CA2153730A1 - Immunostimulating activity of streptococcus pneumoniae serotype 8 oligosaccharides - Google Patents

Abstract

The invention provides compositions comprising an oligosaccharide of S.
pneumoniae serotype 8 useful for stimulating an immune response to an antigen, methods of providing protective immunization against a bacterial pathogen using these compositions, methods of augmenting an immunogenic response to an antigen by administering these S. pneumoniae serotype 8 oligosaccharide compositions along with the antigen, and methods of making the immunostimulatory compositions described above.

Description

21~3730 IMMUNOSTTl~IULATING ACTIVITY OF STREPTOCOCCUS
PNEUMONIAE SEROTYPE 8 OLIGOSACCHA~

Field of the Invention:

This application relates to immllno~timlll~tQry oligosaccharide compositions and methods of making and using them. In particular, the compositions comprise S. pneumococcus serotype 8 oligosaccharides.

1 0 References:
The following le~rences are cited in the application at the relevant portion of the application.

Anderson, P., Infect. Tmmlln. 39:233, 1983 1 5 Anderson, P. W., Tmml-nogenic Conjugates, U.S. Patent No. 4,673,574, 1987.
Anderson, P., Pichichero, M. E., and Insel, R. A., J. Clin. Invest.
76:52, 1985a.
Anderson, P., Pichichero, M. E., and Insel, R. A., J. Pediatr. 107:346, 20 1985b.
Anderson, P. W., Pichichero, M. E., Insel, R. A., Betts, R., Eby, R., and Smith, D. J., J. Tmmlmol. 137:1181, 1986.
Anderson, P. W., Pichichero, M. E., Stein, E.C., Porcelli, S., Betts, R.
F., Connuch, D. M., Korones, D., Insel, R. A., Zabradnick, J. M., and Eby, 25 R., J. Tmmllnol. 142: 2464, 1989.
Avery, O.T. and Goebel, W. F., J. Exp. Med. 50:533, 1929.
Barro, A., Dogan, R., Prued'homme, J. L., Bajart, A., Danue, B. and Fritzell, B., Vaccine, II:1003, 1993.

21~3~30

- 2 -Bixler, G.S. and Pillai, S., The cellular basis of the immlm~ response to conjugate vaccines in "Conjugate Vaccines. Contributions to microbiology and immunology", J. M. Creese and R. E. Lewis, eds., Kargen, Basel, 1989.
Bolan, G., Broome, C. V., Fracklam, R.R." Pitkaytis, B.D., Fraser, D.
5 W., and Schlech, W. F, III, Ann. Internal Med. 104:1, 1986.
Borgano, J. M., McLean, A. A., Vella, P. P., Canepa, I., Davidson, W.
L., and Hilleman, M. R., Proc. Soc. Exp. Biol. Med. 157:148, 1978.
Broome, C. V., Facklam, R. R., and Fraser, D. W., N. Engl. J. Med.
303:549, 1980.
1 0 Bruyn, G.A.W., and van Furth, R., Eur. J. Clin. Microbiol. Infect. Dis., 10:897, 1991.
Chudwin, D. S., Artrip, S. C., Korenbilt, A., Schirr~ , G., and Rao, S., Infect. Tmmlm. 50:213, 1985.
Connelly, K. K., and Starke, J. R., Sem. Resp. Inf. 6: 209, 1991.
1 5 Cryz, S. J., and Furer, E., Conjugate vaccine against infections by gram-negative bacteria, method for its preparation and use, U.S. Patent No.
4,771,127, 1988.
Eby, R., Koster, M., Hogerman, D. and Malinoski, F., Pneumococcal Conjugate Vaccines, in "Modern Approaches to New Vaccines Including 20 Prevention of AIDS", E. Norrby, F. Brown, R. Chanock and H. Ginsberg, eds., Cold Spring Harbor Laboratory Press, New York, 1994.
Fattom, A., Lue, C., Sw, S. C., Mestecky, J., Schiffm~n, G., Brylar, D., Vann., W. F., Watson, D., Kimzey, L. M., Robbins, J.B. and Schneerson, R., Infect. and Tmmlm. 58: 2309, 1990.
Fattom, A., Vann, W. F., Sw, S. C., Schneerson, R., Robbins, J. B., Chu, C., Sutton, A., Vickers, J. C., London, W. T., Curfman, B., Hardagree, M. C., and Shiloach, J. Infect. Tmmlm. 56:2292, 1988.
Forester, H. L., Jahnigen, D. W., and LaForce, F. M., Am. J. Med.
83:425, 1987.

21~730 Gaur, A., Arunan, K., Singh, O. and Talwar, G. P., Int. Tmmllnol.
2:151, 1990.
Giebink, G. S., Koskela, M., Vella, P. P., Haris, M. and Chap, T. L., J.
Inf. Dis. 167: 347, 1993.
Goebel, W. F. and Avery, O. T., J. Exp. Med. 50: 521, 1929.
Gordon, L. K., Polysaccharide-exotoxoid conjugate vaccines, U. S.
Patent No. 4,619,828, 1986.
Gordon, L. K., Haemophilus influenzae b polysaccharide-diphtheria toxoid conjugate vaccine, U.S. Patent No. 4,644,059, 1987.
1 0 Hazelwood, M., Nusrat, R., Kumararatne, D.S., Goodal, M., Raykun~ , C., Wang, D. G., Joyce, H. J., Milford-Wards, A., Forte, M. and Pahor, A., Clin. Exp. Tmmllnol. 93:157, 1993.
Hakamori, S. and K~nn~gi, R., Carbohydrate antigens in higher ~nim~
in "Handbook of experimental immlm~logy - vol. 1", D. M. Weir, L. A.
1 5 Herzenberg, C. Blackwell and L. A. Herzenberg, eds., Blackwell, Oxford, 1986.
Heidelberger, M. and Avery, O. T., J. Exp. Med. 38:73,1923.
Hilleman, M. R., Carlson, A. J., Jr., McLean, A. A., Vella, P. P., Weibel, R. E., and Woodhour, A. F., Rev. Infect. Dis. 3 (suppl):S31, 1981.
Jennings, H. J., and Lugowski, C., Immunogenic polysaccharide-protein conjugates, C~n~ n Patent No. 1,181,344, 1985.
Jennings, H. J., Roy, R., and Gamian, A. J., Modified meningococcal group b polysaccharide for conjugate vaccine, C~n~ n Patent No. 1,261,320, 1989.
Jones, J.K.N. and Perry, M.B., J. Am. Chem. Soc. 79:2787, 1957.
Kenne, L. and Lindberg, B., Bacterial polysaccharides in "The polysaccharides - Vol 2", G. O. Aspinall, Ed., Academic Press, New York, 1983.
Lee, C-J., Banks, S. D. and Li, J. P., Crit. Rev. Microbio. 18:89, 1991.

Lees, A., Finkelman, F., Inman, J.K., Witherspoon, K., Johnson, P., Kennedy, J. and Mond, J.J., Vaccine 12:1160, 1994.
Lock, R. A., ~n~m~n, D., and Paton, J. C., Microbial Pathogen.
12:137, 1992.
Madore, D. V., Jackson, C. L., Phipps, D. C., Penridge Pediatric Association, Popejoy, L.A., Eby, R., and Smith, D. H., Pediatric, 85: 331, 1990.
Malcolm, A. J., Messner, P., Sleytr, U. B., Smith, R. H., and Unger, F.
M., Crystalline bacterial cell surface layers (S-layers) as combined 1 0 carrier/adjuvants for conjugate vaccines, in "Immobilized Macromolecules:
Application Potentials," U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds, Springer-Verlag, London, 1993a.
Malcolm, A. J., Best, M. W., Szarka, R. J., Mosleh, Z., Unger, F. M., Messner, P. and Sleytr, U. B., Surface layers of Bacillus alvei as a carrier for a 1 5 Streptococcus pneumoniae conjugate vaccine in "Adances in Bacterial Paracrystalline Surface Layers, " T. J. Beveridge and S. F. Koval, eds., Plenum Press, New York, 1993b.
Mandell, G. L., "Principles and Practice of Infectious Diseases, "
Churchill Livingston, New York, 1990.
Marburg, S., Jorn, D., Tolman, R. L., Arison, B., McCauley, J., Kniskern, P. J., Hagopian, A., and Vella, P.O., J. Am. Chem. Soc. 108:5282, 1986.
Marburg, S., Tolman, R. L., and Kniskern, P. J., Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such 25 polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and of col'r~ ling covalency, U.S. Patent No. 4,695,624, 1987.
Marburg, S., Kniskern, P. J., and Tolman, R. L., Covalently-modified bacterial polysaccharides, stable covalent conjugates of such polysaccharides and 30 immllnogenic proteins with bigeneric spacers and methods of pl~palillg such `~ 5 21~373~

polysaccharides and conjugates and of confirmin~ covalency, U. S. Patent No.
4,882,317, 1989.
Mufson, M. A., Hughey, D., and Lydick, E., J. Infect. Dis. 151:749, 1985.
Mufson, M. A., Krause, H. E., Schiffm~n, G., and Hughey, D. E., Am. J. Med. Sci. 293: 279, 1987.
Nielsen, S. V., and Henrichsen, J., Scand. J. Infect. Dis. 25: 165, 1993.
Paton, J. C., Lock, R. A., Lees, C-J., Li, J. P., Berry, A. M., Mitchell, T. J., Andrew, P. W., H~n~m~n, D., and Boulnois, G. J., Infect. Tmmlln 1 0 59:2297, 1991.
Peeters, C.C.A.M., Tenbergen-Meekes, A-M., Poolman, J. T., Berutett, M., Zegers, B. J. M. and Rijkers, G. T., Infect. Tmmlln. 59: 3504, 1991.
Penney, C.L., Michon, F., and Jennings, H.J., Improved Vaccine Compositions, WO 92/04951, 1992.
1 5 PelhlluLLel, R. M., Hansburg, D., Briles, D. E., Nicolotti, R. A., and Davie, J. M., J. Immunol. 121:566, 1978.
Porro, M., Oligosaccharide Conjugate Vaccines, C~n~ n Patent No. 2 052 323, 1992.
Porro, M., and Costantino, P., GlycoploLeilleic conjugates having trivalent immlln-)genic activity, U. S. Patent No. 4,711,779, 1987.
Porro, M., Oligosaccharide conjugate vaccines, U.S. Patent Application No. 07/590,649, 1990.
Saunders, L.A.M., Rijkers, G. T., Kuis, W., Tenbergen-Meekes, A. J., de Graff-Meeker, B. R., Hiemstra, I. and Zegers, B. J. M., J. Allergy Clin.
Immunol. 91: 110, 1993 Schidt, R. A., Boyd, J. F., McCracken, J. D., Schiffm~n, G., and Giolma, J. P., Med. Pediatr. Oncol. 11:305, 1983.
Schneerson, R., Barrera, O., Sutton, A., and Robins, J. B., J. Exp. Med.
152:361,1980.

215373~

Schneerson, R., Robbins, J. B., Chu, C., Sutton, A., Vann, W., Vickers, J. C., London, W. T., Curfman, B., and Hardegree, M. C. ,Infect.
Tmmlln 45:582, 1984.
Schneerson, R., Robbins, J. B., Parke, J. C., Bell, C., Schlesselman, J.
5 J., Sutton, A., Wang, Z., Scl-irrlll~l-, G., Karpas, A., and Shiloach, J., Infect.
Tmmlm 52:519, 1986.
Schneerson, R., Levi, L., Robbins, J. B., Bryla, D. M., Schirrlll~l-, G, and Lagergard, T., Infect. and Tllllll~ iLy 60:3528, 1992.
Seid, R. C., Jr., Boykins, R.A., Liu, D. F., Kibrough, K. W., Hsieh, 1 0 C.L., Eby, R., Glycoconj. J. 6: 489, 1989.
Sell, S. H., Wright, P. F., Vaughn, W. K., Thompson, J., and Schiffm~n, G., Rev. Infect. Dis. 3 (suppl):S97, 1981.
Shapiro, E.D., and Clemens, J. D., Ann. Intern. Med. 101:325, 1984.
Shapiro, E. D., N. Engl. J. Med. 316:1272, 1987.
1 5 Shapiro, E. D., Pneumococcal vaccine, In: "Vaccines and Immunotherapy," S. J. Fryz Jr., ed., Pergamon Press, New York, 1991.
Siber, G. R., Weitzman, S. A., Aisenberg, A. C., Weinstein, H. J., and Schiffm~n, G., N. Engl. J. Med. 299:442, 1978.
Simberkoff, M. S., Cross, A. P., Al-Ibrahim, M., Baltch, A. L., 20 Geiseler, P. J., Nadler, J., Richmond, A. S., Smith, R. P., Schiffm~n, G., and Shepard, D. S., N. Engl. J. Med. 315:1318, 1986.
Simberkoff, M. S., Pneumococcal vaccine in adults, In: "Tlllllllll~ ion,"
M. A. Sande, and R. K. Root, ed., Churchill Livingstone, New York, 1989.
Sims, R. V., Steinman, W. C., McConville, J. H., King, L. K., Zwick, 25 W. C., and Schwartz, J. C., 1988, Ann. Intern. Med., 108:653., 1988.
Slack, J., Der-Balian, G. P., Nahm, M. and Davie, J. M., J. Exp. Med.
151:853-1980.
Sorensen, U. B.S., J. Clin. Micro. 31: 2097, 1993.
Sloyer, J. L., Jr., Ploussard, J. H., and Howie, V. M., 1981, Rev.
30 Infect. Dis. 3 (suppl):Sl, 1981.

21S~730 Stein, K. E., J. Inf. Dis. 165: 549, 1992.
Stein, K. E., Int. J. Tech. Assess, Health Care, 10: 167, 1994.
Stein, K. E., Zopf, D. A., Johnson, B. M., Miller, C. B. and Paul, W.
E., J. Immunol. 128: 1350, 1982.
Stein, K. E., Zopf, D. A. and Miller, C. B., J. Exp. Med., 157: 657, 1983.
Steinhoff, M.C., Edwards, K., Keyserling, H., Thoms, M. L., Johnson, C., Madore, D. and Hogerman, D., Pediatr. Infect. Dis. J. 13: 368, 1994.
Tsay, G. C., and Collins, M. S., Vaccines for gram-negative bacteria, U.
1 O S. Patent No. 4,663,160, 1987.

The disclosure of the above publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if the language of each individual publication, patent and patent application were 1 5 specifically and individually included herein.

B~k~round of the Invention:

Immune Responses to Poly.s~r~h~rides Heidelberger and Avery (1923) demonstrated that the type specific antigens of pneumococci are polysaccharides. Bacterial capsular polysaccharides are cell surface antigens composed of identical repeat units which form extendedsaccharide chains. Polysaccharide structures are present on pathogenic bacteria and have been identified on Escherichia coli, Neisseria meningitidis, Haemophilus inJ'luenzae, Group A and Group B Streptococcus, Streptococcus pneumoniae and other species. (Kenne and Lindberg 1983).

Specific blood group determin~ntc and "tumor-associated" antigens are examples of ~ n cell surface carbohydrates. Oncogenically transformed cells often display surface carbohydrates distinctly dirrel~lll from those of non-transformed cells. These glycans consist of only a few monosaccharides (Hakomori and K~nn~gi 1986). The glycan structures by themselves are usually not antigenic, but constitute haptens in conjunction with protein or glycoplotein matrices.

A general feature of saccharide antigens is their inability to elicit significant levels of IgG antibody classes (IgG isotypes) or memory responses, they are considered thymus-independent (TI) antigens. Conjunction of polysaccharide antigens or of immllnologically inert carbohydrate haptens to 1 0 thymus dependent (TD) antigens such as pro~eins enhances their immllnogenicity.
The protein stiml-l~tes carrier-specific T-helper cells which play a role in theinduction of anti-carbohydrate antibody synthesis (Bixler and Pillai 1989).

Much of our current knowledge of TI and TD responses comes from 1 5 studies of pertinent mouse models (Stein et al., 1983; Stein, 1992; Stein, 1994).
TI antigens generally elicit low affinity antibodies of restricted class and do not produce immllnologic memory. Adjuvants have little effect on response to TI
antigens. In contrast, TD antigens elicit heterogeneous and high affinity antibodies with immllni7~tion and produce immllnnlogic memory. Adju~cul~
20 enhance response to TD antigens. Secondary responses to TD antigens shows an increase in the IgG to IgM ratio, while for TI antigens the secondary response IgG to IgM ratio is one-to-one, similar to that of a primary response (Stein et al., 1982; Stein, 1992 and 1994). In mice and hllm~n~, TD antigens elicit predomin~ntly IgGl isotypes, with some amounts of IgG2 and IgG3 isotypes. TI
25 responses to polysaccharides are restricted to IgG3 of the IgG isotypes (Perlmutter et al., 1978; Slack et al., 1980).

Current Pneumococcal Vaccine Pneumococci are ~;ullell~ly divided into 84 serotypes based on their 30 capsular polysaccharides. Although there is some variability of commonly 21~3730 g occurring serotypes with geographic location, generally serotypes 1, 3, 4, 7, 8 and 12 are more prevalent in the adult population. Serotypes 1, 3, 4, 6, 9, 14, 18, 19 and 23 often cause pneumonia in children (Mandell, 1990; Connelly and Starke, 1991; Lee et al., 1991; Sorensen, 1993; Nielsen and Henricksen, 1993).

At present, the most widely used anti-pneumococcal vaccine is composed of purified capsular polysaccharides from 23 strains of pneumococci (Pneumovax~23, Merck Sharp & Dohme). The pneumococcal capsular types included in Pneumovax~23 are 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, lOA, llA, 1 0 12F, 14, l5B, 17F, 18C, l9F, l9A, 20, 22F, 23F, 33F (Danish nomenclature).
These serotypes are said to be responsible for 90 percent of serious pneumococcal disease in the world.

Some controversy exists in the literature over the efficacy of the 1 5 Pneumovax~23 vaccine (Borgano et al.,l978; Broome et al., 1980; Sloyer et al., 1981,; Shapiro and Clemens, 1984; Bolan et al., 1986; Simberkoff et al., 1986;
Forester et al., 1987; Shapiro, 1987; Sims et al., 1988; Simberkoff, 1989;
Shapiro, 1991). The pneumococcal vaccine is effective for induction of an antibody response in healthy young adults (Hilleman et al., 1981; Mufson et 20 al., 1985; Bruyan and van Furth, 1991). These antibodies have been shown to have in vitro opsonic activity (Chudwin et al., 1985). However, there is m~rk~l variability in the intensily of the response and in the persistence of antibody titers to the dirrelenl serotypes (Hilleman et al., 1981; Mufson et al., 1987).

Children under 2 years of age are the group at highest risk of systemic disease, otitis media and acute lower respiratory infection caused by pneumococci, but they do not respond to this vaccine (Sell et al., 1981;
Hazelwood et al., 1993; Saunders et al., 1993). Furthermore, elderly and irnmunosuppressed patients have impaired or varied responses to Pneumovax~23 (Siber et al., 1978; Schildt et al., 1983; Forester et al., 1987; Simberkoff, 1989;

Shapiro, 1991). These population groups do not respond well to the thymus independent polysaccharide antigens of this vaccine. Typical of thymus independent antigens, antibody class switching from an IgM to IgG isotype is notusually observed nor is an ~n~mn~stic response to a booster immllni7.~tion 5 (Borgano et al., 1978).

Recent occurrences of antibiotic resistant strains of bacteria stresses the need to develop efficacious vaccines for the prevention of childhood infection.
Clearly, new vaccines against pneumococci are n~eded, especially for high risk 1 0 groups and children.

Conjugate Vaccines Avery and Goebel were the first to prepare vaccines against bacterial infections (Avery and Goebel 1929; Goebel and Avery 1929). More recently, 1 5 several protein carrier conjugates have been developed which elicit thymus dependent responses to a variety of bacterial polysaccharides. To date, the development of conjugate vaccines to Hemophilus influenzae type b (Hib) has received the most attention. Schneerson et al. (1980) have covalently coupled Hib polysaccharides (polyribitol-phosphate) to diphtheria toxoid. This group has20 also developed a Hib vaccine by deliv~l~ing the polysaccharide with an adipicacid dihydrazide spacer and coupling this material to tetanus toxoid with carbodiimide (Schneerson et al., 1986). A similar procedure was used to produce conjugates cont~ining diphtheria toxoid as the carrier (Gordon, 1986 and1987). A bifunctional spacer was utilized to couple the outer membrane protein 25 of group B Neisseria meningitidis to Hib polysaccharides (Marburg et al., 1986, 1987 and 1989). Finally, Anderson (1983 and 1987) has produced a conjugate vaccine using Hib oligosaccharides coupled by reductive amination to a nontoxic,cross-reactive mutant diphtheria toxin CRMl97.

Reports in the literature differ on the efficacy of these vaccines, and many studies are still in progress. However, oligosaccharide conjugates (Anderson et al., 1985a, 1985b, 1986, 1989; Seid et al., 1989; Madore et al., 1990; Eby et al., 1994) and polysaccharide conjugates (Barra et al., 1993) are reported to be5 immunogenic in infants and elicit a thymus dependent response. Hapten loading is a key factor for conjugate immunogenicity (Anderson et al., 1989; Eby et al.,1994).

Other conjugate vaccines have been developed by Jennings et al. (1985 1 O and 1989), who utilized periodate activation to couple polysaccharides of Neisseria meningitidis to tetanus or diphtheria toxoid carriers. Porro (1987) defined methods to couple esterified N. meningitidis oligosaccharides to carrierproteins. Conjugate vaccines cont~ining polysaccharides of Pseudomonas aeruginosa coupled by the periodate procedure to detoxified protein from the 1 5 same organism (Tsay and Collins, 1987) have been developed. Cryz and Furer (1988) used adipic acid dihydrazide as a spacer arm to produce conjugate vaccines against P. aeruginosa.

21~3730 Polysaccharides of specific serotypes of S. pneumoniae have also been coupled to classical carrier proteins such as tetanus or diphtheria toxoids (Schneerson et al., 1984; Fattom et al., 1988 and 1990; Schneerson et al., 1992), to N. meningitidis membrane protein (Marburg et al., 1987; Giebink et 5 al., 1993) and to a pneumolysin mutant carrier (Paton et al., 1991; Lock et al., 1992; Lee et al., 1994). Technology for coupling S. pneumoniae oligosaccharides to CRMl97 protein has been developed (Porro, 1990). These conjugate vaccines have variable or as yet undetermined immunopotentiation properties. Reproducibility of these coupling technologies with the mailllenallce 1 0 of imml1nogenic epitopes is ~ullelllly the greatest problem in developing effective S. pneumoniae glyco-conjugate vaccines. The optimal immllnogenic oligosaccharide size appears to vary dependent on the serotype, indicating a conformational aspect of certain immlm~genic epitopes (Eby et al., 1994;
Steinhoff et al., 1994).
Vaccines to DTP, tuberculosis, polio, measles, hepatitis, Hib and pneumonia which induce long lasting protection are needed. In order to induce protection in infants to S. pneumoniae, a multi-hapten protein conjugate cont~ining a high level of oligosaccharides of optimal immllnogenic size for each 20 serotype is desired.

Various researchers have proposed enhancement of the immllnogenicity of conjugate vaccines by adjuvant ~lmini~tration. Al~ salt, which is approved for human use, is an example. Carbohydrate moieties, such as beta 25 glucan particles and low molecular weight dextran, have also been reported topossess adjuvant activity. Adjuvax (Alpha-Beta Technology) is an adjuvant composition cont~ining beta glucan particles. Lees et al. (1994) have reported the use of low molecular weight dextran constructs as adjuvants. Penney et al.
(1992) have reported a long chain alkyl compound with immunological activity.

2153~30 Brief Description of the Drawin~:

Figure 1 illustrates the repeat unit structures of the polysaccharides used in the Examples of the invention.

Figure 2 shows the separation profile of Streptococcus pneumoniae serotype 8 capsular polysaccharides through a BioGel P-10 column after acid hydrolysis (0.5 M trifluoroacetic acid, 100C, 20 minutes) resulting in discernible oligosaccharides of one to eight repeat units.
Figure 3 shows the relative size of the repeat units in peaks 1, 2, 3 and 4 of hydrolyzed Streptococcus pneumoniae serotype 8 capsular polysaccharides, as measured by HPLC analysis.

Figure 4 shows the HPLC retention times of the glucose, M-3 maltotriose, M-7 maltoheptose, and M-10 malto-oligosaccharide standards used to determine the relative size of various oligosaccharide repeat units.

Figure 5 is an example of the retention times of ribitol, rhamnose, 20 galactose, fucose and mannose monosaccharide standards used to determine carbohydrate content of the hydrolysed repeat unit.

Figure 6 shows the separation profile of S. pneumoniae serotype 6B
polysaccharide hydrofluoric acid hydrolysates passed over a P-10 BioGel 25 column.

Figure 7 shows the separation profile of S. pneumoniae serotype 6B
polysaccharide TFA hydrolysates passed over a P-60 BioGel column.

2153~3~

Figure 8 shows the separation profile of S. pneumoniae serotype 14 polysaccharide TFA hydrolysates passed over a P-30 BioGel column.

Figure 9 shows a separation profile of S. pneumoniae serotype l9F
5 polysaccharide acetic acid hydrolysates acetic acid passed over a P-10 BioGel column.

Figure 10 shows the separation profile of S. pneumoniae serotype 23F
polysaccharide TFA hydrolysates passed over a P-10 BioGel column.
Figure 11 shows the separation profile of S. pneumoniae serotype 8 polysaccharide cleaved by cellulase passed over a P-10 Bio Gel column.

Figure 12 shows the separation profile of pneumococcal C-substance 15 polysaccharide hydrofluoric acid hydrolysates passed over a P-10 Bio Gel column.

Figure 13 shows the inhibition ELISA results using a mouse antiserum to Streptococcus pneumoniae serotype 8 oligosaccharide protein carrier conjugate.

Figure 14 illustrates the acidification of oligosaccharides for carbodiimide coupling.

Figure 15 shows the separation of reduced and periodate fractions of a 25 polysaccharide (23 valent polysaccharide vaccine-Pneumovax~ 23, Merck, Sharp and Dohme).

Figure 16 demonstrates separation of reduced and periodate fractions of oligosaccharides of serotype 6B of Streptococcus pneumoniae.

2153~3~

Figure 17 demonstrates separation of reduced and periodate fractions of oligosaccharides of serotype l9F of Streptococcus pneumoniae.

Figure 18 depicts the periodate and EDC coupling chemistry reactions.

Figure 19 shows how a mono-hapten 8-oligosaccharide tetanus toxoid conjugate inhibited anti-8 serum binding to a 8 polysaccharide coated ELISA
plate.

Figure 20 depicts the IgG antibody isotypes elicited by S. pneumoniae serotype 8 polysaccharide following immllni7~tion with an 8:14 di-hapten-oligosaccharide-TT conjugate.

Figure 21 shows an increased level of IgGl antibody isotype elicited by 15 polysaccharide following immllni7~tion with an 8:14 di-hapten-oligosaccharide-conjugate, typical of a TD response.

Figures 22A and 22B show IgG isotypes elicited from groups of mice "~l~ni~ed with 14-polysaccharide and oligosaccharide conjugates with and 20 without adjuvant.

Summary of the Invention:

In one aspect, the invention provides compositions comprising: a) a size-25 separated carbohydrate hapten comprising at least one immllnngenic epitope; andb) a carrier, wherein said hapten is covalently coupled to said carrier and wherein said hapten-carrier conjugate is protectively immlmngenic.

In another aspect, the invention provides methods of making conjugate 30 compositions comprising: a) cleaving a bacterial polysaccharide into -oligosaccharides so as to preserve immunogenic epitopes on the resulting oligosaccharides; b) separating the resulting oligosaccharides based on size; c)selecting those oligosaccharides which contain immunogenic epitopes based on inhibition ELISA; d) activating the oligosaccharides selected in step c); and e)5 coupling the activated oligosaccharides to a purified carrier, wherein the resulting composition contains immllnogenic epitopes and is protectively 1mmllnogemc.

In a further aspect, the invention provides methods of providing 10 protective immnni7~tion against a bacterial pathogen comprising :~(lmini~tering to a ,,,~llllll~l in need of such treatment an effective amount of the vaccine composition described above.

In still a further aspect, the invention provides compositions useful for 15 stimnl~ting an immnn~ response to an antigen, said immllnnctimlll~tory composition comprising an oligosaccharide of S. pneumoniae serotype 8 which contains an immlmogenic epitope as determined by inhibition ELISA and a suitable pharmaceutical excipient, wherein said oligosaccharide provides an immunostimlll~tory effect.
In a yet further aspect, the invention provides methods of providing protective immlmi7~tion against a bacterial pathogen comprising ~lmini~tering toa m~mm~l in need of such treatment an effective amount of the composition of the serotype 8 composition described above.
A still further yet aspect of the invention provides methods of augmenting an immunogenic response to an antigen comprising a(lmini.~tering an oligosaccharide of S. pneumoniae serotype 8 which contains an immnnngenic epitope as determined by inhibition ELISA along with said antigen.

17 21~3730 In another further aspect, the invention provides methods of making the immunostimulatory compositions described above, comprising: a) cleaving S.
pneumoniae serotype 8 polysaccharide into oligosaccharides so as to preserve immllnogenic epitopes on the resulting oligosaccharides; b) separating the 5 resulting oligosaccharides based on size; c) selecting those oligosaccharides which contain immllm)genic epitopes based on inhibition ELISA; and d) mixing the selected oligosaccharides with a suitable ph~rm~reutical carrier.

Detailed Description of the Invention:
This invention relates to improved methods for preparing oligosaccharide-protein carrier conjugates. The conjugate product may be composed of various haptens or carriers. Mono, di, and multi-hapten conjugates may be prepared.
Methods to dele~ e the presence of immllnogenic epitopes on the hapten or 15 carrier of the resultant conjugate are described. Such conjugates have utility as vaccines, therapeutic and prophylactic agents, immunomodulators diagnostic agents, development and research tools.

This invention is particularly suited for developing conjugates as vaccines 20 to such bacterial pathogens including, but not limited to Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae B, Group B
Streptococcus, Group A Streptococcus, Bordetellapertussis, Escherichia coli, Streptococcus mutans, Staphylococcus aureus, Salmonella typhi, Cryptococcus neoformans, Pseudomonas aeruginosa and Klebsiella pneumoniae. Conjugates 25 of this invention convert weakly or non-immunogenic molecules to molecules which elicit specific immunoprotective antibody or cellular responses.

Poor immlln~ responses to polysaccharide vaccines (thymus independent antigens, TI) have been observed with high risk groups, such as the elderly and 30 children under 2 years of age. Several investigators are attempting to elicit thymus dependent (TD) responses to a variety of bacterial polysaccharides using protein carriers. Integrity of critical immunogenic epitopes and inconsistency of covalent linkage between the carbohydrate and protein are major limitations withthese conjugate vaccines. The present invention is drawn to the discovery of 5 coupling technology which gives good reproducibility with respect to the carbohydrate to carrier ratio of conjugates. This invention also provides methods to verify the presence of immunogenic epitopes on and oligosaccharide haptens and hapten-carrier conjugates.

Polysaccharide conjugates elicit non-boostable IgM antibody responses, typical of TI antigens. The antiserum produced in response to these polysaccharide conjugates does not have opsonic activity. In the present invention, oligosaccharides prepared by cleavage of polysaccharides from various bacterial strains are size separated and used to produce mono-hapten 15 conjugates. These conjugates elicit IgG antibody isotypes with immllnl~protective, opsonization ability. This antibody response is elicited without the use of any adjuvant. Thus, the methods of the inventions are ideallysuited for producing immunogenic oligosaccharide hapten-carrier conjugates which utilize weakly or non-immunogenic polysaccharides of various strains.
20 The presence of immllnogenic epitopes on these oligosaccharides was found to be critical for eliciting an immunoprotective response.

The number of bacterial antigens needed to develop efficacious anti-pathogen vaccines is expanding. However, repeated a-lmini~tration of tetanus or 25 diphtheria toxoid (often used as carrier proteins in vaccine compositions and as a prophylactic measure following trauma) may cause a phenomenon called carrier-inl1uçe~ epitope suppression. Epitope suppression has been described in the literature with synthetic peptide and saccharide-toxoid conjugates (Gaur et al.,1990; Peeters et al., 1991). Tmmlln~ responses to a hapten coupled to a carrier `_ 2153730 protein can be reduced or absent when the recipient has been previously immllni7e~ with the carrier.

The goal of many researchers is to develop vaccines which elicit 5 protection to the predominant bacterial serotypes which cause acute lower respiratory infection, otitis media and bacteremia in infants, without inducing carrier suppression. The methods of the invention can be utilized to produce multi-hapten conjugates with optimal immunogenic epitopes to each bacterial serotype. These conjugates, which contain lower carrier protein amounts than 10 traditional conjugates, reduce the occurrence of the carrier suppression phenomenon. The reduced antigen load possible using these conjugates ",il~i",i,es the antigenic competition observed with traditional conjugates.

Previously, we reported that crystalline bacterial cell surface layers (S-15 layers) were useful as carriers for the development of prototype conjugatevaccines (Malcolm et al., 1993a) and as a means to avoid the carrier suppression phenomenon (Malcolm et al., 1993b). In our laboratory, we identified several S-layer glycoproteins which elicit non-cross reactive antibody and cellular responses. Vaccines to a variety of diseases can be developed using20 S-layers isolated from various bacterial strains, thereby avoiding carrier suppression observed with tetanus and diphtheria toxoids. However, S-layers are difficult to isolate and purify, as well as costly to produce, making them impractical for wide usage as vaccine carriers. The present invention describes methods to prepare mono, di and multi-hapten oligosaccharide conjugates which 25 reduce the amount of carrier n~cess~ry to elicit specific responses, thereby decreasing the risk of carrier in-1uçe~1 epitope suppression, even when tetanus or diphtheria toxoid is used as the carrier.

One specific application of the technology of the invention is for the 30 development of effective vaccines for the prevention of pediatric pneumoniae 215~730 infections. Another application of the invention is to develop vaccines for protection to strains of Group B Streptococcus, Group A Streptococcus, Haemophilus influenzae B, Streptococcus pneumoniae and N. meningitidis prevalent in infant disease, in the elderly or the immunosuppressed. Other 5 applications include development of conjugates for eliciting protection to various bacterial or virus pathogens.

We have found that the use of conditions which cleave specific linkages (i.e., 1 - 4 linkages) but leave sugar monosaccharides and other immlmologically10 important compounds such as phosphate intact results in improved immllnogenicity of the resulting conjugates.

We have found that oligosaccharide size and conformation is important to m~ximi~e immlmogenicity of conjugate plepal~ion. Dirrel~ oligosaccharide 15 sizes are separated from hydrolyzed polysaccharide mixtures and isolated by size fraction. The monosaccharide content and the relative size of separated oligosaccharides is measured by, for example, HPLC analysis. Dirrerelll size repeat units are tested using inhibition ELISA. We have found that ELISA
inhibition is directly proportional to the immunogenicity of the oligosaccharide20 preparation and the resultant conjugate.

In particular, oligosaccharides prepared from cleavage of polysaccharides of S. pneumococcus strains 3, 6B, 8, 14, l9F and 23; pneumococcal C-substance; and N. meningitidis C-polysaccharide have been used in our 25 laboratory. Preferred repeat units (R.U.) for oligosaccharides are as follows for some S. pneumococcus serotypes and pneumococcal C-substance:
Serotype 3: 4-8 R.U.
6B: 4-10 R.U.
8: 2-8 R.U.
14: 4-6R.U.

21~3730 l9F: 4-10 R.U.
C-substance: 6-10 R.U.
Preferred repeat units for N. meningitidis C-polysaccharide is 6-10 R.U.

Creating charged groups on saccharide haptens has been discovered to facilitate the coupling of the haptens to the carrier. Use of cation or anion exchange columns is effective in allowing coupling of oligosaccharide to carrierat a higher sugar to carrier ratio. This provides more hapten per carrier, and reduces the carrier suppression phenomenon. l~ ce~l fractions of carbohydrate are used for coupling to carrier.

Another important aspect to produce effective conjugate vaccines is the use of purified carrier. Impurities found in a carrier preparation may hllelrelewith coupling procedures. Aggregates of carrier proteins found in a carrier preparation can affect optimum hapten to carrier ratios n~cess~ry to elicit the desired response. Carriers are generally purified using size exclusion column chromatography, although any standard method which removes illl~ulilies and aggregate may be used.

The coupling reaction time and the amount of oligosaccharide, coupling reagent and carrier are critical for obtaining an ideal carbohydrate to carrier conjugate ratio. We have developed methods which quantify carbohydrate to carrier ratios by reproducible assays. Maintenance of pH and temperature conditions determined to be optimal during the coupling reaction is also important to produce an effective conjugate. Likewise, the use of effective blocking reagents which stop the coupling reaction but do not mask the immunogenic groups is important to create effective conjugate compositions.

Use of coupling chemistry which m~int~in~ immllnogenic epitopes on oligosaccharides/polysaccharides is essential. We have found that EDC and 21~3~0 periodate coupling, as described below may be used for coupling oligosaccharides to carriers. In addition to direct coupling of sugar to carrier, various linkers may be used to space the saccharide from the surface of the protein. Appropriate linkers may also provide charged or uncharged moieties as 5 desired. The immunogenicity of coupled sugar-carrier compositions is determined by inhibition ELISA.

Using the methods of the present invention, we have discovered means to produce di-hapten and multi-hapten conjugates which still m~int~in their 10 immunogenic epitopes. Conjugates with various oligosaccharide sequences and/or sizes can be produced. Similarly, conjugates comprising oligosaccharide and polysaccharide combinations may be synthesized. Such conjugates are able to reduce or elimin~te antigenic competition.

Thus, appropffate conjugate design provides the ability to reduce carrier induced epitope suppression. Keys in this regard are the identification and use of immunogenic oligosaccharide epitopes and more effective coupling of sugar to protein. Binding a larger number of immunogenic epitopes per protein molecule means that less carrier is needed to provide protective immuni7~tion.
We have developed methods to quantify immunoprotective antibody response to conjugate compositions by isotyping ELISA, bactericidal and opsonization assays. This allows determination of which conjugates will elicit the ~lv~ffate immlmoglobulin isotype response, i.e., IgG isotypes, when used 25 to protectively i"l~ i,e m~mm~l.c.

Deffnitions:
The following terms have the following m~ning.~ when refelellced herein:

Oligosaccharide means a carbohydrate compound made up of a small number of monosaccharide units. In particular, oligosaccharides may be formed by cleaving polysaccharides.

Polysaccharide means a carbohydrate compound cont~ining a large number of saccharide groups. Polysaccharides found on the outer surface of bacteria or viruses are particularly useful in the present invention.

Carrier means a substance which elicits a thymus dependent immlln~
10 response which can be coupled to a hapten or antigen to form a conjugate. In particular, various protein, glycoprotein, carbohydrate or sub-unit carriers can be used, including but not limited to, tetanus toxoid/toxin, diphtheria toxoid/toxin, bacteria outer membrane proteins, crystalline bacterial cell surface layers, serum albumin, gamma globulin or keyhole limpet hemocyanin.

Tmmum)genic means causing an immlm~ response. An immllnogenic epitope means that portion of a molecule which is recognized by the i~
system to cause an immunogenic response.

Hapten means an antigen, including an incomplete or partial antigen which may not be capable, alone, of causing the production of antibodies. Di-and multi-hapten, for purposes of this application, refer to compositions including two (di) or more (multi) oligosaccharide haptens conjugated to carrier.

Protectively immunngenic or immllnoprotective means stimlll~tin~ an immune response which prevents infection by pathogen.

Immunostimlll~tory means stimlll~ting or enhancing an immlln~ response to weakly immlmogenic haptens or antigens.

2l~373n Neonate means a newborn animal, including an infant.

M~thodology:

5 Preparation and Separation of Cleaved Polysaccharides:
Polysaccharides, available through American Type Culture Collection, Rockville, Maryland or by isolation procedures known in the art, were cleaved into oligosaccharide units using appropliate concentrations of chemicals. These chemicals include, but are not limited to trifluoroacetic acid, acetic acid, 10 hydrofluoric acid, hydrochloric acid, sodium hydroxide and sodium acetate.
Dirr~l~llL time periods and temperatures may be used depending on the particularchemistry and concentration and on the resulting oligosaccharide desired.
Commercially available enzymes (e.g., cellulase and ~-galactosidase) or isolatedbacteriophage-associated endoglycans known in the art can also be used to 15 prepare oligosaccharides from polysaccharides.

Figure 1 shows the repeat unit structures of the polysaccharides used in the Examples of the invention. Other bacterial and viral polysaccharide are known to those of skill in the art, and may be used in the methods and 20 compositions of the present invention. Various polysaccharides can be cleavedincluding, but not limited to, pneumococcal group antigen (C-substance) and capsular polysaccharides of serotypes of Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Group A Streptococcus and Group B
Streptococcus.
After cleavage, the resulting oligosaccharide mixtures are separated by size using P-10 (fractionation range 1,500 - 20,000 molecular weight), P-30 (2,500 - 40,000 molecular weight) and P-60 (3,000 - 60,000 molecular weight) BioGel columns. The presence of carbohydrates in the various column fractions 30 is determined using phenol-sulphuric or sialic acid assays and thin layer _ 21~3730 chromatography (TLC). Carbohydrate-cont~ining column fractions are then analyzed by HPLC.

The presence of immunogenic epitopes on size-separated fractions of 5 cleaved polysaccharides is determined by inhibition ELISA, as described below.If a preparation does not result in oligosaccharide fractions which inhibit in the ELISA test, cleavage procedures may be modified by ch~n~ing enzymes or chemicals, molarity, reaction time or temperature in order to produce immunogenic epitopes.

D~Le~ ination of Immuno~enic Epitopes in Oli~osaccharide Preparation~:
The presence of immunogenic epitopes in column fractions is confirrnP~l 15 by inhibition ELISA and phosphorous assay as set forth in the Examples section.
Oligosaccharide fractions cont~ining immunogenic epitopes (defined as those which produce at least about a 50% reduction in O.D.4"5 at 12.5 ~lg concentration) are selected for coupling to carrier.

20 Couplin~ to Carrier:
The oligosaccharide or polysaccharide to be used for coupling to carrier is acidified or reduced in prepaldtion for EDC or periodate oxidation coupling.
For example, the oligosaccharide preparation may be reduced using a Rexyn~M
101 (H) organic acid cation exchange column to acidify the sugar for EDC
25 coupling. Similarly, sugars may be reduced using standard methods for periodate oxidation coupling. When preparing di-hapten or multi-hapten conjugates, each oligosaccharide is activated individually for EDC or periodate conjugation.

21~37~
-Preferred di-hapten oligosaccharide conjugates include: 3:8-TT, 6:8-TT, 6:14-TT, 8:14-TT, 8:19-TT, 8:23-TT and 14:19-TT.

Carrier:
Various protein, glycoprotein, carbohydrate or sub-unit carriers can be used, including but not limited to, tetanus toxoid/toxin, diphtheria toxoid/toxin, bacteria outer membrane proteins, crystalline bacterial cell surface layers, serum albumin, gamma globulin or keyhole limpet hemocyanin. In the specific examples of this invention, tetanus toxoid was used as the carrier. Tetanus 10 toxoid p~el)alalions routinely contain aggregates and low molecular weight impurities. Purity of carrier is essential for obtaining consistency with coupling reactions. Size exclusion chromatography is used to obtain a purified carrier preparation.
Size separated, immunogenic epitope-cont~ining oligosaccharides are coupled to purified carriers by carbodiimide (EDC) or periodate activation, using the procedures described in the Examples section. Any free hapten oligosaccharides are separated from hapten-carrier conjugates by column chromatography. The carbohydrate to protein ratio of conjugates is d~te~ ed by phenol sulfuric or sialic acid and Lowry protein assays. Typically, conjugates prepared by EDC coupling have a carbohydrate to carrier ratio of 1:2, while conjugates prepared using periodate oxidation coupling have carbohydrate to carrier ratios ranging from 1:5 to l:lO.

Determination of Immunogenic Epitopes on Conjugates:
As stated previously, integrity of critical immllnogenic epitopes is a problem with previously known conjugation technologies. In the present invention, the ELISA inhibition assay is used to determine the potential immunogenicity of various conjugates produced by our conjugation procedures.
We have found that conjugates which demonstrate inhibition in this assay (at least about 50 % reduction in O.D.405 at 6.25,ug concentration) using the 215373~

methods set forth in the Examples, provide protective immunogenicity when used as a vaccine in m~mm~l~. Thus this assay is used to screen for useful conjugate compositions.

5 T"""~ tion to Elicit Immunoprotective Antibody Responses:
Typically, mice are illlllllll~i7.eCl on day 0 (1-primary immuni7~tion) day 7 (2-secondary i""l~l.ni~tion) and day 28 (3-tertiary immuni7~tion) by subcutaneous injection (1001l1 into 2 flank sites) with antigens (polysaccharide-conjugates oligosaccharide-conjugates, uncoupled polysaccharide or 1 0 oligosaccharide, or uncoupled tetanus toxoid) at doses of 0.1, 0.5, 1, 2.5 and 5 ,ug, based on carbohydrate content for EDC conjugates and protein content for periodate conjugates.

Antigens were diluted to various doses in 0.9% NaCl and mice injected 15 with 0.9% NaCl were used as negative controls. Mice were bled 7-10 days post -2 and 3 immuni7~tion to collect serum to assay immlmoprotective antibody responses. A typical immlmi7~tion schedule is shown in Table 1 for S.
Pneumoniae serotype 3 polysaccharide and oligosaccharide-tetanus toxoid conjugates prepared using EDC coupling.
Various other i~ u~ tion schedules are effective, including: day 0 (1), day 14 (2) and day 44 (3); and day 0 (1) day 30 (2) and day 60 (3).

The conjugates of this invention may be used as classical vaccines, as 25 immlmogens which elicit specific antibody production or stimlll~tP specific cell me~ ted i",~"~ iLy responses. They may also be utilized as therapeutic modalities for example, to stimul~te the immunP system to recognize tumor-associated antigens; as immunomodulators for example to stimul~t~
lymphokine/cytokine production by activating specific cell receptors; as 30 prophylactic agents, for example, to block receptors on cell membrane 2ls37~n preventing cell adhesion; as diagnostic agents, for example, to identify specific cells; and as development and/or research tools, for example, to stim~ te cells for monoclonal antibody production.

5 Detelll,hlation of Response:
As previously discussed, antibody responses to TI and TD antigens differ.
In the mouse, the response to a polysaccharide (TI) antigen is usually composed of a one-to-one ratio of IgM and IgG. In general, IgG isotypes are restricted, with IgG3 being over-expressed in anti-polysaccharide serum. IgA isotypes may 10 also be present. TI antigens elicit antibodies with low affinity and imml~nologic memory is not produced.

With TD antigens, increased secondary IgG antibody responses (an an~mn~stic response) are found, with a higher IgG to IgM ratio. Marked levels 15 of IgA are usually not present. The TD antigen elicits a heterogeneous IgG
isotype response, the predominant isotype being IgGl. IgG2a and 2b isotypes can be expressed, while the IgG3 isotype level is usually relatively low. TD antigens elicit immunologic memory and antibody affinity increases with i"~ll"l"i~ions.
Thus, analysis of the immunoglobulin isotypes produced in response to conjugate 20 ~(lmini~tration enables one to determine whether or not a conjugate will be protectively immunogenic.

We have found that the conjugates of the present invention induce a response typical of TD, rather than TI antigens, as measured by direct and 25 isotyping ELISA and opsonization assay.

Conjugates prepared using our EDC coupling methods elicited better antibody responses than conjugates prepared by periodate activation. Doses of l ~g were most immunogenic. Oligosaccharide-conjugates prepared with diphtheria toxoid carriers elicited antibody responses similar to the responses elicited with the oligosaccharide-tetanus toxoid conjugate.

As described previously, several investigators have attempted to increase 5 imml-nngenicity and elicit thymus-dependent antibody protection by coupling polysaccharide material to tetanus and diphtheria toxoids. Results intlir~te that these conjugates are only slightly more immunogenic than uncoupled capsular polysaccharide (CPS). One possible explanation for this may be that pertussis, diphtheria and tetanus toxoids (in alllmimlm salt adjuvant) are often ~imini.~tered 10 as a prophylactic four dose immuni7~tion regime to infants. This regime may tolerize the infant, making the infant incapable of mounting a protective antibody response to a hapten/antigen coupled to these toxoid carriers (carrier suppression). Another possible reason for failure to induce protection may be structural. Protein carriers elicit and augment the immlm~ response to haptens, 15 but in the case of CPS-protein conjugates, the CPS portion is a relatively large TI antigen. The immlln~ system may not recognize the CPS-protein as a conjugate, but simply as two distinct entities, resulting in a thymus-independent response to the CPS and a thymus-dependent response to the carrier.

This appears to be the case in our studies, as shown in Table 2. The immlln~ system recognizes the polysaccharide of our polysaccharide-tetanus toxoid (TT) conjugate as a TI antigen. The potential TD inducing capability of the carrier with respect to antibodies to the polysaccharide is not observed. Wepostulate that the immunogenic epitopes of the carbohydrate haptens (oligosaccharides) must be in close proximity to the TD inducing epitopes of thecarrier in order to convert a TI response to a TD response.

We have also used linker arm technology to prepare conjugates. We have used, for example, 6-amino-n-hexanoic acid as a linker. The resulting 30 conjugates were found to be less effective in eliciting antibody responses than 215:~730 conjugates prepared by directly coupling EDC activated oligosaccharide haptens to carriers. This finding supports our hypothesis that close hapten to carrier proximity is needed to elicit TD responses.

We have also developed methods to determine the level of irnmunoprotective antibody elicited by the conjugates of the present invention using bactericidal or opsonization assays. These tests have shown that the conjugates of the present invention are effective in eliciting protective antibodies, as measured by these assays.
As discussed previously, the epitope-carrier suppression phenomenon has been observed by other researchers and in our laboratory with the S-layer carrier studies (Malcolm et al., 1993b). Our multi-hapten conjugates will reduce or circumvent this suppression, because these conjugates will contain greater mass of immunogenic epitope per molecule of carrier than conventional conjugate vaccines. With our conjugates, the immllne system will not be "overchallenged"
by the carrier. For example, a tri-hapten conjugate prepared by methods of this invention will require only three injections to elicit specific immlmP responses to three different target pathogens. In contrast, using conventional monohapten conjugates, one would need to A(lminicter nine injections to elicit similar responses. This means three times the amount of protein would be required.

Further, i,l""ll~ tion regimes convert an anti-polysaccharide TI
response to a TD response can be designed using the conjugates of the present invention. Economical initial exposure to polysaccharide (e.g., using Pneumovax 23) followed by a single A~mini~tration of a conjugate of the present invention would induce IgG antibody levels (an ~nAmnPstic response). Such an immllni7~tion regime would not induce carrier suppression. In such cases, the immllnf~ system initially e~lllc~tcd to various carbohydrate epitopes and antigens (a TI response) would be induced by multi-hapten conjugates to elicit stronger 21S373~

immllnogenic responses to pathogens frequently causing disease in specific population groups (e.g., serotypes 1, 3, 4, 6, 9, 14, 18, 19 and 23 in infants).

Pharm~e~ltical Compositions:
To elicit antibodies to specific pathogens and/or various carbohydrate moieties the conjugates of the invention may be ~lmini~tered by various deliverymethods including intraperitoneally, hlllallluscularly, intradermally, subcutaneously, orally or nasally.

The formulation of the compositions of the present invention may include suitable ph~rm~ceutic~l carriers. The conjugates of the invention are immunogenic without adjuvant, however adjuvants may increase immunoprotective antibody titers or cell mP(li~te~ "~ y response. Such adjuvants could include, but are not limited to, Freunds complete adjuvant, Freunds incomplete adjuvant, aluminium hydroxide, dimethyldioctadecyl-ammonium bromide, Adjuvax (Alpha-Beta Technology), Inject Alum (Pierce), Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (Ribi Tmmllnochem Research), Titermax (CytRx), toxins, toxoids, glycoproteins, lipids, glycolipids, bacterial cell walls, subunits (bacterial or viral), carbohydrate 20 moieties (mono-, di-, tri- tetra-, oligo- and polysaccharide), various liposome formulations or saponins. Combinations of various adjuvants may be used with the conjugate to prepare the immunogen formulation.

Exact formulation of the compositions will depend on the particular 25 conjugate, the species to be illl"lllni~e~l and the route of ~tlmini~tration.

Such compositions are useful for immuni7ing any animal susceptible to bacterial or viral infection, such as bovine, ovine, caprine, equine, leporine, porcine, canine, feline and avian species. Both domestic and wild ~nim~l~ may be immlmi7ed. Humans may also be immnni7ed with these conjugate compositions.

The route of a(lmini~tration may be any convenient route, and may vary 5 depending on the bacteria or virus, the animal to be immllni7ed, and other factors. PalellLel~l a~lmini.~tration, such as subcutaneous, illll~llluscular, or intravenous ~lmini~tration, is pl~fell~d. Subcutaneous atlministration is most preferred. Oral ~(1mini.~tration may also be used, including oral dosage forms which are enteric coated.
The schedule of ~lministration may vary depending on the bacteria or virus pathogen and the animal to be immnni7ed. Animals may receive a single dose, or may receive a booster dose or doses. Annual boosters may be used for continued protection. In particular, three doses at days 0, 7 and 28 are pler~lled 15 to initially elicit antibody response.

The following examples are not intended to limit the scope of the invention m any manner.

20 Examples Example l:
Plepa~alion and Separation of Polysaccharide Hydrolysates Figure 2 shows the separation profile of Streptococcus pneumoniae 25 serotype 8 capsular polysaccharides through a BioGel P-l0 column after acid hydrolysis (0.5 M trifluoroacetic acid, 100C, 20 minutes) resulting in discernible oligosaccharides of one to eight repeat units. Numbers one to eight correspond to the number of repeat units found in each peak, peak nine contains oligosaccharides of greater than eight repeat units. Oligosaccharides derived 30 from hyaluronic acid were used to standardize the chromatographic system.

Z1~37~0 The relative size of the repeat units in peaks 1, 2, 3 and 4 were measured by HPLC analysis (Figure 3). The HPLC retention times of glucose, M-3 maltotriose, M-7 maltoheptose, and M-10 malto-oligosaccharide (Sigma 5 Chemical Co.) used as standards to determine relative size of various oligosaccharide repeat units is shown in Figure 4. Monosaccharide content of the repeat structure was established by further hydrolysis of the oligosaccharide repeats with 2.0 M trifluoroacetic acid (TFA) at 100C for 2 hours. An example of the retention times of ribitol, rhamnose, galactose, fucose and mannose 10 monosaccharide standards used to determine carbohydrate content of the hydrolysed repeat unit is shown in Figure 5. The chemical structure of one serotype 8 repeat unit was determine to be ~-glucose (1 ~ 4) ~-glucose (1 ~ 4) a-galactose (1 ~ 4) aglucuronic acid (1 ~ 4) by GC-MS and NMR analysis.
This corresponds to the repeating unit structure cited in the lilelalur~ (Jones and 1 5 Perry 1957).

Figures 6 - 10 are examples of separation profiles of S. pneumoniae serotypes 6B, 14, l9F and 23F polysaccharide hydrolysates (TFA, acetic acid or hydrofluoric acid) passed over P-10, P-30 or P-60 BioGel columns.
Figure 11 shows the separation of an enzyme cleaved polysaccharide (serotype 8 cleaved by cellulase). The separation of C-substance oligosaccharides is shown in Figure 12.

25 Example 2:
Inhibition ELISA to Determine Immunogenic Epitopes of Oligosacch~ride Preparations The basic procedure utilized for inhibition ELISA to test for the presence of immunogenic epitopes on oligosaccharide preparations and oligosaccharide or 30 polysaccharide-conjugates was as follows:

- 21537~0 1. Coat 96 well EIA plates (NUNC) with 1 ~g well of the antigen (Ag) using 0.05 M NaCO3 coating butter (100 ~ll/well), incubate at 4C
overnight.
2. On the same day, prepare inhibiting Ag tubes (e.g., various oligosaccharide hydrolysates) using 1 x PBS - 0.01% Tween 20 as diluent.
- Make a 7 fold serial dilution in the tubes (starting from 25 ~g/well to 0.391 ~lg/well in triplicate), the total volume in each tube should be 175 ~11 after serial dilution.
1 0 - Prepare 1:1000 dilution of anti-serum of a specific type (e.g., Diagnostic anti-serum 14 that has been raised in rabbits, Statum Seruminstitut), in 1 x PBS + Tween.
- Add 175 ~1 of this solution to each tube. Total volume in each tube is now 350 ,ul. Incubate the tubes at 4C overnight.

3. Next day, block the EIA plates with 100 ml/well of blocking buffer (1 x PBS + 1% BSA), incubate at room temperature for 1 hour.

4. Flick off the plates and transfer content of each tube to the wells (100 ml/well, incubate at room temperature for 2 hours.

5. Wash the plates 3 times with wash solution (0.01% Tween+ 1 x PBS).

6. Prepare 1:1500 dilution of Goat-anti-rabbit (or anti-species of serotype specific serum used in Step 2) IgG Alkaline Phosphatase conjugate (TAGO) in 1 x PBS + 1% Tween buffer (100 ~Ll/well). Incubate at room temperature for 2 hours.

7. Wash the plates 4 times with wash solution, flick off excess liquid.

8. Dissolve Alkaline Phosphatase substrate tablets (# 104 - Sigma) in the DEA (diethylenl~mine) buffer pH=9.98, 5 ml/tablet, 100 ml/well.

9. Incubate the plates in the dark and read the Absorbance at 405 nm wavelength every 15 minutes.

21~3730 Various commercial and laboratory prepared antiserum can be used in this assay, including, but not limited to, serum produced in mice, rat, rabbit, goat, pig, monkey, baboon and human.

Figure 13 shows the inhibition ELISA results using a mouse antiserum to Streptococcus pneumoniae serotype 8 oligosaccharide protein carrier conjugate (2-4 repeat units coupled using EDC to TT). Inhibition was tested with type 8 oligosaccharides (0.5 M TFA, 100C, 20 minute preparation) of 1, 2, 3, 4, 6, &
8 + repeat units, and with type 8 polysaccharides. From these results, it can beseen that the 1 repeat unit (a 4 monosaccharide chain) does not contain an immunogenic epitope. The 2 repeat unit (8 monosaccharide chain) was capable of inhibiting antibody binding to the ELISA plate, indicating that it contains an immunogenic epitope. The molecular weight of repeat unit 2 was determined to be 1365 by FAB-MS analysis. This correlates well with the theoretical 1 5 molecular weight of 8 saccharides . Repeat units of 3, 4, 6, 8 + and the whole polysaccharide also inhibited antibody binding to the ELISA plate, again indicating that immunogenic epitopes were present in these oligo/polysaccharides .

Table 3 demonstrates similar results found using a rabbit anti-S.
pneumoniae serotype 8 specific serum (Statems Se~ ). Repeat unit 1 did not markedly inhibit binding; repeat units 2, 3, 4, 5, 6, 7, 8+ and whole polysaccharide inhibited binding.

Inhibition ELISA was also used to determine the presence of immunogenic epitopes on oligosaccharides prepared using lirrelelll hydrolysis procedures on various polysaccharides. Table 4 shows results with methods used by the prior art, for example, Porro C~n~ n Patent No. 2 052 323 to hydrolyse S. pneumoniae serotype 6 polysaccharide (0.01 M acetic acid, 100C, 30 hours).
Whole polysaccharide blocked binding at low antigen concentration (effective at 21S373() 0.39 ~lg concentration) while the acetic acid hydrolysate did not. Note that we could not size separate the hydrolyzed preparation because it was "caramelized."

We discovered that different hydrolysing agents (e.g., TFA) and reduced 5 time and temperature produced oligosaccharides with more immllnogenic epitopes, as shown in Table 5. A 0.5 M TFA, 70 C 1 or 2 hour hydrolysate effectively inhibited antibody binding at a 3.13 ~lg concentration, a 4 hour preparation did not. Tables 6 and 7 also illustrate the effect of time for preparing 6B oligosaccharides with or without immunogenic epitopes. A 2 hour 1 0 acetic acid preparation blocked antibody binding (at 3.13 ~lg concentration), the 24 and 48 hour preparations did not. Similarly, a 1.5 hour TFA preparation more effectively blocked binding than a 3 hour preparation.

As shown in Table 8, 0.5 M TFA hydrolysis of S. pneumoniae serotype 1 5 14 at 70C for 7 hours, as disclosed in the prior art (Porro, C~n~ n Patent 2 052 323), is not prefelled. Reduced molar concentrations of TFA (e.g ., 0.1 M) is better for pl~a~ g immunogenic 14 oligosaccharides.

Table 9 illustrates the importance of selecting oligosaccharides which 20 contain immunogenic epitopes for coupling to carrier. The 3 repeat unit structure of serotype 14 oligosaccharide could not inhibit antibody binding, the 4 and 8 repeats, however, contain the immllnogenic epitopes and effectively blocked antibody binding.

Table 10 shows the effect of hydrolysate concentration and reaction time for preparing 14 oligosaccharides cont~ining immunogenic epitopes.
Immunogenic epitopes were conserved by a TFA 7 hour hydrolysis, but destroyed when hydrolysed for 24 hours.

2153~3~

Table 11 illustrates the importance of using optimal heat conditions for producing l9F oligosaccharides cont~ining immunogenic epitopes. Tmmlmogenic epitopes were destroyed by HCl hydrolysis at room temperature, but m~int~inPrl when hydrolysis was performed at 70C.

As shown in Table 12, poor inhibition of antibody binding was observed with 0.25 M TFA, 70C, 3 hr hydrolysates of 23F polysaccharides, (Porro, C~n~ n Patent 2 052 323). Table 13 demonstrates the effect of time on the generation of immunogenic 23- oligosaccharides. Oligosaccharides produced by

10 0.1 M TFA hydrolysis, 70C for 3 hours inhibited antibody binding, oligosaccharides prepared by hydrolysis for 5 hours did not inhibit. Table 14 demonstrates the presence of immunogenic oligosaccharides after 0.5 M TFA
hydrolysis at 70C for 15 minutes or with 5 M acetic acid at 70C for 5 hours.
These hydrolysates effectively inhibited to 0.78 ~lg concentration.
Table 15 demonstrates the utility of the inhibition ELISA to recognize immunogenic oligosaccharides of Neisseria meningitidis serotype C.
Hydrolysates prepared with NaOAc, blocked antibody binding as effectively as the whole polysaccharide.

20 Example 3:
Acidification of Carbohydrate Moieties for Carbodiimide Coupling A Rexyn 101 (H) organic acid cation exchange column (Fisher Scientific) was prepared and washed with dH20. Polysaccharide or oligosaccharide samples 25 dissolved in dH20 (pH neutral) were run over this column and collected at a rate of one drop per six seconds. Acidification was confirmed by pH colour-fixed indicator sticks. Excess dH20 was used to wash the column. Acidified fractions were pooled and lyophilized for use in coupling reactions.

2153~3~

Figure 14 depicts a TFA cleavage between b-D-Glcp (1 ~ 4)b-D-Gal of an oligosaccharide structure resulting in the formation of an aldehyde and hydroxylgroup. Further oxidation of the aldehyde results in a carboxyl group. When this material is passed through a cation exchange column, a COO~ group results.

Example 4:
Coupling Procedures and Quantification Assays Carbodiimide (EDC) Coupling Procedure 1 0 A 1:1 weight ratio of ion charged carbohydrate (polysaccharide or oligosaccharide) sample (e.g., 3 mg) and EDC (3 mg) was dissolved in 2 mls of 0.1 M KH2PO4, a pH of 4.5 was m~int~inPd with lN NaOH or HCl. This mixture was stirred for 1 hour at room temperature. Carrier (3 mg) was added to the EDC activated carbohydrates and then stirred for 4 hours at room 15 temperature. This reaction was stopped by the addition of 200 ~11 of 10%
ammonium bicarbonate, the mixture was then further stirred for 1 hour at room temperature.

The resultant conjugate was dialysed against dH20 overnight using 20 50,000 molecular weight cut off (MWCO) dialysis tubing.

Conjugates were lyophilized and then assayed by Lowry protein, phenol-sulfuric acid, sialic acid and phosphorous assays for composition (methods described below). Typically, conjugates prepared with this coupling methods 25 have a carbohydrate to carrier ratio of 1:2.

Phenol-Sulfuric Acid Assay for Quantification of Carbohydrates Reagent: 5% phenol solution (5.5 mL liquid phenol (90%) added to 94.5 mL
distilled water).

-- 21~30 Standard: Glucose 1 mg/ml stock solution. Prepare 2 to 60 ~g/200 ~11 sample buffer for standard curve.
Procedure: (Adapted from: Handbook of Micromethods for the Biological Sciences. Keleti, G. & W.H. Lederer (eds). 1974. Van Nostrand Reinhold Co., 5 New York.) 1. Place 200 ml samples into very clean tubes.
2. Add 200 ml phenol reagent.
3. Rapidly add 1 mL concentrated sulfuric acid.
4. Vortex well.
1 0 5. Let stand at room temperature for 30 minutes.
6. Color is stable at room temperature for 2 to 30 hours.
7. Read absorbance at 490 nm: blank with tube cont~ining water only as sample in # 1.

Quantitative Estimation of Sialic Acid Reagents:
a. 6 gram of Al2(SO4)3 . 18 H20 dissolved up to 20 ml with distilled water.
b. 1 gram para-dimethylaminobenzaldehyde dissolved up to 20 ml with 6 N
20 HCl. (Store in a dark bottle in the refrigerator).
Standard: N-acetylneuraminic acid at 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 ~Lg/~ll total volume is 350 ~1. Use distilled water to make up to 350 ~1.Method:
1. 200 ml of sample in duplicates. Make up to 350 ml with distilled water.
25 2. Add 700 ml of reagent A to each tube. Shake.
3. Add 350 ml of Ehrlich reagent B.
4. Cover all tubes with marbles.
5. Heat the tubes at 100 C for 30 minutes using Pierce Heating modules.
6. Cool the tubes rapidly to room temperature in an ice bath.
30 7. Read optical density at 530 nm wavelength.

Phosphorous Assay Reagents: a. 2.5 % ammonium molybdate; b. 10% ascorbic acid; c. 70%
perchloric acid; and d. 1 mM sodium phosphate standard.
5 Procedure: (Adapted from: Rouser, G., Siakotos, A. N. and Fleischer, S. 1966.
Lipids 1:85-86) 1. Place samples and standards (0, 25, 50, 100 and 200 ml; 25 to 200 nmoles) into clean tubes.
2. Dry samples in a heater block at 180C for 5 minutes in the fume hood.
1 0 3. Add 450 ml perchloric acid to each tube, cover each tube with a marble and heat at 180c for 30 - 60 minutes.
4. Add 2.5 mL d.H20. after tubes have cooled.
5. Add 0.5 ml ammonium molybdate and vortex immediately.
6. Add 0.5 ml ascorbic acid and vortex immediately.
1 5 7. Place tubes in 95c water for 15 minutes.
8. Read absorbance at 820 nm after tubes have cooled.
9. Samples can be left for several hours before being read.

Lowry Protein Assay 20 Reagents:
a. 2% (w/v) Na2CO3 in 0.1 M NaOH (1 L) b. 0.5% CuS04 in 1% sodium citrate (100 mL) c. Folin-Ciocalteu phenol reagent (2X) d. Bovine serum albumin (1 mg/mL) 25 Procedure:
1. Prepare standard curve which consists of: 0, 12.5, 25, 50, 100 and 200 llg of BSA in a final volume of 200 mL.
2. Bring unknown protein samples to 200 mL with d.H20.
3. Mix reagents A and B 50: 1 (v/v) and add 2 mL to each sample.
30 4. Vortex and let stand at room temperature for 10 minutes.

-- 21~373D

5. Dilute Folin-Ciocalteu phenol reagent 1:1 with d.H20 and add 200 mL to each sample.
6. Vortex and let stand at room temperature for 30 minutes.
7. Read absorbance at 660 nm.

Periodate Oxidation Coupling Procedure Samples of polysaccharide or oligosaccharide (e.g., 3 mg) were dissolved in 3 ml of freshly prepared 60 mM sodium meta-periodate in 50 mM sodium acetate. This preparation was then stirred overnight at 4C. Ethylene glycol (300 10 ~11) was then added to stop the reaction, this mixture was subsequently stirred at room temperature for 1 hour and then lyophilized. Samples dissolved in 1.5 ml of 0.03 M ammonium bicarbonate (pH = 8.0) were run over a P-2 Bio-Gel column. The phenol-sulfuric acid or sialic acid assays were used to determine fractions cont~ining the periodate reduced form of the samples, which were 1 5 subsequently lyophilized.
Figure 15 shows the separation of a reduced polysaccharide (23 valent polysaccharide vaccine-Pneumovax~ 23, Merck, Sharp and Dohme) fraction.
Figures 16 and 17 demonstrate separation of reduced oligosaccharides of serotypes 6B and l9F of Streptococcuspneumoniae, respectively.
Three mg of reduced saccharide and 3 mg of carrier were dissolved in 3 mls of 0.1 M sodium tetraborate decahydrate, pH 8.9. Sodium cyanoborohydride (H + source) was then added to this mixture and stirred for 48 hours at 50C. This reaction was stopped by adjusting the pH to 3 - 4 with 80%
25 acetic acid. This conjugate was then dialysed for 48 hours against dH20 (2 - 3 dH20 changes) using 50,000 MWCO dialysis tubing.

The conjugate was lyophilized, and the composition of the conjugate determined by Lowry protein assay, phenol-sulfuric, sialic acid and phosphorous ` 215373~

assays. Typically, conjugate prepared using this coupling method have carbohydrate to carrier ratios of 1:5 to 1:10.

Figure 18 depicts the periodate and EDC coupling chemistry reactions.

Example 5:
Conjugate Carriers Example 4 describes methods used to produce imml-nogenic oligosaccharide/polysaccharide conjugates from weakly or non-immunogenic polysaccharides .

Tetanus toxoid was purified for use as a carrier by column chromatography. This purified toxoid elicited high levels of IgM (e.g., 50~1g/mlmouse serum) and IgG isotypes (e.g., IgG, 100 ,ug/ml of serum; IgG2a, 38 ~g/ml of serum; IgG2b, 68 ~g/ml of serum; and IgG3, 105 ,ug/ml of serum).
Example 6:
Detellllhlation of Immunogenic Epitopes on Oligosaccharide/Polysaccharide Conjugates:
The inhibition ELISA as described in Example 2 was used. The presence of immunogenic epitopes on a mono-hapten 8-oligosaccharide tetanus toxoid conjugate was confirmed by inhibition ELISA. This conjugate inhibited anti-8 serum binding to a 8 polysaccharide coated ELISA plate (Figure 19). Free tetanus toxoid did not inhibit binding. The presence of immunogenic 8 oligosaccharide on di-hapten 6:8; 14:8 and 19:8 conjugates was also shown.
This figure illustrates the reproducibility of our coupling procedures, as the 8-mono-hapten and di-hapten conjugates equally blocked antibody binding, in(lir~ting that each conjugate contained equivalent amounts of 8 oligosaccharide.

2ls373n Table 16 shows results of inhibition ELISA when 6B polysaccharide, 6B
oligosaccharides, a 6B:8 di-hapten-oligosaccharide tetanus toxoid conjugate or tetanus toxoid alone was used as inhibiting antigens. Tetanus toxoid did not inhibit binding of anti-6B serum to a 6B-polysaccharide coated ELISA plate.
5 Free 6B-oligosaccharide or polysaccharide did inhibit binding. The 6B:8 di-hapten-oligosaccharide-TT conjugate also inhibited binding. This confirms the presence of immnnogenic 6B epitopes on the 6B:8 di-hapten-TT conjugate.

Similarly, a 14:8-di-hapten-TT conjugate inhibited anti-14 serum binding, 10 demonstrating the presence of serotype 14 immllnogenic epitopes (Table 17).
Note that at high concentrations, there was non-specific inhibition by TT alone.We have found that this is an artifact of anti-14 in this assay.

Various oligosaccharide fractions of a 23F hydrolysate were coupled to 15 TT. All contained immunogenic epitopes of the 23F serotype as shown in Table 18.

The immunogenic epitopes of N. meningitidis oligosaccharides (NaOAc preparation) were similarly m~int~in~d when coupled to tetanus toxoid (see Table20 19).

Example 7:
Determining Antibody Isotype Levels Elicited by Thymus Independent (TI) ~n~l Thymus Dependent (TD) Antigens The basic procedure to measure antibody isotype levels is as follows to quantify IgM, IgG and IgA isotypes elicited by various conjugates:
1. Coat EIA plates (NUNC, IMMUNOSORB) with 1 mg/well of Ag in 0.05 M sodium carbonate/sodium bicarbonate buffer pH-9.5, 100 ~l/well.
2. Incubate at 4C overnight.

21~73~
-3. Next day, block plates with 100 ml well of blocking buffer (1 x PBS +
1% BSA). Incubate at room temperature for approximately 1 hour.
4. Prepare 1:25 dilution mouse serum in working-buffer (1 x PBS + 0.1%
Tween). Add 100 ~l/well into the ap~ropliate well, incubate at room 5 temperature for 2 hours.
5. Wash plates 3 x with washing buffer ( 1 x PBS + 0.05 % Tween). Flick off excess liquid by tapping the plates on the bench top.
6. Prepare 1:2 dilution of EIA Grade Mouse Type (Rabbit Anti-Mouse, IgM, IgGI, IgG2a, IgG2b, IgG3 and IgA, Bio-Rad) in working buffer at 100 10 ~l/well. Incubate at room temperature for 2 hours.
7. Wash plates 3 x with washing buffer.
8. Prepare 1:1500 dilution of Goat-anti-Rabbit IgA Alkaline Phosphatase conjugate (TAGO) in working buffer at 100 ~Ll/well. Incubate at room temperature for 2 hours.
15 9. Wash plates 4 x with washing buffer.
10. Prepare enzyme substrate using Sigma # 104 Alkaline Phosphatase Substrate tablets (one tablet/5 mls of 10% diethanolamine substrate buffer), 100,ul/well. Incubate at room temperature in the dark and read every 30 minutes at 405 nm wavelength.
20 11. Convert absorbance readings to mg antibody/ml serum using dose-response curves generated from ELISA responses, of the rabbit anti-mouse isotype antibodies to various concentrations of mouse class and subclass specific immunoglobulin (Zymed Labs. Inc.).

Table 2 shows the antibody elicited in mice when i~ nlllli~cl with S.
Pneumoniae serotype 8 oligosaccharide and polysaccharide conjugates. Only the 8 oligosaccharide-conjugate elicited IgG antibodies of all isotypes, the unconjugated oligosaccharide was not immunogenic, the polysaccharide and the polysaccharide-conjugate elicited antibody isotypes typical of TI responses (mainly IgM, IgA, and IgG3 isotypes). Adjuvant was not necessary to elicit the 21~373~

IgG isotypes with our oligosaccharide-tetanus toxoid conjugate. Conjugates comprising relatively small oligosaccharides, haptens of 2 - 4 repeat units (8 - 16 saccharides), elicited the best antibody responses as measured by direct ELISA.

5 Direct ELISA Protocol 1. Use NUNC Maxisorp Tmmllnoplate.
2. Dilute coating antigen to 1.0 mg/100 ml in carbonate-bicarbonate buffer.
Use glass tubes as antigen will stick to plastic.
3. Add 100 ml to each well of plate. Store overnight at 4C.
1 0 4. Wash 3 x in PBS-.05 % Tween. Shake out excess PBS by tapping on Kimwipes/paper towels.
5. Add 100 ml/well of blocking agent (1 x PBS - 1 % BSA). Incubate for 60 minutes at room temperature.
6. Wash 3 x as in Step 4.
1 5 7. Add 100 ml/well of test antibody applopliately diluted in PBS - .01 %
Tween. Incubate for 90 minutes at room temperature.
8. Wash 3 x as in Step 4.
9. Dilute ;~lk~lin(~ phosphatase conjugated anti-mouse Ig (TAGO Cat # 4653) in PBS-Tween 1/1500. Add 100 Ill/well and incubate for 90 minutes in the dark.
20 10. Wash 3 x as in Step 4.

11. Add 100 ml/well Sigma 104 Phosphatase Substrate (disodium-p-nitrophenyl phosphate tables). Add 2 tablets (5 mg/tablet) of substrate to 10 mLdiethanolamine buffer. Keep in dark as substrate is inactivated by light.

12. Incubate in dark at room temperature. The development of the reaction 25 varies depending on the antibody. Absorbance can be read on the Microelisa Auto Reader (405 nm) at approximate 30 minutes intervals.

Results in Table 20, show a comparison of IgGl and IgG3 levels in mice immllni7ed with 8-conjugate at 3 weeks of age or at 8 weeks of age. Significant 30 IgGl levels were elicited by the 8-oligosaccharide-TT-conjugate in mice ~ 215373() immllni7ed at 3 weeks old (0.273 ~lg/ml) and at 8 weeks old (0.700 ~lg/ml).
Indicative of a TD response, an adjuvant (e.g., FCA) increased specific IgG1 (1.22 ~lg/ml). The 8-polysaccharide induced over-expression of IgG3 and low IgGl, typical of a polysaccharide TI response. The 8-polysaccharide-TT
5 conjugate, considered a "TD antigen", induced only low levels of IgG1, with overexpression of IgG3, characteristic of TI polysaccharide antigens. Also, adjuvant in combination with the 8-polysaccharide-TT conjugate did not enhance IgG1 levels, but did increase IgG3 antibody (TI-like response). Some polysaccharide-conjugates are known to elicit combinations of TI and TD
1 0 antibody response profiles (Stein, 1992; Stein, 1994).

Figure 20 depicts the IgG antibody isotypes elicited by a 8:14 di-hapten-oligosaccharide-TT conjugate to 8 polysaccharide. Like the 8-mono-hapten conjugate, this di-hapten conjugate could induce much higher levels of specific 1 5 IgG1 antibody (a TD response) than a 8-polysaccharide-conjugate or 8-polysaccharide alone. Overexpression of the IgG3 isotype to polysaccharide immunogen is shown. Control mice were injected with tetanus toxoid alone.

Results obtained with serotype 14-oligosaccharide conjugates are shown 20 in Table 21. A 14-oligosaccharide-TT-conjugate prepared by 0.1 M TFA
hydrolysis elicited IgGl, G2a, G2b, and G3 isotypes, the 1 llg dose was the mostimmllnngenic. Oligosaccharide-TT conjugates prepared using carbohydrate fractions of separation peaks 7 and 8 of a 0.5 M TFA hydrolysate elicited lower levels of IgG isotypes. Smaller oligosaccharides (peaks 4 and 5 of the 0.5 M
25 TFA preparation) in conjugate form elicited low levels of IgG isotypes. The 14-polysaccharide-TT conjugate elicited relatively high levels of IgG1 isotypes.
However, serum from mice injected with this polysaccharide conjugate was not immunoprotective (as will be shown in Example 8, Table 24). There appears to be a required threshold level of IgG antibody isotypes to provide 30 immlln~protection to the serotype 14 pathogen. The uncoupled 14 2 1 ~ 3 7 3 ~

polysaccharide, tetanus toxoid alone, or 0.9 % NaCl negative control serum all displayed low levels of all isotypes, equivalent to normal mouse serum (NMS) levels.

Figure 21 shows an increased level of IgG1 antibody isotype to 14-polysaccharide elicited by a 8:14 di-hapten-oligosaccharide-conjugate, typical of a TD response. The 14-polysaccharide induced overexpression of IgG3 (TI
response), the 14 oligosaccharide alone was not immunogenic. Uncoupled tetanus toxoid was a negative control.
As with individual hum~n~, different groups of mice displayed variable responsiveness to oligosaccharide- and polysaccharide-conjugates. In certain groups of mice, variations in the different IgG antibody isotype levels were observed. Figure 22A shows results from a group of "good responser" mice which produced IgGl to a 14-polysaccharide conjugate (a TD-like response).
Nevertheless, a 14-oligosaccharide-conjugate elicited higher IgG1 levels. This conjugate also elicited substantial levels of IgG2b (0-955 ~lg/ml =
oligosaccharide-conjugate; 0.139 ,ug/ml = polysaccharide-conjugate). This response was TD driven as FCA enhanced these IgG2b antibodies, Figure 22B.
(1.509 ~lg/ml = oligosaccharide-conjugate; 0.474 ~g/ml = polysaccharide-conjugate).

The ability of oligosaccharide-conjugates of the invention, to elicit greater TD antibody responses than polysaccharide-conjugates was not limited to S.
25 pneumoniae immunogens. Oligosaccharide-conjugates of Neisseria meningitidis Group C elicited greater levels of IgGI isotype antibody (7.01 ~lg/ml) than the polysaccharide-conjugate (3.60 ~lg/ml) or polysaccharide alone (0.162 ~g/ml).
Interestingly, the IgG3 isotype amounts induced by the oligosaccharide conjugates was also more (13.11 ,ug/ml = oligosaccharide-conjugate; 9.84 ~g/ml 30 = polysaccharide-conjugate; 3.81 ,ug/ml = polysaccharide alone).

-Example 8:
Bactericidal and Opsonization Assays to Measure Immunoprotective Antibodies Elicited by Conju~ates The basic bactericidal and opsonization assays used are as follows:
5 Bactericidal Assay 1. Streak a blood agar plate with desired gram negative bacteria procured from the American Type Culture Collection. Incubate at 37C, overnight.
2. Next day, pick an isolated colony and inoculate it in 1.0 ml of Todd-Hewitt Broth (THB) + Yeast Extraction (YE) media in a sterile test tube.
10 Incubate at 37C overnight.
3. On the following day, measure O.D. of inoculated bacteria at 420 nm wavelength. Use THB+YE media as blank.
4. To a sterile flat bottom 96-well plate, add a sterile 2.5 mm glass bead in each well.
15 5. To each well, add:
a. S ml of bacteria.
b. 10 ml of mouse serum to be tested.
incubate at 37C for 1 hour.
Note: Step # 5 and # 6 are done in triplicate 20 6. After 1 hour incubation, prepare 1:20 dilution of exogenous complement e.g. (Low Tox Rabbit Complement, Cedarlane) sterilely in THB+YE. Add 50 ~l/well. Incubate at 37C for 1 hour.
7. After complement incubation, 50 ml aliquot is plated out on blood agar plates using a glass spreader.
25 8. Wrap all agar plates in plastic bags and incubate at 37C for 12 hours.
9. Next day, count plaque forming colonies.

21~ 3~3 ~

Opsonization Assay l. Streak a blood agar plate with desired gram negative or positive bacteria (procured from the American Type Culture Collection). Incubate at 37 overnight.
2. Next day, pick an isolated colony and mix it with l.0 ml of THB+YE
media in sterile test tube. Incubate at 37C overnight.
3. The next day, prepare l00 U/ml of sterile heparin.
4. I.V. inject l00 ml of sterile heparin into tail of each mouse (5 - l0 mice).
After l0 minutes, bleed mice retro-orbitally into a sterile tube.
5. Measure O.D. of bacteria at 420 nm wavelength. Use THB+YE media as blank. (Use spectrophotometer 4040 to measure O.D.) 6. To a sterile flat bottom 96 well plate with sterile 2.5 mm glass bead in each well, add:
a. 50 ml of heparinized blood.
b. l0mlofserum c. 5 ml of bacteria Do this step in triplicate 7. Wrap plate in tinfoil and incubate at 37C incubator for one hour on a shaker (slow motion.
8. After one hour, a 50 ml aliquot is plated out on blood agar plates using a glass spreader.
9. Wrap all plates in plastic wrapper and incubate at 37C for 12 hours.
l0. Next day, count plaque forming colonies.

Serum from mice immllni7e~ with a S. pneumoniae type 8 oligosaccharide conjugate was found the be immunoprotective as measured by the opsonization assay. Opsonization of S. pneumoniae bacteria me~ tecl by specific anti-capsular antibodies is essential for host defense (Saunders, et al., 1993). This assay is generally considered a reliable indication of immunoprotective capability in vivo. Results from assays show that antibodies to the 8 oligo-conjugate greatly reduce growth of colony forming units of S.
pneumoniae serotype 8 on blood agar plates (Table 22). This reduction was specific, as colony growth of serotypes 3 and 6B (used as specificity controls) were not inhibited. Inllllu~ ation with the unconjugated oligosaccharide or 5 polysaccharide (which is used in the commercially available pneumoniae vaccine) elicited no protection. Protection elicited with the polysaccharide-conjugate was much less (39% reduction) than the protection elicited with the oligosaccharide conjugate (98% reduction). These results demonstrate that our 8 oligo-tetanus toxoid conjugate elicits high levels of immunoprotective antibodies against the 10 serotype 8 S. pneumoniae pathogen. The level of immunoprotective antibody elicited by poly-conjugates was marginal.

As well, the 8-oligo conjugate could elicit an immunoprotective antibody response in mice previously ~(lmini.~tered the whole polysaccharide alone. Mice 15 injected with 2 doses of 8-polysaccharide followed by a tertiary oligo-conjugate injection had immunoprotective antibodies in their serum (70% colony reduction in opsonization assay). As in previous experiments, mice receiving 3 injections of polysaccharide elicited no significant amount of protective antibody. Specific oligosaccharide serotypes coupled to a carrier protein may be beneficial as a 20 booster to augment the immunoprotection of high risk groups, non-responsive or only marginally responsive to the current 23-valent polysaccharide vaccine.

We have performed an immunogenicity study with di-hapten 3 oligo/8 oligo-tetanus toxoid conjugates. Oligosaccharides of both serotypes were 25 prepared by TFA hydrolysis. Mice injected with this multi-hapten conjugate elicited irnmunoprotective antibodies to the 3 and 8 serotypes (96 - 99% colony reduction - Table 23). A 3/8-polysaccharide conjugate elicited little immunoprotective antibody (10 - 12%). The mono-hapten 3 oligo-tetanus toxoid conjugate used in this study was not prepared with oligosaccharides that had been 30 determined to have immunogenic epitopes by inhibition ELISA and was not 215373~

capable of eliciting an immunoprotective response. The mechanism which allows the immllne system to response to epitopes on the 3 oligosaccharide in the di-hapten form is, of course, speculative. However, we suggest that the 8 oligosaccharides stimul~te clones of cell (i.e. accessory or helper cells) which5 can augment the response to the epitopes on the serotype 3 oligosaccharide.

We have discovered that the 8 oligosaccharide structure has adjuvant or adjuvant "like" activity. The relatively simple repeating unit structure of the 8-oligosaccharide (~-glucose (1 ~ 4) ~-Glucose (1 ~ 4)oc-galactose (1 ~ 4) a 10 gluconic acid) may specifically or non-specifically stimul~t~/activate immllnP
cells or induce receptors or factors to enhance a humoral/cellular response to non-immunogenic or weakly immunogenic polysaccharides/oligosaccharides.
Serotype 8 oligosaccharides has adjuvant activity in conjugate form or as an admixture to the vaccine formulation.

Opsonization results of a 14-oligosaccharide-TT conjugate (0.1 M TFA
preparation - Table 24) show good bacterial colony reduction of the 14 serotype (76%). The 14-oligo-TT 0.5 M TFA preparation elicited less immlln~ploLecli~e antibody (54% reduction). The serums from the polysaccharide-TT conjugate, 20 the polysaccharide alone and the tetanus toxoid injected mice showed greatly reduced inhibition capacity (18, 2 and 15% respectively). Serum from control mice (0.9 NaCL injected and NMS) showed no reductive capacity.

Di-hapten-oligosaccharide conjugates also elicited antibody with opsonic 25 activity. A serum to a 8:14-oligo-TT conjugate reduced serotype 14 colony forming units by 65 % (Table 25). This di-hapten conjugate was as immllnogenic as the mono-hapten 14-conjugate (reduction of CFU = 68%). Serum from mice immllni7ed with the polysaccharide-conjugate marginally reduced CFU's by 37% .

- 21~3~3~

F,x~mrle 9:
Circumvention of Carrier Suppression and Reduction of Anti~enic Competition Reduced responses due to antigenic competition when multiple antigens 5 are injected has been reported in the literature under some conditions. Results obtained from immnni7~tion schedules A and D (Table 26) will be used to d~tellllhle if the response to each component of our multi-hapten conjugate is equal to the response elicited by the single mono-hapten conjugates.

The unit mass of carbohydrate antigen of our mono- and multi-hapten conjugates will be equivalent (i.e., 1:2 CHO:protein ratio for EDC conjugates).
The design of our multi-hapten conjugates using reduced antigen load will minimi7e the potential for developing antigenic competition.

Schedules B and E will determine if a primary injection with the conjugate is sufficient to educate the immllnP system to elicit a T dependent response when boosted with uncoupled polysaccharide(s).

Schedules C and F will establish the capability of our conjugates to 20 enhance immunoprotective antibody responses in mice previously primed with polysaccharide(s) alone. If so, a multi-hapten pneumoniae vaccine cont~ining oligosaccharides of 3 to 4 serotypes may be very useful to augment the response to Pneumovax~ 23 in high risk patients.

Groups of mice will be injected by 3 doses (1, 2, 3) of tetanus toxoid (titers to tetanus toxoid to be confirmed by ELISA) followed by 3 injections of various S. pneumoniae oligo or poly-TT conjugates as in G (Table 26).

In all studies, conjugates will be ~lmini~tered orally and by subcutaneous injection.

~ 21~3730 The conjugates of the present invention will stim~ t~ immlm~ responses in infants, in children with imm~tllre immune systems and in the immllnnsuppressed. As models for these situations, we will d~te~ e the imml-nnpotentiating efficacy of our conjugates in young mice, in SCID and nude 5 mice. As described above, these mice will also be pre-sensitized with tetanus toxoid prior to multi-conjugate inoculation to study the carrier suppression phenomenon.

Modification of the above-described modes of carrying out the various 10 embodiments of this invention will be apparent to those skilled in the art following the teachings of this invention as set forth herein. The examples described above are not limiting, but are merely exemplary of this invention, the scope of which is defined by the following claims.

Claims (13)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED AS FOLLOWS:

1. A composition useful for stimulating an immune response to an antigen said immunostimulatory composition comprising an oligosaccharide of S.
pneumoniae serotype 8 which contains an immunogenic epitope as determined by inhibition ELISA and a suitable pharmaceutical excipient, wherein said oligosaccharide provides an immunostimulative effect.

2. The composition of Claim 1 wherein said oligosaccharide is conjugated to a protein carrier.

3. The composition of Claim 1 which does not induce carrier suppression.

4. The composition of Claim 1 which does not induce antigenic competition.

5. A method of providing protective immunization against a bacterial pathogen comprising administering to a mammal in need of such treatment an effective amount of the composition of Claim 1.

6. A method of augmenting an immunogenic response to an antigen comprising administering an oligosaccharide of S. pneumoniae serotype 8 which contains an immunogenic epitope as determined by inhibition ELISA along with said antigen.

7. The method of Claim 6 wherein said administration is selected from the group consisting of oral and parenteral.

8. A method of making a composition according to Claim 1 comprising:
a) cleaving S. pneumoniae serotype 8 polysaccharide into oligosaccharides so as to preserve immunogenic epitopes on the resulting oligosaccharides;
b) separating the resulting oligosaccharides based on size;
c) selecting those oligosaccharides which contain immunogenic epitopes based on inhibition ELISA; and d) mixing the selected oligosaccharides with a suitable pharmaceutical carrier.

9. The method of Claim 8 wherein said cleavage is performed using acid hydrolysis.

10. The method of Claim 8, further comprising the steps of, before step d):
1) activating the oligosaccharides selected in step c); and 2) coupling the activated oligosaccharides to a purified carrier.

11. The method of Claim 10 wherein said activation is acidification on a cation column.

12. The method of Claim 10 wherein said coupling is performed using EDC
or periodate.

13. The method of Claim 10 wherein said coupling provides a predictable ratio of hapten to carrier.