RU2233172C2 - Haemophilus influenzae immunogenic conjugate polysaccharide - porin of meningococcus group b (variants), method for its preparing, pharmaceutical composition and method for immune response induction in animal with regards to haemophilus influenzae - Google Patents

Abstract

FIELD: biotechnology, microbiology, vaccines.

SUBSTANCE: invention can be used in preparing vaccines used for inducing immune response in animals. Invention proposes immunogenic conjugate of polusaccharide from Haemophilus influenzae of type B (Hib) with practically replaced external membrane mature protein of class 2 or 3 from Neisseria meningitides or its fusion form comprising amino acids 1-20 or 1-22 of capside protein

of gene T7 (xPorB). Hib is subjected for oxidation or selective hydrolysis for preparing aldehyde groups. Then this polysaccharide with xPorB is conjugated by reductive amination. The prepared conjugate is used for preparing pharmaceutical composition and induction of immune response in animal with regards to H. influenzae.

EFFECT: improved preparing method, valuable biological and medicinal properties of conjugate.

13 cl, 16 dwg, 5 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of vaccines used to elicit an immune response in an animal. In particular, the invention relates to conjugates of the polysaccharide of H. influenzae, the outer membrane protein of N. meningitidis, pharmaceutical compositions and their use.

State of the art

The microorganism Haemophilus influenzae is a small pleomorphic gram-negative coccobacillus. Isolates are classified into six capsular types (a-f), which differ in the nature of the antigens, and unencapsulated non-typable strains. Haemophilus influenzae can cause meningitis, otitis media (middle ear inflammation), sinusitis, epiglottitis (epiglottis inflammation), septic arthritis, febrile latent bacteremia, cellulitis, pneumonia and empyema, sometimes this organism causes newborn meningitis and septicemia. Other H. Influenzae infections include purulent pericarditis, endocarditis, conjunctivitis, osteomyelitis, peritonitis, epididymal orchitis, glossitis, uvulitis (inflammation of the tongue of the soft palate) and septic thrombophlebitis. In most cases, invasive diseases prior to the introduction of the H. influenzae type b (Hib) conjugate vaccine were caused by type b. Unencapsulated organisms can cause invasive disease in newborns. Unencapsulated strains cause an upper respiratory tract infection, including otitis media, sinusitis and bronchitis, and can cause pneumonia.

The source (micro) of the body is the upper respiratory tract of a person. The transmission method is presumably a transfer from subject to subject through direct contact or by inhalation of droplets of airway secretions containing the body. Often asymptomatic colonization by unencapsulated strains occurs, organisms secrete from the throat in 60-90% of children. However, colonization by type b organisms is rare, ranging from 2 to 5% of children in the era preceding the advent of vaccines, and, obviously, has become even less frequent due to the widespread vaccination with Hib conjugate. The exact period of transmission is unknown, but its duration may be equal to the period of the body in the upper respiratory tract.

Prior to the introduction of effective vaccines, Hib was the most common cause of bacterial meningitis in children in the United States and many other countries. Meningitis and other invasive infections were most common among children aged 3 months to 3 years, and approximately half of the cases were in children under 12 months of age. The dependence of the frequency of invasive type b disease on age varied in countries in different populations; the incidence rate of children under the age of 12 months was the highest in populations with the highest overall incidence rate, which led to a lower incidence rate among middle-aged people. In contrast to meningitis and most other invasive Hib diseases, epiglottitis rarely occurs in children under the age of 12 months, the peak of its occurrence in the era preceding the onset of vaccines was between 2 and 4 years old. Epiglottitis can also occur in older unvaccinated children and adults.

Invasive diseases were more common in boys, African Americans, Alaskan Eskimos, Apache and Navajo Indians, children attending child care centers, children living in overcrowding, and children who were not breastfed. Immunized children, in particular children under the age of 4 years who are in long-term close contact (such as home conditions) with a child with an invasive Hib disease, are at increased risk of getting a serious infection from this organism. Other invasive disease predisposition factors include sickle cell anemia, asplenia, HIV infection, a number of immunodeficiency syndromes, and malignant neoplasms. Children under the age of 1 year with documented invasive infection have an approximately 1% risk of relapse if they are not subsequently vaccinated.

Since 1988, when Hib conjugate vaccines were introduced, the incidence of invasive Hib diseases has decreased by 95% in infants and young children, and the incidence of invasive infections caused by other encapsulated types is currently close to the frequency of infections caused by type b. The result of this success was that the US Public Health Service set a goal to eliminate Hib disease in children under the age of 5 years. Currently, invasive Hib disease occurs mainly in still unvaccinated children and infants who are too small to complete a complete primary vaccination series.

In the United States, four Hib conjugate vaccines are approved. These vaccines consist of a Hib capsular polysaccharide (ie, polyribosylribotolphosphate [PRP] or PRP oligomers) covalently linked to a carrier protein directly or via an intermediate spacer molecule. Protective antibodies are directed against PRP. Conjugate vaccines vary in composition and immunogenicity, which leads to differences in recommendations for their use. For example, PRP-D is recommended only for children 12 months of age and older, while the other three vaccines HbOC, PRP-T and PRP-OMP are recommended for infants starting at two months of age.

Adjuvants are substances that enhance the immune response to antigens and are therefore used in many vaccines and vaccines tested. The immunostimulatory effect of adjuvants is not antigen-specific, since they enhance the immune responses to many antigens of various types. The only adjuvants currently approved by the FDA (Food and Drug Administration) for use in humans are aluminum salts, but many adjuvants used in animal vaccinations and in newer tested vaccines, are of microbial nature (White RG The adjuvant effect of microbial products on the immune response), 1976, Ann. Rev. Microbiol., 30: 579-595), e.g. Freund's adjuvant, Corynebacterium parvum, muramyl dipeptide, toxoids q tetanus, etc. The mechanisms of the immunopotentiating ability of microbial substances are unknown.

The main proteins of the outer membrane of the pathogenic Neisseria (Neisseria gonorrhoeae and Neisseria meningitidis) were tested for the adjuvant potential [P.O. Livingston Approaches to augmenting the IgG antibody response to melanoma ganglioside vaccines, 1993, Ann. N.Y. Acad. Sci., 690: 204-213; P.O. Livingston, M.J. Calves, F. Helling, W.D. Zollinger, M.S. Blake and G.H. Lowell GD3 / proteosome vaccines induce consistent IgM antibodies against ganglioside GD3 (GD3 / proteosome vaccines induce consistent IgM antibodies against ganglioside GD3), 1993, Vaccine, 11: 1199-1204; G.H. Lowell, W.R. Ballow, L.F. Smith, R.A. Wirtz, W.D. Zollinger and W.T. Hockmeyer. Proteosomal lipopeptide vaccines - increasing the immunogenicity of malarial CS peptides (Proteosome - lipopeptide vaccines: enchancement of immunogenecity for malaria CS peptides), 1988, Science, 240: 800-802; G.H. Lowell, L.F. Smith, R.C. Seid and W.D. Zollinger. Peptides bound to proteosomes via hydrophobic feet become highly immunigenic without adjuvant, 1988, J. Exp. Med., 167: 658-663; L.M. Wetzler, M.S. Blake, K. Barry and E.S. Gotschlich. Gonococcal Porin Vaccine Assessment - Comparison of Por proteosomes, liposomes and vesicles isolated from rmp deletion mutants (Gonococcal porin vaccine evaluation: comparison of Por proteosomes, liposomes, and blebs isolated from rmp deletion mutants), 1992, J. Infect. Dis., 166: 551-555] and to elucidate the mechanism that determines their immunopotentiating ability. The proteins of interest are protein IA (PIA) and protein IB (PIB) of gonococcus and proteins of classes 1, 2 or 3 of meningococcus (C1, C2 and C3, respectively) [M. Blake and E. Gotschilich. Functional and immunological properties of pathogenic neisserial surface proteins, 1986. In the monograph by M. Inouye Bacterial Outer Membranes as Model Systems, John Wiley, New York, p. 377-400]. All of them work like porins [E.S. Lynch, M.S. Blake, E.C. Gotschlich and A. Mauro. Studies of porins: Spontaneously transferred from whole cells and reconstituted from sample proteins of Neisseria gonorrhoeae and Neisseria meningitidis), 1984, Biophys., 45: 104-- 107; A. Mauro, M.S. Blake and R. Labarca. The mechanism of voltage gating of conductance in lipid bilayers induced by porin from outer membranes of Neisseria gonorrhoeae, 1988, Proc. Natl. Acad. Sci. USA 85: 1071-1075; J.D.E. Young, M.S. Blake, A. Mauro and Z.A. Cohn. Properties of the major outer membrane protein from Neisseria gonorrhoeae incorporated into model lipid membranes, 1983, Proc. Natl. Acad. Sci. USA, 80: 3831-3835], have significant homology of amino acid sequences relative to each other [S. Butcher, M. Sarvas and C. Runeberg-Nyman. Class 3 porin protein of Neisseria meningitidis: Cloning and Structure of the gene, 1991, Gene 105: 125-128; N. Carbonetti and R. Sparling. Molecular cloning and characterization of the structural gene of protein I, the major outer membrane protein of Neisseria gonorrhoeae, 1987, Proc. Natl. Acad. Sci. USA 84: 9084-9088; E.C. Gotschlich, M.E. Seiff, M.S. Blake and J.M. Koomey. Porin protein of Neisseria gonorrhoeae - cloning and gene structure (Porin protein of Neisseria gonorrhoeae: cloning and gene structure), 1987, Proc. Natl. Acad. Sci. USA 84: 8135-8138; M.J. Ward, P.R. Lambden and J.E. Heckels. Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria, 1992, FEMS Microbiol. Lett., 73: 283-289] and is believed to be part of the porin superfamily of gram-negative bacteria [D. Jeanteur, J.H. Lakely and E. Pattus. The bacterial porin superfamily - sequence determination and structure prediction (The bacterial porin superfamily: sequence alignment and structure prediction), 1991, Molec. Microbiol., 5: 2153-2164].

It was shown that neuris porins, when non-covalently complexed with malaria peptides, enhance the antibody response to these peptides as compared to using peptides or peptides covalently linked to other proteins as immunogens [G.H. Lowell, W.R. Ballow, L.F. Smith, R.A. Wirtz, W.D. Zollinger and W.T. Hockmeyer. Proteosomal lipopeptide vaccines - increasing the immunogenicity of malarial CS peptides (Proteosome-lipopeptide vaccines: enchancement of immunogenecity for malaria CS peptides), 1988, Science, 240: 800-802; G.H. Lowell, L.F. Smith, R.C. Seid and W.D. Zollinger. Peptides bound to proteosomes via hydrophobic feet become highly immunigenic without adjuvant, 1988, J. Exp. Med., 167: 658-663]. In addition, it was shown that peptides isolated from group A streptococcus [G.H. Lowell Proteosomes, hydrophobic anchors, suit particles and liposomes for improved presentation of peptides and protein vaccines (Proteosomes, hydrophobic anchors, iscoms, and liposomes for improved presentation of peptides and protein vaccines), 1990, pp. 141-160 in the monograph GCWoodrow and M .M. Levine New Generation Vaccines, Marcel Dekker, Inc., New York], Influenza virus hemagglutinin [G.H. Lowell Proteosomes, hydrophobic anchors, suit particles and liposomes for improved presentation of peptides and protein vaccines (Proteosomes, hydrophobic anchors, iscoms, and liposomes for improved presentation of peptides and protein vaccines), 1990, pp. 141-160 in the monograph GCWoodrow and M .M. Levine New Generation Vaccines, Marcel Dekker, Inc., New York] or Trypanosome bruceii [G.H. Lowell, L.F. Smith, R.C. Seid and W.D. Zollinger. Peptides bound to proteosomes via hydrophobic feet become highly immunigenic without adjuvant, 1988, J. Exp. Med., 167: 658-663], are more immunogenic in mice when they are introduced into complexes containing Neisseria porins, compared with immunization of mice with peptides alone. Outer membrane vesicles (OMVs) of meningococci, consisting primarily of class 2 protein, were used as a carrier to enhance the immune response against H. influenzae polysaccharide capsule in the recently approved Merck H. influenzae type b vaccine [J.J. Donnelly, R.R. Deck and M.A. Liu. Immunogenicity of Haemophilus influenzae polysaccharide polysaccharide complex - Neisseria meningitidis outer membrane protein complex conjugate vaccine), 1990, J. Immunol., 145: 3071-3079]. Moreover, Livingston has investigated the use of purified neisseria porin as adjuvants in melanoma vaccines. Melanoma cells express human gangliosides GM2 or GD3 on their surface at significantly higher levels than normal melanocytes. To enhance the immune response against GM2 or GD3 and the possible induction of resistance to tumor development in patients with melanoma, GM2 or GD3 was non-covalently bound to purified Neisseria porins, and volunteers with malignant melanoma were immunized with these vaccine constructs. Antibody reactions against GM2 or against GD3 were significantly increased in patients immunized with the porin / GM2 or porin / GDS complexes, compared with patients immunized with these gangliosides alone or these gangliosides complexed with BCG [P.O. Livingston Approaches to augmenting the IgG antibody response to melanoma ganglioside vaccines, 1993, Ann. N.Y. Acad. Sci., 690: 204-213; P.O. Livingston, M.J. Calves, F. Helling, W.D. Zollinger, M.S. Blake and G.H. Lowell GD3 / proteosome vaccines induce consistent IgM antibodies against ganglioside GD3 (1993, Vaccine, 11: 1199-1204]. In addition, tumor burden in patients immunized with porin / CM2 was significantly reduced (personal communication, P. Livingston).

The mechanisms by which Neisseria porins act as adjuvants are unknown. Group from Merck [J.J. Donnelly, R.R. Deck and M.A. Liu. Immunogenicity of Haemophilus influenzae polysaccharide polysaccharide complex - Neisseria meningitidis outer membrane protein complex conjugate vaccine), 1990, J. Immunol., 145: 3071-3079; M.A. Liu, A. Friedman, A.I. Oliff et al. Vaccine carrier isolated from Neisseria meningitidis with mitogenic activity for lymphocytes (A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes), 1992, Proc. Natl. Acad. Sci. USA 89: 4633-4637; J.B. Ulmer, C.J. Burke, C. Shi, A. Friedman, J.J. Donnelly and M.A. Liu. Pore formation and mitogenicity in blood cells by the class 2 protein of Neisseria meningitidis), 1992, J. Biol. Chem., 267: 19266-19271], which developed the Neisseria polysaccharide capsule-OMV meningococcus conjugate vaccine, believed that this could be due to direct stimulation of T cells with a class 2 protein. They initially showed that a class 2 protein could directly stimulate T lymphocytes and as a result, the class 2 protein was renamed the immunity enhancing meningococcal protein (MIEP) [M.A. Liu, A. Friedman, A.I. Oliff et al. Vaccine carrier isolated from Neisseria meningitidis with mitogenic activity for lymphocytes (A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes), 1992, Proc. Natl. Acad. Sci. USA 89: 4633-4637]. However, it was later shown that only denatured class 2 protein in high concentrations (> 50 μg) could stimulate T cells, while the native protein did not produce such an effect [J.B. Ulmer, C.J. Burke, C. Shi, A. Friedman, J.J. Donnelly and M.A. Liu. Pore formation and mitogenicity in blood cells by the class 2 protein of Neisseria meningitidis), 1992, J. Biol. Chem., 267: 19226-19271]. Moreover, since most Neisseria porins are in their native configuration when used as a candidate (test) vaccine or adjuvant, the likelihood that nonspecific stimulation of T cells by denatured porins affects their immunopotentiating ability is low.

In the past few years, details have been clarified regarding the interaction between T and B lymphocytes necessary for antigen recognition, lymphocyte stimulation and antibody formation. In the modern model of stimulation of T-lymphocytes, it is shown that two groups of signals are needed between the antigen-presenting cell (APC) and the T-lymphocyte [K.S. Hathcock, G. Laszlo, H.B. Dickter, J. Bradshaw, P.S. Linsley and R.J. Hodes. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation, 1993, Science, 262: 905-907; C.A. Janeway, Jr. and C. Bottomly. Signals and signs for lymphocyte responses, 1994, Cell, 76: 275-285; R.H. Schwartz. Costimulation of T lymphocytes: The role of CD28, CTLA-4 and B7 / BB1 in interleukin-2 production and immunotherapy (1992); role of CD28, CTLA-4 and B7 / BB1 in interleukin-1 production and immunotherapy Cell 71: 1065-1068]. The first signal (signal 1) arises from the interaction of the main histocompatibility complex (MHC) on antigen-presenting cells (for example, B-lymphocytes, dendritic cells, macrophages, etc.) and a T-cell receptor on T-lymphocytes. The deepening at the MHC complex is usually occupied by an oligopeptide derived from processed antigens (T-cell epitope). The specificity of the reaction is provided by signal 1. A second or co-stimulatory signal (signal 2) occurs when two groups of antireceptors are bound during the interaction of B and T lymphocytes (Fig. 1). Activated T lymphocytes then secrete cytokines, which in turn stimulate effector cells, for example, causing B cells to become antibody-producing cells. It was shown that the induction of costimulation through the interaction of these contraceptors is important for the emergence of immunity to tumor development [M. Azuma, M. Cayabyab, D. Buck, J.H. Phillips and L. L. Lanier. The interaction of CD28 with B7 costimulates primary allogenic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes, 1992, J. Exp. Med., 175: 353-360; S. Baskar, S. Ostrand-Rosenberg, N. Nabavi, L.M. Nadler, G.J. Freeman and L.H. Glimcher Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules, 1993, Proc. Natl. Acad. Sci. USA 90: 5687-5690; L. Chen, S. Ashe, W.A. Brady et al. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4, 1992, Cell 71: 1093-1102; S. Falkow. What is a pathogen? (What is a pathogen?), 1997, ASM News, 63: 359-365; R.H. Schwartz. Costimulation of T lymphocytes: The role of CD28, CTLA-4 and B7 / BB1 in interleukin-2 production and immunotherapy, 1992 Cell 71: 1065-1068; S.E. Townsend and J.P. Allison Tumor rejection after direct costimulation of CD8 + T cells by B7-transfected melanoma cells, 1993, Science, 259: 368-370], prevention of tolerance [C.D. Gimmi, G.J. Freeman, J.G. Gribben, G. Gray and L.M. Nadler Clonal anergy of human T-cells is induced by antigen presentation in the absence of B7 costimuation, 1993, Proc. Natl. Acad. Sci. USA 90: 6586-6590; D.L Mueller, M.K. Jenkins and R.H. Schwartz. Contrasting the clonal expansion of functional clonal inactivation: a costimulatory signaling pathway determines the outcome of T cell antigen receptor occupancy, 1989, Annu. Rev. Immunol., 7: 445-480; P. Tan, C. Anasetti, J.A. Hansen et al. Induction of alloantigen-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7 / BB1), 1993, J. Exp. Med., 177: 165-173] and manifestations of the cytotoxic activity of lymphocytes [M. Azuma, M. Cayabyab, D. Buck, J.H. Phillips and L.L. Lanier. The interaction of CD28 with B7 costimulates primary allogenic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes, 1992, J. Exp. Med., 175: 353-360].

T-lymphocyte antireceptors are CD28 and CTLA-4. Both of them are members of the immunoglobulin superfamily [T.D. Connell, D. Shaffer and J.G. Cannon. Characterization of the repertoire of hypervariable regions in the protein II (opa) gene family of Neisseria gonorrhoeae), 1990, Molec. Microbiol., 4: 439-449]. CD28 is present on resting and activated T cells [M. Azuma, M. Cayabyab, D. Buck, J.H. Phillips and L.L. Lanier. The interaction of CD28 with B7 costimulates primary allogenic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes, 1992, J. Exp. Med., 175: 353-360; L. Chen, S. Ashe, W.A. Brady et al. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4, 1992, Cell 71: 1093-1102; M.K. Jenkins and J.G. Johnson. Molecules involved in T-cell costimulation, 1993, Curr. Opin. Immunol., 5: 361-367; C.H. June, J.A. Bluestone, L.M. Nadler and C.V. Thompson B7 and CD28 receptor families (The B7 and CD28 receptor families), 1994, Immunol. Today, 15: 321-331; P.S. Linsley, W. Brady, L. Grosmaire, A. Aruffo, N.K. Damle and J.A. Ledbetter. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation), 1991, J., Exp. Med., 173: 721-730; P.S. Linsley, E.A. Clark and J.A. Ledbetter. T-cell antigen CD28 mediates adhesion to B cells by interacting with B7 / BB-1 activation antigen (T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7 / BB-1), 1990, Proc. Natl. Acad. Sci. USA 87: 5031-5035; S.D. Norton, L. Zuckerman, K.B. Urdahl, R. Shefner, J. Miller and M.K. Jenkins Ligand CD28, B7, enhances IL-2 production by generating a co-stimulatory signal for T cells (The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells), 1992, J. Immunol., 149: 1556 -1561], while CTLA-4 is expressed only on activated T cells [GJ Freeman, D.B. Lombard, C.D. Gimmi et al. CTLA-4 mRNA and CD28 are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production (CTLA-4 and CD28 mRNA does not correlate with the method of lymphokine production (CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. J. Immunol., 149: 3795-3801; C. Harper, C. Balzano, E. Rouvier, M.G. Mattei, M.F. Luciani and R. Golstein. CTLA-4 and CD28 activated lymphocyte molecules that are very similar in mouse and human sequence, expression of information, gene structure and position on chromosomes (CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location), 1991, J. Immunol., 147: 1037-1044; T. Lindsten, K.P. Lee, E.S. Harris et al. Characterization of CTLA-4 structure and expression on human T cells (1993), J. Immunol., 151: 3489-3499; P.S. Linsley, W. Brady, M. Urnes, L.S. Grosmaire, N.K. Damle and J.A. Ledbetter. CTLA-4 is a second receptor for the B cell activation antigen B7), 1991, J., Exp. Med., 174: 561-569; R.H. Schwartz. Costimulation of T lymphocytes: The role of CD28, CTLA-4 and B7 / BB1 in interleukin-2 production and immunotherapy, 1992 Cell 71: 1065-1068]. The level of CD28 on activated T cells is 20 times higher than CTLA, but the affinity of CD28 for its B-cell counterceptor is much lower [T. Lindsten, K.P. Lee, E.S. Harris et al. Characterization of CTLA-4 structure and expression on human T cells (1993), J. Immunol., 151: 3489-3499; P.S. Linsley, W. Brady, M. Urnes, L.S. Grosmaire, N.K. Damle and J.A. Ledbetter. CTLA-4 is a second receptor for the B cell activation antigen B7), 1991, J., Exp. Med., 174: 561-569]. B-cell antagonists are B7 [G.J. Freeman, A.S. Freedman, J.M. Segil, G. Lee, J.F. Whitman and L.M. Nadler B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells), 1989, J. Immunol., 143: 2714 -2722; C.D. Gimmi, G.J. Freeman, J.G. Gribben et al. B7 surface antigen B cells provides a costimulatory signal that induces the proliferation of T cells and their secretion of interleukin-2 (B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2), 1991, Proc. Natl. Acad. Sci. USA 88: 6575-6579; P.S. Linsley, W. Brady, L. Grosmaire, A. Aruffo, N.K. Damle and J.A. Ledbetter. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation), 1991, J., Exp. Med., 173: 721-730; Z. Razi-Wolf, G.J. Freeman, F. Galvin, B. Benacerraf, L. Nadler, and H. Reiser. Expression and function of the murine B7 antigen, the major costimulatory molecule expressed by peritoneal exudate cells, 1992, Proc. Natl. Acad. Sci. USA 89: 4210-4214; H. Reiser, G.J. Freeman, Z. Razi-Wolf, C.D. Gimmi, B. Benacerraf and L.M. Nadler Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor / CD3 complex), 1992, Proc . Natl. Acad. Sci. USA, 89: 271-275] and the most recently opened B7-2 [M. Azuma, D. Ito, H. Yagita et al. B70 antigen is the second ligand for CTLA-4 and CD28 (B70 antigen is a second ligand for CTLA-4 and CD28), 1993, Nature, 366: 76-79; G.J. Freeman, F. Borriello, R.J. Hodes et al. Discovery of functional alternative CTLA-4 counterceptor in B7-deficient mice (Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice), 1993, Science, 262: 907-909; G.J. Freeman, F. Borriello, R.J. Hodes et al. Murine B7-2, an alternative CTLA-4 counter-receptor that costimulates T cell proliferation and interleukin-2 production), 1993, J. Esp. Med., 178: 2185-2192; G.J. Freeman, J.G. Gribben, V.A. Boussiotis et al. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation), 1993, Science, 262: 909 (Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation). -911; K.S. Hathcock, G. Laszlo, H.B. Dickter, J. Bradshaw, P.S. Linsley and R.J. Hodes. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation, 1993, Science, 262: 905-907]. B7 and B7-2 are representatives of the immunoglobulin superfamily [G.J. Freeman, F. Borriello, R.J. Hodes et al. Mouse B7-2, an alternative CTLA-4 counter-receptor that costimulates T cell proliferation and interleukin-2 production), 1993, J. Esp. Med., 178: 2185-2192; G.J. Freeman, A.S. Freedman, J.M. Segil, G. Lee, J.F. Whitman and L.M. Nadler B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells), 1989, J. Immunol., 143: 2714 -2722; G.J. Freeman, G.S. Gray, C.D. Gimmi et al. Structure, expression, and T cell costimulatory activity of the murine homologue of the human b lymphocyte activation antigen B7 (1991, J. Esp. Med., 174: 625-631] and are present only on activated B lymphocytes [G.J. Freeman, A.S. Freedman, J.M. Segil, G. Lee, J.F. Whitman and L.M. Nadler B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells), 1989, J. Immunol., 143: 2714 -2722]. A number of indirect data indicate the relationship of the new ligand, B7-2, with co-stimulation of T-lymphocytes: 1) the binding of CTLA-4 to activated B cells is only partially inhibited by monoclonal antibody (mAb) against B7 [K.S. Hathcock, G. Laszlo, H.B. Dickter, J. Bradshaw, P.S. Linsley and R.J. Hodes. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation, 1993, Science, 262: 905-907]; 2) lymphocytes isolated from mice defective in B7 expression can nevertheless co-stimulate T cells [G.J. Freeman, F. Borriello, R.J. Hodes et al. Discovery of functional alternative CTLA-4 counterceptor in B7-deficient mice (Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficient mice), 1993, Science, 262: 907-909; G.J. Freeman, F. Borriello, R.J. Hodes et al. Murine B7-2, an alternative CTLA-4 counter-receptor that costimulates T cell proliferation and interleukin-2 production, 1993, J. Esp. Med., 178: 2185-2192]; 3) transfectants expressing only B7-2 can co-stimulate T cells [G.J. Freeman, F. Borriello, R.J. Hodes et al. Mouse B7-2, an alternative CTLA-4 counter-receptor that costimulates T cell proliferation and interleukin-2 production), 1993, J. Esp. Med., 178: 2185-2192; G.J. Freeman, J.G. Gribben, V.A. Boussiotis et al. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation), 1993, Science, 262: 909 (Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation). -911] and 4) a mAb specific for B7-2 may inhibit co-stimulation of T-lymphocytes with B cells [KS Hathcock, G. Laszlo, H.B. Dickter, J. Bradshaw, P.S. Linsley and R.J. Hodes. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation, 1993, Science, 262: 905-907] or B7-2 transfectants [G.J. Freeman, F. Borriello, R.J. Modes et al. Mouse B7-2, an alternative CTLA-4 counter-receptor that costimulates T cell proliferation and interleukin-2 production), 1993, J. Esp. Med., 178: 2185-2192]. The significance of the initially described B7 antigen as a co-stimulatory contraceptor is controversial, because B7-2 expression occurs earlier than B7 expression, and more B7-2 antigens are present on the surface of activated B lymphocytes than B7 [K.S. Hathcock, G. Laszlo, H.B. Dickter, J. Bradshaw, P.S. Unsley and R.J. Modes Identification of an alternative CTLA-4 ligand costimulatory for T cell activation, 1993, Science, 262: 905-907]. A schematic representation of co-stimulation of T-lymphocytes and co-stimulatory contraceptors is illustrated in FIG.

There is preliminary evidence provided by various researchers that microbial products can stimulate B lymphocytes. Liu et al. showed that lipolysaccharide (LPS), a mitogenic influenza virus and an antigen that mimics a viral infection (polyinosine-polycytidylic acid), stimulate B-lymphocytes, which in turn stimulate T-lymphocytes [C.A. Janeway, Jr. and C. Bottomly. Signals and signs for lymphocyte responses, 1994, Cell, 76: 275-285]. Vordermeier demonstrated that purified Salmonella typhi porins (not containing LPS) are potent B cell stimulants but have minimal effect on T lymphocytes [N. Vordermeier and W.G. Bessler. Polyclonal activation of murine B lymphocytes in vitro by Salmonella typhimurium porins (Polyclonal activation of murine B lymphocytes in vitro by Salmonella typhimurium porins), 1987, Immunobiol., 175: 245-251; H. Vordermeier, H. Drexler and W.G. Bessler. Polyclonal activation of human peripheral blood lymphocytes by bacterial porins and defined porin fragments, 1987. Immunol. Lett., 15: 121-126]. In addition, preparations of the outer membrane of meningococci, mainly consisting of meningococcal porins, act as B-cell mitogens and do not stimulate T-lymphocytes [J. Melancon, R.A. Murgita and I.W. DeVoe. Activation of murine B lymphocytes by Neisseria meningitidis and isolated meningococcal surface antigens, 1983, Infect. Immun., 42: 471-479; B.G. Sparks. Immunomodulating activity of meningococcal antigens (Immunomodulating activity of meningococcal antigens), 1983, Can. J. Micribiol., 29: 1611-1618; B.G. Sparks. The double effect of meningococcal antigens on T cell dependent immune response, 1983, Can. J. Micribiol., 29: 1619-1625]. This evidence suggests that the porins of the neysseries and, possibly, the porins of other gram-negative bacteria could be able to stimulate B lymphocytes and increase the expression of B7-2. Increased expression of B7-2 may mediate costimulation of T-lymphocytes, which could be a mechanism by which porins enhance the immune response to other antigens, such as the PRP polysaccharide presented in this context.

SUMMARY OF THE INVENTION

The present invention is directed to a conjugate of H. influenzae type b polysaccharide (Hib) with a practically purified, rearranged matured outer membrane protein of class 2 or 3 Neisseria meningitidis or its fusion form containing amino acids 1-20 or 1-22 capsid protein φ 10 of the T7 gene (rPorB )

The present invention also provides a method for producing a Hib-rPo-B polysaccharide conjugate, comprising

(a) obtaining a Hib polysaccharide;

(b) oxidizing or selectively hydrolyzing said polysaccharide to create aldehyde groups;

(c) obtaining rРоrВ and

(d) conjugation of a polysaccharide containing aldehyde groups with rPoB by reductive amination (hydroamination).

The invention also relates to conjugates prepared according to the methods of the invention. Optionally, the conjugates of this invention may be in combination with DTaP (diphtheria, tetanus, acellular pertussis vaccine).

The invention also relates to pharmaceutical compositions containing conjugates of the invention, optionally containing DTaP and a pharmaceutically acceptable carrier.

The present invention provides a method of inducing an immune response in animals against H. influenzae, comprising administering the conjugates of the invention to an animal in an amount effective to induce said immune response.

The invention partly relates to the unexpected discovery that the Hib-rPorB conjugates of the invention induce significantly stronger immune responses in animals compared to the use of tetanus toxoid and recombinantly produced H. influenzae outer membrane protein P2. Significantly stronger immunogenic responses were also obtained compared to the Hib-CRM conjugate, which is commercially available from Lederle Laboratories, Division of American Cyanamid Company, Pearl River, NY. CRM ₁₉₇ is a non-toxic version of diphtheria toxin obtained by mutation-directed that is isolated from cultures of Corynebacterium diphtheriae C7 (β197) [see Seid RCJr article. et al. Glycoconj. J., (1989) 6: 489-498].

Moreover, the conjugate of the invention is particularly useful in compositions also containing DTaP, since immunological interactions between the components, as well as epitope suppression, are observed using conventional carrier proteins such as tetanus toxoid. The conjugates of the invention overcome this serious limitation in combination vaccine compositions.

List of figures of drawings and other materials

Figure 1 presents a graphical depiction of co-stimulation of a T-lymphocyte.

2 is a graph showing the fermentation profile of B1HB1030.

Figure 3 shows the SDS-PAGE stained gel (polyacrylamide gel electrophoresis using sodium dodecyl sulfate) of purified rPoB used for conjugation.

4 is a bar graph showing rat IgG specific IgG response in rats to Hib conjugates with various carrier proteins.

5-1 to 5-8 are tables showing serum antibodies in Sprague-Dawley rats immunized with Hib-TT, Hib-rPorB and Hib-rP2, determined by ELISA (enzyme-linked immunosorbent assay).

6-1 and 6-2 are data showing the PRP-specific response of IgG in rats to conjugated Hib vaccines with various carrier proteins obtained by ELISA. Two different Hib-rPorB preparations (-1 and -2) were tested. 6-1 in graphical form represent tabular data shown in Fig.6-2.

7 is a graph showing polysaccharide-specific IgG induced by conjugated Hib vaccines in CD-1 mice.

Fig. 8 is a graph showing a polysaccharide-specific IgG induced by conjugated rat Hib vaccines.

Information confirming the possibility of carrying out the invention

The present invention relates to a vaccine for inducing an immune response in an animal containing group B meningococcal outer membrane porin protein coupled to a Hib polysaccharide together with a pharmaceutically acceptable diluent, carrier or excipient, the vaccine being administered in an amount effective to induce an immune animal response to H. influenzae. In a preferred embodiment, the animal is a mammal selected from the group consisting of human, cattle, pigs and sheep, as well as chickens. In another preferred embodiment, the mammal is a human.

By the term "rPoB" is meant a mature rearranged outer membrane protein of class 2 or class 3 of N. meningitidis and its fusion forms containing amino acids 1-20 or 1-22 of the capsid protein φ10 of the T7 gene. Methods of high-level expression of mature rPoB class 2 and class 3 and its fusion forms, repackaging and purification are presented in [H.L. Qi, J.Y. Tai and M.S. Blake Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, 1994, Infect. Immun., 62: 2432-2439] and U.S. Patent No. 5,439,808, the disclosures of which are incorporated herein by reference. Recombinant porin can be expressed at high levels in E. coli according to U.S. Patent No. 5,439,808 or in yeast according to application 08/792302. In a preferred embodiment, rPoB Class 3 is expressed in the host BL21 (DE3) ΔompA, which is transformed with a gene encoding rPoB, is substantially purified and is redistributed according to U.S. Patent No. 5,439,808.

The capsular polysaccharide of H. infeuenzaee can be isolated according to methods well known in the art. See the work of Schneerson et al., J.Exp. Med., 152: 361-376 (1980); Marburg et al., J. Am. Chem. Soc. 108: 5282 (1986); Jennings et al., J. Immunol. 127: 1011-1018 (1981) and Beuvery et al., Infect. Immunol., 40: 39-45 (1983). In a preferred embodiment, the organism is cultured, the microfiltration culture supernatant is subjected to filtration, and the filtrate is passed through a filter separating molecules of a molecular weight of 300,000. Then, the dissolved substance is concentrated, for example, using a filter separating molecules of a molecular weight of 100,000. This material of molecular weight 100000-300000 is then oxidized with mild an oxidizing agent such as metaperiodate is filtered through a filter to separate the molecules of the molecular weight of 30,000, and then concentrated using a filter for the molecule yarnoy weight of 5000 to produce a polysaccharide having aldehyde groups, which can be directly used for conjugation. A preferred polysaccharide has a molecular weight of about 5000-50000. A more preferred polysaccharide has a molecular weight of about 10,000-50000, however, other molecular weight ranges may be used, if desired.

One skilled in the art will recognize that conjugated capsular polysaccharide-carrier protein vaccines can be prepared in a number of different ways. The types of covalent bonds that connect the polysaccharide with a carrier protein and the means for their preparation are well known in the art. Details relating to chemicals with which the two structures can be linked can be found in US Pat. Nos. 5623057, 5371197, 5192540, 4902506 and 4356170, the contents of which are incorporated herein in their entirety. For a review, see Contributions to Microbiology and Immunology, Volume 10, Conjugate Vaccines, editors of volume J.M. Cruse and R.E. Lewis, Jr., 1989 and (29). One such method is reductive amination as described by Schwartz and Gray (Arch. Biochim. Biophys. 181: 542-549 (1977)). This process involves the preparation of a polysaccharide in a form that has terminal reducing groups, and the reaction of a capsular polysaccharide and rPoB in the presence of cyanoborohydride ions or another reducing agent. Reducing groups can be formed by selective hydrolysis or specific oxidative decomposition, or a combination thereof.

The vaccine of this invention contains the Hib-rPorB conjugate in an effective amount, which depends on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, Group B meningococcal porin protein, a fusion protein, or a vaccine of the invention can also be administered by intraperitoneal, intravenous, or intranasal routes. One skilled in the art will appreciate that the amounts to be administered according to any particular treatment protocol can be easily determined without undue experimentation. Suitable amounts are expected to range from 5 to 50 μg / animal, more preferably about 10 μg / animal.

The vaccine of the invention may be used in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. It is preferable to use any inert carrier, such as saline, phosphate buffered saline, or any carrier of the kind in which the conjugate vaccine has suitable solubility. Vaccines can be in the form of single dose formulations or in multiple dose vials that can be used in mass vaccination programs. For methods of preparing and administering vaccines, we refer to the Remington's Pharmaceutical Sciences directory, Mack Publishing Co., Easton, PA, ed. Osol, (1980) and the monograph "New trends and developments in Vaccines" under. ed. Voller et al., University Park Press, Baltimore, MD (1978).

The vaccines of this invention may also contain adjuvants that enhance the production of antibodies specific for H. influenzaee. These adjuvants include, but are not limited to various oil preparations, such as Freund's complete adjuvant (CFA), stearyl tyrosine (ST, see U.S. Patent No. 4,258,029), a dipeptide known as MDP, saponin, aluminum hydroxide, and lymphatic cytokine.

Freund's adjuvant is an emulsion of mineral oil and water, which is mixed with an immunogenic substance. Although Freund's adjuvant is active, it is usually not administered to humans. Instead, an alumina (aluminum hydroxide) or ST adjuvant may be used for administration to humans. The conjugate vaccine can be adsorbed on aluminum hydroxide, from which it is slowly released after injection. The conjugate vaccine can also be encapsulated in liposomes according to U.S. Patent No. 4,235,877 to Fullerton.

In another preferred embodiment, the conjugate of the invention is combined with other immunogens that are used to vaccinate animals. Thus, the conjugate of the invention can be combined with DTaP or DTaP IPV for administration to an animal. DTaP is a combination vaccine against diphtheria, tetanus, acellular pertussis, which is purchased from Amvax, Inc., Beltsville, Maryland. In a preferred embodiment, acellular pertussis is in an oxidized form, such as that of Amvax, Inc.

In another preferred embodiment, the invention relates to a method for inducing an immune response in an animal, which comprises administering to the animal a vaccine according to the invention in an amount effective to induce an immune response. Optionally, the vaccine of the invention may be administered in conjunction with effective amounts of other immunogens, as noted above, to create a plurality of immune responses in an animal.

The following examples serve to illustrate, but not limit the method and compositions of this invention. Other suitable modifications and adaptations of a number of conditions and parameters that are normally found in this field, which are obvious to specialists, are included in the essence and scope of this invention.

EXAMPLES

Example 1. Expression, isolation, re-laying and purification of rРоrВ

Bacterial strains, growth conditions and reagents. Genomic DNA is isolated from strain N. meningitidis of group B 44/76 (serotype 15) using standard procedures and is used as a template for the polymerase chain reaction to amplify a class 3 protein gene, as described elsewhere [H.L. Qi, J.Y. Tai and M.S. Blake Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, 1994, Infect. Immun., 62: 2432-2439]. The amplified product is cloned into the NdeI and XhoI sites of the plasmid pET17b (Novagen, Inc.), which is used to transform competent E. coli DH5a. Plasmid DNA from the selected DH5a clones is isolated and used to transform E. coli BL21 [DE3] -ΔompA. Transformants were selected against the background of carbenicillin and expression was induced by the addition of IPTG to a final concentration of 0.4 mM.

Overexpression of rРоrВ in E. coli and purification procedures by re-laying.

Monitoring of the levels of rPoB protein expressed at various times after induction is carried out by treating SDS-PAGE cell extracts in 8-16% gradient gels using a Novex system (Novex, San Diego, CA) followed by Coomassie staining with blue diamond and densitometric analysis with using an IS-1000 digital imaging system (Alpha Innotech Co., San Leandro, CA). The overexpression of rPoB was isolated by resuspension and lysis of bacterial cells using a Stansted air cell disintegrator (Stansted Fluid Power Ltd.) in TEN buffer (50 mM Tris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid, EDTA), 100 mM NaCl, pH 8 , 0) followed by centrifugation and separation of a precipitate containing rРо-В, aggregated in the form of inclusion bodies (IBs). After washing the pellet with 0.5% deoxycholate in TEN buffer followed by two washing with TEN buffer, the protein is solubilized by resuspension and sonication of IBs for 5 min in a freshly prepared 8 M urea solution using an ultrasonic disintegrator with a water bath. The reassignment of rРо-В into its native conformation is achieved using the reassembly procedure using a detergent. Equal volumes of urea dissolved in urea and 10% Z 3-14 (Calbiochem) were mixed and the resulting porin extract was applied to a Sephacryl S-300 column (5 × 100 cm) (Pharmacia Biotech Inc.), equilibrated with a buffer containing 100 mM Tris-HCl 200 mM NaCl, 10 mM EDTA, 20 mM CaClg and 0.05% Z 3-14, pH 8.0. Fractions containing rPoB were identified by SDS-PAGE, pooled and applied to a Hiload Q-Sepharose HP (2.6 × 20 cm) ion exchange column (Pharmacia) equilibrated with 25 mM Tris-HCl, 200 mM NaCl, 1.0 mM EDTA and 0.05% Z 3-14, pH 8.0. A gradient of 0.2-1.0 mM NaCl was used and eluted with rpo-B as a single peak. Protein concentration is determined by measuring absorbance at 280 nm using an HP Model 8453 UV / Vis fast-scanning spectrophotometer equipped with a diode detector (Hewlett-Packard Company, Palo Alto, CA) using a molar extinction coefficient of 41.960, which is calculated based on the aromatic amino acid content of PorB according to the method of Mach et al. [N. Mach, C.R. Middaugh and R.V. Lewis. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosin in native proteins, 1992, Anal. Biochem. 200: 74-80].

Example 2. The formation, purification and oxidation of polysaccharide PRP

Fermentation of H. influenzae type B in a volume of 14 l is carried out using MAE II medium, as follows. The Eagan H. influenzae type B strain is obtained in a 4 ml seed culture vial from storage under liquid nitrogen in an ultra-low temperature refrigerator and thawed at room temperature for thirty minutes. A 250 ml flask on a shaker with 50 ml of MME II medium (10 mg / L hemin) was inoculated with 1 ml of inoculum culture to obtain inoculum culture I (SI). The SI flask was incubated for 10 hours at 37 ° C and 150 rpm on a thermostatically controlled shaker (Innova 4330, New Brunswick Sci). Twelve ml of SI culture was used to inoculate 600 ml of MME II (10 mg / L hemin) in a Fernbach flask to obtain an SII culture. The SII culture flask was incubated for 9 hours at 37 ° C and 150 rpm on a thermostatically controlled rocking chair. Six hundred ml [4% (v / v) inoculum] SII cultures are used to inoculate 13.4 L of MME II (10 g / L of xylose, 10 mg / L of hemin) in a 20 L BIOFLO IV fermenter (New Brunswick Sc.). An example of a fermentation profile is presented in FIG. 2. After 10 hours of fermentation, collection is started by microfiltration using a hollow fiber cartridge with a pore size of 0.2 μm and a surface area of 0.14 m ² made of polysulfone (Milipore). The dissolved substance is sterile filtered into a 20 L braided bottle and the bottle is placed at 2-8 ° C until further processing. The filtrate is then passed through a filter separating a 300,000 molecular weight substance (MWCO) (Millipore) and the dissolved substance (permeate) passed through the filter is retained. This solute is then applied to a filter (MWCO) 100000 (Millipore) and concentrated to a substance content of more than 20 mg / ml. The stored substance is oxidized at 25 ° C for 2 hours using sodium metaperiodate. The oxidized PRP is ultrafiltered through a 30000 MWCO filter (Millipore) and the solute is retained. This solute is then applied with a filter (MWCO) 5000 (Millipore), concentrated to a final concentration of more than 90 mg / ml, dialyzed against DI water and lyophilized.

Example 3. Preparation of PRP-PorB Conjugate

Purified rPorb used for conjugation is shown in FIG. 3. The previously described oxidized PRP polysaccharide is added to the rPorb solution (at a concentration of 10 mg / ml in 0.25 M HEPES, 0.2 M NaCl and 0.05% Zwittergen 3.14 with a pH of 8.5) to obtain a 10 mg polysaccharide solution / ml The solution was stirred for 1 min after addition of sodium cyanoborohydride at a final concentration of 6 mg / ml. Then the solution is placed in a water bath at 28-30 ° C for 16 to 24 hours. The conjugation reaction is stopped by adding a 2 M ethanolamine solution at pH 8.5 and incubated at 28-30 ° C for an additional 16-24 hours. Then the reaction mixture was applied to a preparative Superdex 200 prep Grade column (Pharmacia), pre-equilibrated, and PBS (phosphate buffered saline) containing 0.01% thimerosal was eluted. The fractions eluted with the free volume of this column, monitored by absorbance at UV-280 nm, were collected, pooled and stored at 4 ° C until analysis. Two chemical analyzes are carried out - to assess the PRP content (analysis using orsine / ferric chloride / hydrochloric acid [G. Reuter and R. Schauer. Determination of sialic acids (Determinatoin of sialic acids), pp. 168-199. In the publication WJ Lennarz and GW Hart, Methods in Enzymology, Vol. 230. Techniques in Glycobiology Biology (Academic Press, New York]) and rPorB Content (Protein Analysis Using Coomassie [M. Bradford. Fast and a sensitive method for the quantification of protein present in microgram amounts using p incipit binding protein-dye (A Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding), 1976, Analyt Biochem, 72:.. 248-254]).

Example 4. Evaluation of the conjugate of PRP-rPorB in rats

Female Sprague-Dawley rats (4-6 weeks old) in groups of 10 animals are subcutaneously injected with 10 μg of conjugated PRP in 0.5 ml of PBS containing 0.01% thimerosal, either in an adsorbed form or adsorbed on aluminum hydroxide (Alhydrogel , Superfos, Denmark) (final elemental concentration of aluminum 1 mg / ml) on days 0, 28 and 49. Blood was taken on days 0, 28, 38, 49 and the animals were bled on day 59.

Measurement of serum antibodies using ELISA. Human serum albumin (HSA) conjugates (Sigma, St. Louis, Mo.) used in ELISA are prepared by reductive amination as described above. The oxidized PRP polysaccharide is added to HSA, followed by reduction with NaBH ₃ CN as described in [HJ Jennings, A. Gamian, F. Michon and FA Ashton. Unique intermolecular bactericidal epitope involving the homosialopolysaccharide capsule on the cell surface of group B Neisseria meningitidis and Escherichia coli, 1989 J. Neisseria meningitidis and Escherichia coli. 142: 3585-3591]. The conjugates were isolated by gel filtration and stored in lyophilized form at -70 ° C. Titles of PRP-specific antibodies are determined by enzyme-linked immunosorbent assay (ELISA). Polystyrene 96-well flat bottom microtiter plates (NUNC Polysorb) (Nunc, Naperville, IL) are coated with PRP-HSA conjugates in PBS (0.01 M sodium phosphate, 0.15 M NaCl, ph 7.4) at a concentration of 0 , 25 μg / well (100 μl / well) by incubation for 1 hour at 37 ° C followed by five times washing with PBS-Tween (0.05% [vol./about.] Tween 20 in PBS). All subsequent incubations are carried out at room temperature. PBS-Tween is used in all recommended washes. Coated tablets were blocked with PBS and 0.1% (w / v) Carnation skimmed milk powder for IgM ELISA at a concentration of 0.15 ml / well for 1 hour, followed by washing. Serum is diluted twice in duplicate in tablets of 100 μl / well and incubated for 1 hour, followed by washing. The conjugate of an antibody (peroxidase-labeled goat anti-rabbit antibody) [Kirkegaard & Perry Lab, Gaithersburg, MD] was added at a concentration of 100 μl / well and incubated for 30 minutes, followed by washing. The dye and substrate solution 1: 1 (Kirkegaard & Perry TMB and peroxide) are added in an amount of 0.05 ml / well and incubated for 10 minutes. Then, the peroxidase reaction was stopped with 1 M H ₃ PO ₄ in an amount of 0.05 ml / well and the result was read on a tablet using a Molecular Devices Emax microplate reader (Molecular Devices, Menlo Park, CA) at a wavelength of 450 nm using 650 nm as a reference wavelength. Background absorption is determined in several serum-free control wells and the average of each plate is calculated. From each dilution of the serum, the mean background absorption value is subtracted, and then the serum absorption values in the replicates are averaged. For subsequent data analysis, a modified Scatchard graph is used, in which the dependence of the absorption (Y axis) on the inverse dilution value (X axis) is plotted (18.22). Under conditions ensuring equilibrium and excess antibodies, a straight line is obtained for each serum dilution series, this line is extrapolated to the X axis to determine the antibody titer. A positive serum control with a predefined antibody titer is used on each plate in order to provide a standard by which all serums are standardized, minimizing variations depending on different plates and days. The results of these analyzes, which compare the PorB-PRP conjugate (with or without aluminum hydroxide) with conjugates constructed from tetanus toxoid, CRM, are shown in FIGS. 4 and 5-1 to 5-8.

Example 5. Comparison of Hib-rPorB, Hib-TT and two commercially available vaccines against Hib

The immunostimulating effects of two Hib-rPorB conjugate preparations (Hib-rPorB-1 and Hib-rPorB-2), Hib-TT conjugate (tetanus toxoid) and two commercially available vaccines, HbOC from Lederle Laboratories, Division of American Cyanamide Company, Pearl, are compared. River, NY (CRM carrier) and PRP-T from Connaught Laboratories, Inc., Swiftwater, PA (tetanus toxoid carrier).

Rats (4-6 weeks old) are immunized with conjugated PRP in doses of 10 μg on days 1, 28 and 49. In addition to pre-immunized (pre-immune) samples, serum samples are taken on 28, 38, 49 and 59 days. The results are presented in Fig.6-1 and 6-2. The IgG titer obtained by ELISA refers to antipolysaccharide antibodies.

A graphical representation of the data in FIGS. 6-1 shows that Hib-rPorB conjugates generate a response of at least two orders of magnitude higher than the response obtained with other conjugate vaccines. Figure 6-2 represent the relevant data are summarized in table. “Responding organisms” (responders) are defined as having ELISA IgG titers greater than 4 times or 4 times relative to the preimmune, where all preimmune values are <50 and sum up to 25 for calculations. Similar experiments to compare Hib-TT, Hib-rPorB-1, and Hib-rPorB-2 were performed in mice using doses of conjugates of 5.0 μg and 0.5 μg. The data is shown in Fig.7.

Finally, eight different Hib-rPorB preparations (AH in Fig. 8) are compared with Hib-TT and Hib-CRM conjugates. Hib-rPorB preparations are stably two orders of magnitude more stimulating than the Hib-TT and Hib-CRM conjugates, as shown in rat ELG ELISA studies using anti-polysaccharide antibodies. These results are shown in Fig. 8.

Now, after a full discussion of this invention, it will be clear to those skilled in the art that the invention can be practiced in a wide and equivalent range of conditions, preparations and other parameters without affecting the scope of the invention or any variant of its implementation. All patents, patent applications and publications cited in this context are fully incorporated by reference in their entirety.

Claims (13)

1. An immunogenic conjugate of the Haemophilus influenzae type b polysaccharide (Hib) with a practically purified, rearranged matured outer membrane protein of class 2 or 3 Neisseria meningitidis or its fusion form containing amino acids 1-20 or 1-22 of the capsid protein φ10 of the T7 gene (rPoroB). 2. The conjugate according to claim 1, characterized in that the polysaccharide has a molecular weight in the range of 5000-50000. 3. The conjugate according to claim 1 or 2, characterized in that the specified rPoR is rPoB class 3. 4. A method of producing a Hib-rPorB polysaccharide conjugate, comprising producing a Hib polysaccharide, producing rРо-В and subsequent conjugation, characterized in that said polysaccharide is subjected to oxidation or selective hydrolysis to obtain aldehyde groups, and the polysaccharide containing aldehyde groups is conjugated, amination. 5. The method according to claim 4, characterized in that said Hib polysaccharide is oxidized, wherein the oxidized polysaccharide has a molecular weight in the range of 5000-50000. 6. The method according to claim 4 or 5, characterized in that the specified rpo-B is rpo-class 3. 7. Immunogenic conjugate of the polysaccharide N. influenzae type b (Hib) with a practically purified rearranged matured class 3 outer membrane protein N. meningitidis or its fusion form containing amino acids 1-20 or 1-22 of the T7 gene φ10 capsid protein obtained according to the method according to item 6. 8. Immunogenic conjugate of Haemophilus influenzae type b polysaccharide (Hib) with a practically purified lane matured outer membrane protein of class 2 or 3 Neisseria meningitidis or its fusion form containing amino acids 1-20 or 1-22 of the capsid protein φ10 of the T7 gene (rРорВ) obtained by reductive amination of the Hib polysaccharide and rPoB, wherein the Hib polysaccharide is oxidized or selectively hydrolyzed to introduce aldehyde groups. 9. A pharmaceutical composition for inducing an immune response in an animal, comprising the conjugate according to one of claims 1, 7 or 8 and a pharmaceutically acceptable carrier. 10. A method of inducing an immune response in an animal with respect to H. influenzae, which method comprises administering to the animal a conjugate according to one of claims 1, 7 or 8 in an amount effective to induce said immune response. 11. The method according to claim 10, characterized in that said conjugate is obtained by reductive amination of the Hib polysaccharide and rРоrВ, wherein the Hib polysaccharide is pre-oxidized to obtain aldehyde groups. 12. The method according to claim 10, characterized in that the polysaccharide has a molecular weight in the range of 5000-50000. 13. The method of claim 10, wherein said rPorB is a class 3 rPorB.

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