Capsular Polysaccharides as Human Vaccines

.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY VOL. 41

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES BY HAROLDJ . JENNINGS Division of Biological Sciences. National Research Council of Canada. Ottawa. Ontario KIA OR6. Canada

I . Introduction ............................. . . . . . . . . . . . . . . . . .155 I1. Structures of Capsular Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . .158 1. Neisseria meningitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 2 . Haemophilus influenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 167 3 . Group B Streptococcus ....................................... 4 . Streptococcus pneumoniae .................................... 170 111. Other Important Structural and Physical Features of Capsular Polysaccharides ...................................... 174 . . . . . . . . . . . . 174 1. Structural Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . .175 2. Determinants and Immunological Specifi 3. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 4 . Molecular Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 5 . Location .................................................. 183 IV . Immune Response to Bacterial Infection ............................ 186 1. Phagocytosis ............................................... 187 2 . Role of Complement ......................................... 187 3 . Humoral Antibodies to Polysaccharide Vaccines .................... 189 V. Polysaccharide Vaccines and Immunity ............................. 191 1. Streptococcus pneumoniae .................................... 191 ............................ 193 2 . Neisseria meningitidis . . . . . . . 3 . Haemophilus injluenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 4 . Group B Streptococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 197 5 . Polysaccharide-Protein Conjugates ............................. 6 . Natural Immunity, and Polysaccharide Serological Cross-reactions . . . . . .200 VI . Bacterial Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 1. Role of the Capsular Polysaccharide ............................. 202 2. Polysaccharide Structure and Pathogenicity ....................... 206

I . INTRODUCTION Vaccination has proved to be one of the most useful scientific developments in the control and eradication of human disease . Early vaccines were based on whole-organism preparations. or on protein toxins isolated from different bacteria and. although these methods 155

Copynght @ 1983 by Academic Press. Inc. All nghts of reproductlon in any form reserved. ISBN 0-12-007241-6

156

HAROLD J. JENNINGS

are still effective, the discovery of a “specific soluble substance” secreted by pneumococcal organisms during growth,’ and the immunogenicity of these substances (capsular polysaccharides),2 opened the door to a new and important development in vaccine technology. In 1923, Heidelberger and Avery3 demonstrated that this substance was, in fact, a type-specific, polysaccharide antigen that was able to precipitate: quantitatively, antibodies produced in animals by injection of the homologous, whole organisms. Heidelberger5 has given an interesting account of these significant, early discoveries of the immunogenicity of polysaccharides. In subsequent, pioneering work, he and his associates6 demonstrated that, when used as human vaccines, these purified, pneumococcal polysaccharides provide type-specific protection against pneumococcal infections. However, at that critical stage of development, the phenomenal success of the newly discovered antibiotics in treating bacterial diseases overshadowed the early promise of polysaccharide vaccines. Since then, the prophylaxis of bacterial disease has been the subject of renewed, intensified research,’ due in large part to the expanding incidence of antibiotic-resistant, bacterial strains6 Also, clinical and epidemiological studies have demonstrated that the antibiotic treatment of infectious diseases caused by encapsulated bacteria does not always prevent their morbidity and m ~ r t a l i t y Thus, .~ “cured” H. inJuenzue type b rneningitidis is the leading cause of acquired mental retardation,9 and epidemiological statistics indicate that deaths due to pneumococcal pneumonia occur at the same rate as in the pre-antibiotic era.’oCurrent interest in the capsular polysaccharides has evolved simultaneously with this resurgence of interest in the prophylaxis of human, bacterial disease, because of their potential as good immunogens in providing protection against bacterial infections. The concept of using a purified polysaccharide immunogen devoid of its accom(1) A. R. Dochez and 0. T. Avery, J . E r p . Med., 26 (1917)477-493. (2) T. Francis, Jr., and W. S. Tillet,]. E r p . Med., 52 (1930) 573-585. (3) M. Heidelberger and 0. T. Avery,]. E x p . Med., 38 (1923) 73-79. (4) M. Heidelberger and F. E. Kendall,]. E x p . Med., 61 (1935) 563-591. (5) M.Heidelberger, Annu. Reo. Microhiol., 31 (1977) 1-12. (6) C. M . McLeod, R. G. Hodges, M. Heidelberger, and W. G. Bernhard,J. E x p . Med., 82 (1945) 445-465. (7) J. B. Rohbins, Zmmunochemistry, 15 (1978) 839-854. (8) M. Finland, Rec;. Infect. Dis., 1 (1979) 4-21. (9) H. W. Sell, R. E. Merril, 0. E. Doyne, and E. P. Zimsky, Pediatrics, 49 (1972)206211. (10) R. Austrian, in R. F. Beers, Jr., and E. G. Bassett (Eds.),The Role of Immunological Factors in Infectious, Allergic and Autoimmune Processes, Raven Press, New York, 1976, pp. 79-89.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

157

panying, complex, bacterial mass is technically elegant. Besides their demonstrated immunogenicity in man, these materials are nontoxic, thus avoiding unpleasant (for example, pyrogenic) and, possibly, other deleterious effects associated with whole-cell vaccines. Another important feature of these purified, polysaccharide immunogens is that they can be chemically and physically defined: criteria that add a greater measure of control over their efficacy as vaccines than can be attained by using whole-cell vaccines. As a measure of the success of these vaccines, up to 1978, 130 million individuals had been immunized with capsular polysaccharides, resulting in a high degree of protection and no fatalities or significant adverse effects.' The purpose of this Chapter is to outline the development of bacterial-polysaccharide vaccines, and also to relate the structures of these capsular polysaccharides to their many roles in the immune response to bacterial infection. Because bacterial disease is host-related, this article will be concerned only with bacterial polysaccharides associated with human disease, particularly those capsular polysaccharides currently being used as human vaccines, or those having some immediate potential as human vaccines. The requirements that mediate this decision include clinical importance, the presence of meaningful epidemiological studies, and the identification of a stable, polysaccharide immunogen. Although Klebsiella pneumoniae" and Staphylococcus aureus'* possess defined, capsular polysaccharides, they have not yet satisfied the first two requirements, and will thus be referred to only briefly. The genus Klebsiella is largely restricted to hospital infections, and, because it has 72 serotypes, more-comprehensive epidemiological studies will be needed before the design of a capsularpolysaccharide vaccine is p ~ s s i b l e . ' ~ In a number of pathogenic bacteria (for example, Salmonella and Shigella), the capsular polysaccharide is replaced by the 0-chain polysaccharide of their lipopolysaccharides. Although these 0-chains have been demonstrated to be the immunological equivalent of the capsular poly~accharides,~~ they will not be covered in this Chapter, because they are unique, in that they can only be isolated from bacteria in their high-molecular-weight form, attached to a highly toxic, and physiologically active, lipid A moiety. This circumstance has generally discouraged the development of lipopolysaccharide vaccines,

-

(11) W. Nimmich, Z. Med. Mikrobiol. Zmmunol., 154 (1968) 117-131. (12) W. W. Karakawa and A. J. Kane,]. Zmmunol., 115 (1975) 564-568. (13) J. Z. Montgomerie, Reo. Infect. Dis., 1 (1979) 736-748. (14) 0.Liideritz, 0.Westphal, A. M. Staub, and H. Nikaido, in G . Weinbaum, S. Kadis, and S. J. Ajl (Eds.),Microbial Torins, Vol. IV, Academic Press, New York, 1971, pp. 145-233.

158

HAROLD J. JENNINGS

as the removal of lipid A results in the non-immunogenicity of the resultant O-chain. However, the conjugation of these 0-chains to nontoxic, protein carriers has obvious significance in the future development of 0-chain A previous review of microbial p~lysaccharides'~ has been updated by reviews on the structure'8 and immunological r e ~ p o n s e of ' ~ polysaccharides; a pertinent and comprehensive review of vaccines for the prevention of encapsulated bacterial diseases has also been published.' The term polysaccharide has been used throughout this Chapter, although, strictly speaking, some of the phosphorylated, capsular antigens bear a close, structural resemblance to teichoic acids.

11. STRUCTURESOF CAPSULARPOLYSACCHARIDES I. Neisseria meningitidis Neisserin meningitidis is a Gram-negative organism that has been classified serologically20into groups A, B, C, 29-e, W-135,X, Y, and Z. This grouping system depends on the presence of capsular polysaccharides that, although identified some time ago in the case of groups A (Ref. 21) and C (Ref. 22), were not compositionally defined until later.'""'" In these studies, it was established that the group A polysaccharide is a partially 0-acetylated, (1+6)-linked homopolymer of 2acetamido-2-deoxy-D-mannopyranosyl and that groups B and C pol ysaccharides are homopolymers of sialic

(15)S. R. Svenson and .4. A. Lindberg,]. Immunol., 120 (1978)1750-1757. (16)H. J. Jorbeck,S. B. Svenson, and A. A. Lindberg, Infect. Immun., 32 (1981)497-

502. (17)K. Jann and 0. Westphal, in M. Sela (Ed.), The Antigens, Vol. 111, Academic Press, New York, 1975,pp. 1-125. (18) L. Kenne and B. Lindberg, in G. 0. Aspinall (Ed.), The Polysaccharides, Vol. 2 , Academic Press, New York, in press. (19)C. T.Bishop and H. J. Jennings, in Ref. 18,Vol. 1, 1982,pp. 291-330. (20) E. C . Gotschlich, T.-Y. Lui, and M. S. Artenstein,J. Exp. Med., 129 (1969)13491365. (21) E. A. Kabat, H. Kaiser, and H. Sikorski,]. E x p . Med., 80 (1944)299-307. (22)R. G. Watson, G. V. Marinetti, and H. W. Scherp,]. Immunol., 81 (1958) 337-344. (23)T.-Y. Lui, E. C. Gotschlich, E. K. Jonssen, and J. R. Wysocki,]. B i d . Chem., 246 (1971)2849-2858. (24) T.-Y. Lui, E. C. Gotschlich, F. T. Dunne, and E. K. Jonssen,]. Biol. Chem., 246 (1971)4703-4712.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

159

3-Deoxy-Dmanno-2-octu~osonicacid (KDO) has also been identified as a component of the group 29-e polysaccharidez5Sz6 and the Escherichia coli K6 capsular poly~accharide.~~ Because of the presence of complex, 3-deoxy-2-glyculosonic acid components and phosphoric diester linkages in these polysaccharides, plus their great fragility, particularly to acid treatment, problems were encountered in the application of more-conventional, chemical techniques to their structural elucidation. This situation prompted a search for new techniques with which to tackle these problems, and one that has been used with great success is l3C-nuclear magnetic resonance (l3C-n.m.r.) spectroscopy. The potential of this technique as applied to polysaccharides had been demonstrated in studies on amyloseZ8and the more-complex polysaccharide heparin.29Subsequent studies on the polysaccharides of N. meningitidis and other bacterial polysaccharides (see later) served to consolidate the method as a powerful technique in structural and conformational investigations of polysaccharides. Since the earlier studies, reports of the application of this technique to other bacterial polysaccharides, and to polysaccharides in general, have been prodigious; these, beyond the scope of this article, have been discussed in a previous Volume of this Series.3oMore-pertinent reviews on the application of 13C-n.m.r. spectroscopy to polysaccharides of human pathogenic bacteria have also been p u b l i ~ h e d . ~ ~ * ~ ~ The structures of the repeating units of these capsular polysaccharides of N. meningitidis are shown in Table I. Although all of the polysaccharides are linear and acidic, and contain acetamido groups, they can be divided into two categories, based on their acidic components: those containing phosphoric diester bonds, and those containing 3deoxy-2-glyculosylonic acid residues. Interestingly, except for the group A polysaccharide, on which some structural information was al(25) A. K. Bhattachaqjee, H. J. Jennings,and C. P. Kenny, Biochem. Biophys. Res. Commun., 61 (1974)439-443. (26) A. K. Bhattacharjee, H. J. Jennings,and C. P. Kenny, Biochemistry, 17 (1978)645651. (27) F. M. Unger, Ado. Carbohydl-. Chem. Biochem., 38 (1981)323-388. (28) D. E. Dorman and J. D. Roberts,]. Am. Chem. Soc., 92 (1970) 1355-1361. (29) A. S. Perlin, N. M. K. Ng Ying Kin, S. Bhattacharjee, and L. F. Johnson, Can. J . Chem., 50 (1972)2437-2441. (30) P. A. J. Gorin,Adu. Carbohydr. Chem. Biochem., 38 (1981) 13-104. (31) H. J. Jennings,A. K. BhattacharJee,D. R. Bundle,C. P. Kenny, A. Martin, and I. C. P. Smith,]. Infect. Dis., Suppl., 136 (1977) s78-s83. (32) W. Egan, in J. S. Cohen (Ed.),Magnetic Resonance in Biology, Vol. 1, Wiley, New York, 1980, pp. 197-258.

HAROLD J. JENNINGS

160

TABLEI Structures of the Capsular Polysaccharides of Neisseria meningitidis Group

structure

References

0

1I

A

+

6)-a~-ManpNAc-l-O-P-O-

9

33

I

OH

I

OAc + 8)aD-NeupAc(2+ + 9)aD-NeupAc(2+ 718

B C

34 34

I

I

OAc + 3)-a~-CalpNAc(l + 7)p~-KDOp(2 + 4(5

29e

26

I

OAc -+ 6)-a~-Galp( 1+ 4)a~-NeupAc(2+ 0

W-135

II

X

+~)~D-G~c~NAc-~-O-P-O-

I

Y

+

Z

OH fi)-a~-Glcp(l-+4)a~-NeupAc(2-+ (contains OAc groups) 0 +

3)-a&CalpNAc(l

II

+

l)glycerol-3-O-P-O-

I

35 33

35 36

OH

ready available,23all of the other structures were deduced entirely by I3C-n.m. r. s p e c t r o s ~ o p y . ~ ~ ~ ~ ~ * ~ ~ - ~ ~ Some of the fundamental principles involved in these structural analyses are outlined here for the group A poly~accharide.~~ The lacn.m.r. spectrum thereof is shown in Fig. 1; although complex, due to the presence of 0-acetyl substituents, it is considerably simplified, to an eight-resonance spectrum (carbonyl signal at 175.8 p.p.m. not (33) D. R. Bundle, I. C. P. Smith, and H. J. Jennings,]. Biol. Chern., 249 (1974) 22752281.

(34) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C. P. Smith, J . B i d . Chern.,250 (1975) 1926-1932. (35) A. K. Bhattacharjee, H. J. Jennings, C. P. Kenny, A. Martin, and 1. C . P. Smith, Can. 1. Biochem., 54 (1976)1-8. (36) H. J. Jennings, K.G. Rosell, and C. P. Kenny, Can.]. Chern., 57 (1979)2902-2907.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

161

p.p.m.

FIG. 1. -Fourier-transformed, %-N.m.r. Spectrum of the Native Polysaccharide Antigen of Croup A Neisseria meningitidis. [Upper, containing ( a )0-acetylated and ( b ) unacetylated residues, and lower, its fully O-deacetylated form.]

shown), on removal of the 0-acetyl groups. This simplicity indicated that the polysaccharide consists of a linear arrangement of a 2-acetamido-mannopyranosyl phosphate repeating-unit. The individual signals in the spectrum of the 0-deacetylated, group A polysaccharide were assigned by using the generally applicable, empirical methodology of comparing the chemical shifts of the signals of the polysaccharide with the corresponding chemical shifts of the anomers of its monomeric or oligomeric constituents. Experience has indicated that the chemical shifts of the monosaccharides are similar to those of the monosaccharide residues within the polysaccharide, except for substituent These effects, caused by the attachment of any substituent to a sugar moiety, cause an increase in the chemical shift of the carbon atom directly involved in the linkage; this increase is usually accompanied by a deckease of smaller magnitude (or, sometimes, an increase) in the chemical shifts (37) H. J. Jennings and I. C. P. Smith, Methods Enzymol., 5OC (1978)39-50. (38) H. J. Jennings and I. C. P. Smith, Methods Carbohydr. Chem., 8 (1980)97-105.

162

HAROLD J . JENNINGS

of the neighboring P-carbon atoms. Thus, these chemical-shift differences serve to determine the position of linkages; similarities in chemical shifts, especially those involving carbon atoms known to be sensitive to change in anomeric configuration, can be employed to determine the configuration of linkages. By using this approach, the l-+6)-linked.33 ( The linkgroup A polysaccharide was shown to be a - ~ age position was also confirmed by the pattern of the two- and threebondi11P-13Ccoupling manifest in the spectrum of the O-deacetylated polysaccharide. Chemical-shift differences between the signals of the native, and the O-deacetylated, group A polysaccharide (see Fig. 1)indicated that the O-acetyl substituents were linked to 0-3 of the 2-acetarnido-2-deoxy-~-mannopyranosyl residues; a comparison of the intensities of' the characteristic methyl signals of the O-acetyl and N-acetyl groups indicated that 70% of these residues were substituted in this way. A similar analysis indicated that the analogous, gronp X polysaccharide is composed of a repeating unit of a-~-(l+4)-linked2acetarnido-2-deoxy-D-glucopyranosyl phosphate.33 The group Z polysaccharide can be included in the same category as the aforementioned polysaccharides, as it also contains phosphoric diesters.36 The structure was shown to be a repeating unit of 10-(2-acetamido-2deoxy-a-D-ga1actopyranosyl)glyceroljoined through phosphoric diester groups at 03 of glycerol and 03 of the 2-amino-2-deoxy-D-galactose residue. However, in this case, the phosphoric diester is not glycosidically linked to the 2-amino-2-deoxy-~-galactoseresidue, and the structure closely resembles that of a teichoic acid. In the second category of meningococcal polysaccharides, those containing 3-deoxy-2-glyculoses, the group B and C polysaccharides are the simplest in structure34;this is illustrated in the 13C-n.m.r.spectrum of the O-deacetylated, group C polysaccharide, shown in Fig. 2. The simple, eleven-resonance spectrum indicated that the group C polysaccharide is a linear polymer of sialic acid. The group B polysaccharide gives a similar, simple, eleven-resonance spectrum. By using the methyl a- and p-D-ketosides of sialic acid as model compounds, and comparing the chemical-shift differences between some of their carbon atoms with those of the sialic residues in the polysaccharides, it was indicated that the group B polysaccharide is (2-+8)-linked, whereas the group C polysaccharide is (2+9)-linked.34 Similarities in the chemical shifts of the configurationally sensitive signals (C-1, C 4 , and C-6) of the polysaccharides with those of the methyl a-D-ketoside, permitted the a-Dconfiguration to be assigned to both polysaccharides. The carboxylate signal (C-1) proved to be extremely useful in these configurational determinations, as it is readily discernible and undergoes a significant, chemical-shift displacement (- 2 p.p.m.) with

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

r

163

I4m7

5

P.P.m FIG.2. -Fourier-transformed, W-N.m.r. Spectrum of the Native Polysaccharide Antigen of Group C Neisseria meningitidis (upper),and its 0-Deacetylated Form (Iower).

change in anomeric config~ration.~~ This characteristic displacement is dependent on the orientation of the carboxylate group (axial or equatorial), and could prove to be generally applicable to 3-deoxy-2glyculose r e s i d ~ e s . ~ ~ , ~ ~ , ~ ~ The 0-acetyl substituents of the native, group C polysaccharide were located by comparing its 13C-n.m.r. spectrum with that of the 0deacetylated, group C polysaccharide (see Fig. 2). The appearance of characteristic muhiplets in some of the signals of the native polysaccharide showed that the 0-acetyl groups are distributed exclusively between 0-7 and 0-8of its sialic acid residues. The group Y and W-135 capsular polysaccharides also contain sialic acid. However, unlike the groups B and C polysaccharides, they are not homopolymers, as they also contain, respectively, D-glucosyl and D-galactosyl residues.35 Structural studies indicated that the group Y (39) H. J. Jennings and A. K. Bhattacharjee, Carbohydr. Aes., 55 (1977) 105-112. (40) H. J . Jennings, K.-G. Rosell, and K. G. Johnson, Carbohydr. Res., 105 (1982)4556.

164

HAROLD J . JENNINGS

polysaccharide has a -+6)-a-D-Glcp-(14)-a-~-NeupAc-(2+repeating unit, whereas that of group W-135 has a -6)-a-D-Galp-(l+4)-a-DNeupAc-(2+ repeating unit.35Interestingly, these two, serologically distinguishable, polysaccharides differ only in the configuration of one hydroxyl group in their respective, disaccharide repeating-units. The group Y polysaccharide contains 0-acetyl groups (1.3mol per sialic acid residue), but the locations of these have not yet been established. The group 29-e polysaccharide is composed of an alternating sequence of 3-deoxy-p-~-manno -2-octulosylonic acid and S-acetamido2-deoxy-a-~-glucopyranosyl residues; linkage is to 0-7 of the former and to 03 of the latter. 0-Acetyl substituents were also located on both 04 and 0-5 of the KDO residues.26It is of interest that, whereas sialic acid has only been found in bacterial polysaccharides, and elsewhere in Nature, as its a-Danomer, there is strong evidence to suggest that KDO probably exists in bacterial polysaccharides in both of its anomeric f ~ r m ~ . ~ ~ , ~ ~ 2. Haemophilus influenzae The Haemophilus influenzae are Gram-negative organisms that can be serologically classified into six types (a through f) on the basis of their type-specific, capsular polysaccharides. Analytical studies indicated that those of types a, b, c, and f a r e poly(sugar phosphates):’ whereas that of type e contains a “hexosamine-uronic acid” component,’2 since characterized as 2-acetamido-2-deoxy-~-mannuronic acid.43*,44 This component sugar is also a constituent of the type d polys a c ~ h a r i d e . Thus, ~ ~ . ~ ~like the meningococcal polysaccharides, the type-specific polysaccharides of H. influenzae may be divided into two groups on the basis of their acidic components, 2-acetamido-2deoxy-D-mannuronic acid replacing the 2-glyculosonic acids of the former. The structures of the type-specific polysaccharides are shown in of type e, are composed Table 11, and, except for one particular of linear arrangements of disaccharide repeating-units. The clinically (41) E. Rosenberg, G . Leidy, J. Jaff, and S. Zamenoff,]. Biol. Chern., 236 (1961) 28412844. (42) A. R. Williamson and S. Zamenoff, J . Biol. Chem., 238 (1963)2255-2258. (43) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 88 (1981)77-84. (44) F.-Y.Tsui, R. Schneerson, and W. Egan, Carbohydr. Res., 88 (1981)85-92. (45) P. Branefors-Helander,L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Carbohydr. Res., 97 (1981)285-291. (46) F.-P. Tsui, R. Schneerson, R. A. Boykins, A. B. Karpas, and W. Egan, Carbohydr. Res., 97 (1981)293-306.

165

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES TABLEI1 Structures of the Capsular Polysaccharides of Haemopkilus influenzae

Type

References

Structure

0 a

ll

+ 4)p~-Glcp(l+4)D-ribito&O-P-O-

48

I

OH 0 b

-+

II

3)p~-Ribf(l-+ l)~-ribito1(5-O-P-O-

I

47,s

OH 0

II

C

d

e

4)pD-GIcpNAc(l+ 3fc~D-Gdp(l-O-P-O3 I t OH OAc + 4)p~-GlcpNAc( 1+ 3)pD-ManpANAc(1-+ -+ 3)p~-GlcpNAc( 1+ 4)p~-ManpANAc( 1+ 3 -+

49,50

45,46 43,44

t

2 p~-Fr~p

f

0

1I

.+ 3)p~-GalpNAc( 1+ 4)a~-GalpNAc( 1-O-P-O-

3

t

OAc

I

51,52

OH

important, type b polysaccharide was the first to have its structure determined4'; I3C-n.m.r. spectroscopy played a prominent role in this early investigation. This technique was also extensively used in subsequent structural determinations on the other H. influenzae, typespecific p o l y ~ a c c h a r i d e s . ~ ~ - ~ ~ ~ ~ ~ - ~ ~ (47) R. M. Crisel, R. S. Baker, and D. E. DormanJ. Biol. Chem., 250 (1975)4926-4930. (48) P. Branefors-Helander,C. Erbing, L. Kenne, and B. Lindberg, Carbohydr. Res., 56 (1977) 117-122. (49) P. Branefors-Helander, B. Classon, L. Kenne, and B. Lindberg, Carbohydr. Res., 76 (1979) 197-202. (50) W. Egan, F.-P. Tsui, P. A. Climenson, and R. Schneerson, Carbohydr. Res., 80 (1980) 305-316. (51) P. Branefors-Helander, L. Kenne, and B. Lindqvist, Carbohydr. Res., 79 (1980) 308-3 12. (52) W. Egan, F.-P. Tsui, and R. Schneerson, Carbohydr. Res., 79 (1980)271-277.

166

HAROLD J. JENNlNGS

Except for the anomeric configuration of the ribofuranosyl residue,53 the repeating unit (1) of the type b polysaccharide was proposed by

Crisel and coworker^.^' They established that the type b polysaccharide is composed of ribose, ribitol, and phosphate in the molar ratios of 1: 1: 1, and, by periodate oxidation studies, that the ribitol is linked at both of its hydroxymethyl groups. The 13C-n.m.r.spectrum ofthe type b polysaccharide exhibited ten individual, carbon signals, indicative of a simple, linear arrangement of the disaccharide repeating-unit. The presence of 13C-31Pscalar couplings in five of these signals was also consistent with the structure proposed. Later studiess3 established the chirality (D) of the ribitol residue, and the anomeric configuration (p-D)of the ribofuranosyl residue. Additional structural studies%*confirmed the structure of the type b polysaccharide. The remaining N. infEuenzae polysaccharides in the phosphoric diester category (a, c, and f) were structurally elucidated by. using similar procedures, and were shown to have other structural similarities; all of them are composed of linear, disaccharide phosphate repeatingunits. The type a polysaccharide, which contains D-glucose, D-ribitol, and phosphate in the molar ratios of 1:1:1was shown to be composed of 4-O-~-D-ghcopyranosyl-D-ribitolresidues linked by phosphoric diesters between 0-5of ribitol and 03 of D - g l u ~ o s eIn . ~ independent ~ studies, two different groups of researchers proposed identical structures for the respective type c (Refs. 49 and 50) and type f (Refs. 51 and 52) polysaccharides. The type c polysaccharide was reported to contain D-galactose, 2-amino-2-deoxy-D-glucose, and phosphate in the (53) P . Branefors-Helander, C. Erbing, L. Kenne, and B. Lindberg,Acta Chem. Scund., Ser. B, 30 (1976)276-277. (54) B. A. Fraser, F.-P. Tsui, and W. Egan. Carbohydr. Res., 73 (19'79)59-65.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

167

molar ratios of 1:1:1. The structure is based on a 34-(2-acetamido-2deoxy-p-D-glucopyranosyl)-a!-D-galactopyranosyl phosphate repeating-unit, and the phosphate is attached to 0-4of the 2-amino-2-deoxyD-glucose residues in the polysaccharide structure. The type c polysaccharide also contains acetyl substituents situated at 03 of 80% of the 2-amino-2-deoxy-D-glucose residues. The structures of the two remaining 2-acetamido-2-deoxy-~-mannuronic acid-containing, H. injluenzae capsular polysaccharides (d and e) were also elucidated independently by the same two groups of re~earchers.~ Both ~ - ~polysaccharides ~ also contain 2-amino-2-deoxyD-glucose, and the structures of both are based on alternating 2-aminoacid residues. 2-deoxy-D-glucose and 2-amino-2-deoxy-D-mannuronic The type d p o l y ~ a c c h a r i d eis~ composed ~~~~ of a +4)-/3-D-GlcpNAcl+ repeating unit, with L-alanine, L-serine, (1+3)-/3-~-ManpANAc-( or L-threonine linked to the carboxylate group of 2-acetamido-2deoxy-D-mannuronicacid by an amide bond, whereas the type e polyis composed of a differently linked, -*3)-/3-D-GlcpNAc(1+4)-p-~-ManpANAc-(l+ repeating-unit. Strain-dependent structure-variations have also been demonstrated for the type e polysaccharide.43 One particular strain of the type e organism produced a polysaccharide possessing the foregoing structure but having additional, terminal p-D-fmctofuranosyl groups linked to 03 of all of the 2-acetamido-2-deoxy-D-mannuronic acid residues. 3. Group B Streptococcus

L a n ~ e f i e l d ~characterized ~-~' two polysaccharide antigens obtained from Group B Streptococcus: a group antigen common to all strains, and the type-specific, capsular polysaccharides that distinguish four major serotypes, namely, Ia, Ib, 11, and 111. The type-specific polysaccharides were originally isolated by extraction of the whole, streptococcal organisms with hot hydrochloric acid, and all had identical constituents: galactose, glucose, and 2-acetamido-2-deoxyglucose.57-62 This extraction procedure produces immunologicallyincomplete antigens that form a lower molecular weight core to the complete, native (55) R. C. Lancefield,]. Exp. Med., 57 (1933) 571-582. (56) R. C. Lancefield,]. E r p . Med., 59 (1934)441-458. (57) R. C. Lancefield,]. E r p . Med., 67 (1938)25-40. (58) R. C. Lancefield and E. H. Friemer,]. Hyg., 64 (1966) 191-203. (59) H. Russell and N. L. Norcross,]. lmmutwl., 109 (1972)90-96. (60)J. A. Kane and W. W. Karakawa, Infect. lmmun., 19 (1978)983-991. (61) J. Y. Tai, E. C. Gotschlich, and R. C. Lancefield,]. E r p . Med., 149 (1979) 58-66. (62) D. L. Kasper, C. J. Baker, R. S. Baltimore, J. H. Crabb, G. Sc hihan, and H. J. Jennings,]. E x p . Med., 149 (1979) 327-339.

168

HAROLD J. JENNINGS

The latter antigens, which can be obtained by neutral or buffered (pH 7.0) extraction of whole organisms,61.62,65,67a contain additional, terminal sialic acid residues. The presence of these residues proved to be essential for the immunological expression of the native antigens. The native types Ia (Ref. 68, 69), Ib (Ref. 61, 69), and I11 (Ref. 63) antigens contain D-galactose, D-glucose, 2-acetamido-2-deoxy-~-glucose, and sialic acid in the molar ratios of 2 : 1:1:1, and constitute a group of isomeric polysaccharides, whereas the type I1 (Ref. 70) native antigen has identical component sugars, but in the ratios of 3 :2 : 1:1. The structures of the repeating units of the types Ia, Ib, 11, and I11 polysaccharides are shown in Table 111. These structures were elucidated by first determining the structures of the simpler, core antigens, as describedm for the type I11 polysaccharide. A comparison of the methylation analysis of the core with that of the native polysaccharide permitted the position of linkage of the terminal sialic acid residues in the native antigen to be established. The anomeric configurations of the sugar residues were determined by W-n.m.r. spectroscopy. Structurally, all of the Group B streptococcal antigens constitute an interesting group of polysaccharides, both in their relationships to each other, and to other biologically important molecules (glycopro~-~~ teins). All of the polysaccharides have in their ~ t r u c t u r e s ~a*common p-~-GlcpNAc-( l+S)-P-~-Galp-(1+4)-p-~-Glcp trisaccharide which forms the repeating unit of the backbone of the type I11 polysaccharide. This was also presumed, in a previously proposed structure:* to be true of the type Ia polysaccharide; however, the evidence on which this structure was based proved not to be definitive, as it was also compatible with an alternative structure in which the 2-amino-2deoxy-0-glucose residue of the trisaccharide becomes a part of the (63)H. J. Jennings, K.-C. Rosell, and D. L. Kasper, Can. ]. Biochem., 58 (1980) 112120. (64)E. H. Friemer,]. E x p . Med., 125 (1967) 381-392. (65) H. W. Wilkinson, Infect. Zmmun., 11 (1975) 845-852. (66) C. J. Baker and D. L. Kasper, Infect. Immun., 13 (1976) 284-288. (67) J. A. Kane and W. W. KarakawqJ. Immunol., 118 (1977) 2155-2160. (67a) C. J. Baker, D. L. Kasper, and C. E. Davies,]. E r p . Med., 143 (1976) 259-5370. (68) H. J. Jennings, K.-G. Rosell, and D. L. Kasper, Proc. Natl. Acad. Sci. U . S . A., 77 (1980) 2931-2935. (69) H . J . Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, Biochemistry, in press. (70) H. J. Jennings, K.-G. Rosell, and D. L. Kasper,]. Biol. Chem., in press. (71) H. J. Jennings, E. M. Katzenellenbogen, C. Lugowski, and D. L. Kasper, unpublished results.

TABLEI11 Structures of the Capsular Polysaccharides of Group B Streptococcus Type

Structure

References

Ia

+ 4)-p-D-Gkp-(1 + 4)-p-D-Gdp-(1 +

69

3

t

p-~GlcpNAc 4

t

Ib

1 c~-~-NeupNAc-(2 + )-P-DGalp + 4)-p-Dklcp-(l + 4)-p-~-Galp-(l+ 3

69

t

1 p-~ClcpNAc 3

t

I1

1 a-D-NeupNAc-(S+ 3)-P-&alp --* 4)-p-D-GlcpNAc-(l+ 3)-B-D-Galp(l+ 4)-p-D-Glcp-(l -* 3 ) - p - ~ - G l ~ p - ( l 2)-8-~-Galp-( + 1+ 6 3

t

I11

1 p-DGdp +4)-p-~-Gl~p 1 --* ( B)-p-D-GlcpNAc-(1 + 3)+-D-G&-( 1 + 4

t

1 a-~-NeupNAc-(2--* 6)-/3-DGdp

70

f

2 a-D-NeupNAc

62

170

HAROLD J. JENNINGS

branches of the type Ia polysaccharide. This was indicated in subsequent, extensive degradation studies on the types Ia and Ib polysaccharides?” the results of which are consistent only with both having trisaccharide branches as shown in Table 111. All of the incomplete core-structures have branches terminating in P-D-galactopyranosyl residues ,63 ,I%-?1 and the fact that the type 111, core antigen has a structure identical to that of the capsular polysaccharide of type 14 S. pneuin the native type Ia m ~ n i a e is ? ~of some serological significance.62*63 (Refs. 68 and 69), Ib (Ref. 69), and I11 (Ref. 63) polysaccharide antigens, the terminal P-D-galactopyranosyi residues of the core antigens are completely masked by sialic acid residues, forming branches that terminate in sialic acid residues. These sialic acid residues are linked to 0-3of the P-D-galactopyranosyl residues of the native type Ia (Refs. 68 and 69) and Ib (Ref. 69) polysaccharides and to 0-6 of the type I11 polysaecharide.63 These branches are of considerable serological importance to the group B streptococcal organism, because of their structiiral homology with some important, human serum-glycoproteins. The terminal 3-0-(N-acetyl-a-o-neuraminy~)-~-D-galactopyranosyl group of the types Ia and Ib antigens is the end group in the human $1 and N blood-group substance^,^^ and the 6-O-(N-acetyl-a-~-neuraminyl)-P-D-galactopyranosylmoiety of the type I11 polysaccharide is also a structural feature of human ~erotransferrin.~~ 4. Streptococcus pneumoniae

Strelitococczis pneumoniae are Gram-positive organisms which, like the group B Streptococcus, have a common, group antigen (C-

s ~ b s t a n c e ) ,and ~ ~ .different ~~ type-specific, capsular polysaccharides. Unlike the organisms previously described, S. pneumoniae have been identified in a prolific number of immunologically distinguishable types based on these capsular polysaccharides. To date, there are at least 84 known type-spe~ificities,~.’O and these have been designated types 1-84 in the American system. However, on the basis of serological cross-reactivity among these polysaccharides, the organisms have also been conveniently classified into serologically related groups (see Table IV) in the Danish system. The pneumococcal polysaccharides are particularly important, because early investigations into (72) B. Lindberg, J. Likmgren, and D. A. Powel1,Carbohydr. Res., 58 (1977) 177-186. (73) J . E. Sadler, J. C. Paulson, and R. L. Hill,/. BioE. Chem., 254 (1979)2112-2119. (74) G . Spik, B. Bayard, B. Fournet, G . Streker, S. Bouquelet, and J . Montreuil, F E B S . k t t . , 50 (1975)296-299. (75) D. E. Brundish and J. Baddiley, Biochem.]., 110 (1968) 573-581. (76) H. J. Jennings, C. Lugowski, and N. M. Young, Biochemistry, 19 (1980) 47124719.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

171

their immunological and structural properties resulted in the acquisition of knowledge fundamental to the development of human, polysaccharide vaccines. Structural studies on these polysaccharides also provided technical and conceptual contributions to the general problem of polysaccharide structure, and it is a tribute to earlier investigators that these contributions were made without the benefit of modem instrumental methods. The structures of the pneumococcal polysaccharides have been re~iewed.'~,''Because of their number and structural complexity (up to 7 sugar components in their repeating units), they can be dealt with only briefly in this chapter, and only the structures of those used in the current, pneumococcal vaccine (see Section V,1) are listed in Table IV. Two of the pneumococcal polysaccharides [types 14 (Ref. 72) and 37 (Ref. 78)] are neutral; in fact, that of type 14 is a rare example of a neutral-polysaccharide capsule involved in human bacterial The rest of the pneumococcal polysaccharides are acidic, and can be classified according to their common acidic components. Types 1 (Ref. 79), 2 (Ref. 80),3 (Ref. 81), 5 (Ref. 18), 8 (Ref. 82),9A (Ref. 83), 9N (Ref. 84), and 9V (Ref. 85) have D-glucuronic acid residues, whereas types 6A (Ref. 86), 6B (Ref. 87), 11A (Ref. SS), 13 (Ref. 89), 15F (Ref. W),17F (Ref. 91), 19F (Refs. 92 and 93),19A (Ref. 94),23F (Ref. 95), 27 (Refs. 96 and 97), 29 (Ref. 98),and (77) 0. L a m and B. Lindberg, Ado. Carbohydr. Chem. Biochem., 33 (1976) 295-322. (78) J. C. Knecht, G. Schiffman, and R. Austrian,J . Exp. Med., 132 (1979) 475-487. (79) B. Lindberg, B. Lindqvist, J. Lonngren, and D. A. Powell, Carbohydr. Res., 78 (1980) 111-117. (80) L. Kenne, B. Lindberg, and S. Svensson, Carbohydr. Res., 40 (1975) 69-75. (81) R. E. Reeves and W. F. Goebel,]. Biol. Chem., 139 (1941) 511-519. (82) J. K. N. Jones and M. B. Perry,]. Am. Chem. Soc., 79 (1957) 2787-2793. (83) L. G. Bennet and C. T. Bishop, Can. J . Chem., 58 (1980) 2724-2727. (84) H. J. Jennings, K . G . Rosell, and D. J. Carlo, unpublished results. (85) M. B. Perry, V. Daoust, and D. J. Carlo, Can.]. Biochem., 59 (1981) 524-533. (86) P. A. Rebers and M.Heidelberger,J. Biol. Chem., 139 (1941) 511-519. (87) L. Kenne, B. Lindberg, and J. K. Madden, Carbohydr. Res., 73 (1979) 175-182. (88) D. A. Kennedy, J. G. Buchanan, and J. Baddiley, Biochem.]., 115 (1969) 37-45. (89) M. J. Watson, J. M. Tyler, J. G. Buchanan, and J. Baddiley, Biochem.]., 130 (1972) 45-54. (90) M. B. Perry, D. R. Bundle, V. Daoust, and D. J. Carlo, Mol. Immunol., 19 (1982) 235-246. (91) M. B. Perry, personal communication. (92) H. J. Jennings, K . 4 . Rosell, and D. J. Carlo, Can.J. Chem., 58 (1980) 1069-1074. (93) H. Ohno, T. Y. Yadomae, and T. Miyazaki, Carbohydr. Res., 80 (1980) 297-304. (94) C.-J. Lee and B. A. Fraser,J. B i d . Chem., 255 (1980) 6847-6853. (95) M. B. Perry, V. Daoust, and R. Lowe, unpublished results. (96) L. C. Bennet and C. T. Bishop, Can.]. Chem., 55 (1977) 8-16. (97) L. C. Bennet and C. T. Bishop, lmmunochemistry, 14 (1977) 693-696. (98) E. V. Rao, M. J. Watson, J. G. Buchanan, and J. Baddiley, Biochem.]., 111 (1969) 547-556.

TABLEIV Structures of Some of the Capsular Polysaccharides of Streptococcus pneumoniae Contained in the Current, Pneumococcal Vaccine Structureb

Type"

w

1 2

References

79

-+ B)a-Sugp(1 + 4)aD-GalpA(1 S)aD-GalpA(1 + S)a~-Rhap(l S)a~-Rhap(l+ 3)P~-Rhap(l+ 4)aD-GIcp(l -+ 2 -+

-+

4

80

-+

t

&a

3 4

1 a ~ - G l c p A ( l - +6 ) a ~ G l c p + 4)PD-GlCp(l S)pD-GIcpA(1 -+ + S)a~-FucpNAc(l-+ S)aD-GalpNAc(l -+ 4)P~-ManpNAc(l -+

-+

H,C 5

-+

Z)PD-GkpA(1 -+ B)aL-FucpNAc(1 -+ 4

x

4 ) a ~ - G a l (-+ l

81 100

CO,H 18

t

-+

2)aD-Gdp(1 -+

a-Sugp(l 3)crD-Glcp(1

1 0 4)P~Glcp II 3)a~-Rhap( 1 -+ 3)-ribitol-(5-O-P-O-

-+

-+

I

OH

86

12F (12)

+ 4)a~-FucpNAc( 1+ 3)p~-GalpNAc(l+ 4)p~-ManpANAc(l+

3

3

t

t

1

1 aDGalp a ~ - G l c p (+ l 2)aDGkp + 4)pD-Gkp(l+ G)pD-GlcpNAc(l+ 3)p~-Galp(l + 4

14

82 a4 101

72

t

1

0

2

II

19F (19)

+ 4)pD-ManpNAc(1+ 4)aD-Gkp(1--* 2)a~-Rhap( 1-O-P-O-

23F (23)

+ 4)p~-Gfcp(l + 4)pD-Galp(l + 4)a~-Rhap(l+

I

OH

I

P

a

2

t

1 aL-Rhap

US. typing system in parentheses. *Sug = 2-Acetamido-l-arnino-2,4,6-trideoxygalactose.

92,93

95

174

HAROLD J . JENNINGS

34 (Ref. 99) contain phosphate. Type 27 also contains p y r u ~ a t e ~as ~,~' an additional acid component, and type 4 contains pyruvate as the sole acid component." Type 1 2 F contains 2-acetamido-2-deoxy-~-mannuronic acid as its only acidic component.101 The structure of the type 3 polysaccharide was the first to be established, by Reeves and Goebel,8' and thus this polysaccharide became a model for many immunological investigations. The concept of a repeating unit was also established in this work, and this was elegantly confirmed in later, chemical-degradation studies by Rebers and Heidelberger,H6in which they isolated the tetrasaccharide repeating-unit of the type 6A polysaccharide in crystalline form in 94% yield. Another early study!' in which partial hydrolysis with acid was employed, established the structure of the type 8 polysaccharide as having a tetrasaccharide repeating-unit containing cellobiouronic acid. Other, extremely valuable, early degradative studies were carried out b y Baddiley and coworkers on the types 11A (Ref. 88), 13 (Ref. 89),29 (Ref. 98), and 34 (Ref. 99)polysaccharides; they were able to establish that each was composed of a pentasaccharide phosphate repeating-unit, and to locate the phosphoric diester linkages in these polysaccharides. Finally, the combined use of gas-liquid chromatography and mass spectrometry by Lindberg and coworker^'^^^'^^ has had a profound impact on the structural analysis of polysaccharides. This was demonstrated in work on the type 2 polysaccharide,8° and in subsequent, structural elucidations of other pneumococcal polysaccharides (see Table IV). 111. OTHER IMPORTANT STRUCTURAL AND PHYSICAL OF CAPSULAR POLYSACCHARIDES

FEATURES

1. Structural Heterogeneity There is now abundant structural, spectroscopic, and biosynthetic evidence to suggest that, except for the possibility of minor structural irregularities, the fundamental structures of bacterial polysaccharides consist of fairly small, regular repeating-sequences of from 1to 7 saccharide units. Thus, as the polysaccharides are multivalent antigens, the effect of minor irregularities in their structures would, in general, (99)G. J. F. Chittenden, W. K. Roberts, J. G . Buchanan, and J. Baddiley, Biochem.J., 109 (1968) 597-602. (100) P.-E. Jansson, B. Lindberg, and U. Lindquist,Carbohydr. Res., 95 (1981) 73-80. (101) K. Leontein, B. Lindberg, and J. L(inngren,Can.]. Chem., 59 (1981) 2081-2085. (102) B. Lindberg, Methods Enzymol.,28B (1972) 178-195. (103) B. Lindberg, Methods Enzymol., 5OC (1980) 3-34

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

175

be unimportant to their immunological specificity. However, minor structural irregularities cannot be ignored in other immunological properties, as it has been demonstrated that the presence of extremely small proportions of attached lipid can have a profound effect on the immunogenicity of polysaccharides (see Section 111,4). A larger degree of structural heterogeneity has been documented for some bacterial, capsular polysaccharides, and this is mostly introduced by the distribution of 0-acetyl substituents on these polysaccharides. Only 70% of the 2-amino-2-deoxy-D-mannose residues of the group A menand this hetingococcal polysaccharide have 0-acetyl s~bstituents,3~ erogeneity is also exhibited by the group C meningococcal polysac~ h a r i d e . 3In ~ the latter, an a-D-(2+9)-linked homopolymer of sialic acid, the molar ratio of 0-acetyl to sialic acid is 1.2:l-0. These 0acetyl groups are distributed on the sialic acid residues, in a complex pattern, at 0-7 and 08 (monosubstitution and disubstitution), and some of the sialic acid residues remain unsubstituted (see formulas 25). Interestingly, in the group C polysaccharide, the pattern of O-acetyl substitution is dependent on the conditions used to grow the group C organisms31; this could be important in the production of human polysaccharide vaccines. However, it is unlikely that the extent of this structural variation would be sufficient to impart complete, serological specificity to the variant group C polysaccharide and to preclude its use as an effective, group C meningococcal vaccine, because, even the 0-deacetylated group C polysaccharide can function as an effective, group C meningococcal vaccine in man (see Section V,2).

-

2 R=R'=H 3 R = R' = COCH, 4R=H,R=COCH, 5 R=COCH,,R'=H

The Four Different, N-Acetylneuraminic Acid Residues in the Native Polysaccharide Antigen of Croup C Neisseria meningitidis.

2. Determinants and Immunological Specificity An important step in understanding the immunology of polysaccharides consists in establishing which part of the polysaccharide is re-

176

HAROLD J. JENNINGS

sponsible for its immunological specificity. This part of the polysaccharide is called a determinant, and, from early studies by Goebel,lW it became apparent that determinants constitute only a small part of the large polysaccharide molecule. Antibodies made to the type 3 pneumococcal polysaccharide are strongly inhibited by cellobiouronic acid, the disaccharide repeating-unit of the polysaccharide; conversely, antibodies made to a cellobiouronic acid-protein conjugate cross-react with the type 3 pneumococcal polysaccharide. This procedure of using compounds of low molecular weight that are representative of parts of the polysaccharide structure, in order to inhibit the classical, antibody-antigen (polysaccharide) precipitin reaction of Heidelberger and Kendall? was used extensively by Kabat in ~ t u d i e s ' ~on ~ Jthe ~ linear dextran-antidextran reaction. This model system is probably representative of all linear polysaccharides, with the possible exception of those terminating in nonreducing sialic acid groups (see later); as such, it is pertinent to polysaccharide vaccines, as most of the polysaccharides involved are linear. In these definitive studies, this procedure furnished information on the location and size of determinant groups, and on the heterogeneous nature of antibodies in terms of their specificities. By using a series of oligosaccharides of the isomaltose series with human antidextran sera, it was found that the inhibitory power of the oligosaccharides increases with molecular size until it becomes more or less constant at the hexasaccharide, and this was interpreted as a measure of the optimum size of the combining site of the antibody molecule. From the relative, inhibitory powers of each oligosaccharide, it was possible to calculate the contribution, to the binding energy, of each successive D-glucose residue; it was found that, although the terminal mglucose unit contributed most to this binding energy, each succeeding D-glucose unit also made incrementally smaller contributions. The nonreducing, terminal D-glucosyl groups were called immunodominant, although, in fact, they remain a part of the larger determinant. Another important finding in these studiesIo7was the heterogeneous nature of the antibodies in regard to their serological specificities. The antibodies were adsorbed onto Sephadex, and fractionated by elution with isomalto-oligosaccharidesof' different molecular sizes. Inhibition studies on the different fractions indicated that antibodies having a (104) W. F. Goebel,]. Exp. Med., 68 (1938)469-484. (105)E. A. Kabat,]. Immunoi., 84 (1960) 82-85. (106) E. A. Kabat, Experimental Immunochemistry, 2nd edn., Charles C. Thomas, Springfield, Illinois, 1967, pp. 241-267. (107) J. Gelmer and E. A. Kabat, Immunochemistry, 1 (1964)303-316.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

177

specificity both to small (disaccharide)and large (hexasaccharide) determinants were present in the antidextran serum. Branched polysaccharides differ from linear polysaccharides in having many more terminal, glycosyl residues on each polysaccharide molecule. This fact, together with the more exposed, and, thus, more accessible, position of these residues, tends to make them immunodominant, although not exclusively so, as populations of antibodies having specificities to the backbone of the polysaccharide can also be formed. Although the immunodominance of terminal p-D-glucosyluronic acid groups was recognized in early studies,lWthis phenomenon was more definitively resolved in classical studies on the serological determinants of the lipopolysaccharides of SaZmoneZZa; these have been reviewed." As with the capsular polysaccharides, the 0chains of the lipopolysaccharides consist of a linear arrangement of oligosaccharide repeating-units,the majority of which contain unique, terminal 3,6-dideoxyhexosyl groups in each repeating unit. These terminal saccharides are, to a large degree, responsible for the specificity of antibodies made to the Salmonella organisms; however, antibodies having a specificity for the backbone are also detected in these antisera. In serological studies on branched Dmannans, Ballou and coworkerslOgJ1O determined that the participation of the backbone D mannosyl residues in the immunological response to these branched D-mannans is dependent on the length of the branch structure. When the side chains extend to a tetrasaccharide unit, they are able completely to inhibit antibodies made to the homologous Dmannan. Interesting exceptions to the general rule of the immunodominance of branch saccharides have become apparent as regards the capsular polysaccharides of Group B Streptococcus. These are structure-related, and are important in both the human immunologicalresponse to (see Section 111,3),and the virulence of (see Section VI,Z), the Group B streptococcal organisms."' The type I11 polysaccharide has a 6-0(N-acetyl-cu-Dneuraminyl)-P-wgalactopyranosyl branch,= whereas those of types Ia and Ib have terminal 3-0-(N-acetyl-a-Dneuraminy1)p-Dgalactopyranosyl ~ n i t s .Neither ~ ~ . ~ of ~ these terminal-branch disaccharides is immunodominant, and this can probably be attributed to structural homology between the type 111, and the types Ia and Ib polysaccharides and human serum glycoproteins (serotransferrin and (108) M. Heidelberger, Fortschr. Chem. Org. Natumt., 18 (1960) 503-536. (109) C. E. Ballou,]. BioZ. Chem., 245 (1970) 1197-1203. (110) C. E. Ballou, P. N. Lipke, and N. C. Rashke,J. Bacteriol., 117 (1974)461-467. (111) H. J . Jennings, C. Lugowski, and D. L. Kasper, Biochemistry, 20 (1981) 45114518.

178

HAROLD J. JENNINGS

the M and N blood-group substances, respectively)."' The production of antibodies to these determinants would be highly unfavorable, and, consequently, is probably suppressed by the human immune-system. Following this reasoning, it is highly improbable that terminal sialic acid residues can be a part of any determinant responsible for significant amounts of antibody, although these residues exercise confonnational control over these determinants"' (see Section 111,3). This would also imply that nonreducing, terminal sialic acid residues of any linear polysaccharide (for example, groups B and C meningococcal polysaccharides) would not be immunodominant. Many of the bacterial polysaccharides contain small, noncarbohydrate substituents that could be regarded as branches, and that can also be important in the serological reactions of polysaccharides. These substituents have been r e ~ i e w e d , ' ~and , ' ~ the most important, in terms of the formulation of current, polysaccharide vaccines (see Section V,2), is the 0-acetyl substituent. These substituents can be immunodominant, but are not exclusively so; other populations of antibodies having specificities for other sectors of the polysaccharide are usually formed.

3. Conformation It has been established that the primary structures of the capsular polysaccharides are responsible for their serological specificity. Obviously, conformational factors must also piay a role in this specificity. Rees112showed that polysaccharides, like proteins, can have ordered (helical) structures in which interchain and intrachain associations are both involved, and that these polysaccharides undergo temperatureinduced, order-disorder transitions. Rees'I3 also found that the secondary structures are responsible for the physical and biological properties of these polysaccharides. Of special interest to the present discussion is the fact that the ordered conformation of the capsular polysaocharide from the Gram-negative organism Xanthomonus campestris, a plant pathogen, is necessary, in order that the bacteria may bind to the surface of the plant-host cell^."^ A similar dependence of specific binding to antibody molecules on the ordered (helical) structure of capsular polysaccharides has not been established. However, similar, order-disorder transitions have been detected in a number of (112) D. A. Rees, Biochem. I., 126 (1972) 257-273. (113) D. A. Rees, M T P Int. Reu. Sci., Org. Chem., Ser. One, 7 (1973) 251-283; M T P Int. Ret;. Sci., Biochem., Ser. One, 5 (1975) 1-42. (114) E. R. Morris, D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke,J. MoZ. Biol., 110 (1977) 1-16.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

179

the capsular polysaccharides from Klebsiella, which would suggest that these polysaccharides have some helical character in solution at lower temperature^."^ Helical structures for some of these polysaccharides have also been reported from study of their X-ray fiber, diffraction pattern^.'^^,"^ Interestingly, the K8 polysaccharide has a two-step, temperature transition, the first step of which is due to the breaking of interactions between its terminal Dglucosyluronic acid groups and its backbone saccharides that results in a change in the backbone conformation of the poiysaccharide.l15 Although helical structure in solution has not been demonstrated for capsular polysaccharides associated with human vaccines, it has been identified in oriented fibers and films of the capsular polysa~charides."~,~~~ In an X-ray fiber diffraction study, the pneumococcal type 3 polysaccharide was found to exist in an extended, left-hand, helical conformation, hydrogen bonding being involved in maintenance of this secondary structure."* This was confirmed by X-ray studies of oriented films of both the types 3 and 8 pneumococcal poly~accharides."~The former exists as a two-fold helix, and the latter as a three-fold helix. Extended conformations in solution have also been assigned to the groups A (Ref. 33), X (Ref. 33), and Z (Ref. 36) polysaccharides of N. meningitidis on the basis of large, three-bond (31P-13C)coupling-constants in their 13C-n.m.r. spectra, but the presence of any helical content in solutions of these polysaccharides has not been established. Laser light-scattering techniques were applied to the group C meningococcal polysaccharide, and these studies indicated that it behaves like a random coil in solution.lZ0This conclusion is consistent with data obtained from 13Cn.m.r.-relaxation studies on the same polysaccharide that indicated32 that a fair degree of flexibility is exhibited by the group C polysaccharide in solution. This situation could prove to be representative of the majority of capsular polysaccharides in solution, although, as in the case of amylosic chain conformations,121the occurrence of regions of helical content in these polysaccharides is also a distinct possibility. (115)C. Wolf, V. Elsasser-Beile, S. Stirm, G. G . S. Dutton, and W. Burchard, Bio-

polymers, 17 (1978) 731-748. (1161 D. H. Isaac, K. H. Gardner, E. D. T. Atkins, V. Elsasser-Beile, and S. Stirm, Carbohydr. Res., 66 (1978)43-52. (117) D. H. Isaac, E. D. T. Atkins, H. Niemann, and S. Stirm, I n t . ] . Biol. Macromol., 3 (1981) 135-139. (118) R. H. Marchessault, K. Imada, T. L. Bluhm, and P. R. Sundararajan, Carbohydr. Res., 83 (1980)287-302. (119) W. T. Winter and I. Adelsky, Biopolymers, 20 (1981) 2691-2694. (120) T. Tsunashima, K. Mom, B. Chu, and T.-Y. Lui, Biopolymers, 17 (1978)251-265. (121) R. C. Jordan, D. A. Brant, and A. Cesbo, Biopolymers, 17 (1978) 2617-2632.

180

HAROLD J. JENNINGS

Because interactions between antibodies and polysaccharides are restricted to comparatively small regions of the polysaccharides, the orientation of the glycosidic linkages between the individual saccharide units is a hndamental parameter; this is the linkage orientation,122,123 and is defined by the torsion angles (A4 and A+) between these saccharides. Although Se1alz4has shown that antibodies made to peptide sequences did not recognize the same sequences when they formed part of a protein helical structure, this type of conformational specificity is not the general rule for polysaccharides. In fact, regions of conformational similarity are found in polysaccharides of different structures, and this is manifested in the extensive, serological crossreactivity of polysaccharides (see Section V,6). These regions of conformational similarity have been demonstrated in X-ray studies of oriented films of the cross-reacting types 3 and 8 pneumococcal polysaccharide^."^ The common cellobiouronic acid unit adopts the same conformation in both polysaccharides, despite the fact that it is the repeating unit of the former and is separated by a 4-O-a-~glucopyranosyl-cu-D-galactopyranosyl spacer in the structure of the type 8 polysac'~~ that disaccharides charide (see Fig. 3). Rees and S k e r ~ - e t tshowed can generally be fitted into polysaccharide structures without significant changes in their torsion angles, and this is the basis of the use of

FIG. 3.-Conformations of the Types 3 (Lower) and 8 (Upper) Polysaccharides of Streptococcus pneumoniae, Showing the Common Disaccharide Unit. (122) D. A. Rees,J. Chem. SOC., B , (1969)217-226. (123) D. A. Rees,j. Chem. SOC., B , (1970)877-884. (124) M. Sela, B. Schecter, I. Schecter, and F. Barek, Cold Spring Harbor Symp. Quant. Biol., 32 (1967) 537-545. (125) D. A. Rees and R. J. Skerrett, Carbohydr. Res., 7 (1968) 334-348.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

181

hard-~phere,'~~-'~' and other, more sophisticated, calculations,128in predicting the conformations of homog1ucans,lz6diheteroglycans,126 and even more-complex, saccharide sequence^.^^^^^^^ Not all disaccharides maintain their torsion angles in different structural environments, however, and this results in the type of conformationally controlled, and highly serologically-specific, determinants demonstrated by Jennings and coworkers111J31 in the type 111 capsular polysaccharide of group B Streptococcus. The repeating unit of this capsular polysaccharide is shown in Fig. 4. The conformation of the determinant of this polysaccharide, responsible for the population of antibodies involved in the protection of humans against type I11 group B streptococcal infections, is dependent on its terminal sialic acid groups, even though these are not immunodominant (see Section V,4) I I

I

.'

/O

FIG.4.-Proposed Conformation of the Repeating Unit of the Type I11 Polysaccharide Antigen of Group B Streptococcus. (126) D. A. Rees,]. Chem. SOC., B, (1969) 217-226. (127) D. A. Rees and W. E. Scott,J. Chem. SOC., (1971) 469-479. (128) D. A. Rees and P. J. C. Smith,]. Chem. SOC., Perkin Trans. 2, (1975)836-840. (129) R. U. Lemieux, K. Bock, L. T. Delbaere, S. Koto, and V. S. Rao, Can.]. Chern., 58 (1980) 631-653. (130) K. Bock, S. Josephson, and D. R. Bundle,]. Chem. SOC., Perkin Trans. 2, (1982) 59-70. (131) H. J. Jennings, C. Lugowski, K.-G. Rosell, and D. L. Kasper, in D. A. Brant (Ed.), Solution Properties of Polysaccharides, A.C.S. Symp. Ser., 150, American Chemical Society, Washington, D. C., 1980, pp. 161-172.

182

HAROLD J. JENNINCS

and are, most probably, not even a part of the determinant. By using '"C-n.m.r.-spectroscopicdata on the native, and modified-native, type I11 polysaccharides, it was possible to determine that the terminal sialie acid groups exert conformational control over the torsion angles of the penultimate 2-acetamido-2-deoxy4O-~-D-ga~actopyranosy~-~-Dglucopyranosyl unit of the polysaccharide. It is possible that this control is achieved by interactions (possibly hydrogen bonding) between these terminal sialic acid groups and the backbone of the type I11 polysaccharide. It is also possible, and, indeed, probable, that substituent groups smaller than sialic acid groups, such as 0-acetyl, pyruvate, and phosphate, could also confer immunospecificity in polysaccharide determinants by a similar mechanism. 4. Molecular Size One of the most important physical parameters in the effectiveness of capsular polysaccharides as vaccines is their molecular size. This fact was discovered b y Kabat and B e ~ e r ,who ' ~ ~ demonstrated that the molecular weight of native dextrans that are highly immunogenic in man is of the order of several million. They then proceeded to ascertain the dextran of lowest molecular weight that could still retain its immunogenicity; this was achieved by monitoring the increase in serum-antibody titers following the injection of humans with dextrans having different ranges of molecular weight. They found that, in the molecular weight range of 90,OOO and above, the dextrans remained excellent immunogens, whereas, at values of 50,000and below, they exhibited poor immunogenicity. In subsequent, similar experiments on the pneumococcal, type 3 capsular polysaccharide, Howard and cow o r k e r ~ injected '~~ fractions of different molecular weight of the type 3 polysaccharide into mice; the immunogenicity of each fraction was determined by monitoring the number of plaque-forming cells in the spleens of the mice. It was found that the number of such cells was directly related to the molecular size of the fraction; the native polysaccharide was easily the most effective immunogen. In studies on the capsular polysaccharides of N . meningitidis, Gotschlich and coworkersznwere able to obtain the group A and C polysaccharides in their high-molecular-weight form by precipitation directly from the liquid culture by means of Cetavlon. These highmolecular-weight polysaccharides were highly immunogenic in man. However, the group A polysaccharide isolated from cultures concen(132) E. A. Kabat and A. E. Bezer,Arch. Biochem. Biophys., 78 (1958)306-310. (133) J. G . Howard, H. Zola, G. H. Christie, and B. M. Courtenay,]. Immunol., 21 (1971)535-546.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

183

trated by rotary evaporation had lower molecular weights (less than 50,000), and proved to be non-immunogenic in humans. This depolymerization of the group A polysaccharide was attributed to the action of specific enzymes in the culture medium during evaporation. Lui and coworkerslMthen made the interesting and important observation that estimations of the molecular size of the group A polysaccharide by reducing end-group analysis were considerably lower than those obtained by gel filtration; this phenomenon was also reported ~ ~ the earlier for the group X polysaccharide of N . m e n i n g i t i d i ~ . 'On basis of their results, Lui and coworkers postulated134that some form of aggregation was occurring between individual polysaccharide chains, resulting in a macromolecular structure, and that lipid components could be responsible for this aggregation. In subsequent experiments by Gotschlich and coworkers,136aggregation was found to occur in the groups A, B, and C capsular polysaccharides of N. meningitidis and in the type K92 polysaccharide of E. coli. Experiments were then carried out to determine the nature of this aggregation. They showed,136as previously postulated, that a small proportion of lipoidal material was attached to all of these polysaccharides (8040% of di-0-palmitoylglycerol and 10-20% of di-0stearoylglycerol), and that these diO-acylglycerols were glycosylically attached to the reducing eqd of the polysaccharides by phosphoric diester bonds (see Fig. 5). This small proportion of lipid was sufficient to impart micellar behavior to the individual chains of the polysaccharides. These results couid be significant in our perceptions of capsular polysaccharides, in that an apparently minor component can have such a profound effect on their physical (molecular size) and immunological (immunogenicity) properties. In addition, this minor, lipoidal component could be the entity by which these polysaccharides are actually attached to the outer membrane of the bacterium (see Section 111,5).

5. Location

The importance of capsular polysaccharides in the immune response to bacterial infection is due to their location on the outer surface of the bacteria. They are at the interface of the many host-bacte(134) T.-Y. Lui, E. C. Gotschlich, W. Egan, and J. B. Robbins,]. Inject. Dis., Suppl., 136 (1977) S71-S77. (135) D. R. Bundle, H. J. Jennings, and C. P. Kenny,]. Biol. Chem., 249 (1974)47974801. (136) E. C. Gotschlich, B. A. Fraser, 0.Nashimura, J. B. Robbins, and T.-Y.Lui,J. Biol. Chem., 256 (1981) 8915-8921.

r

1

Ac AC

0

FIG.5.-Proposed Structure of the Lipid Functional Group of the Group C Meningococcal Polysaccharide.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

185

ria interactions, and constitute the principal antigens in most of the pathogenic, Gram-negative and Gram-positive organisms. However, the outer membranes of encapsulated bacteria are complex, and other can also play antigens, such as protein^^"^^ and lipopolysaccharide~,~~~ a minor, but important, role in the human immune-response to bacterial infection. Physical experiments have established the surface location of capsular polysaccharides on bacteria; from early experiments, previously reviewed,19using the optical microscope to visibilize the interactions of capsules and specific antibody (quellung reaction) with India ink, to improved techniques using electron microscopy to study the latter intera~ti0ns.l~~ Electron microscopy has also been used to study the capsules of bacteria following the incubation of the bacteria with ferritin-conjugated, specific antibody.140When applied to the group B Streptococcus, the surface location of the type Ia, Ib, Ic, 11, and 111 capsules was experimentally confirmed; types Ib and Ic also have additional, surface-protein antigens.141 The definition of a capsular polysaccharide is arbitrary; it is generally described as an envelope of mucilaginous material surrounding the bacterium. A fundamental question concerning these capsules is whether they are actually attached to the bacteria. Although the detection of free, capsular polysaccharide in culture filtrates is indicative of only weak forces of attraction, this conclusion need not necessarily be correct, as the release of capsular polysaccharide from the bacteria could be due to enzymic activity.142Evidence that covalent bonding could be involved in the attachment of some capsular polysaccharides to bacteria was reported by Tai and coworkers61;they had to use muralytic enzymes in order to isolate the type Ib, group B streptococcal polysaccharide. The identification of end-group diO-acylglycerol phosphate moieties on the group A, B, and C meningococcal and type K92 E. coli polysaccharides has also raised the possibility that these esters could be involved in the anchoring of the capsular polysaccharides to the outer membranes of their respective bacteria.13s Endgroup phosphoric esters have also been detected, by 31P-n.m.r. spectroscopy, in the H. injuenzae capsular polysaccharides; these esters (137) T. M. Buchanan, in M. Inouye (Ed.), Bacterial Outer Membranes, Wiley, New York, 1979, pp. 475-514. (138) H. J. Jennings,A. K. Bhattacharjee, L. Kenne, C. P. Kenny, and G. Calver, Con.J . Biochem., 58 (1980) 128-136. (139) M. E. Bayer and H. Thurow.]. Bacterial., 130 (1977) 911-936. (140) J. Swanson, K. C. Hsu, and E. C. Gotschlich,]. Exp. Med., 130 (1969) 1063-1075. (141) D. L. Kasper and C. J. Baker,]. Infect. Dis., 139 (1979) 147-151. (142) F. A. Troy, Annu. Reu. Microbial., 33 (1979) 519-560.

186

HAROLD J . JENNINGS

could have been part of an original linkage to the outer membrane of the bacteria.143 Iv. IMMUNE RESPONSE TO BACTERIALINFECTION

In order to understand the function of capsular polysaccharides as human vaccines, it is necessary to describe the human immuneresponse to infections caused by encapsulated bacteria. This response consists of a highly complex interplay of different cells and molecular components, and, of necessity, will only be described in an abbreviated and simplified way. More-extensive information on this subject can be obtained in books and review^.'^^-^^^ Once micro-organisms have penetrated subepithelial tissue and have invaded the human circulatory system, the immune mechanism is the last line of defense against proliferation of the bacteria and the eventual establishment of the disease state in the host. The fact that infections are common indicates that the host defenses do not constitute an impenetrable barrier for micro-organisms. In fact, we are all engaged in a constant struggle with invasive bacteria, a struggle in which various strategies employed by micro-organisms play an important role. As surface components of the bacteria, capsular polysaccharides are implicated in the complex, host uerszis micro-organism interactions, and, in particular, are responsible both for the stimulation of the human immune-system against the invading bacteria and for the virulence of the encapsulated bacteria (see Section V1,l). Two excellent reviews have been written on the intriguing subject of bacterial strategy and the human, immunological-defense ~ y s t e m . ~ " The * ' ~ ~process of eliminating the invading, encapsulated bacteria from the circulatory system is based on three factors; phagocytosis, the activation of complement, and the production of humoral antibodies. Cell-mediated immunity, as opposed to humoral immunity, is less important in the critical, acute and early stages of infections due to encapsulated bacteria, but is probably important in long-term immunity. (143)W.Egan, H. Sclineerson, K. E. Werner, and G. ZonJ. Am. Chem. Soc., 104 (1982) 2898-2910. (144) 31. C. Raff; Nature, 242 (1973) 19-23. (145) E. S. Golub, The Cellular Basis of the Immune Response, Sinauer Assocs., Sunderland, Mass., 1977. 1146) W. E. Paul, in J. B. Robbins, R. E. Horton, and E. M. Krause, New Approaches f o r Inducing Natural Immunity to Pyogenic Organisms, Proceedings of a Symposium, DHEW Publ., No. (NIH) 74553. (147) P. J. Baker and B. Prescott, in J. A. Rudbach and P. J. Baker (Eds.),Development in Immunology, Vol. 2, Elsevier-North Holland, New York, 1979, pp. 67-104. (148) P. Densen and G. L. Mandell, Rev. Infect. Dis., 2 (1980)817-838.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

187

1. Phagocytosis The engulfment and digestion of micro-organisms, termed phagocytosis, is the function of specialized cells in the human circulatory system.149J50 These cells consist of two major types, the largest and most complex of which are the macrophages. The macrophages constantly monitor the subepithelial tissues and all circulatory fluids, and have an important, immediate function, in that they are able to adhere directly to microbes by some kind of rather primitive, recognition mechanism. They are also able to adhere to microbes by a more specific recognition mechanism involving the prior coating of the bacteria with specific antibody, or the third component (C3) of complement. These molecules function as ligands between the macrophages and the bacteria by adhering to specific, macrophage receptor-sites to the Fc portion of the antibody, or to the C3 component of complement. Macrophages are also important in cell-mediated immunity and in the instigation of the immune response to invading micro-organisms. They achieve this by stimulating antibody-producing cells (see Section IV,3) to produce antibodies having specificities for surface components of micro-organisms to which the macrophages have previousIy adhered. Polymorphs are small, less complex cells that are of extreme importance to the immune system, as they are highly specific and short-lived, and can be rapidly produced by the body and delivered to the tissues by chemotactic response. Like the macrophages, they operate in association with complement and antibody through their respective C3 and Fc receptors. 2. Role of Complement The complement system has been r e v i e ~ e d ' ~ it~ is ~ 'composed ~~; of a series of proteins, Cl-C9, present in normal human serum, that serve as important mediators in the host defense. The terminal components, C3-C9, are involved in the destruction of invading microorganisms, but, in order to achieve this, they have to be activated. This activation process can be divided into two pathways, the alternative pathway and the classical pathway, although both pathways can occur simultaneously in the host defense-mechanism. Surface carbohydrates of micro-organisms are able to activate the al(149)C.A. Mims, The Pathogenesis of Infectious Disease, Academic Press, New York, 1977. (150) M. J. Taussig, Processes in Pathology, Blackwell, Oxford, 1979. (151)H. J. Muller-Eberhard and R. D. Schrieber,Adu. Zmmunol., 29 (1980)1-53. (152)J. A. Winkelstein, Reu. Infect. Dis., 3 (1981)289-298.

188

HAROLD J. JENNINGS

ternative pathway, directly generating C3 convertase activity; those important to human immunity to bacterial infection are the cell-wall mucopeptide of Gram-positive o r g a n i ~ m s , ~in ~ ~which - l ~ ~the teichoic acid moiety is probably f ~ n c t i o n a l , ' ~and * ' ~ the ~ lipopolysaccharides of Gram-negative organisms.'58 The convertase splits C3, to give C3b, which binds to the microbe, and a chemotactic agent that serves to attract polymorphs. The polymorphs then migrate towards the site of infection, and are able to adhere readily to the C3b-coated microbes, because of their surface C3b-receptors. The exact, structural basis of the activation of the alternative pathway is as yet but little understood, but it performs an important, immediate function in the destruction of invading microbes, because of the speed at which this defense mechanism can be deployed as compared to the classical pathway. Although antibody is not required for the activation of the alternative pathway, there is evidence to show that it can participate functionally in this mechanism.15z The classical pathway generally requires the presence of antibody ( 1 s ) having a specificity for a surface component of the bacterium, in order that activation may occur. The antibody adheres to the bacterium, and the resulting, immune complex, through a conformational change in the Fc portion of the antibody molecule, activates the first component of complement (Cl).This, in turn, activates C2 and C4, to yield C3b from C3, in which respect, this pathway now converges with that of the alternative pathway. In fact, the generation of C3b at this stage can lead to the activation of the alternative pathway.152Interestingly, in a few isolated cases, the classical pathway can be activated without the participation of antibody by some molecular structures; this has been reported for the lipid A moiety of lipop~lysaccharides,~~~ for a polysaccharide found in ant venom,'6oand for some synthetic oligosaccharides when linked to an 8-methoxycarbonyloctanol carrier.161 (153) J. A. Winkelstein and A. Tomasz,]. Zmmunol., 118 (1977)451-454. (154) P. H. Quinn, F. J. Crossan, J. A. Winkelstein, and E. R. Moxon, Inject. Zmmun., 16 (1977)400-402. (155) J. W. Tauher, M. J. Polley, and J. B. Zabriskie,J. E r p . Med., 143 (1976) 13521366. (156) J. A. Winkelstein and A. Tomasz,J. Zmmunol., 120 (1978) 174-178. (157) B. A. Fiedel and R. W. Jackson, Infect. Zmmun., 22 (1978)286-287. (158) C. Galanos and 0. Luderitz, Eur. J . Biochem., 65 (1976)403-408. (159) D. C. Morrison and L. F. Kline,]. Zmmunol., 118 (1977)362-368. (160) D. R. Schultz, P. I. Arnold, M . X . Wu, T. M. Lo, J. E. Volkanakis, and M. Loos, Mol. Zmmunol., 16 (1979) 253-264. (161) D. R. SchuIk and P. I. Amold,J. Zmmunol., 126 (1981) 1994-1998.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

189

Capsular polysaccharides are actively involved in the mediation of complement, in that they are able to suppress the activation of the immediate, alternative-pathway mechanism, thus forcing the immune system to use the classical pathway; this is an important factor in the virulence of bacteria (see Section VIJ).

3. Humoral Antibodies to Polysaccharide Vaccines

An important distinction must be made between the humoral response to a pure, capsular polysaccharide, and to the same polysaccharide when it is an integral part of the bacterium. Thus, the immunity received on recovery from infection by encapsulated bacteria, in terms of the polysaccharide antigen, differs from that generated by purposeful immunization with purified capsular-polysaccharide vaccines. Fortunately, with the exception of infants, the polysaccharide vaccines still stimulate protective-antibody levels in humans, despite these differences. In infants, due to the immature nature of their immune systems, these polysaccharide vaccines are of only marginal benefit.7 Some insights into the nature of these different responses in humans can be found in studies on the cellular basis of the immune response to polysaccharides. However, for the purposes of this Chapter, it would be inappropriate to provide a lengthy description of this incompletely understood mechanism; in-depth reviews of this burgeoning field of research can be referred t ~ . ~ ~ - ~ ~ ~ , ~ ~ For most antigens, the production of antibody (immunoglobulin) is based on the cooperative interaction of two types of lymphocyte, called T-cells (thymus-derived) and B-cells (bone marrow-derived). The T-cells, preprimed with macrophage-presented antigen, stimulate the B-cells to secrete copious quantities of antibody. However, on the basis of animal studies, such polysaccharide antigens as the type 111 pneumococcal polysaccharide have been considered to be T-cellindependent, as they are capable of triggering B-cells to produce antibody (IgM) in T-cell-deficient mice.16' These studies also indicated (162) P. J. Baker, H. C. Morse, S. C. Gross, P. W. Stashak, and B. PrescottJ. Infect. Dis., Suppl., 136 (1977) s20-~24. (163) D . E. Mosier, N. M. Zidivar, E. Goldings, J. Mond, 1. Sher, and W. E. Paul, J . Infect. Dis., Suppl., 136 (1977) s14-s20. (164) H. Braley-Mullen, Immunology, 40 (1980) 521-527. (165) E. C. Gotschlich, I. M . Goldschneider, M. L. Lepow, and R. Gold, in E. Huber and R. M. Krause (Eds.),Antibodies in Human Diagnosis and Therapy, Raven Press, New York, 1977, pp. 391-402. (166) P. J. Baker, D. F. Amsbaugh, P. W. Stashak, G. Caldes, and B. Prescott, Reu. Infect. Dis., 3 (1981) 332-341. (167) J. G. Howard, G. H. Christie, B. M. Courtenay, E. Leuchars, and A. J. S. Davies, Cell. Immunol., 2 (1971)614-626.

190

HAROLD J . JENNINGS

that the same type of antibody (IgM) is produced in normal mice by the same polysaccharide z ~ n t i g e n . ' ~ ~ - ' ~ ~ The majority of antibodies can be broadly classified into three main categories ( I s , IgM, and I d ) on the basis of their different structure and functions in the immune response, and IgG is the most important type of antibody in promoting the classical pathway of the complement system."O Further experiments in mice demonstrated that, although pol ysaccharides are capable of functioning as T-cell-independent antigens in athymic mice, in normal mice, polysaccharides do stimulate T-cell 'activity, although the T-cell response differs markedly (more restricted) from that generated by the injection of mice with whole b a ~ t e r i a . 'T-Cells ~ ~ , ~ ~can ~ modulate the immune response in mice b y either an effector or a suppressor mechanism, and, unlike the situation for the whole bacteria, polysaccharides cause the suppressor mechanism to be d~rninant.'~'~''~ Also, unlike the response of whole bacteria in mice, polysaccharide antigens fail to induce a memory (amnestic) re~ponse.'~" If the results of the foregoing experiments in mice were projected to the human situation in general, the use of polysaccharides as efficacious, human vaccines would show little promise. However, the immunological response to polysaccharides is species-dependent, and, in humans, with the exception of young infants, a fuller range of antibody types is produced. Thus, polysaccharides stimulate the production of IgG antibodies in humans, in addition both to IgM and IgA ant i b ~ d i e s , 'but, ~ ~ as in the mouse experiments, they fail to exhibit a sizable, amnestic response to subsequent, booster injection^.^ Although the presence of this effect would be a decided advantage in immunoprophylaxis, it is not detrimental, because efficacious, antibody levels in humans are maintained for up to 8 years follbwing the injection of pneumococcal poiysa~charides.'~~ Human infants have immature, immune systems in relation to polysaccharide vaccines, (168) B. Anderson and €1.Blombren, Cell. Zmmunol., 2 (1971) 411-424. (169) C . F. Mitchell, F. C. Grumet, and H. D. McDevittJ. E x p . Med., 135 (1972) 126135. (170) E. C. Gotschlich, I . M. Coldschneider, and M. S. Artenstein,J. E x p . Med., 129 (1969) 1367- 1384. (171) P. J. Baker, N. D . Reed, P. W. Stashak, D. F. Amsbaugh, and B. Prescott,]. E x p . Med., 137 (1973) 1431-1441. (172) P. J . Baker, T r Q n S p h f .Rer;., 26 (1975) 3-20. (173) A. Basten and J. G. Howard, Contemp. T o p . Zmmunobiol., 2 (1973)265-291. (174) W. J . Yount, M. M. Domer, H. J. Kunkel, and E. A. Kabac]. E x p . Med., 127 (1968) 633-646. [175) M. Heidelberger, M. M. Dilapi, M. Siegel, and A. W. Walter,]. lmmunol., 65 (1950) 535-541.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

191

and do not produce the necessary IgG antibodies (see Sections V,5 and 6). This behavior is similar to that exhibited by mice, and, if it is permissible (not yet proved) to use these experiments in mice as models, this immaturity could be attributed to T-suppressor-cell functi~n.’~~

v. POLYSACCHAFUDE VACCINES AND

IMMUNITY

1. Streptococcus pneumoniae

Pneumonia was, and still remains, among the leading causes of death in the United States. It is responsible for lower-respiratory-tract infections in humans, and is also the most common cause of otitis media (a bacterial infection of the middle ear) in children. The high rate of mortality caused by this disease prompted a search for a preventive approach to its control, an investigation in which the pneumococcal, capsular polysaccharides were the first, purified polysaccharides to be used as human vaccine^.^*'^^*'^^ This followed directly from the discovery by Francis and TilleP that the intradermal injection of type 1 and 2 pneumococcal polysaccharides induced serum antibodies in humans. These results led to the demonstration by Heidelberger and his associates: in a large field-trial under epidemic conditions, that a vaccine composed of types 1,2,5,and 7 polysaccharides is efficacious against disease caused by S. pneumoniae. These studies also confirmed the type-specific protection induced in humans by these capsular polysaccharides. Other successful field-trials were subsequently carried out; in one of these, Heidelberger and demonstrated that a hexavalent, polysaccharide vaccine administered in a single injection induced the corresponding, satisfactory, serotypeantibody levels, which persisted for up to 8 years.175This success rapidly led to the commercial licensing of hexavalent, pneumococcalpolysaccharide vaccines. However, interest in the prophylaxis of pneumococcal pneumonia waned at this time, due to the advent of the “sulfa” drugs, and then the unprecedented success of antibiotic therapy. This development engendered such a complacent attitude towards pneumococcal infections that even the accurate serotyping of disease isolates was discontinued in most medical centers. However, subsequent epidemiological studies of pneumococcal pneumonia by conclusively showed that, despite the success of antibi(176) R. Austrian, Reu. Infect. Dis., Suppl., 3 (1981) sl-sl7. (177) M. Heidelberger, C. M. McLeod, and M. M. DilapiJ. E r p . Med., 88 (1948)369372. (178) R. Austrian, Am. J . Med. Sci., 57 (1959) 133-139.

192

HAROLD J. JENNINGS

otic therapy, the disease occurs with the same frequency, and the same mortality rate, as in the pre-antibiotic era. This fact, together with the emergence of antibiotic-resistant strains: led to consideration of reviving the preventative approach to control of the disease; the eventual development and licensing of a pneumococcal-polysaccharide vaccine179gave fruition to the earlier, interrupted research. Because of the diversity of pneumococcal-capsular types (84 have so far been identified), and a reluctance to use them all in a single, multivalent vaccine, the final composition of the vaccine was the result of a compromise in which the number of serotype polysaccharides was limited to fourteen, presumably with minimum loss of effective coverage. This decision was based on epidemiological s t ~ d i e s , ' ~con~,'~~ ducted in the United States, which indicated that 80-9O% of pneumococcal, bacteremic infections are caused by fourteen serotypes (see Table V). However, because of the restricted nature of these epidemiological studies, this vaccine can only be regarded as a core vaccine to which other serotypes may have to be eventually added. This flexibility will probably be required, in order to allow for geographical variations in pneumococcal serotypes, for time-related changes in the prevalence of serotypes in disease isolates, and, also, for the possibility of the tailoring of pneumococcal vaccines in response to agerelated, epidemiological studies (infant ~accine).'.''~ A further factor that enabled the limitation of the number of serotypes used in the vaccine was the occurrence of some structural homology in the pneumococcal polysaccharides, resulting in extensive cross-reactions and cross-immunity in their serological properties. This is exemplified in the Danish serotyping system, which designates capsular types within groups, based on this cross-reactivity. For example, the two types recognized as 6A (Ref. 86) and 6B (Ref. 87), that is, types 6 and 26 in the U. S. system, differ structurally by only one linkage position in a tetrasaccharide phosphate repeating-unit, the a-L-rhamnopyranosyl residues of type 6B being linked to 04 of the ribitol residues instead of to 03 of the same residues, as in the case of type 6A (see Table IV).This structural similarity, accompanied by a degree of conformational retention in the structure, allows for the production of antibodies common to both types. Because of this crossreactivity (see Section V,6), only type 6A is used in the vaccine. Other significant cross-reactions that occur within the polysaccharides used in the vaccine are types 19F (Refs 92 and 93) and 19A (Ref. 94),of which, type 19F is used in the vaccine, and types 9A (Ref. 83), 9V (Ref. (179) R. E. Weibel, P. P. Vella, A. A. McLean, A. F. Woodhour, W. L. Davidson, and M. R. Hilleman, Proc. SOC. E x p . Biol. Med., 156 (1977) 144-150.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

193

TABLEV Distribution of Pneumococcal Types Responsible for Bacteremic Infection in the United States During 1968-1973 Pneumococcal type" 1 2 3 4 5 6A (6) 7 8 9N (9) 12 14 18 19F (19) =F (23) All other Total

Number of isolates

Percentage of isolates

293 10 237 320 70 160 213 325 132 182 2.40 159 134 117

9.1 0.3 7.3 9.9 2.2 5.0 6.7 10.1 4.1 5.6 7.4 4.9 4.2 3.6 19.6 100.0

633 3225

American type-designation is given in parentheses.

85), and 9L and 9N (Ref. 84), of which, type 9N is used in the vaccine (see Table V). The choice of these serotypes (6A, 19F, and 9N) was made because, of all the serotypes in each cross-reacting group, these occur more frequently in disease isolates. 2. Neisseria nzeningitidis

Meningococcal disease (purulent meningitis) commonly occurs in children, but is also observed in adults. Without antibiotic treatment, the mortality rate is high (85%), and, even with this treatment, cured patients can suffer serious and permanent neurological deficiencies.ls5 These facts, together with the emergence of antibiotic-resistant strains: prompted the rapid development of a commercial vaccine. This vaccine was developed almost simultaneously with the pneumococcal vaccine. In contrast to the pneumococcal vaccine, however, the composition of the meningococcal vaccine was greatly simplified, due to the fact that fewer polysaccharides were required. Based on their capsular polysaccharides, there are only eight different serogroups of N.meningitidis (A, B, C, 29e, W-135, X,Y,and Z), of which, groups A, B, and

194

HAROLD J. JENNINGS

C account for more than 90% of meningococcal d i s e a ~ e . ' ~The ~-~*~ high-molecular-weight, group A and C polysaccharides raise titers of

bactericidal antibody in adults,16j although, in young children, their use has been only marginally (see Section V,5). The group A and C polysaccharide vaccines have also been used in numerous, successful, human field-trials.165-1R' Interestingly, because of the lack of a suitable animal model in which to test the efficacy of these vaccines, the standards for their licensure and release were, for the first time, based purely on physiochemical criteria. However, one major problem in the design of a comprehensive, trivalent pol ysaccharide, meningococcal vaccine inclusive of group B is the poor immunogenicity of the group B polysaccharide in man.lW2 Two major reasons have been proposed to account for this phenomenon. One is that the rr-~-(2-+8)-linkedsialic acid homopolymer is rapidly depolymerized in human tissue, because of the action of neuraminidase; the other is that this structure is recognized as "self" by the human immune-system, and, in consequence, the production of antibody having a specificity for this structure is suppressed. The weight of evidence is in favor of the latter explanation. A neuraminidase-sensitive variant of the group C polysaccharide [an a-D-(2+9)-linked, sialic acid homopolymer] having no 0-acetyl groupsIR3still proved to be highly immunogenic in man.Is4In addition, in experiments using tetanus toxoid conjugates of the group B polysaccharide, it was demonstrated that, when conjugated at the nonreducing sialic acid group (thereby producing a neuraminidase-resistant, group B polymer), its immunogenicity was not enhanced.185Finally, the Escherichia coli K92 capsular polysaccharide contains alternating sequences of CPD( 2 4 ) -and -(2-+9)-linked sialic acid.1*6This polysaccharide proved to be immunogenic, but produced only antibodies that cross-reacted with the group C polysaccharide [a-~-(2+9)-linked]. The immune mechanism avoids the production of antibody having a specificity for the cy-1)-(2+8)linkage.'"*'Rg (180) E. C. Gotschlich, in Ref. 10, pp. 91-101. (181) R. Gold, M. L. Lepow, I. M. Goldschneider. and E. C. Gotschlich,]. Infect. Dis., S U ? I ~ >136 ~ . , (1977) S31-S35. (182) F . A. Wyle, M. S. Artenstein, D. L. Brandt, D. L. Tramont, D. L. Kasper, P. Altieri, S. L. Berman, and J. P. Lowenthal,]. Infect. Dis., 126 (1972)514-522. (183) M . A. Apicellq]. Infect. Dis.,129 (1974) 147-153. (1%) s.1. P. Glode,E. B. Lewin,A. Sutton,C. T. Le,E.C. Gotschlick,and J. B. Robbins, J . In-fect.Dis., 139 (1979)52-59. (185)H. J. Jennings and C. Lugowski,]. lmmunol., 127 (1981) 1011-1018. (186) W. Egan,T.-Y. Lui, D. Dorow, J. S. Cohen, J. D. Robbins, E. C. Gotschlich, and J. B. Robbins, Biochemistry, 16 (1977)3687-3692. .

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

195

Attempts to surmount this problem have included the use of ( a ) other surface-components of the group B organism (type-specific prot e i n ~ ) , ' ~(b) ~ . 'group ~ ~ B polysaccharide conjugates (see Section V,5), and (c) cross-reacting polysaccharides as alternative vaccines. In the last category, a proposal has been made to use a cross-reacting, E. coli p o l y s a c ~ h a r i d eThe . ~ ~ E. ~ coli K1 polysaccharide is ~ t r u c t u r a l l y 3 ~ ~ ~ ~ and serologically'" identical to the meningococcal group B polysaccharide (see Section V,6), but a form variant (OAc+)of this organism was isolated that produces a polysaccharide randomly 0-acetylated at 0-7 and 0-9 of its sialic acid residues.lgl This form variant (OAc+)of the E. coli K1 organism is more immunogenic in rabbits than the OAcvariant, and produces antibodies having specificities for both the 0acetylated and the nonacetylated polysa~charides.'~~ Human trials using the 0-acetylated K1 polysaccharide as a vaccine are now needed, in order to determine whether this approach to the problem will show any promise.

3. Haemophilus infiuenzae Although there are six capsular types of H. influenzae, the most serious disease is caused'9z by type b. This organism is the cause of meningitis, which occurs exclusively in infants, and even survivors of this disease can suffer severe, and permanent, neurological defect^.^ In consequence, an extensive amount of work has been dedicated to finding a vaccine for this organism, and the capsular polysaccharide was a prime candidate. Heidelberger and coworkers's3 demonstrated that protective antibodies in hyperimmune rabbit antisera could be removed by absorption with the purified H. influenzae type b polysaccharide. Since then, Anderson and coworkers,194and Parke and cow o r k e r ~ , 'were ~ ~ able to induce, in adults, long-lived, complement(187) C. E. Frasch and E. C. Gotschlich,]. E x p . Med., 140 (1974) 87-104. (188) W. D. Zollinger and R. E. Mandrell, Infect. Immun., 18 (1977)424-433. (189) J. B. Robbins, R. Schneerson, J. C. Parke, T.-Y. Lui, Z. T. Handzel, I. Brskov, and F. Brskov, in Ref. 10, pp. 103-120. (190) D. L. Kasper, J. L. Winkelhake, W. D. Zollinger, B. Brandf and M. S. Artenstein, J . Immunol., 110 (1973) 262-268. (191) F. Brskov, A. Sutton, R. Schneerson, L. Wenlu, W. Egan, G . E. Moff, and J. B. Robbins,J. E r p . Med., 149 (1979)669-685. (192) J. C. Parke, R. Schneerson, J. B. Robbins, and J. J. Schlesselman,J. Infect. Dis., S ~ p p l . 136 , (1977) S25-~30. (193) H. E. Alexander, M. Heideiberger, and G . Leidy, Yale 1. Biol. Med., 16 (1944) 425-438. (194) P. Anderson, G . Peter, R. B. Johnston, L. H. Wetterlow, and D. H. SrnithJ. Clin. Inoest., 51 (1972) 39-44.

196

HAROLD J . JENNINGS

mediated, bactericidal antibodies by using the purified type b polysaccharide as a vaccine. However, the development of this pure, polysaccharide vaccine was retarded when it was discovered that the p l y saccharide induced only short-lived immunity in older infants, and little or no protection in younger infants.lS5Current research to obviate this problem has focussed on the use of ( a ) type b polysaccharide-protein conjugates (see Section V,5) and (b) cross-reacting organisms. The latter approach would involve the deliberate colonization of infants with the non-pathogenic, cross-reacting E. coli (see Section V,6).

4. Group B Stseptococcw Group B Streptococcus is a major cause of bacterial meningitis in new-born infant^.'^^'^^ The organisms can be into four distinct serotypes (Ia, Ib, 11, and HI), of which, type I11 is the most important in human disease.Is7 The original isolation of the type 111 capsular polysaccharide was achieved by using acid-extraction proced u r e ~ ~ ~this . ~ resulted '; in the isolation of an immunologically incomplete, core antigen that originated from a native polysaccharide containing labile, terminal sialic acid residuesw The complete, native, type I11 antigen could be isolated by growing the type I11 organism under pH-controlled conditions and isolating the polysaccharide by mild extraction-procedures.62The core antigen is structurally identical to the capsular polysaccharide of type 14 S . pneumoniae,63and, on the basis of serological experiments in animals using the latter organism, it was suggested that antibodies to the core polysaccharide could be functional in the production of protective antibodies against type 111, group B Streptococcus organisms.1s8However, confirmatory evidence for the essential participation ofthe native, type I11 polysaccharide in the development of human immunity to the disease was obtained when it was demonstrated that, in human sera, only antibody directed to the native antigen correlated most highly with opsonic (bactericidal) a ~ t i v i t y . ~ ~ * ' ~ ' I n a disease that is restricted to the newborn, the use of immunoprophylaxis is impractical, due to the time lag before effective levels of (195) I>. H . Smith, G . Peter, D. L. Ungram., A. L. Harding, and P. Anderson, Pediatrics, 52 (1973) 637-645. (1%) T. C . Eickhoff, J . 0. Klein, A. K. Daly, D. Ingall, and M. Finland, New Engl. /. Med., 271 (1964) 1221-1228. (197) C. J. Baker,Ado. Intern. Med., 25 (1930) 475-499. (198) G. W.Fisher, G . M. Lowell, M. H. Cumrine, and J. W. Bass,]. E x p . Med., 148 (1978) 776-786.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

197

protective antibodies are produced. Therefore, a different vaccination strategy is envisaged for this disease, one in which the target population for the polysaccharide vaccine would be pregnant mothers deficient in antibodies specific for the native, type 111, group B streptococcal polysaccharide. The type I11 polysaccharide is immunogenic in and the baby could acquire immunity by the placental transfer of antibody ( 1 s ) . It has been demonstrated that babies born of mothers having high levels of type I11 polysaccharide-specific antibody are less liable to infectionse2than others. 5. Polysaccharide-Protein Conjugates

The utilization of the previously described, capsular polysaccharides as human vaccines is only partially successful, due to the fact that they are poor immunogens in young children. This situation is highly undesirable, as this section of the population experiences the highest incidence of disease (particularly meningitis) caused by these pathogenic bacteria.' The immunological basis of this phenomenon, discussed in Section IV,3, is the inability of young children to generate a mature and amnestic response involving the production of IgG antibodies to these purified-polysaccharide antigens. A possible solution would be to enhance the immunogenicity of these pure polysaccharides by converting them into thymus-dependent antigens. One method of achieving this objective would be to conjugate them to a protein carrier. The feasibility of this approach is well established. Fifty years ago, Goebel and AverylWcoupled the type 3 pneumococcal polysaccharide to horse serum-globulin by the diazotization of p aminobenzyl ether substituents on the polysaccharide. They demonstrated that this polysaccharide conjugate,2O0and a similar conjugate made with the oligosaccharide repeating-unit (cellobiouronic acid) of the type 3 pneumococcal polysaccharide,2°0were able to induce polysaccharide-specific antibody in rabbits previously unresponsive to the pure polysaccharide. GoebelzO1*zOz also established that the cellobiouronic acid conjugate was able to confer immunity to pneumococcal infection in mice. Other investigators confirmed these results by using the type 3 pneumococcal polysaccharide covalently linked to proteinzwand to erythrocytes,204and noncovalently linked in ionic as(199) W. F. Goebel and 0. T. Avery, ]. E x p . Med., 54 (1931)431-436. (200) 0. T. Avery and W. F. Goebel,]. E x p . Med., 54 (1931)437-447. (201) W. F. Goebel,]. Em. Med., 72 (1940)33-48. (202) W. F. Goebel,]. E r p . Med., 69 (1939)353-364. (203) W. E. Paul, D. H. Katz, and B. Benacerraf,]. lmmunol., 107 (1971)685-688. (204) H. Braley-Mullen,]. lmmunol., 113 (1974) 1909-1920.

198

HAROLD J. JENNINGS

sociation with methylated bovine serum albumin.205In some of these methods, the coupling techniques employed were far too drastic for the highly sensitive polysaccharides currently used as human vaccines. Also, the carrier proteins and the coupling methods employed in the synthesis of these conjugates resulted in the formation of conjugates having constituents or structural features (for example, aromatic groups) highly undesirable for use in human vaccines. More-comprehensive studies on polysaccharide-protein conjugates directed specifically to their use as human vaccines have now been reported; the development of simple, and efficient, coupling procedureszo6has resulted in the formation of linkages to compounds containing more-innocuous, and more-acceptable, structural features. The H. injuenzue type b polysaccharide was conjugated to a number of proteins by Schneerson and coworkers207by using an adipic dihydrazide spacer between the molecules. These conjugates were relatively nontoxic, and, in contrast to the pure polysaccharide, functioned as thymic-dependent (T-cell-dependent) antigens. They produced polysaccharide-specific, serum antibodies in mice and other animals, and the level of these antibodies could be augmented by reinjection of the conjugate. The group C meningococcal polysaccharide was also successfully converted into a thymic-dependent antigen b y Beauvery and coworkers,2°M who linked it directly through the carboxyl groups of its sialic acid residues to the amino groups of tetanus toxoid (amide linkages), using l-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The foregoing conjugation methods employed random activation of the many functional groups of the polysaccharides, and more-specific coupling was obtained in the formation of artificial SaZmoneZZa typhimurium and Pseudomonas aeruginosa vaccines. Svenson i n d Lindberg15*.209 synthesized the former by coupling the smaller molecularsize octa- and dodeca-saccharides, obtained by treatment of the 0chain of the Salmonella typhimurium lipopolysaccharide with phage enzymes, to bovine serum albumin. A carboxyl group, unique to the oligosaccharide, was generated on their reducing (end-group) rham(205) 0. J. Plescia, W. Braun, and N. C. Palczuk, Proc. Natl. Acad. Sci. U . S. A., 52 (1964)279-285. (206) C. P. Stowell and Y. C. Lee, Ado. Carbohydr. Chem. Biochem., 37 (1980) 225281. (207) R. Schneerson, 0.Barrena, A. Sutton, and J. B. Robbins,]. E r p . Med., 152 (1980) 361 -376. (208) E. C. Beauvery, F. Miedema, R. W. Van Delft, and J. Nagel, in J. B. Robbins, J. C. Hill, and J. C. Sadoff (Eds.), Seminars in Infectious Disease. Bacterial Vaccines, Vol. 4, Thieme-Stratton, New York, 1982, pp. 268-274. (209) S. B. Svenson and A. A. Lindberg, J . Immunol. Mkthods, 25 (1979)323-335.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

199

nose residues, through which they were linked to the protein by amide linkages by using 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride. The conjugates elicited good antibody responses in rabbit^,'^,^'^ but not in mice,2l0although it was demonstrated that the rabbit antibody was able passively to protect the mice against infection by live, homologous Salmonella organisms.210In subsequent work, Seid and Sadoff prepared a tetanus toxoid conjugate of the (larger molecular size) whole, base-treated lipopolysaccharide of type 5 Pseudomonas aeruginosa by the formation of amide linkages between the carboxyl groups of its few KDO residues and the amino groups of 1,4-diaminobutane spacers pre-attached to the protein. The conjugate proved considerably less toxic than the original lipopolysaccharide, and preliminary, immunological results indicated that IgG antibodies having a specificity for the 0-chain can be readily induced in mice by using this conjugate.211Except for the potentiaI loss of alkali-labile substituents on lipopolysaccharide 0-chains, this method could prove to be applicable to all bacterial lipopolysaccharides, and it has obvious potential in the synthesis of human vaccines. In an attempt to extend the monofunctional-group approach to the conjugation of the meningococcal polysaccharides, and thus to produce conjugates more chemically defined, Jennings and L ~ g o w s k i ' ~ ~ inserted a unique, terminal, free aldehyde group into the groups A, B, and C polysaccharides. This was achieved by controlled, periodate oxidation of the native, group B and C polysaccharides and of the group A polysaccharide premodified by reduction of its terminal, reducing 2-acetamido-2-deoxy-~-mannose residue (see Fig. 6). These monovalent molecules were then specifically coupled to tetanus toxoid by reductive amination, using sodium cyanoborohydride, without activating the other functional groups in the polysaccharide. When used as vaccines in mice and rabbits, the group A and C polysaccharide-tetanus toxoid conjugates produced high-titer antisera having bactericidal activity against the homologous organisms, indicating the potential of these conjugates as human vaccines. In contrast, the group B polysaccharide-tetanus toxoid conjugate failed to elicit detectable, polysaccharide-specific antibodies in these anim a l ~Inhibition . ~ ~ ~ experiments indicated that the antibody produced was not specific for the polysaccharide, but was highly specific for the linkage between the lysine residues of tetanus toxoid and the nonreducing (end-group) heptulosylonic acid group of the oxidized group B polysaccharide. (210) S. B. Svenson and A. A. Lindberg, Infect. lmmun., 32 (1981) 490-496. (211) R. C. Seid, Jr., and J. C. SadofT,/. B i d . Chem., 256 (1981) 7305-7310.

200

HAROLD J. JENNINGS

6 R=R'=H IR=R'=COCH, 8 R=H,R'=COCH, 9 R = COCH,, R' = H

1

L R = OCCH, or H

FIG.6.-Shucture ofthe Group C (uppermost),B (middle), and Terminally Reduced Group A (lowest) Polysaccharide Antigens OfNeisseria meningitidis, Depicting the Positions of Cleavage on Oxidation by Periodate.

6. Natural Immunity, and Polysaccharide Serological Cross-reactions Adult-animal sera contain antibodies to a variety of polysaccharides, including those of human pathogenic bacteria, indicating that, to confer immunity, disease is not required. Robbins and were able to detect antibodies having a specificity for Vibrio cholerue (212)J. B.Robbins, R. Schneerson,M. P. Glode, W. Vann,M. S. Schiffer,T.-Y. Lui, J. C . Parke, and C. Huntley,]. Cell. Clin. Zmmunol., 56 (1975)141-151.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

201

in animals, as the animals were allowed to mature without the possibility of contact with the homologous organism. In addition, in serological studies on a healthy human population, there is frequently detected the presence of antibodies having a specificity for the capsular polysaccharides of groups A, B, and C N. meningitidis and type b H. influenme that cannot be satisfactorily explained by asymptomatic Similar findings were reported for antibodies to the pneumococcal type 3 polysaccharide in children.213These populations of antibodies result from exposure to cross-reacting antigens among the nonpathogenic bacteria found in the nasopharyngeal or gastrointestinal Serological cross-reactions among polysaccharides are a well documented phenomenon, due in large part to the extensive work conducted in this area by Heidelberger and This phenomenon is due to the ability of polysaccharide antigens to promote the formation of heterogeneous populations of antibodies, and to the special property of polysaccharides to retain domains of structural and conformational similarity despite some structural differences. This is well illustrated by the cross-reactions exhibited between the different pneumococcal polysaccharides (see Sections III,3 and VJ). However, cross-reactions between polysaccharides from different species of other organisms have been used to great advantage in probing polysaccharide structures. Thus, an antiserum of one polysaccharide of known structure becomes a reagent to monitor for similar structural features in other polysaccharides. Heidelberger and coworkers have used this type of analysis extensively, and the results have constituted the subject of several reviews.108*214-219 In addition to its analytical value, this phenomenon is probably the basis of the important mechanism by which humans develop natural imrn~nity.~ In fact, it has also been postulated that exposure to these cross-reacting, T-cell-dependent organisms is the most satisfactory explanation for the eventual maturation of the polysaccharide immuneresponse in infants.165It can be shown, for instance, that there is an age-related increase in natural antibodies to the group A meningococcal polysaccharide in children, even though the group A organism is

(213) M. Finland,]. Infect. Dis., 128 (1973) 76-124. (214) M. Heidelberger, Res. lmmunochem. lmmunobiol., 3 (1973) 1-40. (215) M. Heidelberger, in J. B. G. Kwapinski (Ed.), Research in Zmmunochemistry, Vol. 3, University Park Press, Baltimore, 1973. (216) M. Heidelberger, Annu. Reo. Biochem., 36 (1967) 1-12. (217) J. M. Tyler and M. Heidelberger, Biochemistry, 7 (1968) 1384-1392. (218) M. Heidelberger and W. Nimmich,J. lmmunol., 109 (1972) 1337-1344. (219) M. Heidelberger and W. Nimmich, Immunochemistry, 13 (1976) 67-80.

202

HAROLD J. JENNINGS

rarely isolated in the United States.22oFrequently found in normal human flora are cross-reacting organisms that could be responsible for natural immunity to groups A, B, and C N. meningitidis and type b H. infiuenzae. Robbins and coworker^^*^^^ and Egan and coworkers222 identified some of these important organisms; they are listed in Table VI. Serological studies indicated that the capsular polysaccharides of these organisms are responsible for the cross-reactions, and this is confirmed by the structural similarity of some of these polysaccharides (see Table VI) to those from N. meningitidis (see Table I) and H. influenzcre (see Table 11). Schneerson and rob bin^^^^ clearly demonstrated the feasibility of this mechanism by deliberately feeding nonpathogenic E. coli possessing the K-100 capsule to human-adult volunteers, Colonization readily occurred, and antibodies specific for the H. infiuenzae type b polysaccharide were induced.

VI. BACTERIAL VIRULENCE

1. Role of the Capsular Polysaccharide We are constantly in contact with a wide range of micro-organisms in our external environment, but few of these prove to be virulent. The virulence of bacteria is dependent on their ability to invade the human host and to evade the host's immune system, thus allowing the bacteria to propagate within the host. The varied strategies employed by the bacteria to evade the host's immune system have been re~iewed.':'~.'*~ Although the mechanisms behind these different strategies are incompletely understood, research in this area is encouraging, and suggests that an understanding of the pathogenesis of infectious diseases at the molecular and macromolecular level will eventually be possible. Because of their surface location, capsular polysaccharides are important agents in bacterial pathogenesis, as they interact directly with the host's immune system. The initial event in the pathogenesis of most bacterial infections is the attachment of the bacteria to the mucosal surface. This probably occurs by a receptor mechanism that exhibits a high degree of cellular specificity. Capsular polysaccharides have not been implicated in this (220) I. M. Goldschneider, M. L. Lepow, E. C. Gotschlich, F. T. Mauck, F. Bache, and M. Randolph,]. Infect. Dis., 128 (1973)769-776. (221) J. B Robbins, R. L. Myerowitz, J. K.Whishant, M. Argaman, R. Schneerson, Z.T. Handzel, and E. C. Gotschlich, Infect. Zmmun., 6 (1972)651-656. (222) W. Egan, F.-P. Tsui, and H.Schneerson,]. Biol. Chem., submitted for publication. (223) R. S. Schneerson and J. B. Robbins, New Engl.]. Med., 292 (1975) 1093-1095.

TABLEVI

Polysaccharides of Bacteria, Frequently Found in Human Flora, That Cross-react with the PolysaccharideCapsules of Human Pathogenic Bacteria Pathogen Neisseria meningitidis Group A Croup B Group C

Cross-reacting organism Bacillus pumilis Streptococcus fecalis Escherichia coli K 1 Escherichia coli K92

Structure

References

2-acetamido-2-deoxymannosyl phosphate residues

22 1 7 191 186

+ g)cyD-NeupAc(z+

Haemophilus influenme Type b

and its OAc' variant

+ 8)a~-NeupAc(2 + g)aD-NeupAc(2+

0 Escherichia coli KlOO Staphylococcus aureus Bacillus pumilis Bacillus subtilis Lactobacillus plantarum

+ 3)p~-Ribf( 1-+

II

2)D-ribitol(5-O-P-

I

OH teichoic acids containing ribitol phosphate

222

7

204

HAROLD J. JENNINGS

mechanism, which probably involves interactions between the glycose moieties of the surface glycoprotein of human cells and surface proteins (pili or fimbriae) of the b a ~ t e r i a . ' Capsular ~ ~ , ~ ~ ~polysaccharides, however, are very much involved in the pathogenesis of bacteria following the penetration of these bacteria into body tissue. There is abundant accumulated evidence to demonstrate that capsular polysaccharides are important virulence factors in disease caused by Neisseria meningitidis, Haemophilus influenxae, group B Streptococcus, and Streptococcus pneu~oni~e.7~14E~'s5.225~227 This is also the case for the capsular polysaccharides of other pathogenic bacteria, including those in which the high-molecular-weight 0-chains of their lipopolysaccharides are functionally equivalent to the capsular polysaccharides.17 The importance of capsular polysaccharides in pneumococcal infections was demonstrated fifty years ago, when it was shown that the enzymic depolymerization of the capsular polysaccharide on the surface of type 111 S. pneumoniae organisms considerably decreased the virulence of these organisms in mice.z28The property of the capsular polysaccharide that enhances the virulence of bacteria is its ability to mediate the host's immune system. Except for a few cases of molecular mimicry (see Section VI,2), the major mechanism involved in this mediation is its function as an inhibitor of the fast-acting, alternative pathway of complement-induced phagocytosis, thus forcing the immune system to utilize the slower, classical pathway. The complement system is briefly explained in Section IV,2. Because the classical pathway has a requirement for polysaccharide-specific antibody, and because the process of producing this antibody takes a few days, the host is compromised during the initial, acute stages of bacterial infection, and is liable to die, or to acquire serious morbidity effects. Thus, the rationale behind vaccination with capsular polysaccharides is to maintain a long-lasting, effective level of polysaccharide-specific antibody in the host. The function of the polysaccharide capsule in inhibiting the alternative pathway is most satisfactorily and simply explained by the fact that it masks the underlying, bacterial structures (for example, teichoic acids), which are known to be powerful activators of the alternative p a t h ~ a y . ' ~ " -However, '~ although this mechanism is no doubt (224)E. H. Beachey,]. Infect. Dis., 143 (1981) 325-345. (225) R. Bortolussi, P. Ferrieri, B. Bjorksten, and P. G. Quie, Infect. Immun., 25 (1979) 293-298. (226) C . M. McLeod and M. R. Krauss,]. E r p . Med., 92 (1950) 1-9. (227) C. J . Howard and A. A. Glynn, Immunology, 20 (1971) 767-777. (228)0.T. Avery and R. Dubos,J. E x p . Med., 54 (1931) 73-89.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

205

operative, the concept of the capsule as a simple, physical barrier is inadequate to explain all of the experimental results and observations. For instance, why are not all encapsulated bacteria pathogenic? To understand this mechanism filly, it will first be necessary to comprehend the molecular events leading to the complement-mediated phagocytosis of bacteria. Although most of these events have been elucidated, the critical mechanism whereby the alternative pathway is first ~ . ~ ~ from ~ , ~ the~point ~ , of ~ ~ ~ activated still remains o ~ s c u ~ However, view of the capsular polysacckaride, evidence has been accumulated that helps to reveal some of its functional aspects in the inhibition of complement-mediated phagocytosis of bacteria. Evidence that the capsule creates a simple, physical barrier to the underlying surface of the bacteria can be found in the fact that only encapsulated bacteria are pathogens, and, for any given pathogenic strain of bacteria, its virulence is directly related to the amount of capsu1e.176,226,231 However, the amount of capsule is not the only criterion on which virulence is based, because although heavily encapsulated type 3 pneumococci are extremely virulent in mice, type 37 pneumoIn addition, cocci, having the same degree of encapsulation, are type 12 pneumococci, having very small capsules, are extremely virulent in humans.176On the basis of these results, plus the known species-specificity of bacterial pathogenesis, other physical, compositional, or structural properties must be postulated to account for the role of the polysaccharide capsules in bacterial pathogenesis. Experimentation leading to the delineation of this role is hampered by the complexity of the bacterial surface and by lack of knowledge as to whether the polysaccharide capsules function alone as mediators of the complement system. However, the use of molecular cloning-techniques has considerably simplified the problem, and has demonstrated that not all capsular polysaccharides have the same function in bacterial pathogenesis. Moxon and Vaughn232demonstrated that the polysaccharide capsule of type b H. injluenzue is necessary, and, indeed, sufficient, to perform this function. Type b and d transformants were made from the same, unencapsulated, H. influenme strain, thus giving the transformants DNA homology, except for the regions that determine serotype specificity. As in the clinical situation, the type b (229)D.T.Fearon and K. F. Austen, Proc. Natl. Acad. Sci. U . S . A.,74 (1977)16831687. (230)R. D. Schrieber, M. K. Pangburn,P. H. Lesavre, and H. J. Muller-Ebenhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978)3948-3952. (231)A. A. Glynn, W. Brumfitt, and C. J. Howard, Lancet, (1977)514-516. (232)E. R. Moxon and K. A. Vaughn,]. Infect. Dis., 143 (1981)517-524.

206

HAROLD J. JENNINGS

transformant proved to be more invasive and virulent in rats than that of type d. This experimental result is also consistent with the structural basis of bacterial pathogenicity. In similar studies by Silver and coworkers,233the cloned genes responsible for synthesis of the polysaccharide capsule of the highly pathogenic E. coZi K 1 organisms were able to synthesize the identical capsule in the non-pathogenic E. coZi K12 organisms. In this case, however, the capsule alone was not able to induce the same virulence properties in the transformed organism nonnally associated with the E. coli K1 bacteria, indicating that the E . coli K1 capsular polysaccharide probably functions in concert with other surface components of the K1 organisms.

2. Polysaccharide Structure and Pathogenicity

That polysaccharide structure could be involved in bacterial pathogenesis can be deduced from the observation that, of all the encapsulated bacteria, only a few are virulent in man, and interestingly, two of these different species of bacteria share a common capsular polysaccharide. Group B N. meningitidis and K1 E . coZi produce the same aD-(2+8)-linked sialic acid homopolymer (see Table V) as their only common surfacecomponent, and both are a major cause of meningitis in children. Some experimental evidence234is also consistent with the structure of polysaccharide capsules’s being implicated in the virulence of bacteria, which stems from their differing abilities to inhibit the activation of complement by way of the alternative pathway. In measuring the survival time of the six serotypes (a, b, c, d, e, and f ) of H . influenme in antibody-free sera containing complement, only the type b organisms were able to survive complement-mediated phagocytosis for any appreciable length of time. Although it has, to date, not been possible to identify any common structural feature among all the polysaccharide capsules of bacteria associated with the most pathogenic human disease, there is one common feature in many of them. The capsular polysaccharide of type I11 group B Streptococcus has terminal sialic acid residues in its struct~ree,6~ asqdo ~ ~ the groups B and C N. rneningitidis and K 1 E . C O Z ~ . ~ ~ ~ ~ ~ The ability of terminal sialic acid residues to inhibit the activation of complement by way of the alternative pathway has been well docu(233)R. P. Silver, C. W. Finn, W. F. Vann, W. Aaronson, R. Schneerson, P. J. Kretschmer, and C. F. Garon, Nature, 289 (1981)696-698. (234) A. Sutton, R. Schneerson, S. Kendail-Morris, and J. B. Robbins, Infect. Immun., 35 (1982)95-104.

CAPSULAR POLYSACCHARIDES AS HUMAN VACCINES

207

mented for erythrocyte-membrane ~ u r f a c e s . ~ ~Edwards ~ - ~ ~ ' and cow o r k e r ~also ~ ~observed ~ this phenomenon on bacterial surfaces. Normally, type I11 group B streptococcal organisms are potent inhibitors of the alternative pathway, but, when they are grown in neuraminidase, which removes the terminal sialic acid residues, they are converted into alternative-pathway activators. F e a r ~ described n ~ ~ ~ a similar experiment for the conversion, using neuraminidase, of sheep erythrocytes into activators of the alternative pathway. In an attempt to delineate the structure-function relationship of the terminal sialic acid residues of the type I11 group B streptococcal polysaccharide with this inhibition, Edwards and coworkers238chemically modified the polysaccharide on the surface of the bacteria. Reduction of the carboxylate groups of the sialic acid residues to hydroxymethyl groups also changed the surface of the type I11 organisms to become alternative-pathway activators, thus indicating that removal of these terminal residues in order to expose underlying, structural features is not required for activation to occur. F e a r ~ nalso ~ ~demonstrated ~ that removal of only C-8 and C-9 of the glycerol end-chain of terminal sialic acid residues of sheep erythrocytes is sufficient to convert them into activators; in this experiment, the charge on the sialic acid residues was retained, thus indicating, by extrapolation to the type I11 group B streptococcal polysaccharide, that the charge alone is not responsible for its inhibitory properties. All of these experiments involving the chemical modification of terminal sialic acid residues gave results that are consistent with the hypothesis that any change in the integrity of the sialic acid residue could alter its capacity to modulate the complement system, and this is also consistent with the work of Varki and K ~ r n f e l dwho , ~ ~showed ~ that the extent of 0-acetylation of sialic acid residues is directly related to the capacity of murine erythrocytes to activate the alternative-complement pathway. However, this hypothesis may be oversimplistic, and there still remains the possibility that there are involved more-complex, structural features in which the sialic acid residues could provide tertiary conformation to those surface structures. Certainly, such conformationally controlled determinants (235) D. T. Fearon, Proc. Natl. Acad. Sci. U . S. A., 75 (1978) 1971-1975. (236) M. K. Pangbum and H. J. Muller-Eberhard,Proc. Natl. Acad. Sci. U . S . A., 75 (1978) 2416-2420. (237) M. D. Kazatchkine, D. T. Fearon, and K. F. Austen,J. Immunol., 122 (1979) 7581. (238)M. S. Edwards, D. L. Kasper, H. J. Jennings, C. J. Baker, and A. NicholsonWeller,J. Zmmunol., 128 (1982) 1278-1283. (239) A. Varki and S. Komfeld,J. E r p . Med., 152 (1980)532-544.

208

HAROLD J. JENNINGS

have been identified in the type I11 group B streptococcal polysaccharide.76.131 Another mechanism whereby capsular polysaccharide could mediate the human immune-system is by molecular mimicry. If a bacterium were able to coat itself with molecules having a structure similar to that of those found in the host’s tissue, the production of antibodies having a specificity for these structures would be suppressed, as they would be recognized as part of “self” by the host. Some organisms (schistosomes) are able to acquire human blood-group determinants on their surfaces in order to circumvent the deleterious effects of the host’s immunological response.%O However, known examples of this type of molecular mimicry in bacteria are very few. Although the capsular polysaccharides of both types Ia and Ib, and type I11 group B Streptococcus have a high degree of structural homology with the respective M and N human blood-group s ~ b s t a n c eand s~~ human ~ ~ serotran~ferrin,6~ there is no evidence that this structural homology interferes with the production of polysaccharide-specific antibody in humans.62The best example of molecular mimicry is probably that of the a-D-(2+8)-linked sialic acid homopolymer, which serves as the capsule for both groups B N . meningitidis and K1 E . coli (see Table VI). This polysaccharide is poorly immunogenic in humans,ls2 and there is evidence to suggest that this poor immunogenicity could be attributed to its structural homology with human glycoprotein.lS5Already highly pathogenic, were it not for the production of antibodies against other surface components of both organisms, they would, indeed, be superpathogens.

(240) 0.L. Goldring, J. A. Clegg, S . R. Smithers, and R. J. Teny, Clin. Enp. Zmmunol., 26 (1976)181-187.