of the polysaccharides was probed by measuring the ‘%Z n m.r. spin-lattice relaxation times (NT,) of the CH and CH2 carbons where N is the number of attached hydrogens. This parameter for a particular 13C nucleus can be related to a molecular correlation time TR which is a measure of the speed of motion of that part of the molecule in solution. Hence, if all the NT, values are equal it is possible to deduce that the molecular fragment is tumbling in solution as a rigid body. Extension of the theory is possible to include the detection of additional internal degrees of flexibility (segmental motion), and measurement at two n.m.r. field strengths is necessary to remove ambiguity in the results24. Measurement of the NT, values at two magnetic field strengths showed that there is a field dependence, and this was used to confirm the correlation times. The results for the B, (0-AC-)C and K92 polysaccharides are listed in Table 22”,2h. The constancy of the NT, values for C-4, C-6, C-7 and C-8 of the B polysaccharide repeating unit indicates that there is no segmental motion for the backbone of this species. At a field strength of 5.9 T, C-9 of the B polysaccharide has a NT, longer than that of the rigid backbone, but at 9.4 T this becomes shorter than those of the carbons in the rigid part. This demonstrates that C-9 has some extra degree of motion over and above the overall molecular tumbling. For the (0-AC-)C polysaccharide, increased T1 values are observed for C-7, C-8 and C-9, something not observable for the (0-Ac+)C polysaccharide, which consists of a mixture of non-O-, 7-O-, 8-O-, and 7,8-di0-acetylated residues. These longer NT, values demonstrate extra segmental motion for C-7, C-8 and C-9, all involved in the chain backbone. The NT, values for C-3 are generally longer than those for the rigid parts of the molecule and this may be a result of increased molecular flexibility due to ringpuckering motions. If it is assumed that the molecule is tumbling isotropically, and this is likely to be true for a coiled polymer, then there is a single rotational correlation time, sR. Using the NT, values for the rigid parts of the polysaccharide one obtains for the B polysaccharide TR = 6.7 X lo-‘s at 5.9 T and zR = 7.1 X lo-' s at 9.4 T. The model developed by Doddrell et a1.24 has been applied to the internal rotation (q;) of the pendant -CH20H (C-9) of the serogroup B polysaccharide and applying this specifically to the B polysaccharide Table 2
‘% n.m.r. spin-lattice relaxation times (NT,), seconds
Carbon atom
B(5.9 T)
B(9.4 T)
(O-AC-)C (5.9 T)
K92 (8.8 T) (2+8)_u_ (2+g)_a_
c3 c4 c5 C8 c7 C8 c9
0.16 0.16 0.17 0.17 0.15 0.16 0.19
0.56 0.44 0.37 0.43 0.42 0.43 0.35
0.17 0.14= 0.16 0.14 0.19= 0.17 0.20
0.27 0.21 0.21 0.22 0.21 0.22 0.33
0.25 0.21 a 0.22 0.22 0.24a 0.23 0.28
aAssignments may be reversed
Vaccine,
Vol. 5, March
1987
13
Meningococcal group B vaccine: M. R. Life/y et al
gives, at 5.9 T, zo =1.3 x 10p”‘s; and at 9.4 T, r. = 0.7 x lo-” s. The consistency of these correlation times is obvious from the constancy obtained for the B polysaccharide at the two field strengths. The agreement for r. is clearly not so good as for ra, but the results at both field strengths indicate that the -CH20H group has an internal rotational correlation time 50-100 times faster than the overall molecular tumbling. The molecules under consideration are homopolymers of sialic acid and. hence the NT, values measured represent an average for all the monosaccharide units throughout the chain, including end groups. Consequently, increased flexibility at the ends of the polymer chain could invalidate the motional model used. This situation is unlikely, because of the high molecular weight which effectively gives the end groups little relative weight. Also, if a particular carbon in different sialic acid residues along the chain possessed a variety of NT, values, it is unlikely that the relaxation data for that carbon would fit the observed, single exponential decay. An important conclusion of this study is the relative flexibility of the B and C polysaccharides in solution. The lack of segmental motion for the former (except for C-9) is compatible with some form of three-dimensional structure, whereas the C polysaccharide gives indications of greater flexibility in solution. Immunologically, these results suggest that the B polysaccharide bears conformational epitopes and that group C determinants are sequential. Similar experiments on the molecular dynamics of the B and C polysaccharides have been reported by Egan et ~1.‘~. ‘, and their results for the molecular correlation times, ra, are in the same range as those derived by us. An exact comparison cannot be drawn because of the different experimental conditions used. For example, their solution concentration was four times higher than in our study, with a related increase in viscosity. However, by measuring spectra at 37°C compared to our 27”C, this may have been somewhat counterbalanced. In addition, their experiments were performed at magnetic field strengths of 2.4 T and 6.4 T and are thus probing a slightly different time-scale. The main difference between the two sets of results is that we observe differences in the 13C relaxation times for the side-chain carbons of the (0-AC-)C polysaccharide. It appears that the previous study used the (0-Ac+)C polysaccharide, which is a mixture of partially acetylated species, giving rise to a loss of peak resolution and chemical shift changes on 0-acetylation that lead to assignment difficulties. This may account for their inability to differentiate NT, values for the side-chain and hence derive relative flexibility information on the two polymers. It should be remembered that n.m.r. NT, measurements only probe fluctuations that occur on the timescale of the n.m.r. observation frequency (2-4 X 10” ss’), and that slower segmental motion may not affect NT,. Indeed, by using the Stokes-Einstein-Debye equation, the absolute magnitudes of the experimental NT, values, ours2s.2h and those of Egan et uI.“.~*, indicate that all the polysaccharides possess considerable internal flexibility at other frequencies. Like the B polysaccharide, the (2+8)-a-linked (B) residues of the K92 polysaccharide showed a constancy 14 Vaccine, Vol. 5, March 1987
of ‘% relaxation-times for C-4/8, which indicates that there is no segmental motion for the backbone of these residues. However, C-9(B), the pendant -CH20H group, had a longer NT, than the rigid backbone, demonstrating some extra degree of motion besides the overall molecular tumbling. The (2-+9)-a-linked (C) residues of the K92 polysaccharide also showed a constancy of NT, values for C-4/8, and only C-9 showed any significant additional flexibility. These results differ from those obtained for the meningococcal (0-AC-)C polysaccharide where the NT, values indicated flexibility along the whole of the C-7, 8, 9 side-chain. Use of the model of isotropic reorientation leads to a correlation time, ra. of 4.0 x lo-” s, and an internal rotation correlation time for the C-9(B) -CH*OH group, ro(B), of 0.6 x lO_"'s. Furthermore, since it appears that only C-9 of the (2+9)-a-linked (C) residues showed any significant additional flexibility, it is possible to use the model of Doddrell et d2’, allowing one degree of internal motion, on the C-9(C) group, giving Q;(C) = 0.8 x lo-“’ s. Thus, the C-9 groups of both (2+8)-u-linked (B) and (2+9)-a-linked (C) residues have an internal rotational correlation time -SO times faster than the overall molecular tumbling. The K92 polysaccharide is characterized by internal or segmental motion of only the C-9(B) and C-9(C) parts of the molecule, which rotate -50 times faster than the overall molecular tumbling. Since C-9(C) is involved in the ketosidic linkage, however, this suggests that the polysaccharide chains will have a considerable degree of freedom of motion, similar to that predicted for the (0-AC-)C polysaccharide, but different to the relative rigidity envisaged for the B polysaccharide. N.m.r. studies ofpolysaccharide conformation. More definitive structural information on the differences between B and C polysaccharides comes from ‘H n.m.r. studies of the carbohydrate conformations. Initially the monomer NeuNAc was studied as the data for this compound were in the literature although it is important to remember that this relates to the more stable p anomer in aqueous solution2”. Since then the conformations of both anomers of NeuNAc have recently been shown to be identica13”. Chemical shifts (6) and spin coupling constants (.I) were obtained for N-acetylneuraminic acid (NeuNAc) by first-order analyses in our work2s. These are listed in Table 3 and are in close agreement with those published. The assignments are as previously given and were confirmed by double resonance and two-dimensional chemical shift correlation experiments. The ring coupling constants, .13a,l. J 1.5., and J5,(, are typical of axial-axial hydrogen interactions and confirm the assignment of the ring conformation as ‘C.5. The coupling constants observed for the C-7-C-9 side-chain are averages over internal rotation that is fast on the n.m.r. timescale, although the barriers to rotation are such that the side-chain exists in staggered conformations. Haasnoot et al.“, have formulated predictive rules for vicinal H-H coupling constants in carbohydrate systems based on the gauche or antiperiplanar nature of the coupled hydrogens and the relative disposition of the oxygen substituents. These predictions explain the magnitudes of the observed coupling constants given in Table 3 giving detailed conformational information
Meningococcal group B vaccine: M. R. Life/y et TRANS
GAUCHE
C7-C8
;l*lI)OH
C-8-C-7
GAUCHE
(y$
al.
C-7-C-8
y$;
H8
“‘1
/“”
1 I
/
‘;fj$:c
HOHZC,,, ;;@:c
,;;;‘&:Ac
H8l I I I ’ ac OH (H)OH2C / N-AcNeu Figure 2
OH
OH
H8
P B polysaccharide
and C polysaccharide
The conformations of the sialic acid sidechains for (2+8)-u- and (2-9)u-linked
Table 3 ‘H n.m.r. data for 8-N-acetylneuraminic acid, saccharide (0-AC-)C polysaccharide and K92 polysaccharide Hydrogen
NeuNAc
B
(0-AC-)C
Chemical shifts (S)= 3a
3e 4 5 6 7 8 9 9’
1.89 2.32 4.08 3.95 4.07 3.57 3.77 3.63 3.86
Coupling constants (Hz) J 3a.3e 13.06 J 3a.4 J 3e.4
11.62
J4.5 J5.6
10.21 10.37
J 6.7
J78 J 8.9
Js.9, J 9.9’
4.97
1.31
9.12 6.30 2.63 11.76
1.72 2.62 b b b 3.87 4.11 4.05 b
1.72 2.74 b b 3.82 3.62 3.98 3.87 3.75
12.1 11.8 4.5 b
12.3 11.5 4.5 b
b
10.1 I .2 9.0 6.2 2.3 10.3
residues as deduced from the n.m.r. spectroscopic studies
B polyK92
“6
248
2-9
1.79 2.70 3.73 3.85 3.87 3.85 4.18 3.75 4.13
1.82 2.88 3.84 3.86 3.64 3.67 4.00 3.92 3.77
<3 8.4 5.2 3.1
aFrom Me&I, taking the N-acetyl resonance as 6 2.07. %esolution insufficient to allow determination of the n.m.r. parameters
the important deduction being that H-7 and H-S are antiperiplanar. The total confirmation is shown in
with
Figure 2.
The ‘H n.m.r. spectrum of the (0-AC-)C polysaccharide is shown in Figure 3, and the n.m.r. parameters, where they can be resolved, are also given in Table 3. Because of the viscous nature of the solution, the relaxation times (T2) of the nuclei are shorter and this results in broader lines. This effect was lessened by measuring the spectrum at 70°C. Computer resolution enhance’ment3* was necessary and under these conditions, by analysis of the coupling patterns and
“9
nn
hR
“7
Figure 3 360 MHz’H n.m.r. spectrum of (O-AC-)C polysaccharide, excluding H3a and H3e
through the use of spin decoupling, the bands due to H-7 and H-S are assigned. The signal for H-8 is easily assigned because, apart from H-4, it is the only nucleus coupled to three other protons. Thus, irradiating the signal at 6 3.98 caused no effect at either of the H-3 resonances but removed the 9.0 Hz coupling on the band at 6 3.62, thereby confirming the assignments of H-7 and, H-S. The coupling constant is typical of the antiperiplanar arrangement of H-7 and H-8 as in NeuNAc, and is similar to the monosaccharide value, indicating the same total conformation. The B polysaccharide was examined under the same conditions as for the (0-AC-)C polysaccharide, and the ‘H n.m.r. spectrum is shown in Figure 4. The assignments of the resonances again follow from application of the techniques used on the other polysaccharide. The H-3 resonances show coupling constants similar to those of the other substances and confirm that the ring conformation is unchanged. The other protons give rise to three separate bands with considerable overlap. At highest frequency is a two-proton band, followed by a signal from a single hydrogen, and a four-proton complex band at lowest frequency. On resolution enhanceVaccine,
Vol. 5, March
1987
15
Meningococcal group B vaccine: M. R. Life/y et al. b.“‘s,b,“~
and this provides additional support for the strict threedimensional requirements for an immunological response with the B polysaccharide. The determination of the conformations of the units in the K92 polysaccharide required the assignment of the ‘H n.m.r. resonances in a very complicated spectrum with much peak overlap. This was achieved only through the use of a two-dimensional “C/‘H correlation n.m.r. experiment, taking advantage of the fact that the ‘%Z spectrum is easily assignable. In this experiment the results are plotted as a contour map. A peak appears where the ‘-‘C chemical shift of the signal of a CH,, fragment intersects with its ‘H chemical shifts’“. An expansion of the region containing C-4 to C-9 for both (2+8)-aand (2-9)~u-linked residues is shown in Figure 5. Having assigned the ‘H shifts it was then possible to use computer resolution enhancement to obtain the ‘H coupling constants to yield the conformational information’h. In the following argument H-4(B) and C-4(B) will, for example, represent H-4 and C-4. respectively, of the (2-G)-a-linked residues in the K92 polysaccharide. The N-acetyl methyl and C-3 methylene groups are trivially assigned and the expanded contour plot (Figure 5) enables the assignment of the other ‘H chemical shifts. The digital resolution in the projection of the ‘H spectrum precludes further analysis. but recourse to a resolution-enhanced one-dimensional spectrum (Figure 6) allows the relevant coupling constants to be extracted. In addition, the assignments and, particularly, the ‘H shifts of the signals of the hydrogens attached to C-4(B), C-4(C), and C-7(C) which have very similar “C shifts, were confirmed by doubleresonance difference experiments. Figlue 6 shows that the resonances at d 4.18 and 6 4.00, assigned to H-g(B) and H-g(C). respectively, each have three coupling constants, as expected. The resonance due to C-O(B) at F 4.13 showed a large coupling constant (-12 Hz) consistent with the expected geminal H-9( B)-H-9’( B) coupling. and 21
H7
“8
HO
wt.4
Figure 4 and H3e
360 MHz n.m.r. spectrum of B polysaccharide, excluding H3a
ment, the signal at 6 4.11 can be seen to possess three couplings, indicating that it must be due to H-4 or H-8. The couplings do not match those for H-3a and, thus, the band cannot be due to H-4. The band assigned to H-8 shows a quartet structure, indicating three moderate couplings of similar magnitude (3-4 Hz). The next band at 6 4.05 shows two couplings, one of = 12 Hz typical of a geminal interaction, and is therefore assigned to one of the non-equivalent protons in the C-9 methylene group (the two protons are expected to have different chemical shifts because of the chiral nature of the molecule). The single proton resonance at 6 3.87 has only one coupling constant of 3.5 Hz and, as this is repeated in the band due to H-8, it is assigned to H-7, with J7.x 3.5 Hz. The coupling constant Jh,, is not resolved and, as in previous molecules, must be
6B 6C
6C 78 48
9B
58 5C
3.67(7C) 3.75
3.67
Figure 5 Two-dimensional contour plot of the correlation between ‘% and ‘H chemical shifts of the K92 polysaccharide, with assignments as marked, excluding C3(H2) and the N-AC methyl signals
16 Vaccine,
Vol. 5, March
1987
Meningococcal group B vaccine: M. R. Life/yet al.
larger than the monosaccharide polysaccharide2h
repeating
unit of the
Stability of the three-dimensional structure Since there are no a determinants in the B polysaccharide with a poor immune response, we have assumed that the three-dimensional structure is unstable, thus resulting in poor immunogenicity. A potential source of antigenic instability has been suggested for the B polysaccharide through the formation of internal esters or lactones under mild conditions, resulting in a considerable loss in antigenicityX4. This internal esterification in the highly water soluble B polysaccharide was observed because reaction of the polymer with a carbodiimide or with 48% HF resulted in a water insoluble product. Infrared spectra of the modified polymers showed a major band near 1750 cm-‘, consistent with ester formation (Figure 7b); this band virtually disappeared after mild alkali treatment (Figure 7a). More relevantly, esterification was shown to occur by incubating the B polysaccharide below pH 6 and infrared spectra showed that the degree of esterification increased as the pH was lowered. “C n.m.r. spectroscopy of the fully esterified polymer provided conclusive evidence for cross-linking between the carboxy1 group of one residue and HO-9 of an adjoining residue (Figure 8) to form a six-membered intramolecular ester or lactone ringZ4. Following the development of a gas-liquid chromatographic procedure” to give more sensitive and precise measurement of the degree of esterification of B polysaccharide, it was shown’” that the salt form of the polymer underwent no ester formation whereas about 80% esterification occurred in the free acid form. In contrast, the C polysaccharide, whether 0-acetylated or not, underwent no ester formation either in the salt or free acid form. Under more forcing conditions of activation of carboxyl groups with a carbodiimide, and in the absence of 0-acetyl groups, the C polysaccharide underwent partial internal esterification (about 50%) in contrast to the full esterification of the B polymer. “C n.m.r. spectroscopy showed that the activated carboxyl groups condense with an adjacent HO-8 group (Figure 8) to form a six-membered Intramolecular esterzjkation.
priori reasons to equate conformational
4.20
4.15
,.,o
4.05
3.95
4.00
3.90
3.85
3.80
3.75
PPM
Figure 6 360 MHz ‘H n.rn.r. spectrum of the K92 polysaccharide, excluding H3a and H3e from both residues of the repeating unit
smaller coupling, with second-order effects, to the resonance assigned to H-8(B). Analysis of the H-8(B) multiplet showed three couplings, viz J7.x (B), Jx.<)(B), and Jx,u8 (B), each of =4 Hz. In a decoupling experiment, irradiation of the H-8(B) resonance collapsed the adjacent H-9(B) resonance to a geminally coupled doublet, and perturbed the spectrum at 6 3.85 and 6 3.75. Using double-resonance difference spectroscopy, the resonance at 6 3.75 showed the geminal coupling constant, thus confirming it as that of H-9’(B). It follows that the resonance at 6 3.85 is due to H-7(B), which, in the decoupled spectrum, appears as a broad singlet, showing that J6,, (B) is
a
8
b
n
z, 8 9
Figure 7 Infrared spectrum of B polysaccharide before (a) and after (b) carbodiimide treatment
Vaccine,
Vol. 5, March
1987
17
Meningococcal group B vaccine: M. R. Life/y et
Figure 8
al
The mechanism of internal esterification of B polysaccharide (left) and (0-AC-)C polysaccharide (right)
internal ester ring analogous to that formed in the B polysaccharide. A good correlation was observed between the degree of esterification and the antigenicity of the B polysaccharide by countercurrent immunoelectrophoresis (CIE)“4. At pH 5.5, the Na+ salt of the polymer underwent 2-3% esterification and little reduction in antigenicity, whereas at pH 5 and -9% esterification the antigenicity was dramatically lowered (Table 4). These results emphasize the ease with which esterification can occur in the B polysaccharide, thus causing destabilization of the three-dimensional structure of the polymer and resulting in loss of antigenicity. The n.m.r. approach to molecular conformation has been coupled with theoretical molecular mechanics calculations in order to probe the conformations about chemical bonds for which no coupling constants are available and in order to explain the propensity of the (2-8)~a-linkages to undergo internal esterification2”. NeuNAc models were joined together to form disaccharides linked either (2-+8)-u- or (2&9)-a- using the graphics facilities of the Wellcome Molecular Modelling system. Atom centred partial atomic charges, calculated by the CNDO method were included and the geometry was fixed at the known crystal structure with the ring and side chain conformational information from n.m.r. as an additional constraint. The molecular mechanics energy calculations are based on variations in bond length, bond angle, torsion angle, van de Waals contacts and coulombic interactions and the minimum energy conformation obtained. The only unknowns remaining are the two torsional angles at the anomeric linkage and these were optimized to give minimum energy forms by sequential bond rotations. These final conformations are shown in Figure 9 for the B and C type linkages, respectively. Examination of these models shows that the (2-+8)-a-linkages impart a geometry in which the carboxyl group in the free acid form is perfectly arranged to undergo internal esterification
Table 4 Correlation between degree of esterification and the antigenicity of B polysaccharide
PH
(“/)
Minimal concentration giving positive band by C.I.E. (pg ml-‘)
3.0 4.0 5.0 5.5 6.0 Salt form
53 30 8.6 2.5
>I00 >I00 >I00 0.4 0.4 0.4
Incubation
18
Degree of esterification
Vaccine, Vol. 5, March 1987
Minimal concentration giving full inhibition by C;I.E. (pg ml-‘) ND ND 400 80 40 20
0
O‘i
\
0
0
Figure 9 Calculated conformations about the glycosidic linkage for NeuNAc disaccharides with a (2+8)-a-(left) and a (2+9)-u-(right) linkage. Derivation of the rest of the structures is described in the text
with the adjacent HO-9 group. Conversely, it can be seen that the relative disposition of the carboxyl group and the HO-8 in the (2-+9)-a-linkages is most unfavourable for internal esterification. A second source of antiTemperature dependence. genie instability of the three-dimensional structure of B polysaccharide has been s;Egested by the experiments of Mandrel1 and Zollinger- who observed a considerable decrease in avidity of group B specific antibodies with an increase in temperature from 4 to 37°C. whereas that of anti-C antibodies decreased only marginally. We interpret these results in terms of a loss of the three-dimensional structure and therefore of the specific determinants of the B polysaccharide with the increase in temperature, whereas the continuous determinants of the C polysaccharide remain unaffected. However, it must be stressed that there is no n.m.r. evidence for a temperature-dependent conformational change of the B polysaccharide. Neuraminidase sensitivity. The sensitivity of the B polysaccharide to neuraminidases may contribute to decrease immunogenicity further”‘. although there is admittedly little experimental evidence to prove or disprove this point. Unpublished experiments in our laboratory have indicated that the B polysaccharide (or colominic acid) is rapidly eliminated from liver and spleen after intravenous injection, in comparison with the elimination of B512 dextran of a similar molecular weight. Reluctance to accept the relevance of neurami-
Meningococcal group B vaccine: M. R. Life/y et al.
nidase degradation of B polysaccharide in decreasing irnmunogenicity has been due to the argument that (O-AC-)C polysaccharide, which is a good immunogen in adults, is also sensitive to neuraminidases and should therefore be degraded as rapidly as the B polysaccharide in viva. However, the difference in the antigenit stabilities of the two polysaccharides caused by their contrasting determinant specificities makes it probable that the epitopes on the B polysaccharide will be destroyed far more rapidly than those on the (O-AC-)C polysaccharide. Cross-reactive brain determinants Although there is a consensus of opinion that tolerance to the B polysaccharide in the host is due to crossreactive tissue components, there seems to be some confusion over which of these components are responsible. We, and others, believe that the immune response is suppressed most effectively when directed against the ubiquitous cell surface sialogangliosides and sialoproteins, containing only short chains (about 24 residues) of (2+8)-a-linked sialic acid residues identical to the repeating unit of the B polysaccharide; hence, the immune response is directed with some success against conformational determinants on the polysaccharide. Finne and co-workers37%38, however, have shown that a polysialoglycoprotein present in brain tissue, and referred to as N-CAM (neural cell adhesion molecule) protein”‘, contains long chains of (2-43)~a-linked sialic acid residues. This polysialoglycoprotein, found in developing fetal brain tissue, but declining rapidly after birth, was originally reported to cross-react with a horse anti-B serum”, a result which has been confirmed by us using mouse anti-B monoclonal antibodies4’. They have suggested that immunological tolerance to the B polysaccharide may be due to this cross-reactive brain antigen, and have urged caution in efforts to develop an effective vaccine containing the B polysaccharide component. They have argued the breakdown of natural tolerance caused by an ‘artificial’ vaccine might initiate an autoimmune process and might have adverse effects due to interference in the normal physiological function of the polysialoglycoproteins in brain tissue. They have also voiced their concern over immunotherapeutic approaches involving passive administration of antibodies against group B meningococci or E. coli Kl. This is a view which we (and others) do not share. Zollinger et al.“‘, have already stated their opposition to the likelihood of cross-reaction in vivo between anti-B polysaccharide antibodies and brain polysialoproteins; the vaccines they administered (with no ill-effects) were not artificial but were in a ‘natural’ hydrophobic complex with OMPs. Moreover, vaccine-induced antibodies to B polysaccharide or antibodies from convalescent patients, which were predominantly of the immunoglobulin M(IgM) class and of low avidity, appeared to be equivalent to the naturally occurring anti-B antibodies present in 80-90% of adults. Animals hyperimmunized with group B meningococci or with a B polysaccharideOMP complex showed no ill-effects, despite high circulating antibody levels. In addition, we have found4” that monoclonal antibodies against not only the B polysaccharide but also against the polysaccharide from another pathogenic serogroup (W135) of N. meningitidis bind to human fetal brain tissue. Yet the W13.5
polysaccharide is one component of a commercially available tetravalent vaccine which has proved to be safe and effective in children42 and adults4”. It has not been adequately explained how tolerance to cross-reactive brain polysialo roteins would operate in the adult. It has been argued’ Pthat the lack of an IgG anti-B response in experimental animals and humans is because the polysialoproteins would be readily accessible to IgG (but not IgM) antibodies with B specificity. Since, however, as Finne et al. 1’,37338report, the polysialoproteins decline rapidly after birth, then, unless similar molecules are present in other organs, the maturing immune system would not be in contact with such an antigen. Although IgG responses have been described as more susceptible to tolerance than their IgM counterparts, such a state would not be maintained without the persistence of the tolerogen. The immunological cross-reaction observed between B polysaccharide and the brain polysialoprotein does not necessarily imply any untoward pathological consequences, especially if the antigen is embryonic and/or separated from the immune system by the blood-brain barrier. Although molecular mimicry caused by sharing of antigens between parasite and host has been detected before, and potential immunological implications for the host described, there is very little experimental evidence that cross-reactive antibodies are harmfu121,4L47, and it is certainly within the bounds of reason to expect, with the increasingly sophisticated and sensitive techniques available, that many further examples of cross-reactions between the extremely complex and continually altering brain tissue components and foreign antigens will arise without adverse consequences for the host. However, by stating this we do not imply that such cross-reactivities should be ignored. On the contrary, it is our opinion that they should be the subject of much more research and careful monitoring.
Enhancement of immunogenicity of the B polysaccharide Introduction We interpret the poor immunogenicity of B polysaccharide as due to suppression of the immune response when directed against the ubiquitous short chains of (2+8)-u-linked sialic acid determinants, such as those present in sialogangliosides, and the response is therefore directed against conformational or discontinuous determinants present on the B polysaccharide. The polysaccharide adopts a preferred three-dimensional structure in solution which facilitates internal esterification, thereby radically altering the antigenic structure of the native molecule, and the sensitivity of the polymer to neuraminidases contributes to decrease immunogenicity further. As a consequence, purified B polysaccharide does not induce specific antibodies’, and the response against whole bacteria, or the polysaccharide in a complexed form, is relatively short lived and consists almost exclusively of IgM antibodies4s.4y. These factors may also explain the failure of previous attempts to raise an immune response with (2+8)-alinked sialic acid oligosaccharides covalently coupled to a tetanus toxoid Carrie?‘. The stabilization of the three-dimensional structure, held together in a rather Vaccine, Vol. 5, March 1987
19
Meningococcal group B vaccine: M. 17.Life/y
et al.
precarious way, seems to be a basic requirement therefore for immunogenicity of the B polysaccharide and this has been at the core of our efforts to manufacture an effective vaccine against group B disease. B polysaccharide-outer membrane protein (OMP) complex Noncovalent B polysaccharide complexes with outer membrane proteins (OMPs) from N. meningitidis have been prepared in several laboratories. Zollinger et al.4X studied the effect in a small group of human volunteers of two vaccine preparations both containing B polysaccharide complexed to type 2 OMPs. One vaccine was a partially purified B polysaccharide, which had a sialic acid:protein ratio of 1.6. The second vaccine preparation was a combination of a partially purified B polysaccharide mixed with an outer membrane complex of protein and lipopolysaccharide (LPS); the mixture was subsequently processed to remove LPS and the final vaccine had a sialic acid:protein ratio of 1.1. Both vaccine lots gave encouraging results in that an enhancement of immunogenicity of the B polysaccharide component was observed, even though the antibodies were restricted mostly to the IgM class and were relatively short lived. Raised antibody titres to the type 2 OMP were also observed4s. The results support the view that the three-dimensional structure of the B polysaccharide can, to some extent, be stabilized by maintaining the polysaccharide in a complex with OMPs. Frasch and co-workers”’ prepared outer membrane complexes of serotype 2 proteins and LPS, which were subsequently treated with Brij-96, a nonionic detergent, to selectively remove the LPS. The resulting serotype 2 protein vaccine was soluble and relatively more immunogenic in mice” than previously prepared particulate OMP vaccines”. The immunogenicity of the serotype 2 protein vaccine could be further improved by mixing with an e ual weight of the purified B or C polysaccharides2~’ 7. The disadvantages of such a vaccine are threefold: (i) the LPS content was relatively high (=10%)5’.5s; (ii) most of the B polysaccharide (~80%) did not complex with the protein”‘, and it was not surprising, therefore, that anti-B responses in mice were very low”‘; and (iii) there are many antigenically distinct OMPs within group B meningococci2, whereas the B polysaccharide is always present. A vaccine based on OMPs would require multiple serotype roteins for complete protection. More recently 49 , we developed a methodology for preparing the B polysaccharide complexed to type 2 or
type 6 OMPs, which had several advantages: (i) they were naturally formed complexes prepared in high yield directly from culture supernatants by a mild method; (ii) they were low in nucleic acid (<2%) and lipopolysaccharide (<0.5%) content; and (iii) the B polysaccharide and protein components were exclusively associated in a high molecular weight (>2 x 10’) complex held together by hydrophobic interactions, which probably occur through binding of hydrophobic regions on the protein with a phospholipid moietysh covalently attached to the reducing end residue of the B polysaccharide chains. Using the methods of chain length analysis’s discussed earlier in this review, this lipid moiety was shown to be present on all of the B polysaccharide chains, which were estimated to have an average length of ~200 residues. Immunization of mice with a B polysaccharide-type 6 OMP complex resulted in a mean 3 to 4-fold rise in anti-B titres4” which were mostly of the IgM class (Figure 10). In most experiments, however, the anti-B response declined rapidly after the first week and was barely detectable by day 21. Immunized mice were, however, protected against challenge with group B N. meningitidis strains of the same (type 6) or of a different serotype (type 2a) from that used for immunization (Table 5) when the immunizing dose was 1000 ng per
days 7
21 10wlmoure
7
14
21
,
14
7
1vglmoure
21
O.l~glmoura
Figure 10 Anti-B responses (solid-phase radioimmunoassay) of CBA mice immunized with 10, 1, or 0.1 pg of group B polysaccharide-type 6 OMP complex given intraperitoneally (0) or subcutaneously (E4). The values obtained with non-immunized mice are indicated to the left of the graph. Results are expressed as the geometric mean of five mice + standard errors
Table 5 Protection of mice from challenge with N. meningitidis after primary and secondary immunization with a B polysaccharide-type and purified B polysaccharide Death/total after challenge’with Primary immunizationa
Secondary immunizatior?
7619
1000 ng complex 100 ng complex 100 ng complex 1000 ng B polysaccharide 100 ng B polysaccharide None
None None 1 .ng complex None None None
118d 518e 118d 818 818 819
Ww
B, twe2)
6 OMP complex strain 7622 kvoupB,twe 6) 118’ 418e ND 818 719 719
aPrimary immunization was given intraperitoneally on day 0. bSecondary immunization was given intraperitoneally on day 10. ‘Challenge with IO4 CFU of the corresponding strain, in combination with iron dextran. Data are recorded as deaths per total 72 h after challenge and 21 days after immunization. “x2 shows significance at p < 0.01. eNot significant: p > 0.1
20
Vaccine, Vol. 5, March 1987
Meningococcal group B vaccine: M. R. Life/y et
mouse, although no significant protection was afforded with an immunizing dose of 100 ng per mouse. However, primary immunization with 100 ng followed by secondary immunization with as little as 1 ng complex was sufficient to give almost complete protection (Table 5). No protection was observed following immunization with the purified B polysaccharide. The results suggest, firstly, that the anti-B antibodies are responsible for prevention of death in mice, since challenge with either the homologous or a heterologous strain of group B meningococci resulted in clear protection, and, secondly, priming with the complex leads to the buildup of memory cells although the secondary anti-B response, similar to the primary response, contains mostly IgM and very little IgG antibody. As mentioned above, immunization of mice with the purified B polysaccharide resulted in a lack of protection. Although the classification of the polysaccharide as a poor immunogen is well documented, this designation must be qualified by defining the term immunogen. Immunogenicity is most commonly defined only as implying the induction of circulating antibodies; however, we have shown that the purified B polysaccharide is immunogenic if the term encompasses the induction of immune memory. We found that neither primary nor secondary immunization of mice with the purified B polysaccharide resulted in a significant antibody response. However, when primary immunization with B polysaccharide was followed one month later by a second immunization with 10s formalin-fixed group B meningococci, a clear dose-dependent secondary response was obtained with an optimum at 0.1 pg of B polysaccharide per mouse 4y. Therefore, although B polysaccharide is non-immunogenic in the sense that it does not induce humoral responses, it is perfectly capable of inducing immune memory. Stabilization with aluminium ions One of the factors suggested in this review to be responsible for the poor immunogenicity of the B polysaccharide is the intrinsic instability of the polymer due to its tendency to undergo internal esterification under mildly acidic conditions”4, thus resulting in the loss of conformational or discontinuous antigenic determinants that are characteristic of the native polysaccharide. Neuraminidase sensitivity has also been implicated in the oor immunogenicity of the polymer12. We have shown P’ that the Ca2+ salt of B polysaccharide undergoes internal esterification at a slower rate than the Naf salt, a fact that has been ascribed to coordination of Ca2+ ions to the HO-9 of sialic acid residues in the polymer. Because intramolecular esterification occurs in B polysaccharide through condensation of -CO?H and HO-9 groups of adjacent residues”4, cations, binding principally to the -C02H group, but
Table 6
1:0.2 1:0.3
also by coordination of -OH groups, were therefore of potential use in inhibiting the rate of ester formation in the polymer. This rationale was used to select cations to examine: (i) their ability to complex with the B polysaccharide; (ii) their effect on esterification and neuraminidase susceptibility of the B polysaccharide; (iii) their effect on stabilization of the main epitope(s) of the B polysaccharide; and (iv) the resultant immunogenicity of the polysaccharide. Evidently, each of these possibilities is meaningful only when the previous ones have been answered in the affirmative. Binding occurred spontaneously between B polysaccharide and A13+ ions”, either by equilibrium dialysis or by direct incubation. It seemed clear that sequestration of Al”+ by the polysaccharide occurred in preference to Ca*+ binding and that this complex was dissociated by neither ethanol precipitation nor gel filtration. The nature of the binding is still unclear, but nuclear magnetic resonance data, although preliminary, suggest that, apart from the obvious interaction with the carboxylic groups, additional interactions take place involving -OH groups at C-95x. Binding between A13+ and B polysaccharide resulted in a clear resistance to neuraminidase digestion (Figure 21). Moreover, at acid pH, Al”+ forms of B polysaccharide, as compared with the Ca2+ and Na+ salts, showed markedly reduced rates of esterification (Table 6). This, in itself, resulted in a persistent antigenicity of B polysaccharide measured both by immunoprecipitation (counterimmunoelectrophoresis) and by radioimmunoassay”‘. Since the antigenic stability was enhanced through complexation with Al 3+ ions, the effect of Al”+ complexes of the B polysaccharide on the immune response in mice was tested. The results were disappointing with no significant increase in the anti-B titres when compared with the preimmunization levels or with antibody levels in mice immunized with the Ca2+ salt of the B polysaccharide. As already mentioned in this section,
60
60 TIME
100
120
140
OF INCUBATION
160
160
200
220
(hl
Figure 11 Rate of release of sialic acid by neuraminidase digestion of B polysaccharide complexed to Al ‘+ ions; the polymers had molar ratios of sialic acid/A13’ of I:0 (U), 1:0.14 (0) and 1:0.31(A)
Effect of A13+ binding on esterification in B polysaccharide at low pH
Molar ratio of sialic acid/A13+ in B polysaccharide 1 :o 1 :o.i
al.
Esterification (%) after incubation for 24 h with molar ratio of sialic acid/H+ of 1:0.2
1:0.4
1:o.a
12.9 3.6 <0.5 <0.5
27.1 15.0 7.4 2.3
48.9 41.0 27.7 15.6
Vaccine,
Vol. 5, March
1987
21
Meningococcal group B vaccine: M. R. Life/y et al.
the lack of antibody production does not necessarily reflect a lack of effective interaction with the immune system, since immunization with purified B polysaccharide leads to immune memory. The interaction of native B polysaccharide with the immune system was apparently transient and of an abortive nature, and complexing with Al”+ did not alter this situation. However, since B polysaccharide is known to be a far better immunogen when associated with meningococcal outer membrane proteins, the effect of binding of Al”+ ions to a B polysaccharide-type 6 OMP complex on the immune response in mice was tested”‘. Animals were immunized intraperitoneally with 10 ug doses of the group B-type 6 protein complex given with either 10-s M Alz(SO&, with 100 ug AI(O or with no addition of Al’+ ions. The anti-B response examined seven days later (Table 7) clearly showed a 2 to 3-fold increase when A12(S0 4)_Twas added, without alteration of the anti-OMP titres, and almost a lo-fold increase with Al(OH)3, both results being highly significant statistically. This potentiation was not apparent when the same experiment was performed with a meningococcal group C polysaccharide-OMP complex administered in identical fashion to the group B complex. These results were interpreted in terms of stabilization of B polysaccharide in its native form rather than an adjuvant effect, based on the following observations: (i) administration of protein-B polysaccharide complexes in alhydrogel (as an adjuvant) boosted both anti-B and antiprotein responses, whereas Al’+ as a salt stimulated the production of anti-B antibodies alone; and (ii) Al”+ forms of protein-C polysaccharide complexes did not show this enhancing effect. Why is it, then, that purified B polysaccharide, as the Al”+ salt, did not seem to stimulate antibody production? The answer to this question probably lies in the way phagocytic cells handle, process, and present antigens to B cells. The rapid clearance of the B polysaccharide from liver and spleen following intravenous injection suggests that this relatively short-lived antigen can stimulate the production of memory cells but not antibody, which requires B polysaccharide stabilization to preserve the conformational determinants and, possibly, B and T cell proliferation factors together with T helper function. Probably none of these activities are stimulated by polysaccharide alone, but protein-polysaccharide complexes do so. Al”+ is not the only cation capable of enhancing the anti-B response in protein-B polysaccharide complexes. Results obtained in our laboratory with ruthenium red as a complexing agent have indicated similar chemical stability and antigenic stimulation. It cannot be established at the moment whether reduction of Table 7
Immunization of CBA mice with a B polysaccharid&ype
internal esterification or increased resistance to neuraminidases is more important in promoting the immunogenicity of B polysaccharide, and, although we favour the former as being the most significant, it is possible that both phenomena play a role. Whatever the explanation may be, it is important that these findings suggest the interesting possibility of using Al”+ to enhance the immunogenicity of N. meningitidis group B vaccines for immunization of humans.
Immunobiology of the B polysaccharide response Generally, the immune response to purified polysaccharides is thymus-independent with production of IgM antibodies, and no enhancement of the response is observed upon subsequent immunizations. There are exceptions to these rules, however, since a booster response can be induced in young children to the meningococcal A polysaccharide”“, and high levels of IgG antibodies can be induced in mice with the group C polysaccharide6’. We must bear in mind, however, that unlike many thymus-independent antigens that persist for a very long time in the lymphoid organs, A polysaccharide (and B polysaccharide as well) is unstable and degrades relatively fast in viva. The induction of an immune response to polysaccharides can be enhanced by injecting the polysaccharide (or oligosaccharide thereof) coupled, either covalently or non-covalently, to protein carrier@‘. This usually results in a shift from a thymus-independent to a thymus-dependent antigen, a more robust response than that of the polysaccharide alone, and the induction of IgG antibodies and memory cells, as demonstrated, amongst others. for the group C polysaccharide”‘,“2,6’. The unique immunological characteristics of B polysaccharide, however, make unsafe any prediction of its behaviour deduced from the properties of other similar polysaccharides. In fact, coupling (2-S)u-linked sialic acid oligosaccharides (from B polysaccharide) to protein carriers proved unsuccessful”“. For this reason, we have studied in detail the immunobiology of the B polysaccharide response in mice. Athymic mice were shown@ to be capable of producing IgM antibodies to the B polysaccharide when immunized with the polysaccharide complexed with type 6 OMPs, whereas no immunogenicity could be demonstrated with the purified polysaccharide. When athymic mice were given T cells obtained from the spleen of normal mice, the primary, anti-B immune response to the B polysaccharide-type 6 OMP complex in Al(OH)3 (given five days after transfer) was low and comparable to a control group of athymic mice receiving no cells. However, when the animals were reimmunized in the same way 15 days later, the anti-B
6 OMP complex administered in combiration with Al&SO&
Group
Antigen at day Oa
Additive to antigen
Anti-B response at day 7b
1 2 3 4 5 6
+ -
None None 1O-3 M AIP(SO& 1O-3 M AIZ(SO& 100 pg AI 100 pg AI(
1.6(1.38) 0.49 (1.04) 3.92 (1.16) 0.65 (1.20) 14.7 (1.83) 0.46 (1.04)
+ + -
or AI(
p valueC
<0.025
Ten trg polysaccharide-type 6 OMP complex given intraperitoneally in 5% lactoseO.01 M sodium phosphate buffer pH 7.3 (0.5 ml). Control mice (groups 2,4 and 6) received lactose-phosphate buffer pH 7.3 (0.5 ml). ‘Geometric average of five mice per group expressed in pg lgiml serum, with standard error in parentheses. %alculated by the student’s 1test when comparing groups 3 and 5 with group 1
22
Vaccine,
Vol. 5, March
1987
Meningococcal group B vaccine: M. R. Life/y Table 8 Effect of T-cell transfer upon the anti-B response of BALB/c nu/nu mice immunized with a B polysaccharide-type 6 OMP complex Anti B titrea Day
Action
Group 1
Group 2
-5
Cell transfer 2.3 x10* Balblc T cells
+
-
0.1 (1.01)
0.13 (1.04)
d.46 (1.19) 0.32 (1.09)
d.,, (1.15) 0.35 (1.27)
8f.03 (1.64) 1.23 (1.58)
0+.56(1.52) 0.55 (1.53)
-1 0 +7 +14 +15 +22 +29
Primary vaccination*
Secondary vaccination b
aGeometric average of five mice per group expressed in pg Ig/ml serum, with standard error in parenthesis. ‘7en Kg B polysaccharide-type 6 OMP complex + 100 ttg AI given intraperitoneally in PBS (0.2 ml)
titre at day 22 was 14 times higher than in control mice (Table 8). These results demonstrate clearly that a primary immune response to the B polysaccharidetype 6 OMP complex is thymus-independent; this in itself is rather surprising, since the presence of large polysaccharide chains presented in combination with protein carriers should be able to stimulate both thymus-independent and thymus-dependent responses. Secondary responses are quite different, however, and here there is a substantial shift towards a thymusdependent component. In order to clarify whether the secondary response depended on the accumulation of T and/or B memory cells, lethally irradiated mice were injected with either immune or non-immune T-cells or B-cells. Both donors and recipients were immunized with the B polysaccharide-type 6 OMP complex given with a purified meningococcal group Y polysaccharide (control, thymus-independent antigen) and Al(O The response to B, Y and type 6 OMP was measured 7 days after immunization. The results can be summarized as follows: (i) Transfer of immune T cells (but not immune B cells) resulted in an enhancement of the response for both B polysaccharide and type 6 OMP. (ii) Transfer of immune as compared to non-immune cells resulted in a slight reduction in the response to Y polysaccharide. These experiments demonstrate the presence of memory T cells for both B polysaccharide and type 6 OMP, but no memory B cells could be found. The truly thymus-independent antigen, Y polysaccharide, performed as expected with no build-up of memory. This series of experiments also demonstrated the failure of T-suppressor cells to explain the poor immune response to the purified B polysaccharide since: (i) athymic mice respond poorly to the purified polysaccharide; (ii) addition of normal spleen cells to athymic mice does not decrease the response to the polysaccharide-OMP complex; and (iii) cyclophosphamide treatment of normal mice does not result in an increased anti-B response. Bearing in mind that the epitope on B polysaccharide is most probably conformational and very likely to be chemically unstable, one would tend to regard the response to B polysaccharide as ‘abortive’, in the sense that it is triggered by the antigen only temporarily without reaching the period when it becomes antigen
et al.
independent, i.e. a plasma cell. This could be the reason why the B lymphocytes (specific for this antigen) undergo antigen-driven mitogenic cycles and only partial differentiation, without ever reaching full maturation to produce IgG antibodies or to maintain a sustained production of IgM. Therefore, modifications that stabilize the antigen, like aluminium salts of the polysaccharide, may improve its immunogenicity without changing completely its ‘abortive’ nature. Also it could explain why anti-B antibodies are of low affinity. This is probably the reason why the secondary response of mice, although substantially more robust than the primary one, does not result in a shift towards the production of IgG antibodies specific for B polysaccharide. It is unlikely, however, that a situation such as restricted IgM production is going to apply rigidly to every other strain of mice or other animal species, since it has been reportedh5 that NZB mice produce IgG antiB antibodies and other murine strains, as well as New Zealand albino rabbits, have been found to produce IgG antibodies when immunized with the B polysaccharide-type 6 OMP complex in our laboratories. Two injections of the same vaccine given two to three weeks apart resulted in a substantial increase of the anti-B and anti-OMP antibodies, and to a lesser extent this was also observed when a different serotype was used for the second immunization”4. These facts ought to be considered if planning immunization strategies using similar vaccines in human volunteers. Conclusions In this article, we have attempted to show that an effective vaccine requires stabilization of the threedimensional structure of the B polysaccharide, which our proposed vaccine seems to be able to do to an extent, partly through association with OMP proteins49, and partly through interactions with Al”+ ions5’. The poor immunogenicity of the B polysaccharide has been studied at a molecular level through the use of n.m.r. spectroscopy25; with the aid of molecular modelling using computer graphics, this has led to a detailed understanding of one of the underlying mechanisms involved in destabilization of the three-dimensional structure of the polysaccharide, namely, esterification’5,‘4. Although studies of the secondary and tertiary structure of proteins have been conducted*’ and the relationship to immunogenicity delineated, this is the first report, to our knowledge, of such an undertaking with a polysaccharide. It must be emphasized, however, that this study is, as yet, incomplete, since there are questions which remain unanswered. What, for instance, is the complete three-dimensional structure of B polysaccharide? Where is the location and what is the nature of the conformational or discontinuous determinant(s) on the polymer? Can the determinant(s) be stabilized sufficiently to give a longer lasting immune response, and can a shift from IgM to IgG antibodies be induced? Evidently, these questions are interrelated and, in attempts to give answers to them, results using one approach will inevitably provide information which may be used to confirm or modify results from another. It is also evident that in answering these, further questions are posed: Do conformational/ discontinuous epitopes occur on other polysaccharides? Can these epitopes be stabilized to allow an immune Vaccine,
Vol. 5, March
1987
23
Meningococcal group B vaccine: M. R. Life/y et al.
response to be mounted? Perhaps mimicking of unstable epitopes by designing synthetic compounds of carbohydrate or non-carbohydrate origin may be the basis of future vaccines. An obvious deterrent to the study of polysaccharide three-dimensional structures has been the need to develop suitable techniques. The breathtaking speed with which n.m.r. spectroscopy has advanced in recent years in structural and conformational studies of carbohydrates, however, has made this technique invaluable for future studies of carbohydrate conformation in relation to immunogenicity. Continuous determinants have been successfully delineated on a number of polysaccharides’7,‘“.66,67 through the use of monoclonal or polyclonal antibodies and specific carbohydrate inhibitors, which mimic the primary structure and are generally smaller than the hexasaccharide. However, this approach cannot be applied to discontinuous determinants on the polymer with any confidence, precisely because the carbohydrate inhibitors cannot be readily predicted. The determination of polysaccharide secondary and tertiary structures through the use of n.m.r. and other spectroscopies, X-ray diffraction and theoretical predictions will undoubtably aid in this respect. At present, it is difficult to speculate on the methods which may be used to stabilize particular discontinuous determinants, although these should become more evident as information on the determinants is generated. In this article, for example, the meningococcal group B polysaccharide has been shown to have discontinuous determinants which are destabilized by internal esterification involving -CO?H and HO-9 groups of adjoining residues (see Figure 8). It was logical to predict that blocking of either or both of these groups would inhibit ester formation and therefore stabilize the determinants on the polymers. It is hoped that in the next few years, we will see more examples of discontinuous determinants on oligoand polysaccharides. This, in turn, will give impetus to the question of the relationship between discontinuous determinants and their immunology and, finally, to the stabilization or synthesis of these determinants as potential immunogens. The schematic diagram in Figure 12 illustrates many of the factors involved in the immune response to the meningococcal group B polysaccharide. The polymer may be degraded by neuraminidases or antigenically modified by esterification under mild conditions. Continuous determinants, i.e. short chains of (2+8)-w-linked sialic acids, within the polysaccharide do not appear to be recognized as ‘foreign’ by the immune system due to the ubiquitous presence of sialogangliosides and sialoproteins on cell surfaces of the host, bearing identical sialic acid structures. Therefore, discontinuous determinants, held together in a precarious way, on the three-dimensional structure of the polysaccharide are important for immunogenicity and must be stabilized in order for the B polysaccharide to interact effectively with the immune system; this achieved, an anti-B response is mounted. It has been speculated that anti-B antibodies, which cross-react with polysialoglycoproteins (N-CAM proteins) found in fetal and neonatal brain tissue, may initiate an autoimmune process’ ‘. It is highly unlikely, however, that interaction of N-CAM proteins with the immune system occurs (discussed earlier in this review), and we do not regard this as a
24
Vaccine,
Vol. 5, March
1987
Figure 12 A schematic diagram of factors involved in the immune response to B polysaccharide, as explained in the text
serious drawback in efforts to manufacture an effective vaccine containing the group B polysaccharide. However, we must stress that careful monitoring of any new vaccine is essential, and to ignore this cross-reactivity would be irresponsible. We believe that the potential cross-reactivity of foreign antigens with brain components, particularly at the early developmental stage. should be the subject of further close examination and research. Immunization of mice with a meningococcal group B polysaccharide-type 6 OMP complex given with Al(OH)3 resulted in an increased anti-B response5’. Memory T-cells were stimulated and. on secondary immunization with the same vaccine, a boosted response to both B polysaccharide and type 6 OMP components of the vaccine was observed’“. This secondary response did not require OMP serotypes to be the same for primary and secondary immunization, suggesting that the potential protective value of T-helper cells induced could be maintained even when the vaccine and field strains do not share the same serotype. Moreover, a secondary anti-type 6 OMP response could be induced when mice. primed with the B polysaccharide-type 6 OMP complex. were challenged with a B polysaccharide-type 2a OMP complexh4. Although small (16%) in comparison with the homologous challenge, it was highly significant 0, < 0.0005 for the student’s f-test) with a 2 to 3-fold antibody increase over non-immunized controls. Our chemical, molecular and immunological studies have led us to propose reasons for the poor immunogenicity of B polysaccharide and from this has emerged a candidate vaccine for use in humans. Volunteer studies are now in progress.
Acknowledgements Many of our colleagues at the Wellcome Research Laboratories have made significant contributions to this endeavour and we gratefully acknowledge their invaluable help. In particular we thank Mrs J. Esdaile, Miss U.T. Nowicka (immunology), Mr D.C. Brown (bacteriology), Dr A.S. Gilbert (IR). Dr R.C. Glen, Dr
Meningococcal group 8 vaccine: M. R. Life/y et
J.G. Vinter (molecular modelling) and Mrs J.M. Williams (n.m.r.). We also thank Dr A.J. Beale for his encouragement and support. 22
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