KR19990007777A - Immune and immunostimulatory oligosaccharide compositions and methods of making and using them - Google Patents

Abstract

The present invention provides an immunoglobulin composition and a method for preparing and using the same. In particular, the composition consists of the oligosaccharide covalently coupled to a carrier, which appears to contain the conjugate specific immunoepitope produced therein and induces a protective immune response. The present invention also relates to a composition comprising an oligosaccharide of S. pneumoniae serotype 8 useful for stimulating an immune response to an antigen, a method of imparting protective immunization against bacterial pathogens using this composition, A method of increasing the immune response to an antigen by administering an oligosaccharide composition of pneumoniae serotype 8 and a method of producing the immunostimulatory composition as described above.

Description

Immune and immunostimulatory oligosaccharide compositions and methods of making and using them

Immune response to polysaccharides

Heidelberger and Avery [1923] demonstrated that type-specific antigens of Streptococcus pneumoniae are polysaccharides. Bacterial capsular polysaccharides are cell surface antigens composed of identical repeating units that form an extended sugar chain. The polysaccharide structure is present in pathogenic bacteria and is found in E. coli, Neisseria meningitidis, Hemophilus influenzae, Group A and Group B streptococcus, Streptococcus pneumoniae and other species (Kenne and Lindberg 1983). ≪ / RTI >

Specific blood type determinants and tumor-associated antigens are examples of mammalian cell surface carbohydrates. Carcinogenic transformed cells often show surface carbohydrates that are significantly different from those of unmodified cells. These glycans are composed of a small number of monosaccharides (Hakomori and Kannagi 1986). The glycan structure itself is not normally antigenic but binds to a protein or glycoprotein matrix to form a hapten.

A common feature of sugar antigens is that they do not induce significant levels of IgG antibody class (IgG isotype) or memory response, so they are considered thymus-independent (TI) antigens. Binding to thymus-dependent (TD) antigens, such as proteins in carbohydrate hapten, which are polysaccharide antigens or immunologically inert, enhance their immunity. The protein stimulates carrier-specific T-helper cells that act on induction of anti-carbohydrate antibody synthesis (Bixler and Pillai 1989).

Our current knowledge of TI and TD responses comes from a number of studies of related mouse models [Stein et al., 1983; Stein, 1992; Stein, 1994]. TI antigens generally induce a limited class of low-affinity antibodies and do not produce immune memory. Adjuvants have little effect on the response to TI antigen. In contrast, TD antigens induce heterologous high affinity antibodies by immunization and produce immune memory. The adjuvant enhances the response to the TD antigen. The second-order reaction to the TD antigen shows an increase in the ratio of IgG to IgM, while the second-order reaction to the TI antigen is 1: 1, with an IgG: IgM ratio similar to that of the first reaction [Stein et al., 1982; Stein, 1992 and 1994]. In rats and humans, TD antigens induce mainly IgG ₁ isotypes with some IgG ₂ and IgG ₃ isotypes. The TI response to polysaccharides is confined to IgG ₃ among IgG isotypes (Perlmutter et al., 1978; Slack et al., 1980].

Current pneumococcal vaccine

Pneumococci are currently classified into 84 serotypes based on their capsular polysaccharide. Serum types 1, 3, 4, 7, 8, and 12 are generally more prevalent in adult populations, although there are a number of serotype variations that are common to geographical locations. 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, wherein that - it consists of a capsule polysaccharide purification of the 23 strains of Streptococcus pneumoniae vaccine Streptococcus pneumoniae [pneumophila BAX 23 ^[trademark] (Pneumovax 23), Merck Sharp and Dome (Merk Sharp Dohme)]. The capsule type of S. pneumoniae contained in the NemoBacs 23 was 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, 33F (Danish Nomenclature). These serotypes are said to cause 90% of the world's severe pneumonia.

There are some contradictions in the literature on the efficacy of the Nemovax 23 vaccine (Borgano et al., 1978; Broome et al., 1986; Simberkoff et al., 1986; Forester et al., 1987; Shapiro, 1987; Sims et al., 1988; Simberkov, 1989; Shapiro, 1991]. Pneumococcal vaccines are effective in inducing antibody responses in healthy adults [Hilleman et al., 1981; Mufson et al., 1985; Bruyan and van Furth, 1991). However, there is a distinct variety in the persistence of antibody titers and the intensity of responses to other serotypes (Hillman et al., 1981; Muffson et al., 1987].

Infants younger than 2 years old are most vulnerable to systemic diseases, otitis media and acute lower respiratory tract infections caused by pneumococci but do not respond to this vaccine (Sell et al., 1981; Hazelwood et al., 1993; Saunders et al., 1993]. Furthermore, patients whose immune suppression of old age shows impaired or diverse responses to Nemovascus 23 (Siber et al., 1978; Schildt et al., 1983; Forrester et al., 1987; Simberkov, 1989; Shapiro, 1991]. These groups do not respond well to the thymus-independent polysaccharide antigen of this vaccine. Antibody switching from IgM to IgG isotype, typical of thymus-independent antigens, is not generally observed and no anamnestic response to booster immunization is observed [Borghano et al., 1978].

Recently, the emergence of antibody resistant strains has highlighted the need for the development of efficacious vaccines for the prevention of childhood infections. Clearly, new vaccines against pneumococcal are needed especially for groups and children who are at greatest risk.

Conjugate vaccines

Averry and Goebel produced vaccines for bacterial infections for the first time [Avery and Goebel 1929; Gurubel and Avery 1929]. Recently, several protein carrier conjugates have been developed that cause thymus-dependent responses to polysaccharides in a variety of bacteria. Up to now, the development of a conjugate vaccine against type b (Hib) has received the greatest attention in Hemophilus influenza. Schneerson et al., 1980) covalently coupled Hib polysaccharide (polyribitol-phosphate) to diphtheria toxoid. They also developed a Hib vaccine by detaching the polysaccharide with adipic acid dihydrazide spacer and coupling it to the tetanus toxoid along with carbodiimide [Shyne, et al., 1986]. A similar method was used to produce a co-pore containing diphtheria toxoid as a carrier (Gordon, 1986 and 1987). A bifunctional spacer was used to couple the outer membrane protein of B group Nyseria meningitidis to Hib polysaccharides (Marburg et al., 1986, 1987 and 1989). Finally, Anderson (1983 and 1987) produced conjugated vaccines using Hib oligosaccharides coupled to the non-toxic cross-reactive mutant diphtheria toxin CRM ₁₉₇ by reductive amination.

Reports in the literature are different for the efficacy of these vaccines, and many studies are still underway. However, oligosaccharide cofactors [Anderson et al., 1985a, 1985b, 1986, 1989; Seid et al., 1989; Madore et al., 1990; Eby et al., 1994] and polysaccharide conjugate [Barra et al., 1993] have been reported to cause immune and thymic-dependent responses in infants. Haptic loading is an important factor for pore immunity (Anderson et al., 1989; Eby et al., 1994].

Other conjugate vaccines were developed by Jennings et al. (Jennings et al., 1985 and 1989), which used periodate activation to couple the polysaccharide of Nyseria meningitidis to the tetanus or diphtheria toxoid carrier. Porro (1987) identified a method for coupling an esterified N. meningitidis oligosaccharide to a carrier protein. Tsay and Collins, 1987), which has been linked to the toxin removal protein of Pseudomonas aeruginosa by the peritrodite method (Tsay and Collins, 1987). Cryz and Furer [1988] used adipic dihydrazide as a spacer arm to generate a conjugate vaccine against P. aeruginosa.

The specific serotype polysaccharides of S. pneumoniae also include classical carrier proteins such as tetanus or diphtheria toxoid [Srinierson et al., 1984; Fattom et al., 1988 and 1990; Shinneyerson et al., 1992), N. meningitidis membrane proteins (Marburg et al., 1987; Giebink et al., 1993] and a neomolingic mutant carrier (Paton et al., 1991; Lock et al., 1992; Lee et al., 1994). Techniques for coupling S. pneumoniae oligosaccharide to CRM ₁₉₇ protein have been developed [Po et al., 1991]. These conjugate vaccines have immunity enhancing properties, although not mutable or yet to be determined. Regeneration of these coupling techniques with the maintenance of immune epitopes is currently the biggest problem in developing effective S. pneumoniae glyco-conjugated vaccines. The size of the optimal immunoglobulin seems to depend on the serotype that represents the morphological aspect of a given immunologic epitope (Evy et al., 1994; Steinhoff et al., 1994).

A vaccine is needed to induce long-term, sustained protection against DTP, tuberculosis, polio, measles, hepatitis, Hib and pneumonia. To induce protection against S. pneumoniae in infants, a multi-dentin protein conjugate containing a high level of oligosaccharide of optimal immunity size for each serotype is required.

Several investigators have suggested that conjunctival injection enhances the immunity of conjugate vaccines. An example is aluminum salts approved for use in humans. Carbohydrate moieties such as beta glucan particles and low molecular weight dextrins have also been reported to have adjuvant activity. Ajugas (alpha-beta technology) is an adjuvant composition containing beta glucan particles. Lee et al. [1994] reported the use of low molecular weight dextrin constructs as adjuvants. Penny et al. (1992) reported long chain alkyl compounds with immunological activity.

The present invention relates to immunosuppressive and immunostimulatory oligosaccharide compositions and methods of making and using the same. In particular, the immunogenic composition comprises an oligosaccharide covalently coupled to a carrier which elicits the conjugate autoimmune protective response generated therefrom. The immunostimulatory composition consists of the pneumococcal serotype 8 oligosaccharide covalently coupled to the carrier or as a mixture in the formulation.

references

The following references are incorporated herein by reference.

Anderson, P., Infect. Immun. 39: 233, 1983

Anderson, P. W., Immunogenic Conjugates, U.S. Pat. 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, 1985b.

Anderson, P. W., Pichichero, M. E., Insel, R. A., Betts, R., Eby, R., and Smith, D. J., J. Immunol. 137: 1181, 1986.

E., Zabradnick, J.M., and Eby, R., J. Immunol., 2000, pp. 315-323, 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.

Bixier, G.S. and Pillai, S., The cellular basis of the immune 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., Frackiam, R. R., Pitkaytis, B. D., Fraser, D. 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.

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., Schiffman, G., and Rao, S., Infect. Immun. 50: 213,1985.

Connelly, K. K., and Starke, J. R., Sem. Resp. Inf. 6: 209, 1991.

Cryz, S. J., and Fuerer, E., Conjugate vaccine against infections by gramnegative bacteria, method for its preparation and use, U.S. Pat. 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 Prevention of AIDS, E. Norrby, F. Brown, R. Chanock and H. Ginsberg , eds., Cold Spring Habor Laboratory Press, New York, 1994.

Fattom, A., Lue, C., Szu, S. C., Mestecky, J., Schiffman, G., Brylar, D., Vann., W. F., Watson, D., Kimzey, L. M., Robbins, and Schneerson, R., Infect. and Immun. 58: 2309, 1990.

Fattom, A., Vann, WF, Szu, SC, Schneerson, R., Robbins, JB, Chu, C., Sutton, A., Vickers, JC, London, WT, Curfman, B., Hardagree, MC, and Shiloach, J. Infect. Immun. 56: 2292, 1988.

Forester, H. L. Jahnigen, D. W., and LaForce, F. M., Am. J. Med. 83: 425,1987.

Gaur, A., Arunan, K., Singh, O. and Talwar, G. P., Int. Immunol. 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. Pat. 4,619,828, 1986.

Gordon, L. K., Haemophilus influenzae b polysaccharide-diphtheria toxoid conjugate vaccine, U.S. Pat. Patent No. 4,644,059, 1987.

C., Wang, DG, Joyce, HJ, Milford-Wards, A., Forte, M. and Pahor, A., Clin . Exp. Immunol. 93: 157,1993.

Hakamori, S. and Kannagi, R., Carbohydrate antigens in higher animals, in Handbook of experimental immunology - vol. 1, D. M. Weir, L. A. Herzenberg, C. Blackwell and L.A. Herzenberg, eds., Blackwell, Oxford, 1986,

Heidelberger, M. and Avery, O. T., J. Exp. Med. 38:73, 1923.

Milleman, 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, Canadian Patent No. 1,191,344, 1985.

Jennings, H. J., Roy, R., and Gamian, A. J., Modified meningococcal group b polysaccharide for conjugate vaccine, Canadian Patent No. 1,261,320, 1989.

Jones, J.K.N. and 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, CJ, 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., Hansman, 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., Crystaline bacterial cell surface layers (S-layers)

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.

Malcom, AJ, Best, MW, Szarka, RJ, Mosleh, Z., Unger, FM, Messner, P. and Sleytr, UB, Surface layers of Bacillus alvei as a carrier for a vaccine in Streptococcus pneumoniae vaccine in Adces in Bacterial Paracrystalline Surface Layers, TJ Beveridge and SF 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: 5782,1986.

Marburg, S., Tolman, R. L. and Kniskern, P. J., Covalently-modified polyanionic bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers, and methods of preparing such polysaccharides and conjugates and conforming covalency. 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 immunogenic proteins with bigeneric spacers and methods of preparing suchpolysaccharides and conjugates and conforming covalency, 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., Schiffman, 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.

P., Berry, A. M. Michell, T. J., Andrew, P. W., Hansman, D., and Boulnois, G. C., Lock, R. A., Lees, C-J. J., Infect. Immun. 59: 2297,1991.

Peeters, C. A. M., Tenbergen-Meekes, A-M., Poolman, J. T., Berutett, M., Zegers, B. J. M. and Rijkers, T., Infect. Immun. 59: 3504,1991.

Penney, C. L., Michon, F., and Jennings, H. J., Improved Vaccine Compositions, WO 92/04951, 1992.

Perlmutter, R. M., Hansburg, D., Briles, D. E., Nicolotti, R. A., and Davie, J. M., J. Immunol. 121: 566, 1978.

Porro, M., Oligosaccharide Conjugate Vaccines, Canadian Patent No. 2 052 323, 1992.

Porro, M., and Costantino, P., Glycoproteinic conjugates having trivalent immunogenic activity, U.S. 4, 711, 779, 1987.

Porro, M., Oligosaccharide conjugate vaccines, U.S. Pat. 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., McCarcken, J. D., Schiffman, 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.

Schneerson, R., Robbins, J. B., Chu, C., Sutton, A., Vann, W., Vickers, J. C. London, W. T., Curfman, B. and Hardegree, M. C., Infect. Immun. 45: 582, 1984.

Schneerson, R., Robbins, J. B., Parke, J. C., Bell, C., Schlesselman, J. J., Sutton, A., Wang, Z., Schiffman, G., Karpas, A., and Shiloach, J., Infect. Immun. 52: 519, 1986.

Schneerson, R., Levi, L., Robbins, J. B., Bryla, D. M. Schiffman, G. and Lagergard, T., Infect. and Immunity 60: 3528,1992.

Seid, R. C., Jr., Boykins, R.A., Liu, D.F., Kibrough, K.W., Hsieh, C.L. Eby, R., Glycoconj. J. 6: 489, 1989.

Sell, S. H., Wright, P. F., Vaugh, W. K., Thompson, J., and Schiffman, G., Rev. Infect. Dis. 3 (suppl): S97, 1981.

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 Schiffman, G., N. Engl. J. Med. 299: 442, 1978.

Schiffman, G., and Shepard, D. S., N. Engl., N., N., N., Richmond, A., S., Simberkoff, M., Cross, A. P., Al-Ibrahim, M., Baltch, A. L., J. Med. 315: 1318, 1986.

Simberkoff, M. S., Pneumococcal vaccine in adults, in: Immunization, M. A. Sande, and R. K. Root, ed., Chrchill Livingstone, New York, 1989.

Sims, R. V., Steimman, W. C., McConville, J. H., King, L. K., Zwick, 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.

Solyer, J. L., Jr., Ploussard, J. H., and Howie, V. M. 1981, Rev. Infect. Dis. 3 (suppl): S1, 1981.

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, 1992.

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.S. Pat. 4,663,160, 1987.

The disclosures of these publications, patents, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated into its language.

1 shows a repeating unit structure of a polysaccharide used in an embodiment of the present invention.

FIG. 2 shows the results of the analysis of the streptococcus pneumoniae serotype (SEQ ID NO: 2), which is a fractional oligosaccharide of 1 to 8 repeating units after passage through a bio gel P-10 column after acid hydrolysis (0.5 M trifluoroacetic acid, 8 < / RTI >

Figure 3 shows the relative sizes of the repeating units at peaks 1, 2, 3 and 4 of the hydrolyzed Streptococcus pneumoniae serotype 8 capsular polysaccharide as measured by HPLC analysis.

Figure 4 shows the HPLC delay times of glucose, M-3 maltotriose, M-7 maltotriose, and M-10 malto-oligosaccharide standards used to determine the relative sizes of the various oligosaccharide repeating units.

FIG. 5 is an example of the delay times of the standard of ribitol, rhamnose, galactose, fucose, and mannose monoglyceride used to measure the carbohydrate content of hydrolyzed repeat units.

Figure 6 shows the separation of S. pneumoniae serotype 6B polysaccharide hydrofluoric acid hydrate through a P-10 bio-gel column.

Figure 7 shows the separation of S. pneumoniae serotype 6B polysaccharide TFA hydrate through a P-60 bio gel column.

Figure 8 shows the separation of S. pneumoniae serotype 14 polysaccharide TFA hydrate through a P-30 bio gel column.

Figure 9 shows the separation of S. pneumoniae serotype 19F polysaccharide acetic acid hydrate through a P-10 bio gel column.

Figure 10 shows the separation of S. pneumoniae serotype 23F polysaccharide TFA hydrate through a P-10 bio gel column.

Figure 11 shows the degree of cleavage of polysaccharides cleaved by the cellulase of S. pneumoniae serotype 8 that passed through a P-10 bio gel column.

Figure 12 shows the degree of separation of the polysaccharide hydrofluoric acid hydrate of the C-material of Streptococcus pneumoniae through the P-10 bio-gel column.

13 shows the results of inhibition ELISA using mouse antiserum against the oligosaccharide protein carrier conjugate of Streptococcus pneumoniae serotype 8.

Figure 14 shows the acidification of the oligosaccharide to the carboxydecoupling.

15 shows the separation of a reduced fraction and periodate fraction of a polysaccharide (23 polysaccharide vaccine - Newcastle 23, Merck, Sharp & Dome).

Figure 16 shows the separation of the reduced and oligosaccharide fractions of the oligosaccharides of serotype 6B of Streptococcus pneumoniae.

Figure 17 shows the separation of the reduced fraction and the periodate fraction of the oligosaccharide of serotype 19F of Streptococcus pneumoniae.

Figure 18 depicts periodate and EDC coupling chemistry.

Figure 19 shows how the mono-hapten 8-oligosaccharide tetanus toxoid conjugate inhibited anti-8 serum binding to 8 polysaccharides coated on an Eliza plate.

Figure 20 shows IgG antibody isotype induced by polysaccharide of S. pneumoniae serotype 8 after immunization with 8:14 di-hapten-oligosaccharide-T conjugate.

Figure 21 shows the level of increase in polysaccharide-induced IgG ₁ antibody isotype after immunization with a typical 8:14 di-hapten-oligosaccharide-T conjugate in a TD response.

Figures 22A and 22B show IgG isotypes induced from a population of rats immunized with 14-polysaccharide and oligosaccharide conjugates with or without adjuvant.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising a) a carbohydrate hapten separated by size consisting of at least one immunogenic epitope and b) a carrier covalently coupled to hapten wherein the hapten-carrier conjugate has protective immunity do.

In another aspect, the present invention provides a method of producing a polypeptide comprising the steps of: a) cleaving a polysaccharide of a bacterium into oligosaccharides so that the resulting oligosaccharide has an immunogenic epitope; b) separating the resulting oligosaccharide by size; c) selecting an oligosaccharide containing an immunogenic epitope based on an inhibition ELISA; d) activating the oligosaccharide selected in step c); And e) coupling the activated oligosaccharide to the purified carrier (so that the resultant composition contains an immunogenic epitope and has protective immunity).

In another aspect, the invention provides a method of imparting protective immunity against bacterial pathogens comprising administering an effective amount of a vaccine composition described above to a mammal in need of such treatment.

In another aspect, the invention relates to an immunostimulatory composition comprising an oligosaccharide of S. pneumoniae serotype 8 containing an immunostimulatory epitope as determined by an inhibitory ELISA and a pharmaceutical excipient suitable for providing an immunostimulatory effect of said oligosaccharide Which is useful for stimulating an immune response against a < RTI ID = 0.0 > anti-human < / RTI >

In another aspect, the invention provides a method of imparting protective immunity against bacterial pathogens comprising administering to a mammal in need of such treatment an effective amount of a serotype 8 composition as described above.

In another aspect, the invention provides a method of increasing an immune response to an antigen comprising administering, together with the antigen, an oligosaccharide of S. pneumoniae serotype 8 containing an immunogenic epitope determined by an inhibitory ELISA do.

In another aspect, the present invention provides a method for producing oligosaccharides comprising: a) cleaving polysaccharides of S. pneumoniae serotype 8 into oligosaccharides so that the oligosaccharide produced has an immunogenic epitope; b) separating the resulting oligosaccharide by size; c) selecting an oligosaccharide containing an immunogenic epitope based on an inhibition ELISA; And d) mixing the selected oligosaccharide with a suitable pharmaceutical carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improved method for preparing an oligosaccharide-protein carrier conjugate. The coplanar product may consist of various haptens or carriers. Mono, di, and multi-haptic copiers can be manufactured. Methods for determining the hapten of the resulting conjugate or an immunological epitope on the carrier are described. Such conjugates have utility as vaccines, therapeutic and prophylactic agents, immune modulator diagnostic agents, development and research tools.

The present invention relates to a method for the treatment of Streptococcus pneumoniae, Nisseria meningitidis, Hemophilus influenzae B, Group B streptococcus, Group A streptococcus, Bordetella pertussis, Escherichia coli, Streptococcus mutans but are not limited to, mutans, Staphylococcus aureus, Salmonella typhi, Cryptococcus neoformans, Pseudomonas aeruginosa and Klebsiella pneumoniae But are not limited to, vaccines against bacterial pathogens.

Inadequate immune responses to polysaccharide vaccines (thymus-independent antigens, TI) have been observed in high-risk populations such as elderly people and children under 2 years of age. Some researchers are attempting to elicit thymus-dependent (TD) responses against a variety of bacterial polysaccharides using protein carriers. Conservation of important immune epitopes and the inconsistency of covalent linkages between carbohydrates and proteins are the major limitations of these co-injected vaccines. The present invention is derived from the discovery of a coupling technique that gives good reproducibility with respect to the carbohydrate-to-carrier ratio of the blank. The present invention also provides a method for verifying the presence of an immunogenic epitope on oligosaccharide haptens and hapten-carrier conjugates.

Polydannic conjugates induce a non-boostable IgM antibody response typical of TI antigens. The antiserum produced by the reaction to this polysaccharide conjugate does not have the activity of opsonin. In the present invention, oligosaccharides prepared by cleaving polysaccharides from various bacterial strains are separated by size and used to produce mono-conjugated co-pores. These conjugates induce IgG antibody isotypes with immunoprotective and opsonizing abilities. This antibody response is induced without the use of any adjuvant. Thus, the method of the present invention is ideally suited for producing immunogenic oligosaccharide-carrier conjugates using weakly immune or non-immunogenic polysaccharides of various strains. The presence of immune epitopes on these oligosaccharides has been found to be important in inducing an immunoprotective response.

The number of bacterial antigens required to develop efficacious anti-pathogenic vaccines is increasing. However, repeated administration of tetanus or diphtheria toxoid (carrier protein in vaccine compositions and often used as a preventative measure after trauma) may cause a phenomenon called carrier-induced epitope suppression. Epitope suppression or synthetic peptides and sugar-toxoid conjugates in the literature (Gaur et al., 1990; Peeters et al., 1991]. The immune response to the hapten coupled to the carrier protein may decrease or disappear when the recipient is already immunized by the carrier.

The goal of many researchers is to develop a vaccine that does not induce carrier inhibition, but also protects against serotypes of major bacteria causing acute lower respiratory infections, otitis media, and bacteremia in infants. The method of the present invention can be used to generate multi-dentin co-pores with optimal immuno-epitopes for each bacterial serotype. These conjugates, which contain a smaller amount of carrier protein than conventional conjugates, reduce the incidence of carrier inhibition. The possible reduced antigen load using these conjugates minimizes the antigen competition observed in conventional conjugates.

Previously, we reported that a crystalline bacterial surface membrane (S-membrane) was used as a carrier for the development of a prototype pore vaccine [Malcolm et al., 1993a] and as a means of avoiding carrier inhibition phenomena [ Malcolm et al., 1993b]. In our laboratory, we have identified several S-manganese glycoproteins that cause comparative response antibodies and cellular responses. Vaccines against a variety of diseases can be developed using S-membranes identified in a variety of bacterial strains, thus avoiding carrier inhibition observed in the tetanus and diphtheria toxoids. However, S-membranes are not practical for extensive use as vaccine carriers because they are costly to produce and difficult to identify and purify. The present invention relates to a mono-, di- and multi-hapten oligosaccharide conjugate which reduces the risk of carrier-induced epitope suppression even when tetanus or diphtheria toxoid is used as a carrier by reducing the amount of carrier required to induce a specific response Describe the method of manufacture.

One specific application of the technique of the present invention is to develop an effective vaccine for the prevention of pediatric pneumonia infection. Other applications of the present invention include protection against infantile diseases, Group B streptococci, Group A streptococci, H. influenzae B, Streptococcus pneumoniae and N. meningitidis strains predominant in older or immunosuppressed To develop a vaccine for.

We have found that the improved immunity of the resultant conjugate can be obtained by using a condition in which a particular linkage (ie, 1-4 linkages) is cleaved and other immunologically important compounds such as sucrose monosaccharide and phosphate are left intact.

We have found that the size and shape of oligosaccharides are important for maximizing the immunity of the formulations. Oligosaccharides of different sizes can be generated from the hydrolyzed polysaccharide mixture and separated by size fractions. The monosaccharide content and the relative size of the separated oligosaccharides are measured, for example, by HPLC analysis. Repeating units of different sizes are tested using an inhibition ELISA. We found that ELISA inhibition is directly proportional to the immunity of the oligosaccharide preparation and the resulting porphyry.

In particular, in our laboratory, polysaccharides of S. pneumoniae strains 3, 6B, 8, 14, 19F and 23; C-substance of Streptococcus pneumoniae; And oligosaccharides obtained by cleavage of N. meningitidis C-polysaccharide. The preferred repeat units (R.U.) for oligosaccharides are as follows for some S. pneumoniae serotypes and the C-material of Streptococcus pneumoniae:

Serotype 3: 4-8 R.U.

6B: 4-10 R.U.

8: 2-8 R.U.

14: 4-6 R.U.

19F: 4-10 R.U.

C-material: 6-10 R.U.

A preferred repeat unit for N. meningitidis C-polysaccharide is 6-10 RU.

It has been found that generating a charged group on sugar heptane facilitates the coupling of haptens to the carrier. The use of a cation or anion exchange column is effective for coupling oligosaccharide to the carrier by increasing the ratio of sugar: carrier. This provides more haptens per carrier and reduces carrier suppression. Reduced fractions of carbohydrates are used for coupling to the carrier.

Another important aspect of generating effective pore vaccines is the use of purified carriers. Impurities present in the carrier formulation may interfere with the coupling process. The aggregation of the carrier protein in the carrier preparation may affect the optimal ratio of the hapten: carrier required to elicit the desired reaction. The carrier is generally purified using size exclusion column chromatography, but any standard method of removing impurities and aggregates may be used.

The coupling reaction time, and the amount of oligosaccharide, coupling and carrier, are important for obtaining the ideal ratio of carbohydrate to carrier conjugate. We have developed a method for quantifying the ratio of carbohydrate to carrier by reproducible assays. The maintenance of pH and temperature conditions determined to be optimal during the coupling reaction is also important to generate an effective blank. Likewise, the use of an effective blocking agent that disrupts the immunogen while interrupting the coupling reaction is also important in producing an effective pore composition.

The use of coupling chemistry to maintain the immunoaffinity of the oligosaccharide / polysaccharide is also essential. We have found that EDC and periodate coupling can be used for the coupling of oligosaccharides to a carrier as described below. In addition to coupling the sugar directly to the carrier, various linkers can be used to float the sugar from the surface of the protein a certain distance. Suitable linkers may also provide charged or uncharged moieties as desired. The immunogenicity of the coupled sugar-carrier composition is determined by the inhibitor ELISA.

Using the method of the present invention, we have found a means of generating di-haptens and multi-haptens that still maintain an immunological epitope. Co-pores having various oligosaccharide sequences and / or sizes may be produced. Similarly, a conjugate consisting of a complex of oligosaccharide and polysaccharide can be synthesized. Such copulas can reduce or eliminate competition between antigens.

Thus, a suitable coplanar design provides the ability to reduce carrier induced epitope suppression. The point in this regard is the identification and use of more effective coupling of sugars to immunoglobulin epitopes and proteins. The binding of a greater number of immune epitopes per protein molecule means that fewer carriers are needed to provide protective immunization.

We have developed a method for quantifying the immunoprotective antibody response to a conjugate composition by isotyping ELISA, sterilization and opsonization assays. This makes it possible to determine when a conjugate will protectively immunize a mammal, which conjugate will cause the proper immunoglobulin isotype reaction, i.e., the response of the IgG isotype.

Justice:

The following terms have the following meanings when referred to herein.

Oligosaccharide means a carbohydrate compound composed of a small number of monosaccharide units. In particular, the oligosaccharide can be formed by cleaving the polysaccharide.

Polysaccharide means a carbide compound containing a plurality of sugar groups. Polysaccharides present on the outer surface of bacteria or viruses are particularly useful in the present invention.

Carriers are substances that cause a thymus-dependent immune response that can be coupled to hapten or antigen to form a pore. In particular, a variety of proteins, glycoproteins, carbohydrates or sub-unit carriers can be used and can be selected from the group consisting of tetanus toxoid / toxin, diphtheria toxoid / toxin, outer membrane protein of bacteria, cell surface membrane of crystalline bacteria, serum albumin, gamma globulin or keyhole Including, but not limited to, lymphatic hemocyanin.

Immunity means to cause an immune response. Immunity Epistax is a part of a molecule that is recognized by the immune system and causes an immune response.

Haptens refer to antigens that contain incomplete or partial antigens (which alone may not cause antibody production) that cause antibody production. Di- and multi-haptens for such applications refer to compositions comprising two (di) or multiple (multi) oligosaccharide haptens conjugated to a carrier.

Protective immune or immunoprotective means stimulating an immune response that prevents infection by pathogens.

Immunostimulation means stimulating or promoting an immune response to a weakly immune hapten or antigen.

Neonate refers to a newborn animal, including infants.

methodology

Preparation and separation of cleaved polysaccharide

Polysaccharides obtainable from the American Type Culture Collection, Rockville, MD, or by known separation methods are cleaved into oligosaccharide units using appropriate concentrations of chemicals. Such chemicals include, but are not limited to, trifluoroacetic acid, acetic acid, hydrofluoric acid, hydrochloric acid, sodium hydroxide, and sodium acetate. Different time intervals and temperatures can be used depending on the specific chemistry and concentration, and depending on the desired oligosaccharide. Commercially available enzymes (e. G., Cellulase and beta -galactosidase) or identified bacteriophage-related endoglycans, which are known or known in the art, can also be used to prepare oligosaccharides in polysaccharides.

1 shows a repeating unit structure of a polysaccharide used in an embodiment of the present invention. Other bacterial and viral polysaccharides are known to those skilled in the art and may be used in the methods and compositions of the present invention. But are not limited to, pneumococcal group antigens (C-substances) and capsular polysaccharides of Streptococcus pneumoniae, Nyseria meningitidis, Hemophilus influenza, Group A streptococcus and Group B streptococcus serotype The various polysaccharides can be cleaved.

After cleavage, the resulting oligosaccharide mixture is separated in size using P-10 (fraction range molecular weight 1,500-20,000), P-30 (molecular weight 2,500-40,000) and P-60 (molecular weight 3,000-60,000) bio gel column . The presence of carbohydrates in the various column fractions is determined using phenol-sulfuric acid or cyanic acid assays and thin-film chromatography (TLC). The carbohydrate-containing column fractions are then analyzed by HPLC.

The presence of an immunogenic epitope of the fraction separated by the size of the cleaved polysaccharide is determined by the inhibitory ELISA described below. If the sample does not produce an oligosaccharide fraction that is inhibited in the Eliza test, the method of cleavage can be modified by changing the enzyme or chemical, molarity, reaction time or temperature to produce an immune epitope.

Determination of immune epitopes in oligosaccharide samples

The presence of immune epitopes in the column fractions is confirmed by inhibitory ELISA and phosphorus assays as described in the Examples section. An oligosaccharide fraction containing an immunogenic epitope (defined as causing at least about 25% reduction, preferably about 50% reduction, at OD ₄₀₅ at a concentration of 12.5 μg) is selected for coupling to a carrier.

Coupling to carrier

The oligosaccharide or polysaccharide to be used for coupling to the carrier is acidified or reduced in formulations for EDC or periodate oxidative coupling. For example, oligosaccharide samples can be reduced using a column of Alexin ^TM 101 (H) organic acid cation exchange column that acidifies the sugars for EDC coupling. Similarly, sugars can be reduced using standard methods for periodate oxidative coupling. When preparing di-haptens or multi-haptens, each oligosaccharide is activated individually for EDC or periodate cotylation.

Preferred di-hindered 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

A variety of proteins, glycoproteins, carbohydrates or sub-unit carriers can be used and can be selected from the group consisting of tetanus toxoid / toxin, diphtheria toxoid / toxin, bacterial outer membrane protein, cell surface membrane of crystalline bacteria, serum albumin, gamma globulin, But are not limited to, non-mossy. In a specific embodiment of the present invention, tetanus toxoid is used as the carrier. Tetanus toxoid preparations usually contain aggregates and low molecular weight impurities. The purity of the carrier is essential for obtaining the consistency of the coupling reaction. Size exclusion chromatography is used to obtain purified carrier samples.

The size-separated immunogenic epitope-containing oligosaccharide is coupled to a carrier purified by carboxymide (EDC) or periodate activation using the method described in the Examples section. Any free hindered oligosaccharides are separated from the hapten-carrier conjugate by column chromatography. The percentage of carbohydrate to protein in the conjugate is determined by phenol sulfate or cyanic acid and the Rauri protein assay. Typically, the conjugate prepared by EDC coupling has a carbohydrate: carrier ratio of 1: 2, whereas the conjugate prepared using periodate oxidative coupling has a carbohydrate: carrier ratio of 1: 5 to 1:10.

Determination of immune epitopes on a blank

As described above, the maintenance of important immunological epitopes is a problem of conventionally known conjugation techniques. In the present invention, ELISA inhibition assays are used to determine the potential immunity of the various coforms generated by our conjugation method. When conjugates showing inhibition (causing at least about 25% reduction, preferably about 50% reduction at OD ₄₀₅ at 6.25 농도 concentration, preferably about 50% reduction) in this assay using the methods described in the examples, are used as vaccines in mammals Thereby providing protective immunity. Thus, this assay is used to screen for useful pore composition.

Immunization causing immunoprotective antibody response

Typically, rats were dosed with 0.1, 0.5, 1, 2.5, and 5 ug of antigen (polysaccharide-conjugate, oligosaccharide-conjugate, conjugate-conjugate) based on the carbohydrate content for the EDC conjugate and the protein content for the periodate conjugate. (Primary immunization), 7 days (secondary immunization), and 28 days (tertiary immunization) with subcutaneous injection (100 μl on both sides) of the unmodified polysaccharide or oligosaccharide Lt; / RTI >

Antigens are diluted in 0.9% NaCl for various doses and rats injected with 0.9% NaCl are used as negative control. Blood is collected from 7-10 day old mice after secondary and tertiary immunization and serum is collected to quantify immunoprotective antibodies. A typical immunization schedule is shown in Table 1 for S. pneumoniae serotype 3 polysaccharide and oligosaccharide-tetanus toxoid conjugate prepared using EDC coupling.

Several different immunization schedules are available and include the following schedules:

0 (first), 14 (second) and 44 (third); And

0 (first), 30 (second) and 60 (third) days.

The conjugates of the present invention can be used as classical vaccines as immunogens to induce specific antibody production or to stimulate specific cell-mediated immune responses. They may also be used as therapeutic agents, such as by stimulating the immune system to recognize tumor-associated antigens; As an immune modulator, such as stimulating the production of lymphokines / cytokines by activating specific cell receptors; As a prophylactic agent for inhibiting receptors on the cell membrane to prevent cell adhesion; As a lysing agent for identifying specific cells, and as a development and / or research tool for stimulating cells for the production of monoclonal antibodies.

Determination of reaction

As discussed above, antibody responses to TI and TD antigens are different. In rats, the response to a polysaccharide (TI) antigen usually consists of a 1: 1 ratio of IgM: IgG. In general, and IgG isotypes are restricted, the IgG _3, wherein - is expressed-in over-polysaccharide serum. IgA isotypes may also be present. TI antigens induce low affinity antibodies, and immune memory is not produced.

By the TD antigen, the increased secondary IgG antibody response (anti-amyotrophic response) was found to have a high IgG: IgM ratio. Significant levels of IgA usually do not exist. TD antigens induce heterologous IgG isotype reactions and the predominant isotypes IgG ₁ , IgG _2a and 2b isotypes can be expressed, but the level of IgG ₃ isotype is usually relatively low. TD antigens induce immune memory, and antibody affinity increases with immunization. Thus, analysis of the immunoglobulin isotype generated by the response to the administration of the conjugate makes it possible to determine whether the conjugate has protective immunity.

We have found that the conjugates of the present invention induce a typical response to the TD antigen rather than the TI antigen, as measured by direct ELISA, sterilization and opsonization assays and their isotyping.

Conjugates prepared using the EDC coupling method induced a better antibody response than the conjugate produced by periodate activation. The dose of 1 占 퐂 was the most immunogenic. The oligosaccharide-conjugate prepared from the diphtheria toxoid carrier induced an antibody response similar to that induced by the oligosaccharide-tetanus toxoid conjugate.

As described above, attempts have been made to increase the immunity and to induce thymus-dependent antibody protection by coupling the polysaccharide to tetanus and diphtheria toxoid. The results show that these conjugates are slightly more immune than uncoupled capsular polysaccharides (CPS). A possible explanation for this may be that the pertussis, diphtheria and tetanus toxoid (in aluminum salt adjuvant) are often administered as a preventative 4-dose immunization regimen for infants. This regimen may tolerate infants and prevent infants from causing a protective antibody response (carrier suppression) to the haptens / antigens coupled to these toxoid carriers. Another possible reason for failure to induce protection may be structural. Although the protein carrier induces and enhances the immune response to hapten, the CPS-portion is a relatively large TI antigen in the CPS-protein conjugate. The immune system may recognize the CPS-protein as a cofactor, but merely recognize it as two distinct entities, resulting in a thymus-independent response to CPS and a thymus-dependent response to the carrier.

This seems to be the case in our study, as shown in Table 2. The immune system recognizes the polysaccharide-tetanus toxoid (TT) conjugate as a TI antigen. The potential TD inducing ability of the carrier on antibodies to polysaccharides is not observed. We infer that the immune epitope (oligosaccharide) of carbohydrate hapten should have close approximation with the TD induction epitope of the carrier to convert the TI reaction to the TD reaction.

We also used linker arm technology to fabricate the blank. We used, for example, 6-amino-n-hexanoic acid as a linker. The resulting conjugates were found to be less effective in inducing antibody responses than conjugates prepared by coupling EDC-activated oligosaccharide haptens directly to a carrier. These results support our hypothesis that close approximation of hapten versus carrier is needed to induce TD responses.

We have also developed a method for determining the level of immunoprotective antibodies induced by the conjugates of the present invention using sterilization or opsonization assays. These tests have shown that they are effective in inducing protective antibodies, as measured by the conjugate self-assay of the present invention.

As discussed above, epitope-carrier inhibition has been observed in other laboratories and our laboratories with S-membrane support studies [Malcolm et al., 1993b]. Our multi-hacton conjugates will reduce or eliminate this inhibition because they will contain more mass of immunogenic epitopes per carrier molecule than conventional pore vaccines. By our picture, the immune system will not be overly stimulated by the carrier. For example, the tri-hacton conjugate produced by the method of the present invention will require only three injections to produce a specific immune response to three different target pathogens. In contrast, using a conventional mono-holten coplanar would require the administration of nine injections to induce a similar response. This means that the amount of protein needs three times.

Furthermore, an immunization regime that converts an anti-polysaccharide TI response to a TD reaction can be designed using the co-injector of the present invention. A single dose of the inventive conjugate following economical initial exposure to polysaccharides (e.g., using Nemovacus 23) will induce IgG antibody levels (anticonvulsant response). Such an immunization regimen will not induce carrier inhibition. In such cases, initially the learned immune system for a variety of carbohydrates and antigens (TI response) has been identified in a special population group (e.g. serotypes 1, 3, 4, 6, 9, 14, 18, 19, 23) will be induced by multi-hapten graphene in order to induce a stronger immune response to pathogens that frequently cause disease.

Pharmaceutical composition

In order to induce antibodies against particular pathogens and / or various carbohydrate moieties, the conjugates of the present invention may be administered by a variety of delivery methods including intraperitoneal, intramuscular, endothelial, subcutaneous, oral or pervious.

The formulations of the compositions of the present invention may comprise suitable pharmaceutical carriers. The conjugate of the present invention is immunogenic without an adjuvant, but an adjuvant may enhance the immunoconjugate antibody titre or the cell mediated immune response. Such adjuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide, dimethyl dioctadecyl-ammonium bromide, adjuvant (alpha-beta technology), inject alum (Pierce), monophosphoryl lipid A (Bacterial or viral), carbohydrate moieties (such as mono (mono) aminomethyl), MPL + TDM (ribimycinemecom Research), CytRx, toxins, toxoids, glycoproteins, lipids, -, di-, tri-, tetra-, ortho- and polysaccharides), various liposome formulations or saponins.

The precise formulation of the composition will depend on the specific coenzyme, the species to be immunized and the route of administration.

Such compositions are useful for immunizing any animal susceptible to bacteria or viruses such as cows, sheep, goats, horses, rabbits, pigs, dogs, cats and birds. Both livestock and wild animals can be immunized. Humans can also be immunized with these formulations.

The route of administration may be any convenient route and may vary depending on the bacteria or virus, the animal to be immunized and other factors. Parenteral administration such as subcutaneous, intramuscular or intravenous administration is preferred. Subcutaneous administration is most preferred. Oral administration may also be used, including intestinally coated oral dosage forms.

The dosing schedule may vary depending on the bacterial or viral pathogen and the animal to be immunized. The animal may be given a single dose or may be given a booster dose or multiple doses. In particular, administration three times on days 0, 7 and 28 is preferable for inducing the first antibody reaction.

The following examples do not limit the scope of the invention in any way.

Example 1

Production and separation of polysaccharide hydrolyzate

Figure 2 shows that Streptococcus pneumoniae sera, in which fractional oligosaccharides of 1 to 8 repeating units are produced after passage through a bio gel P-10 column after acid hydrolysis (0.5 M trifluoroacetic acid, 100 캜, 20 min) 8 < / RTI > of the capsule polysaccharide. 1 to 8 correspond to the number of repeating units present at each peak, and peak 9 contains more oligosaccharides than 8 repeating units. Oligosaccharides derived from hyaluronic acid were used to standardize chromatographic systems.

The relative sizes of the repeating units of peaks 1, 2, 3, and 4 were measured by HPLC analysis (FIG. 3).

The HPLC delay times of glucose, M-3 maltotriose, M-7 maltotriose, and M-10 malto-oligosaccharide (Sigma Chemical Co.) used for measuring the relative sizes of various oligosaccharide repeat units are shown in FIG. have. The monosaccharide content of the repeating structure was established by further hydrolyzing the oligosaccharide repeating unit with 2.0 M trifluoroacetic acid (TFA) at 100 DEG C for 2 hours. An example of the delay time of the standard of the ribitol, rhamnose, galactose, fucose and mannose monoglyceride used to measure the carbohydrate content of the hydrolyzed repeat unit is shown in Fig. The chemical structure of one repetitive unit of serotype 8 was identified by GC-MS and NMR analysis as? -Glucose (1? 4)? -Glucose (1? 4)? -Galactose (1? 4) 4). This corresponds to the repeating unit structure cited in the literature [Jones and Perry, 1957].

Figures 6-10 show the degree of separation of S. pneumoniae serotype 6B, 14, 19F, and 23F polysaccharide hydrolyzate (TFA, acetic acid or hydrofluoric acid) through a P-10, P-30 or P- For example.

Fig. 11 shows the degree of separation of polysaccharide (serotype 8 cleaved by the cellulase) cleaved by the enzyme. The separation of the C-material oligosaccharide is shown in FIG.

Example 2

Inhibition Eliza to Determine Immunoglobulin Epitope of Oligosaccharide Samples

The basic method used for the inhibitory ELISA to test for the presence of an oligosaccharide sample and an immunogenic epitope on an oligosaccharide or polysaccharide-conjugate was as follows.

1. Coat a 96 well EIA plate (NUNC) with 1 ug wells of antigen (Ag) using 0.05 M NaCO ₃ coating buffer (100 ug / well) and store at 4 캜 overnight.

2. On the same day, prepare inhibitive Ag test tubes (eg, various oligosaccharide hydrolyzates) using 1X PBS-0.01% Tween 20 as a diluent.

The total volume of each test tube should be 175 μl after a series of dilutions, making a series of dilutions in the test tube 7 times (from 25 μg / well to 0.391 μg / well in triplicate).

- Prepare anti-sera of a particular type (eg, diagnostic anti-serum 14 produced in rabbit staphylococcal streptose) diluted 1: 1000 in 1X PBS + Tween.

- Add 175 μl of this solution to each test tube. The total volume of each tube is now 350 μl. Store the test tubes at 4 ° C overnight.

3. The next day, the EIA plate is blocked with 100 [mu] l / well of blocking buffer (1X PBS + 1% BSA) and stored at room temperature for 1 hour.

4. Lightly discard the plate and transfer the contents of each tube to the well (100 μl / well) and store at room temperature for 2 hours.

5. Wash plate three times with wash solution (0.01% Tween + 1X PBS).

6. IgG alkaline phosphate conjugate (TAGO) with goat-anti-rabbit (or serum-specific serotype specific sera used in step 2) was diluted 1: 1 with PBS 1% Tween Buffer (100 쨉 l / well) 1500 dilution is prepared. Store at room temperature for 2 hours.

7. Wash plate 4 times with wash solution and discard excess liquid.

8. Alkaline phosphate substrate purification (# 104-sigma) is dissolved in DEA (diethylenamine) buffer pH = 9.98, 5 ml / tablet, 100 μl / well.

9. Store the plate in the dark room and read the absorbance at 405 nm every 15 minutes.

Antisera prepared in various commercial and laboratory laboratories may be used in this assay and include, but are not limited to, murine (murine), murine (rat), rabbit, goat, porcine, monkey, .

Figure 13 shows the inhibition ELISA results using rat antiserum against Streptococcus pneumoniae serotype 8 oligosaccharide protein carrier conjugate (2-4 repeat units coupled to tetanus toxoid using EDC). The inhibition was tested with type 8 oligosaccharide (0.5M TFA, prepared at 100 ° C for 20 minutes) and type 8 polysaccharide in 1, 2, 3, 4, 6, 8+ repeating units. From these results, it can be seen that one repeating unit (chain per 4 units) does not contain an immunological epitope. Two repeat units (chain per 8 units) were able to inhibit binding of the antibody to the ELISA plate, which means that it contains an immunological epitope. The molecular weight of the repeating unit 2 was determined to be 1365 by FAB-MS analysis. This has a good correlation with the theoretical molecular weight of 8 per molecule. Repeating units 3, 4, 6, 8 + and whole polysaccharides also inhibited the binding of the antibody to the Eliza plate, indicating the presence of immunoepitopes in these oligos / polysaccharides.

Table 3 shows the results for the rabbit anti-S. Similar results were obtained using pneumococcal serotype 8 specific serum (Stemens serylin stitut). Recurring unit 1 did not significantly inhibit binding, and repetitive units 2, 3, 4, 5, 6, 7, 8 + and total polysaccharides inhibited binding.

Inhibition elicitors are also used to determine the presence of immunoepitopes on oligosaccharides prepared using different hydrolysis methods for various polysaccharides. Table 4 shows the results by the method used in the prior art, for example the method described in Canadian Patent No. 2052323, for the hydrolysis of S. pneumoniae serotype 6 polysaccharide (0.01 M acetic acid, 100 < 0 & . Whole polysaccharides inhibited binding at low concentrations (effective at 0.39 μg) of antigen, but not acetic acid gas broth. It should be noted that the hydrolyzed samples could not be separated by size because they were caramelized.

We found that, as shown in Table 5, other hydrolytic reagents (eg, TFA) and oligosaccharides with reduced time and temperature have more immune epitopes. 0.5M TAF, 70 ° C, 1 or 2 hours hydrolyzate effectively inhibited antibody binding at 3.13 μg concentration, and 4 hours production was not. Tables 6 and 7 also illustrate the effect of time for the preparation of 6B oligosaccharides with or without immune epitopes. Preparation of 2-hour acetic acid inhibited antibody binding (at 3.13 [mu] g concentration), and 24 and 48 hours production was not. Similarly, 1.5 hour TFA production inhibited binding more effectively than 3 hour production.

As shown in Table 8, 0.5 M TFA hydrolysis of S. pneumoniae serotype 14 for 7 hours at 70 DEG C was undesirable, as disclosed in the prior art [Capo, Canadian Patent No. 2052323]. A reduced TFA molarity (e.g., 0.1 M) is better for the preparation of immunoglobulin 14 oligosaccharides.

Table 9 illustrates the importance of oligosaccharide screening containing an immune epitope for coupling to a carrier. The three repeating unit structures of the serotype 14 oligosaccharides were unable to inhibit antibody binding, but the 4 and 8 repeat units contained immunostimulatory epitopes, effectively blocking antibody binding.

Table 10 shows the effects of concentration and reaction time of the hydrolyzate to prepare 14 oligosaccharides containing immunoepitopes. The immunopotope was preserved by TFA, 7 hours hydrolysis, but was destroyed by hydrolysis for 24 hours.

Table 11 illustrates the importance of using optimal heat conditions to generate 19F oligosaccharides containing immunoepitopes. The immune epitaxial was destroyed by HCl hydrolysis at room temperature, but was retained when hydrolyzed at 70 ° C.

As shown in Table 12, insufficient inhibition of antibody binding was observed in 0.25M TFA, 23F polysaccharide, 70 ° C, 3 hours hydrolyzate [Capo, Canadian Patent 2052323]. Table 13 shows the effect of time on the production of immunogenic 23-oligosaccharides. Oligosaccharides were produced by hydrolysis of 0.1 M TFA and inhibited antibody binding at 70 ° C for 3 hours, but did not inhibit oligosaccharides prepared by hydrolysis for 5 hours. Table 14 shows the presence of immune oligosaccharide after hydrolysis at 70 ° C for 15 minutes or 5M acetic acid treatment at 70 ° C for 5 hours. The hydrolyzate was effectively inhibited to a concentration of 0.78 mu g.

Table 15 shows the inhibitory potency of the inhibitory oligosaccharide for recognizing the immunogenic oligosaccharide of Nasielia meningitidis serotype C. The hydrolysates prepared with NaOAc inhibited antibody binding as effectively as whole polysaccharides.

Example 3

Acidification of Carbohydrate Residues for Carboyl Mead Coupling

The reksin 101 (H) organic acid cation exchange column (Fisher Scientific) was prepared by washing with dH ₂ O. A polysaccharide or oligosaccharide sample dissolved in dH ₂ O (neutral pH) was passed through the column and collected at a rate of one drop per 6 seconds. Acidification was confirmed by pH color-attached indicator bars. Excess dH ₂ O was used to wash the column. The acidified fractions were collected and lyophilized for use in the coupling reaction.

Figure 14 depicts the formation of aldehydes and hydroxyl groups as a result of TFA cleavage between [beta] -D-Glcp (1 → 4) beta-D-Gal of the oligosaccharide structure. When the aldehyde is further oxidized, a carboxyl group is formed. When this material passes through a cation exchange column, a COO- group is formed.

Example 4

Quantitative analysis and coupling method

Carboymide (EDC) coupling method

A sample of ion-charged carbohydrate (polysaccharide or oligosaccharide) (eg, 3 mg) and EDC (3 mg) in a 1: 1 mass ratio was dissolved in 2 ml of 0.1 M KH ₂ PO ₄ and maintained at pH 4.5 with 1 N NaOH or HCl . The mixture was stirred at room temperature for 1 hour. Carriers (3 mg) were added to the EDC-activated carbohydrate and then stirred at room temperature for 4 hours. The reaction was stopped by the addition of 200 [mu] l of 10% ammonium bicarbonate, and the mixture was further stirred at room temperature for 1 hour.

The resulting conjugate was dialyzed against dH ₂ O overnight using a MWCO dialysis tubing with a molecular weight of 50,000.

After the lyophilized lyophilizate, the composition was quantified by lauric protein, phenol-sulfuric acid, cyanic acid and phosphorus determination method (the method described below). Typically, conjugates prepared with this coupling method have a carbohydrate to carrier ratio of 1: 2.

Determination of phenol-sulfuric acid for the determination of carbohydrates

Reagent: 5% phenol solution (5.5 mL of liquid phenol (90%) added to 94.5 mL of distilled water).

Standard: 1 mg / ml stock solution of glucose. For the standard curve, 2 to 60 μg / 200 μl sample buffer is prepared.

Methods: (Handbook of Micromethods for the Biological Sciences, Keleti, GWH Lederer (eds.) 1974. Van Nostrand Reinhold Co., Adopted in New York)

1. Place 200 μl sample in a very clean test tube.

2. Add 200 μl of phenol reagent.

3. Add 1 mL of concentrated sulfuric acid rapidly.

4. Make a good vortex.

5. Leave at room temperature for 30 minutes.

6. The color is stable for 2 to 30 hours at room temperature.

7. Read the absorbance at 490 nm: As a # 1 sample, use a test tube containing only water as a blank.

Quantitative evaluation of cyanic acid

reagent:

a. Al ₂ (SO ₄ ) ₃ 6 grams. 18H ₂ O dissolved in 20 ml of distilled water.

b. 1 g of para-dimethylaminobenzaldehyde (stored in a dark bottle in the refrigerator) dissolved to 20 ml with 6N HCl.

Standard: 350 μl of N-acetylneuraminic acid in a total volume of 0, 2.5, 5. 10, 15, 20, 25, 30, 40, 45 and 50 μg / Distilled water is used to make 350 μl.

Way:

1. Make two 200 μl samples. To 350 μl in distilled water.

2. Add 700 μl of reagent A to each test tube. It shakes.

3. Add 350 μl of Ehrlich reagent B.

4. Cover all test tubes with marbles.

5. Heat the test tube at 100 ° C for 30 minutes using the Pierce Heating Module.

6. Quickly cool the test tube from ice bath to room temperature.

7. Read the optical density at a wavelength of 530 nm.

Phosphorus determination method

Reagents: a. 2.5% ammonium molybdate; b. 10% ascorbic acid; c. 70% perchloric acid; And d. 1 mM sodium phosphate standard.

Methods: (adopted from Rouser, G., Siakotos, AN and Fleischer, S. 1966. Lipids 1: 85-86)

1. Place samples and standards (0, 25, 50, 100 and 200 μl; 25 to 200 nmol) in a clean test tube.

2. Dry the sample in a heater block in a fume hood at 180 ° C for 5 minutes.

3. Add 450 μl of perchloric acid to each test tube, cover each test tube with marbles, and heat at 180 ° C for 30-60 minutes.

4 is added to 2.5 mL dH ₂ O after the test tube is cooled down.

5. Add 0.5 ml of ammonium molybdate and vortex immediately.

6. Add 0.5 ml ascorbic acid and vortex immediately.

7. Place the test tube in 95 ° C water for 15 minutes.

8. After the tube has cooled, read the absorbance at 820 nm.

9. You can leave the sample for several hours before reading.

Rauri protein determination method

reagent:

a. 2% (w / v) Na ₂ CO _{3 in} 0.1 M NaOH (1 L).

b. 0.5% CuSO ₄ in a 1% sodium citrate (100mL).

c. (2X) < / RTI > < RTI ID =

d. Bovine serum albumin (1 mg / mL)

Way:

1. Prepare a standard curve consisting of BSA 0, 12.5, 25, 50, 100 and 200 μg in a final volume of 200 μl.

2. Add 200 μl of unknown protein sample to dH ₂ O.

3. Mix reagents A and B at 50: 1 (v / v) and add 2 mL to each sample.

4. Leave the vortex at room temperature for 10 minutes.

5. Dilute the folin-thiocaltiophenol reagent with dH ₂ O and add 200 μl to each sample.

6. Leave the vortex at room temperature for 30 minutes.

7. Read the absorbance at 660 nm.

Iodate oxidation coupling method

A sample of polysaccharide or oligosaccharide (e.g., 3 mg) was dissolved in 3 ml of freshly prepared 60 mM sodium meta-periodate in 50 mM sodium acetate. The sample was then stirred overnight at 4 < 0 > C. Ethylene glycol (300 mu L) was then added to stop the reaction, and the mixture was continuously stirred at room temperature for 1 hour and then lyophilized. A sample dissolved in 1.5 mL of 0.03 M ammonium bicarbonate (pH = 8.0) was passed through a P-2 bio-gel column. A phenol-sulfuric acid or cyanic acid assay was used to determine the fraction containing the periodate-reduced form of the sample, and the sample was then lyophilized.

Figure 15 shows the separation of the reduced polysaccharide (23 polysaccharide vaccine - Newcastle 23, Merck, Sharp & Dome) fraction. Figures 16 and 17 show the separation of reduced oligosaccharides of serotypes 6B and 19F of Streptococcus pneumoniae, respectively.

3 mg of the reduced sugar and 3 mg of the reduced polysaccharide were dissolved in 3 ml of 0.1 M sodium tetraborate decahydrate, pH 8.9. Sodium cyanoborohydride (H + feed) was then added to the mixture and stirred at 50 < 0 > C for 48 hours. This reaction was stopped by titrating the pH to 3-4 with 80% acetic acid. This conjugate was then dialyzed against dH ₂ O (2-3 dH ₂ O changed) using 50,000 MWCO dialysis tubing.

The co-pores were lyophilized and the co-pore composition was determined by quantification of Laurie protein, phenol-sulfuric acid, cyanic acid and phosphorus determination method. Typically, the conjugate produced using this coupling method has a ratio of carbohydrate to carrier of 1: 5 to 1:10.

Figure 18 shows the periodate and EDC coupling chemistry.

Example 5

Coaxial carrier

Example 4 describes a method used to produce an immunoglobulin / polysaccharide conjugate from a weakly immune or nonimmune polysaccharide.

Tetanus tosidide was purified for use as a carrier in column chromatography. This purified toxoid may be used in combination with high concentrations of IgM (eg, 50 μg / ml rat serum) and IgG isotype (eg, IgG, 100 μg / ml of serum; IgG _2a , 38 μg / ml of serum; IgG _2b , 68㎍ / ㎖; IgG _3, was induced 105㎍ / ㎖) of serum.

Example 6

Determination of Immune Epiphyte on Oligosaccharide / Polysaccharide Bone

An inhibitor ELISA as described in Example 2 was used. The presence of immunoepitopes on the mono-hapten 8-oligosaccharide tetanus toxoid conjugate was confirmed by inhibitory ELISA. This conjugate inhibited the binding of anti-8 serum to ELISA plates coated with 8 polysaccharides (Figure 19). The liberated tetanus toxoid did not inhibit binding. It also showed the presence of immunogenic 8-oligosaccharides on di-haptens 6: 8, 14: 8 and 19: 8 conjugates. This figure illustrates the regeneration of our coupling method, such that 8-mono-hapten and di-hectene conjugate autoantibody binding are equally inhibited, indicating that each conjugate contains the same amount of 8 oligosaccharides.

Table 16 shows inhibitory ELISA results when 6B polysaccharide, 6B oligosaccharide, 6B: 8 di-stent-oligosaccharide tetanus toxoid conjugate or tetanus toxoid alone was used as the inhibitory antigen. Tetanus toxoid did not inhibit the binding of anti-6B serum to the 6B-polysaccharide-coated Eliza plate. Free 6B-oligosaccharides or polysaccharides inhibited binding. 6B: 8 di-hapten-oligosaccharide-TT conjugate also inhibited binding. This confirms the presence of an immunogenic 6B epitope on the 6B: 8 di-Hertene -TT co-pic.

Similarly, the 14: 8-di-hapten -TT conjugator inhibits anti-14 serum binding, which indicates the presence of serotype 14 immunogenic epitopes (Table 17). It should be noted that there was nonspecific inhibition by TT only at high concentrations. We found that this is an anti-14 artifact in this assay.

The various oligosaccharide fractions of 23F hydrolyzate were coupled to TT. As shown in Table 18, all 23F serotypes of immune epitopes were contained.

Immunity of N. meningitidis oligosaccharide (NaOAc) Episotope remained similar when coupled to tetanus toxoid (see Table 19).

Example 7

Determination of antibody isotype levels induced by thymus-independent (TI) and thymus-dependent (TD) antigens

The basic method to measure the levels of antibody isotypes to quantify IgM, IgG and IgA isotypes induced by various types of coenzymes is as follows.

1. EIA plates (NUNC, IMMUNOSORB) with 1 μg of antigen (Ag) are coated in 0.05M sodium carbonate / sodium bicarbonate buffer pH 9.5 (100 μg / well).

2. Store at 4 ° C overnight.

3. On the next day, the plate is blocked with 100 [mu] l / well buffer (1X PBS + 1% BSA) for blocking buffer. Store at room temperature for about 1 hour.

4. Prepare a 1:25 dilution of rat serum with buffer solution (1X PBS + 0.1% Tween). Add 100 μl / well to appropriate wells and store at room temperature for 2 hours.

5. Wash plates three times with wash solution (1X PBS + 0.05% Tween). Discard the excess liquid by gently tapping the plate on the bench top.

Prepare the second dilution: 1 6. action-buffer by EIA Grade Mouse Type of 100㎕ / well (Rad rabbit _{anti-Mouse, IgM, IgG 1, IgG 2a} , IgG 2b, IgG 3, and IgA, Bio). Store at room temperature for 2 hours.

7. Wash plate three times with wash solution.

8. Prepare a chlorine-anti-rabbit IgA alkaline phosphate conjugate (TAGO) with 100 μl / well of 1: 1500 dilution in buffer of action. Store at room temperature for 2 hours.

9. Wash plates 4 times with buffer solution.

10. Prepare the enzyme substrate using 100 μl / well of # 104-Sigma Alkaline Phosphate Substrate Purification (1 tablet / 5 ml of 10% diethanolamine substrate buffer). Store in the dark at room temperature and read the absorbance at 405 nm every 30 minutes.

11. Absorbance values were determined using a dose-response curve generated from Eliza reaction of rabbit anti-rat isotype antibody to various concentrations of murine and mouse subspecies immunoglobulins (Zymed Labs. Inc.) ml) < / RTI > per day.

Table 2 shows antibodies induced in mice when immunized with S. pneumoniae serotype 8 oligosaccharide and polysaccharide conjugate. 8 oligosaccharide-conjugate induced all isotype IgG antibodies, non-conjugated oligosaccharides were not immune, and polysaccharides and polysaccharide-conjugates were the most common antibody isotype (predominantly IgM, IgA, and IgG ₃ isotype) Lt; / RTI > The adjuvant is not required to induce IgG isotype with our oligosaccharide-tetanus toxoid conjugate. The conjugate consisting of relatively small oligosaccharides, heptene (with 8 to 16 sugars) with 2 to 4 repeating units, showed the best antibody response when measured by direct ELISA.

Direct Eliza Protocol

1. Use a NUNC Maxisorp Immune Plate.

2. Dilute the coated antigen to 1.0 μg / 100 μl in carbonate-titanate buffer. Use a glass test tube as the antigen may adhere to the plastic.

3. Add 100 μl to each well of the plate and store overnight at 4 ° C.

4. Wash three times with PBS-0.05% Tween. Remove excess PBS by gently patting it with a Kim Wipe / paper towel.

5. Add 100 μl / well of a stopper (1 × PBS-1% BSA). Store at room temperature for 60 minutes.

6. Wash three times as in 4.

7. Add 100 μl / well of test antibody diluted appropriately to PBS-0.01% Tween. Store at room temperature for 90 minutes.

8. Wash 3 times as in 4.

9. Alkaline phosphate conjugated anti-rat Ig (TAGO catalog # 4653) is diluted in PBS-Tween 1/1500. Add 100 μl per well and store in the dark for 90 minutes.

10. Wash 3 times as in 4.

11. Add 100 μl per well of Sigma 104 phosphatase substrate (disodium p-nitrophenyl phosphate tablet). Two tablets (5 mg / tablet) of substrate were added to 10 ml of diethanolamine buffer. Store in dark conditions as substrates can be inactivated by sunlight.

12. Store in a dark place at room temperature. The progress of the reaction varies depending on the antibody. Absorbance readings can be read with a Micro-ELISA auto reader (405 nm) at intervals of about 30 minutes.

The results in Table 20 are the comparison of IgG ₁ and IgG ₃ levels in 3-week-old mice and 8-week-old rats immunized with 8-fold. Significant levels of IgG ₁ were found to be 0.273 μg / ml in mice immunized at 3 weeks with 8-oligosaccharide-TT-conjugates and 0.700 μg / ml in mice immunized at 8 weeks. As an index of the TD reaction, adjuvant (e.g., FCA) increased had a specific IgG ₁ (1.22㎍ / ㎖). 8-polysaccharide induced over-expression of the typical low IgG ₁ and IgG ₃ TI reactions. The 8-polysaccharide-TT-conjugate, considered as a TD antigen, induced overexpression of IgG ₃ , a characteristic of TI polysaccharide antigen, and low levels of IgG ₁ . In addition, adjuvants conjugated with 8-polysaccharide-TT conjugate did not enhance IgG ₁ levels, but increased IgG ₃ antibody (TI-like response). Some polysaccharide-conjugates are known to induce a combination of TI and TD antibody response profiles [Stein, 1992; Stein, 1994].

Figure 20 shows the IgG antibody isotype induced by a ratio of 8:14 to di-hapten-oligosaccharide-TT co-porter versus 8-polysaccharide. Like the 8-mono-haptens conjugate, the di-hecten conjugate was able to induce higher levels of specific IgG ₁ antibody (TD response) than 8-polysaccharide-conjugated or 8-polysaccharide alone. Overexpression of the IgG ₃ isotype appears to the polysaccharide immunogen. Control rats were injected with only tetanus toxoid.

The results obtained from the serotype 14-oligosaccharide conjugate are shown in Table 21. The 14-oligosaccharide-TT conjugate produced by 0.1 M TFA hydrolysis produced isotype of IgG ₁ , G _2a , G _2b and G ₃ , and the most immunogenic dose was 1 μg. The oligosaccharide-TT conjugate prepared using the carbohydrate fraction of the 7th and 8th separation peaks of 0.5M TFA hydrolyzate induced low levels of IgG isotype. Small oligosaccharides in the form of pores (4 and 5 peaks of 0.5 M TFA sample) induced low levels of IgG isotype. The 14-polysaccharide-TT conjugate induced a relatively low level of IgG ₁ isotype. However, sera from rats injected with these polysaccharide conjugates did not have immunoprotective ability (as seen in Example 8, Table 24). Here, it seems that a threshold level of IgG antibody isotype is required to give immunity to 14 pathogens of serotype 14. Uncoupled 14 polysaccharides, tetanus toxoid alone or 0.9% sodium chloride negative control serum all showed low levels of all isotypes at the same level as the normal rat serum (NMS) level.

Figure 21 shows the increased level of IgG ₁ antibody isotype for a typical 8:14 di-hapten-oligosaccharide-T conjugate induced 14-polysaccharide in a TD response. 14-polysaccharide induced over-expression of IgG was ₃ (TI response), the 14 oligosaccharide alone was not immunogenic. The untethered tetanus toxoid was a negative control.

As in individual humans, different groups of mice exhibited different responses to oligosaccharide- and polysaccharide-conjugates. Diversity in the level of other IgG antibody isotype was observed in some groups of rats. Figure 22A shows the results from a group of good responders rats producing IgG ₁ against 14-polysaccharide co-pores (TD-like response). Nevertheless, the 14-oligosaccharide-conjugate caused higher IgG ₁ levels. This conjugate also induced significant levels of IgG _2b (0.955 ug / ml = oligosaccharide-conjugate; 0.139 ug / ml = polysaccharide-conjugate). This response was due to TD induction (1.509 [mu] g / ml = oligosaccharide-conjugate; 0.474 [mu] g / ml = polysaccharide-conjugate) as FCA increased these IgG _2b antibodies (Figure 22B).

The ability of the oligosaccharide-conjugate of the invention to induce a greater TD antibody response than the polysaccharide-conjugate was not limited to S. pneumoniae immunogens. The oligosaccharide-conjugate of Nacelia meningitidis Group C induced more levels of IgG ₁ isotype antibody (7.01 μg / ml) than the polysaccharide-conjugated (3.60 μg / ml) or polysaccharide alone (0.162 μg / . Interestingly, the amount of IgG ₃ isotype induced by oligosaccharide conjugate was also higher (13.11 μg / ml = oligosaccharide-conjugate; 9.84 μg / ml = polysaccharide-conjugate; 3.81 μg / ml = polysaccharide alone).

Example 8

Sterilization and opsonization assays for measuring immunoprotective antibodies induced by conjugates

The basic sterilization and opsonization assays used are as follows.

Sterilization method

1. Strain the desired gram negative bacteria (obtained from the American Type Culture Collection) on blood agar plates. Store at 37 ° C overnight.

2. On the following day, one isolated colony is picked and inoculated into 1.0 ml of the Todd-Hewitt Broth (THB) + Yeast Extraction (YE) medium in the sterile test tube. Store at 37 ° C overnight.

3. The next day, measure the OD of the inoculated bacteria at a wavelength of 420 nm. THB + YE medium is used as the blank.

4. Add sterilized 2.5 mm glass beads to each well of a sterile, flat 96-well plate.

5. Add the following to each well:

a. 5 μl of bacteria

b. 10 [mu] l of rat serum to be tested

Store at 37 ° C for 1 hour.

Note: Steps 5 and 6 should be three sets.

6. After 1 hour of storage, prepare a 1:20 dilution of the external replenisher (eg Lowotox rabbit replenisher, cedarane) in THB + YE in sterile condition. 50 μl per well is added. Store at 37 ° C for 1 hour.

7. After storing the replenishment solution, use a glass disperser to plate 50 μl of the aliquot on the blood agar plate.

8. All agar plates are wrapped in plastic bags and stored at 37 ° C for 12 hours.

9. Count plaques forming colonies the next day.

Opsonization determination method

1. Streak the desired Gram negative and positive bacteria (obtained from the American Type Culture Collection) on blood agar plates. Store at 37 ° C overnight.

2. The next day, take a single isolated colony and mix with 1.0 ml of THB + YE medium in a sterile test tube. Store at 37 ° C overnight.

3. Prepare 100 U / ml of sterilized heparin the following day.

4. 100 μl of sterile heparin is injected intravenously into the tail of each rat (5-10). Blood is collected in a sterile test tube after 10 minutes.

5. Measure the OD of the bacteria at a wavelength of 420 nm. THB + YE medium is used as the blank. (Use Spectrophotometer 4040 for OD measurement)

6. Add the following to a sterile, flat 96 well plate containing sterile 2.5 mm glass beads in each well:

a. 50 μl of heparinized blood

b. 10 μl of serum

c. 10 μl of bacteria

This step will be three.

7. Cover plates with tinfoil and store in a 37 ° C incubator on a shaker (slow motion) for 1 hour.

8. After 1 hour, 50 μl aliquots are plated on blood agar plates using a glass spreader.

9. All plates are wrapped in plastic wrap and stored at 37 ° C for 12 hours.

10. Count the plaques forming the colonies the next day.

Serum from rats immunized with S. pneumoniae type 8 oligosaccharide conjugate was found to be immunoprotective, as determined by opsonization assays. Obsonization of S. pneumoniae bacteria mediated by specific anti-capsule antibodies is essential for host defense [Saunders et al., 1993]. This method of quantification is generally regarded as a reliable indicator of in vivo immunity protection. The quantification results show that 8 oligo-conjugates significantly reduce colony growth forming units of S. pneumoniae serotype 8 in blood agar plates (Table 22). This decrease is not specific because serotype 3 and 6B (used as a specificity control) did not inhibit colony growth. Immunization with non-conjugated oligosaccharides and polysaccharides (used in a commercial polysaccharide-conjugated vaccine) did not induce any protection. The polysaccharide-conjugated protection (39% reduction) was much less than the oligosaccharide conjugate-induced protection (98% reduction). These results show that our 8 oligo- tetanus toxoid conjugate induces high levels of immunoprotective antibodies against serotype 8 S. pneumoniae pathogens. The level of immunoprotective antibodies induced by poly-cofactors was modest.

The 8-oligo conjugate induces an immunosuppressive antibody response in mice pre-dosed with whole polysaccharide alone. Rats injected with oligo-cofactor three times after 8 doses of polysaccharide had immunoprotective antibodies in their serum (70% colony reduction in the opsonized dose). As in previous experiments, rats injected with three injections of polysaccharide did not induce significant amounts of protective antibodies. Oligosaccharides of specific sera coupled to carrier proteins may be advantageous as boosters to boost immune protection in high risk populations that are unresponsive to or present only a mild response to the current 23-mer polysaccharide vaccine.

We conducted an immunogenicity study with di-haptene 3 oligo- / 8 oligo-tetanus toxoid conjugate. Oligosaccharides of both serogroups were prepared by TFA analysis. Rats injected with this multi-hapten confocal mouse resulted in immunoprotective antibodies against serotypes 3 and 6 (96-99% colony reduction - Table 23). The 3/8-polysaccharide conjugate did not induce nearly immunoprotective antibodies (10-12%). The mono-haptenic 3-oligo-tetanus toxoid conjugate used in this study was not made of oligosaccharides determined to have immunological epitopes by inhibitory elicitors and was not capable of inducing an immunoprotective response. The mechanism by which the immune system responds to epitopes on the tri-oligosaccharide form of the di-hapten form is of course net theoretical. However, we suggest that 8 oligosaccharides stimulate clones of cells (i. E., Accessory or helper cells) that can increase the response to epitopes on serotype 3 oligosaccharides.

We have found that 8 oligosaccharide structures have an activity such as adjuvant or adjuvant. A comparatively simple repeating unit structure of 8 oligosaccharides (? - glucose (1 → 4) β-glucose (1 → 4) α-galactose (1 → 4) α gluconic acid) specifically or nonspecifically induces Or may induce receptors or elements to enhance a body fluid / cell response to a non-immunogenic or weak polysaccharide / oligosaccharide. Serotype 8 oligosaccharides have adjuvant activity either in conjugated form or as a mixture for vaccine formation.

The opsonization results of the 14-oligosaccharide-TT conjugate (0.1 M TFA preparation-Table 24) show good bacterial colony reduction of 76% for serotype 14. The 14-oligo-TT 0.5M TFA sample induced less immunoprotective antibody (54% reduction). Serum from the polysaccharide-TT conjugate, polysaccharide alone and tetanus toxoid injected mice showed greatly reduced inhibition (18, 2, and 15%, respectively). Serum from the control rats (0.9 M NaCl injection and NMS) did not show any reduced ability.

The di-hapten-oligosaccharide conjugate also induced antibodies with opsonic activity. Serum for the 8: 14-oligo -TT conjugate reduced serotype 14 colony forming units to 65% (Table 25). This di-hoto conjugate was as immunogenic as the mono-hapten 14-conjugate (CFU reduction = 68%). Serum from mice immunized with polysaccharide-conjugate slightly reduced CFU to 37%.

Example 9

Cleavage of carrier and antigen competition

A reduced response due to antigen competition when multiple antigens are injected is reported in the literature under some circumstances. The results from immunization schedules A and D (Table 26) will be used to determine whether the response to each constituent of our multi-hapten blank is identical to that caused by mono-hapten blank alone.

The unit mass of the carbohydrate antigens of our mono- and multi-hapten grains will be equivalent (ie, the CHO: protein for the EDC conjugate is 1: 2). Our multi-haptic conjugate design with reduced antigen loading will minimize the potential for antigen competition.

Schedules B and E determine if the primary injection of the conjugate is sufficient to educate the immune system to induce a T-dependent response when it is boosted to an un coupled polysaccharide.

Schedules C and F will establish the ability of our conjugate to enhance the immunosuppressive antibody response in mice pre-infused with only polysaccharides. Thus, a multi-conjugated pneumoniae vaccine containing three to four serotypes of oligosaccharides may be very useful in increasing the response to Nimobax 23 in high-risk patients.

The rat population was treated with a variety of S. pneumoniae or poly-TT conjugates as in G after three doses (primary, secondary, tertiary) of tetanus toxoid (titer of tetanus toxoid identified by Eliza) (Table 26).

In all studies, the conjugate will be administered orally and subcutaneously.

The conjugates of the present invention will stimulate an immune response in infants, children and immune suppressed patients with an immature immune system. As a model for this situation, we will determine the immunosuppressive efficacy of our conjugates in young rats, SCID, and nude mice. As described above, these rats will also be pre-sensitized with tetanus toxoid prior to multi-void injection to study carrier backbone phenomena.

Modifications of the above-described aspects of carrying out the various aspects of the invention will be apparent to those skilled in the art in accordance with the teachings of the invention set forth in this disclosure. The above-described embodiments are not limitative but merely exemplary of the present invention, and do not limit the scope defined by the following claims.

Typical immunization schedules for conjugate vaccines Day of administration Date of collection Paintball ^* 0 17 PSC-TT (A-123) 7 38 28 0 17 PSC (A-123) 7 38 28 0 17 OSC-TT (A-124) 7 38 28 0 17 OSC (A-124) 7 38 28 0 17 TT 7 38 28 0 17 0.9% NaCl 7 38 28 N / A Normal rat Never inject Groups of 5 rats were injected subcutaneously with 0.1, 0.5, 1.0, 2.5 or 5.0 占 퐇 of a coenzyme (100 占 퐇 in each side) in 200 占 퐇 of 0.9% NaCl. PSC-TT (A-123) is a polysaccharide-tetanus toxoid conjugate (Lot # A123). OSC-TT (A-124) is an oligosaccharide (4-8 repeating unit) -tetanus tosidomethacrylate (Lot # A-124). PSC (A-123) is a polysaccharide (lot # A-123). OSC (A-124) is an oligosaccharide (4-8 repeating units) (lot # A-124). TT is tetanus toxoid.

Mean concentrations of rats serum antibodies to various serotypes 8 S. pneumoniae conjugates (after tertiary immunization) Antiserum Immunoglobulin isotype class (占 퐂 / ml) IgA IgG ₁ IgG _2a IgG _2b IgG ₃ IgM 8-Oligosaccharide-Tetanus Toxoid Pore 3.3 2.7 0.08 0.07 1.7 8.4 Nonconjugated 8-oligosaccharides 0.3 0.05 0.02 0.02 0.8 0.8 8-polysaccharide-tetanus toxoid pore 10.8 0.13 0.07 0.07 1.75 14.6 Non-conjugated 8-polysaccharide 5.75 0.08 0.05 0.06 2.75 10.4

Inhibition ELISA using type-8 oligosaccharide Inhibitory antigen concentration ([mu] g) O.D. 405 nm 0.5 M TFA, 100 캜, 20 minutes, 8-PS 1 R.U. 2 R.U. 3 R.U. 4 R.U. 5 R.U. 6 R.U. 7 R.U. .8+ R.U. 25.0 0.013 2.575 1.015 0.680 0.733 0.698 0.564 0.489 0.177 12.5 0.011 2.583 1.610 1.109 1.190 0.974 1.008 0.827 0.427 6.25 0.031 2.542 2.042 1.751 1.878 1.395 1.303 1.243 0.783 3.13 0.105 2.591 2.359 2.127 2.456 1.845 1.827 1.628 1.204 1.57 0.228 2.585 2.566 2.264 2.748 2.282 2.234 1.944 1.744 0.781 0.468 3.602 2.643 2.313 2.761 2.597 2.531 2.219 2.014 0.391 0.821 2.919 2.829 2.549 3.000 2.814 2.837 2.318 2.865

Inhibition with type-6B oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm 6B-PS 6B (0.01 M acetic acid, 100 DEG C, 30 hours) 25.0 0.018 0.300 12.5 0.023 0.502 6.25 0.025 0.776 3.13 0.054 0.952 1.57 0.109 1.090 0.781 0.207 1.192 0.391 0.331 1.266

Inhibition using other TFA preparations of type-6B oligosaccharides Inhibitory antigen concentration ([mu] g) O.D. 405 nm 6B-PS 6B (0.5M TFA, 70 DEG C, 1 hour) 6B (0.1 M TFA, 70 DEG C, 2 hours) 6B (0.1M TFA, 70 DEG C, 4 hours) 25.0 0.016 0.223 0.211 1.897 12.5 0.017 0.281 0.372 2.417 6.25 0.069 0.717 0.702 2.276 3.13 0.370 1.096 1.216 2.674 1.57 0.958 2.411 1.793 2.673 0.781 0.959 2.595 2.243 2.817 0.391 1.216 2.540 2.502 2.563

Inhibition using Type 6B oligosaccharide and other acetic acid production Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm 6B-PS 6B (2M acetic acid, 70 < 0 > C, 2h 6B (2M acetic acid, 70 DEG C, 24 hours) 6B (2M acetic acid, 70 DEG C, 48 hours) 25.0 0.016 0.341 1.530 2.452 12.5 0.017 0.631 1.849 2.534 6.25 0.069 0.982 2.319 2.613 3.13 0.370 1.504 2.579 2.716 1.57 0.958 2.034 2.748 2.807 0.781 0.959 2.473 2.751 2.732 0.391 1.216 2.533 2.597 2.626

Inhibition ELISA using type-6B oligosaccharide Inhibitory antigen concentration ([mu] g) O.D. 405 nm 6B-PS 6B (0.05M TFA, 70 DEG C, 1.5 hours) 6B (0.05M TFA at 70 DEG C for 3 hours 25.0 0.100 0.297 0.594 12.5 0.140 0.485 0.916 6.25 0.125 0.816 1.371 3.13 0.396 1.180 1.852 1.57 0.563 1.702 2.195 0.781 1.060 2.286 2.644 0.391 1.616 2.515 2.551

Inhibition with type-14 oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm 14-PS 14 (0.1M TFA, 70 DEG C, 3 hours) 14 (0.5M TFA, 70 DEG C, 7 hours) 25.0 0.020 0.072 0.201 12.5 0.022 0.229 0.339 6.25 0.022 0.362 0.745 3.13 0.065 0.631 1.032 1.57 0.161 1.028 1.341 0.781 0.299 1.277 1.614 0.391 0.644 1.691 1.714

Inhibition with type-14 oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) OD, 405 nm 0.5M TFA, 100 캜, 15 min. 3 R.U. 4 R.U. 8 R.U. 25.0 2.011 0.596 0.337 12.5 1.966 1.078 0.517 6.25 1.936 1.425 0.712 3.13 1.891 1.620 0.924 1.57 2.021 1.889 1.242 0.781 2.055 1.923 1.595 0.391 2.089 1.877 1.727

Inhibition with type-14 oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) OD, 405 nm 14-PS 14 (0.01M TFA, 70 DEG C, 7 hours) 14 (0.05M TFA, 70 DEG C, 7 hours) 14 (0.05M TFA, 70 DEG C, 24 hours) 25.0 0.038 0.075 0.163 0.610 12.5 0.055 0.110 0.283 0.986 6.25 0.093 0.191 0.503 1.406 3.13 0.197 0.318 0.857 2.225 1.57 0.330 0.528 1.367 2.562 0.781 0.623 0.945 2.095 2.622 0.391 1.053 1.462 2.429 2.569

Inhibition with type-19F oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) OD, 405 nm 19F-PS 19F (0.2 M HCl, non-heating, 3 hours 19F (0.2 M HCl, 70 DEG C, 3 hours) 25.0 0.347 2.235 0.253 12.5 0.380 2.926 0.328 6.25 0.460 3.286 0.495 3.13 0.669 3.415 0.691 1.57 0.805 3.405 1.064 0.781 1,000 3.491 1.414 0.391 1.508 3.549 1.827

Inhibition with type-23F oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) OD, 405 nm 23F-PS 23F (0.5M TFA, 70 DEG C, 1 hour 23F (0.25M TFA, 70 DEG C, 3 hours) 25.0 0.033 0.385 0.435 12.5 0.033 0.586 0.592 6.25 0.027 0.686 0.743 3.13 0.037 0.832 0.870 1.57 0.062 0.952 0.933 0.781 0.128 1.085 1.029 0.391 0.240 1.155 1.099

Inhibition with type-23F oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) OD, 405 nm 23F-PS 23F (0.1 M TFA, 70 < 0 > C, 3h 23F (0.1M TFA, 70 DEG C, 5 hours) 25.0 0.487 0.187 2.107 12.5 0.364 0.261 2.517 6.25 0.355 0.339 2.623 3.13 0.341 0.545 3.009 1.57 0.358 0.770 2.689 0.781 0.401 0.998 2.624 0.391 0.432 1.366 2.887

Inhibition with type-23F oligosaccharides Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm 23F-PS 23F (0.5M TFA, 70 DEG C, 1 hour) 23F (0.5M TFA, 70 DEG C, 15 min) 23F (5M acetic acid, 70 DEG C, 5 hours) 23F (2M acetic acid, 70 DEG C, 24 hours) 25.0 0.127 0.309 0.159 0.160 0.241 12.5 0.111 0.529 0.149 0.144 0.405 6.25 0.118 0.923 0.179 0.243 0.750 3.13 0.148 1.480 0.242 0.410 1.172 1.57 0.288 2.118 0.430 0.852 1.662 0.781 0.480 2.534 0.712 1.352 2.361 0.391 0.940 2.557 1.218 1.909 2.430

N. Meningitidis-C inhibition with polysaccharides Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm N. Meningitisdis -CPS N. menin GT display _{(0.1M NaOA C, 50 ℃,} 6 hours) N. meningitidis-C (1M NaOAc, 50 C, 6 hours) 25.0 0.304 0.269 0.269 12.5 0.240 0.245 0.258 6.25 0.369 0.406 0.412 3.13 0.836 1.088 1.151 1.57 2.402 2.593 2.512 0.781 2.679 2.839 2.749 0.391 2.434 2.448 2.504

Inhibition with type-6B coplanar ELIZA Inhibitory antigen concentration ([mu] g) O.D. 405 nm 6B-PS 6B: 8-TT 6B-OS T.T. 25.0 0.019 0.070 0.037 2.314 12.5 0.022 0.117 0.046 2.289 6.25 0.033 0.205 0.081 2.209 3.13 0.058 0.339 0.145 2.173 1.57 0.127 0.533 0.287 2.208 0.781 0.248 0.816 0.534 2.237 0.391 0.434 1.234 0.856 2.249

Inhibitor Eliza with Type-14 Balloons Inhibitory antigen concentration ([mu] g) O.D. 405 nm 14-PS 14: 8-TT 14-OS T.T. 25.0 0.004 0.029 0.026 0.216 12.5 0.006 0.043 0.037 0.51- 6.25 0.017 0.075 0.068 0.769 3.13 0.043 0.141 0.131 1.018 1.57 0.078 0.258 0.247 1.242 0.781 0.160 0.447 0.400 1.362 0.391 0.299 0.669 0.655 1.475

Inhibition with type -23F coater Inhibitory antigen concentration ([mu] g) O.D. 405 nm 23F-PS-TT 23F-TT (F: 27-47) 23F-TT (F: 32-51) 23F-TT (F: 32-65) 23F-TT (F: 31-51) T.T. 25.0 0.027 0.348 0.235 0.397 0.053 1.073 12.5 0.038 0.433 0.390 0.463 0.093 1.017 6.25 0.055 0.556 0.547 0.604 0.161 1.094 3.13 0.104 0.659 0.665 0.698 0.255 1.097 1.57 0.189 0.806 0.823 0.769 0.371 1.098 0.781 0.215 0.944 0.861 0.954 0.548 1.085 0.391 0.412 1.029 1.017 0.978 0.696 1.089

N. Meningitidis-C inhibition with polysaccharides Eliza Inhibitory antigen concentration ([mu] g) O.D. 405 nm N. Meningitisdis -CPS N. meningitidis -CPS-TT (F: 37-49) N. meningitidis -CPS-TT (F: 37-49) T.T. 25.0 0.038 0.068 0.250 2.222 12.5 0.025 0.042 0.221 1.880 6.25 0.028 0.120 0.297 1.917 3.13 0.058 0.209 0.529 1.894 1.57 0.108 0.212 0.610 1.807 0.781 0.371 0.511 0.762 1.813 0.391 0.393 0.623 1.101 1.849

Antiserum 8 Mean concentration of IgG ₁ and IgG ₃ antibodies to S. pneumoniae conjugate (after tertiary immunization) Immunoglobulin isotype (μg / ml) IgG ₁ IgG ₃ 1. Immunization 3 weeks of age 8-O-TT 0.273 0.468 8-P-TT 0.036 0.213 8-P 0.052 3.18 TT 0.034 - 8 weeks of age 8-O-TT 0.700 1.96 8-P-TT 0.159 2.89 8-P 0.007 2.17 TT 0.003 0.74 8 weeks age + FCA 8-O-TT 1.22 1.74 8-P-TT - 3.73 8-P 0.055 5.06 TT - 0.295

The mean concentrations of rats serum antibodies to various serotype 14 S. pneumoniae monocytes (after the third immunization) Antisera: IgM IgG ₁ IgG _2a IgG _2b IgG ₃ IgA 14-oligo-TT (0.1 M TFA) 6.50 50.99 0.178 0.158 37.65 0.078 1 μg 14-oligo-TT (0.5 M TFA peak 45) 2.8 - 0.009 0.018 1.51 - 1 [mu] 14-oligo-TT (0.5 M TFA peak 78) 3.39 12.35 0.155 0.143 9.32 0.020 1 [mu] g 14-poly-TT 5.11 15.25 0.038 0.053 8.95 0.001 1 [mu] g 14-poly 4.70 - 0.008 0.031 9.20 0.077 1μ T.T. 3.37 - 0.024 0.060 8.36 0.031

Serum Liquid 8 Obsonisation test for antiserum against S. pneumoniae % Reduction rate of colony forming units (CFU) Antisera: Type 8 8-oligosaccharide tetanus toxoid pore 98 Nonconjugated 8-oligosaccharides 10 8-polysaccharide tetanus toxoid pore 39 Non-conjugated 8-polysaccharide 11

The opsonization results of testing antisera against serotype 3 and 8 S. pneumoniae % CFU reduction rate Antisera: Type 3 Type 6B Type 8 3/8-oligosaccharide tetanus toxoid pore 96 0 99 3/8-polysaccharide tetanus toxoid pore 10 0 12 PBS control 0 0 0

Serum Liquid 8 Obsonisation test for antiserum against S. pneumoniae % CFU reduction rate Antisera: Type 14 14-O-TT (0.1 M TFA) 76 14-O-TT-10.5 M TFA peak 7 8) 54 14-poly-TT 18 14-polysaccharide 2 Tetanus-toxoid 15 0.9% NaCl 0 NMS 0

≪ RTI ID = 0.0 > A < / RTI > di-heptene S. pneumoniae % CFU reduction rate Antisera: Type 14 14-O-TT 68 8: 14-O-TT 65 14-P-TT 37 Tetanus toxoid 0 0.9% NaCl 0 NMS 0

Immunization system A: 1 ˙ mono-hapten-oligosaccharide conjugate 2 ˙ mono-hapten-oligosaccharide conjugate 3 ˙ mono-hapten-oligosaccharide conjugate B: 1 mono-hapten-oligosaccharide conjugate 2 ˙ uncoupled polysaccharide 3 ˙ uncoupled polysaccharide C: 1 ˙Uncoupled Polysaccharide 2 ˙ Mono-Hapten-Oligosaccharide Polymer 3 ˙ Mono-Hapten-Oligosaccharide Polymer D: 1 ˙ Multi-hapten-oligosaccharide pore 2 ˙ Multi-hapten-oligosaccharide pore 3 ˙ Multi-hapten-oligosaccharide pore E: 1˙i multi-hapten-oligosaccharide cofactor 2˙ uncoupled polysaccharide 3˙ uncoupled polysaccharide F: 1˙ uncoupled polysaccharide 2 ˙quick multi-hapten-oligosaccharide 3 π multi-hapten-oligosaccharide coplanar G: 1 Tetanus toxoid 2 Tetanus toxoid 3 Tetanus toxoid 4 Mono- or multi-hapten-oligosaccharide conjugate 5 Mono- or multi-hapten-oligosaccharide conjugate 6 ˙ Mono- or multi-hapten-oligosaccharide conjugate Photo Frame

Claims (47)

a) at least one carbohydrate hapten separated by size consisting of at least one immunogenic epitope; And b) carrier Wherein the hapten is covalently coupled to the carrier and the hapten-carrier conjugate is protective immunity. 2. The composition of claim 1, wherein the hapten is an oligosaccharide of bacterial or viral polysaccharide. 3. The composition of claim 1, wherein the presence of the immunogenic epitope is determined using an inhibition ELISA. 3. The composition of claim 2, wherein the oligosaccharide is produced by acid hydrolysis of the polysaccharide. 2. The composition of claim 1, wherein said protective immunity is determined by an isotype ELISA. 2. The composition of claim 1, wherein said protective immunity is determined by a sterile or opsonized assay. The method of claim 2, wherein the polysaccharide is selected from the group consisting of S. pneumoniae serotypes 1, 2, 3, 4, 5, 6B, 7, 7F, 8, 9N, 9V, 10A, 11A, 12, 12F, 17F, 18C, 19F, 19A, 20, 22, 23F and 33F. 2. The composition of claim 1 consisting of two or more haptens. The composition of claim 1 which does not induce carrier suppression. 2. The composition of claim 1 which does not induce antigenic competition. The composition of claim 1, further comprising an adjuvant. a) cleaving the polysaccharide of the bacterial or viral into oligosaccharides so that the resulting oligosaccharide has an immunogenic epitope; b) separating the resulting oligosaccharide by size; c) selecting an oligosaccharide containing an immunogenic epitope based on an inhibition ELISA; d) activating the oligosaccharide selected in step c); And e) coupling the activated oligosaccharide to the purified carrier (so that the resultant composition has protective immunity). 13. The method of claim 12, wherein the cleavage is performed using acid hydrolysis. 13. The method of claim 12 wherein the activation is acidification on a cationic column. 13. The method of claim 12, wherein the coupling is performed using EDC or periodate. 13. The method of claim 12, wherein the coupling provides a predictable ratio of hapten to carrier. 13. The method of claim 12, wherein steps (a) to (d) are repeated using one or more polysaccharides of a bacterium or virus to produce a bifunctional or multivalent blank. 18. The method of claim 17, wherein the polysaccharide of the bacterium is selected from the group consisting of S. pneumoniae serotype 1, 2, 3, 4, 5, 6B, 7, 7F, 8, 9N, 9V, 10A, 11A, 12, 15B, 17F, 18C, 19F, 19A, 20, 22, 23F and 33F. A method of imparting protective immunity against bacterial pathogens comprising administering an effective amount of the composition of claim 1 to a mammal in need of such treatment. 20. The method of claim 19, wherein said administration is selected from the group consisting of oral and parenteral administration. 20. The method of claim 19, wherein the mammal is neonate. 20. The method of claim 19, wherein the mammal is selected from the group consisting of immunosuppressed mammals and older mammals. Use of an oligosaccharide of S. pneumoniae serotype 8 containing an immunostimulatory epitope determined by an inhibitory ELISA and a pharmaceutical excipient suitable for providing an immunostimulatory effect of said oligosaccharide is useful for stimulating an immune response to an antigen Composition. 24. The composition of claim 23, wherein the oligosaccharide is conjugated to a protein carrier. 24. The composition of claim 23 which does not induce carrier inhibition. 24. The composition of claim 23 which does not induce antigen competition. 26. A method of imparting protective immunity against bacterial pathogens comprising administering an effective amount of the composition of claim 23 to a mammal in need of such treatment. Comprising administering to a subject with an antigen an oligosaccharide of S. pneumoniae serotype 8 containing an immunogenic epitope as determined by an inhibitory ELISA. 29. The method of claim 28, wherein said administration is selected from the group consisting of oral and parenteral administration. a) cleaving the polysaccharide of S. pneumoniae serotype 8 to oligosaccharides so that the resulting oligosaccharide has an immunogenic epitope; b) separating the resulting oligosaccharide by size; c) selecting an oligosaccharide containing an immunogenic epitope based on an inhibition elicitor; And d) mixing the selected oligosaccharide with a suitable pharmaceutical carrier. 31. The method of claim 30, wherein said cleavage is performed using acid hydrolysis. 31. The method of claim 30, wherein step d) 1) activating the oligosaccharide selected in step c); And 2) coupling the activated oligosaccharide with the purified carrier. 33. The method of claim 32, wherein said activation is acidification on a cationic column. 33. The method of claim 32, wherein the coupling is performed using EDC or periodate. 33. The method of claim 32, wherein the coupling provides a predictable ratio of hapten to carrier. A method of enhancing the immunity of an antigen in a mammal comprising administering an oligosaccharide of S. pneumoniae serotype 8 containing an immunogenic epitope as determined by an inhibitory ELISA with an antigen that enhances the immunological effect of the oligosaccharide. 37. The method of claim 36, wherein the oligosaccharide is conjugated to a carrier. 37. The method of claim 36, wherein said oligosaccharide is S. pneumoniae serotype 8 administered as a mixture to a pharmaceutical or vaccine preparation. 38. The method of claim 36, 37 or 38 wherein said administration is selected from the group consisting of oral and parenteral administration. The oligosaccharide of S. pneumoniae serotype 8 containing the? -glucose (1? 4)? -glucose (1? 4)? -galactose (1? 4) Wherein the cell is stimulated specifically or nonspecifically. 41. The method of claim 40, wherein the oligosaccharide is conjugated to a carrier. 42. The method of claim 41, wherein the oligosaccharide is administered as a mixture to a pharmaceutical or vaccine preparation. 43. The method according to any one of claims 40, 41 or 42, wherein said administration is selected from the group consisting of oral and parenteral. (1 → 4)? -glucose (1 → 4) β-glucose (1 → 4) Wherein the adjuvant is administered to the mammal. 45. The method of claim 44, wherein the oligosaccharide is conjugated to a carrier. 45. The method of claim 44, wherein the oligosaccharide is administered as a mixture for a pharmaceutical or vaccine preparation. 46. The method according to any one of claims 44, 45 or 46, wherein said administration is selected from the group consisting of oral and parenteral.