WO2007081583A2 - Anti-pneumococcal preparations and methods of use - Google Patents

Abstract

Disclosed are methods for treating, reducing or preventing diseases and infections caused by pneumococci. The methods rely on the use of a cell wall polysaccharide- (CWPS-) containing composition (e.g., a CWPS teichoic acid polymer preparation) of pneumococcus and an adjuvant, which may be chemically conjugated or simply admixed. The methods are particularly useful against nasopharyngeal colonization and invasive disease due to encapsulated pneumococci.

Description

ANTI-PNEUMOCOCCAL PREPARATIONS AND METHODS OF USE

FIELD OF THE INVENTION

[001 ] The present invention relates to compositions and methods for preventing illnesses associated with pneumococcal infection. The compositions contain an antigen such as teichoic acid polymer of the pneumococcus (or a variant or derivative thereof, as described below), and they can be formulated for intramuscular administration.

BACKGROUND OF THE INVENTION

[002] Pneumococcus is a pathogenic, Gram-positive bacterium that can be classified as having one of about 90 antigenic serotypes, depending on the capsular polysaccharide expressed external to the bacterial cell wall. Pneumococcus causes a variety of undesirable conditions, including pneumonia, middle ear infections (otitis media), bacteremia, and bacterial meningitis, and it can exacerbate other conditions, such as chronic bronchitis, sinusitis, arthritis, and conjunctivitis.

[003] Currently, few pneumococcal vaccines based upon the capsular polysaccharides are available for use in humans. The vaccines intended for immunologically mature humans include a combination of unconjugated pneumococcal polysaccharides. These are PNEUMO V AX® 23 (Merck Sharp & Dohme, West Point, PA), which includes 23 different purified pneumococcal polysaccharides, and PNU-IMMUNE® 23, which is a similar vaccine produced by Wyeth- Lederle Vaccines (Pearl River, NY). These vaccines represent up to 90% of the serotypes that cause invasive pneumococcal infections in the United States, including the six serotypes that most frequently cause invasive drug-resistant pneumococcal infection. A vaccine specifically designed for infants, PREVN AR®, consists of seven serotypes of pneumococcal polysaccharides (4, 6B, 9V, 14, 18C, 19F, and 23F according to the Danish nomenclature) that have been conjugated to the protein CRM-197 (also produced by Wyeth-Lederle Vaccines).

[004] Unfortunately, vaccines based upon capsular polysaccharides are not ideal. For example, unconjugated pneumococcal polysaccharide vaccines are not very effective in young children. Additionally, although the conjugated vaccine is effective in infancy, the number of serotypes that can be included in those vaccines is limited; serotype replacement may occur (serotype replacement has been reported in clinical trials of PREVNAR® for otitis media); and production is costly. Thus, there is a need for simpler, more economical compositions (and methods of administering those compositions) that confer better protection on subjects of all ages against more serotypes, or perhaps all serotypes, of pneumococcus.

SUMMARY OF THE INVENTION

[005] The present invention provides methods for inhibiting pneumococcal bacterial colonization, reducing the ability of the pneumococcus bacteria to cause disease in a subject and methods for inducing an immune response against pneumococci in a subject. In one embodiment, the method comprises parenteral administration to the subject a composition comprising a teichoic acid-containing cell wall polysaccharide (CWPS) preparation of pneumococcus in a pharmaceutically acceptable carrier.

[006] In one embodiment, the disease is pneumonia. In one embodiment, the disease is otitis media. In one embodiment, wherein the disease is meningitis. In one embodiment, wherein the disease is bacteremia. In one embodiment, the subject is administered a dose of CWPS equal to or greater than 50 micrograms. In another embodiment, the subject is administered two parenteral doses of CWPS equal to or greater than 50 micrograms per dose.

[007] The immune response elicited by the techoic acid-containing CWPS composition may be CD4⁺ T cell mediated. [008] In one embodiment, the teichoic acid-containing CWPS preparation administered to a subject further comprises an adjuvant.

[009] In one embodiment, the CWPS preparation administered to a subject includes fragments of the pneumococcal cell wall peptidoglycan.

[0010] In one embodiment, the CWPS preparation administered to a subject includes a glycolipid endgroup such as described for lipoteichoic acid, Forssman or F antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a published representation of the teichoic acid polymeric structure of pneumococcal CWPS (reproduced from Szu et al., Infection and Immunity 54:448-455, 1986). Variants based upon the number of phosphorylcholine groups have recently been reported (Karlsson et al, Eur. J. Biochem. 275:1091) and are within the scope of the invention.

[0012] Figure 2 shows reduced nasopharyngeal colonization in mice immunized with CWPS and CT compared to mice immunized with CT alone.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention is based, in part, on the surprising discovery that parenteral administration of CWPS compositions provide protection from pneumococcal colonization. Previous efforts documented in the prior art were unsuccessful in inducing protection from pneumococcal disease and did not study pneumococcal colonization.

[0014] As noted above, pneumococcus is a pathogenic, Gram-positive bacterium in which serotype is based on the capsular polysaccharide expressed external to the bacterial cell wall. Compositions that contain a teichoic acid polymer present in pneumococci of all capsular serotypes has been evaluated as a protective antigen, and compositions including preparations of the polymer or its components have been tested as a possible vaccine in animal models (Briles et al, J. Exp. Med. 153:694-705, 1981; Wallick et al., J. Immunol. 130:2871-2875, 1983; Szu et al, Infection and Immunity 54:448-455, 1986; Sorenson etal., Infection and Immunity 56: 1890- 1896, 1988; Musher et at., J. Infect. Dis. 161:736-740, 1990; Nielsen et al, Microb. Pathogen. 14:299-305, 1993; Briles et ai, Clinical Microbiology Reviews 11:645-657, 1998). U.S. Pat No. 20030157133 issued to Drabick discloses lipotechoic acid compositions from Gram-positive bacteria for use as immunizing antigens. Yet, the lipotechoic acid structures from the different bacteria are widely different structures and pneumococcus-derived lipotechoic acid is not specifically disclosed. Furthermore, Drabick fails to disclose successful protection of mice from bacterial colonization.

[0015] "Teichoic acid" is a general term for a class of polymer in bacteria in which the common structural element is the presence of glycerol or ribitol residues within the polymer backbone. The polymers otherwise differ structurally and immunologically among species. Gram-positive species in general contain a form called lipoteichoic acid that contains a glycolipid end-group whereby the polymer is bound to the cell membrane; some species contain also a teichoic acid bound to the cell wall (in Microbiology, B.D. Davis et al., 4^th edition 1990, J.B. Lippencort Co., Philadelphia).

[0016] Pneumococci can contain several morphologic forms of the polymer. One is associated with the cell wall and has been called cell wall polysaccharide or CWPS. Preparations of CWPS may thus contain residual fragments of the cell wall peptidoglycan. However, certain strains of pneumococci were experimentally generated in which the original serotype capsular polysaccharide was deleted and replaced with a capsule-like external layer consisting of the teichoic acid polymer, hyperproduced. Commercially available "CWPS" preparations are typically made from such strains, although the morphologic origin is the capsule and not the cell wall. In yet another form, originally called Forssman or F antigen, the teichoic acid has a glycolipid end group that causes the polymer to associate with the (phospholipid) cell membrane of the pneumococcus; this form has more recently been called the pneumococcal lipoteichoic acid or LTA. The teichoic acid polymer (Fig. 1) of these several forms is identical (Fisher, "Pneumococcal Lipoteichoic Acid and Teichoic Acid" in S. pneumoniae - Molecular Biology and Mechanisms of Disease, pp. 155-177, A. Tomasz, Ed., Mary Ann Liebert, Inc., Larchmont NY, 2000). Typical preparations can contain polymer without and with the peptidoglycan fragments or the glycolipid group. For brevity, any such preparations are referred to herein as CWPS.

[0017] Serum antibodies to phosphorylcholine, a component of CWPS, may protect mice against parenteral challenge with capsulated serotype 3 pneumococci (Briles et al., supra). In addition, compositions including phosphorylcholine conjugated to a carrier protein and mixed with Freund's adjuvant may protect mice against an intravenous challenge with serotype 1 or serotype 3 pneumococci (Wallick et al., supra). Moreover, serum transferred from treated mice passively protected untreated mice (Wallick et al., supra). In a similar study, parenterally injected CWPS that was coupled to a protein carrier failed to protect mice against challenges with type 3 or 6 A pneumococci, however, and antibodies that were raised in rabbits with that composition (i.e., CWPS coupled to a protein carrier and mixed with Freund's adjuvant) failed to passively protect mice against similar challenges (Szu et al., supra). Other investigators also concluded that serum antibodies to CWPS, whether directed against phosphorylcholine or another component of CWPS, fail to provide protection against capsulated pneumococci (Sorensen et al., Musher et al., and Nielsen et al., supra). Given these findings, interest in CWPS as an immunizing agent for pneumococcal infection has all but disappeared.

[0018] While the compositions described above have been tested in animals, a few pneumococcal vaccines based upon the capsular polysaccharides are available for use in humans. The vaccines intended for immunologically mature humans include a combination of unconjugated pneumococcal polysaccharides. These are PNEUMOV AX® 23 (Merck Sharp & Dohme, West Point, PA), which includes 23 different purified pneumococcal polysaccharides, and PNU-IMMUNE® 23, which is a similar vaccine produced by Wyeth-Lederle Vaccines (Pearl River, NY). These vaccines represent up to 90% of the serotypes that cause invasive pneumococcal infections in the United States, including the serotypes that most frequently cause invasive drug-resistant pneumococcal infection. A vaccine specifically designed for infants is PREVN AR®, which consists of seven serotypes of pneumococcal polysaccharides (4, 6B, 9 V, 14, 18C, 19F, and 23F according to the Danish nomenclature) that have been conjugated to the protein CRM- 197 (also produced by Wyeth-Lederle Vaccines).

[0019] Unfortunately, vaccines based upon capsular polysaccharides are not ideal. For example, unconjugated pneumococcal polysaccharide vaccines are not effective nor are they indicated for use in children under the age of two, those at highest risk for infection (Douglas et al., J. Infect. Dis. 148: 131-137; Ahonkai et al., New Engl. J. Med. 301:26-27, 1979; and Sell et al., Rev. Infect. Dis. 3_:S97-S107, 1981). Furthermore, while the conjugated vaccine is effective in infancy, this vaccine also has several limitations. First, the current conjugate vaccine comprises only 7 of the 100 capsular types; while studies are being performed with 10- or 13- valent vaccines, it is clear that the number of serotypes that can be included in these complex vaccines is limited. This represents a potential limitation since serotype replacement, whereby pneumococcal strains with serotypes not included in the vaccine increase as a consequence of immunization of a population, has already been reported in clinical trials and epidemiologic studies. In one study performed after the introduction of PREVNAR (Malley et al., data presented at the Society for Pediatric Research, San Francisco, 2005), up to 50% of pneumococci isolated from cerebrospinal fluid of US children with meningitis were of serotypes not included in the vaccine., Finally, production of these conjugated vaccines is costly and complex, resulting in frequent shortages. Thus, there is a need for simpler, more economical compositions (and methods of administering those compositions) that confer better protection on subjects of all ages against more serotypes, and preferably all serotypes, of pneumococcus. . [0020] The compositions and methods described herein are based, in part, on a CWPS- containing preparation of pneumococcus formulated for parenteral, e.g., intramuscular or subcutaneous, administration that when administered to a subject by that route, can reduce the risk of disease associated with pneumococcus. As discussed further below, administration can begin before a subject has a pneumococcal disease, i.e., the preparation can be administered prophylactically, after suspected exposure to a pneumococcal disease, but while the patient is still apparently healthy, or after a subject has apparently recovered from a pneumococcal disease, i.e., the preparation can be administered to reduce the likelihood of recurrent infection or disease. In alternative embodiments, the CWPS-containing compositions can include, or can be administered with, an adjuvant, e.g., an adjuvant suitable for the type of patient being treated, e.g., human patients, and known to increase immunogenicity by parenteral routes, e.g., intramuscular, subcutaneous or intradermal routes. In other embodiments, the antigens can be conjugated or conjugated to a carrier and, optionally, administered with an adjuvant.

[0021] Antigens: Pneumococci can contain several morphologic forms of the polymer teichoic acid, a published structure of which is shown in Figure 1. One form of teichoic acid is associated with the cell wall, and that form has been called cell wall polysaccharide (or CWPS). Preparations of CWPS, including those useful in the physiologically acceptable compositions of the present invention, may thus contain residual fragments of the cell wall peptidoglycan. In certain strains of experimentally generated pneumococci, the original serotype capsular polysaccharide was deleted and replaced with a capsule-like external layer including the teichoic acid polymer; some commercial sources of "cell wall polysaccharide" are in fact isolated from such capsular layers. In yet another morphological form, originally called Forssman or F antigen, the teichoic acid moiety includes a glycolipid end group that facilitates interaction between the polymer and the phospholipid cell membrane of pneumococcus. This form of CWPS has more recently been called lipoteichoic acid or LTA. The teichoic acid polymer of these several forms is essentially identical. Further, teichoic acid that is substantially identical to any of the forms described here can be a part of the CWPS -containing compositions of the present invention. For example, typical preparations can contain teichoic acid polymers with or without the peptidoglycan fragments or the glycolipid groups.

[0022] The compositions of the invention include CWPS or a portion, fragment, or derivative thereof (such as described above) that is immunologically active. CWPS (or a portion, fragment, or derivative thereof) is "immunologically active" when, upon administration, e.g., parenteral administration, e.g., intramuscular administration, to a mammal, it evokes an immune response (either humoral or cellular) in the mammal (all that is required is an immune response sufficient to benefit the patient). The portion or fragment of CWPS may be all or a portion of the CWPS backbone (see, e.g., Figure 1) or all or a portion of the phosphoryl choline moiety that extends from the backbone (see, e.g., Figure 1).

[0023] In a particular embodiment, the teichoic acid-containing CWPS preparation comprises a repeating unit of

wherein R=H or COCH₃.

[0024] CWPS can be obtained from a commercial source (e.g., Statens Seruminstitut, Copenhagen, Denmark) (some adjuvants, such as toxin-derived adjuvants are also commercially available)), or it may be extracted or purified from, for example, pneumococcal cells. Strains of bacteria (e.g., pneumococci and those that produce native toxins as well as detoxified derivatives) can be obtained from the American Type Culture collection (ATCC; Manassas, VA). Strains of pneumococcus that hyperproduce CWPS as a capsule-like structure can also be obtained from the ATCC, and CWPS can be purified by published techniques. For example, CWPS can be isolated from culture supernatants as well as from bacterial cells from the base extraction technique described in U.S. Patent No. 6,248,570 (the contents of which is incorporated herein by reference). The CWPS produced by that method may lack covalent attachment to extraneous peptidoglycan. CWPS-containing compositions that include LTA can be extracted from pneumococci by a chloroform-methanol procedure and purified by hydrophobic affinity chromatography (Fischer, Pneumococcal Lipoteichoic Acid and Teichoic Acid In: S. pneumoniae — Molecular Biology and Mechanisms of Disease, pp. 155-177, A. Tomasz, Ed., Mary Ann Liebert, Inc., Larchmont, NY, 2000, which is hereby incorporated by reference in its entirety).

[0025] Other purification methods rely on treating the source cells with mutanolysin, which cleaves the bacterial wall and frees cellular components. The cell lysates can be treated with additional enzymes to remove proteins and nucleic acids, and purification can be carried out by differential precipitation and chromatography (Wessels et al, Infect. Immunol. 57:1089-1094. 1989; Wessels et al., J. Clin Invest. 86:428-433, 1990).

[0026] Conjugation: As noted above, antigenic components can be conjugated (i.e., linked by covalent bonds) to other molecules to increase immunogenicity. The antigenic components can also be conjugated to lipid or glycolipid molecules or to protein adjuvants or carrier proteins by methods known in the art ("proteins" and "peptides" are both polymers of amino acid residues and either may be used in the present invention; the term protein is used here only because it is more commonly applied to the higher molecular weight polymers used as adjuvants and carriers). CWPS or a portion, fragment, or derivative thereof that is immunologically active can be thus conjugated by methods that take account of the particular structure of CWPS which, like most proteins, contains free amino groups and which contains an easily hydrolyzed phosphodiester linkage (Fischer et al., Eur. J. Biochem. 215:851-857, 1993). Amino acids amenable to conjugation may also extend from a teichoic acid backbone.

[0027] One suitable conjugation procedure is described in Szu et al. (supra.), in which the reagent N-succinimidyl-3-(2-pyridyldithio)-proprinate (SPDP) is first coupled separately to the free amino groups of the CWPS and the protein, and the two adducts are coupled by disulfide bond exchange. In another suitable method, CWPS is selectively cleaved by periodate oxidation giving immunogenic fragments with aldehyde termini, the free amino groups of the fragments are reversibly blocked with the reagent "t-BOC", the aldehyde groups are coupled to the free amino acid groups of the protein by reductive amination (Anderson et al., J. Immunol. 137:1181- 1186, 1987), and the blocking groups are then removed. While specific conjugation methods are described herein, the invention is not so limited. Any suitable mode of conjugation may be employed to conjugate the CWPS component with an adjuvant or carrier.

[0028] CWPS -containing conjugates include conjugates in which a protein or peptide is linked to the CWPS through one or more sites on the CWPS. Accordingly, the CWPS- containing compositions can include conjugate molecules that are monomers, dimers, trimers and/or more highly cross-linked molecules, the CWPS cross-linking multiple proteins.

[0029] Adjuvants: Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvant may be administered prior to or subsequent to antigen administration, but generally, activation of antigen presenting cells by adjuvant occurs prior to presentation of antigen. Alternatively, adjuvant and antigen may be separately presented within a short interval of time but targeting the same anatomical region, e.g., the same draining lymph node field. Ideally, the adjuvant selected will: (1) lack toxicity; (2) stimulate a long- lasting immune response; (3) remain stable despite long-term storage; (4) elicit humoral and perhaps cellular responses to CWPS; (5) act synergistically with other adjuvants; (6) selectively interact with populations of antigen presenting cells (APC); specifically elicit appropriate THl or TH2 cell-specific immune responses; and (8) electively increase appropriate antibody isotype levels (for example IgA) against antigens. Of course, adjuvants having fewer than all of these characteristics can still be used.

[0030] Adjuvants include, for example, chemokines (e.g., defensins, HCC-I , HCC4, MCP-I, MCP-3, MCP4, MIP-Ia, MIP-I β, MIP-I δ, MIP-3α, MIP-2, RANTES); other ligands of chemokine receptors (e.g., CCRl, CCR-2, CCR-5, CCR6, CXCR-I); cytokines (e.g., IL- l β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17 (A-F), IL-18; IFNα, IFN-γ; TNF-α; GM-CSF); TGF)-β; FLT-3 ligand; CD40 ligand; other ligands of receptors for those cytokines; ThI cytokines including, without limitation, IFN- γ, IL-2, IL-12, IL-18, and TNF; Th2 cytokines including, without limitation, IL-4, IL-5, IL-10, and IL-13; and Th 17 cytokines including, without limitation, IL-17 (A through F), IL-23, TGF-β and IL-6; immunostimulatory CpG motifs in bacterial DNA or oligonucleotides; derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL); muramyl dipeptide (MDP) and derivatives thereof (e.g., murabutide, threonyl-MDP, muramyl tripeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alani- ne-2-( 1 '-2'-dipalmitoyl-sn-glycero- 3hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE)); MF59 (see Int'l Publication No. WO 90/14837); poly[di(carboxylatophenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA); RIBI (GSK), which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion; OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); heat shock proteins and derivatives thereof; Leishmania homologs of elF4a and derivatives thereof; bacterial ADP- ribosylating exotoxins and derivatives thereof (e.g., genetic mutants, A and/or B subunit- containing fragments, chemically toxoided versions); chemical conjugates or genetic recombinants containing bacterial ADP-ribosylating exotoxins or derivatives thereof; C3d tandem array; lipid A and derivatives thereof (e.g., monophosphoryl or diphosphoryl lipid A, lipid A analogs, AGP, AS02, AS04, DC-Choi, Detox, OM- 174); ISCOMS and saponins (e.g., Quil A, QS-21, Stimulon® (Cambridge Bioscience, Worcester, MA)); squalen©; superantigens; or salts (e.g., aluminum hydroxide or phosphate, calcium phosphate). See also Nohria et al. Biotherapy, 7:261-269, 1994; Richards et al, in Vaccine Design, Eds. Powell et al., Plenum Press, 1995; and Pashine et al, Nature Medicine, 11 :S63-S68, 4/2005) for other useful adjuvants.

[0031 ] Adjuvant may be chosen to preferentially induce antibody or cellular effectors, specific antibody isotypes (e.g., IgM, IgD, IgAl, IgA2, secretory IgA, IgE, IgGl, IgG2, IgG3, and/or IgG4), or specific T-cell subsets (e.g., CTL, ThI, Th2, Th 17 and/or T_DTH). For example, antigen presenting cells may present Class Il-restricted antigen to precursor CD4+ T cells, and the ThI , Th2, or ThI 7 pathway may be entered. T helper cells actively secreting cytokine are primary effector cells; they are memory cells if they are resting. Reactivation of memory cells produces memory effector cells. ThI characteristically secrete IFN-γ (TNF-β and IL-2 may also be secreted) and are associated with "help" for cellular immunity, while Th2 characteristically secrete IL4 (IL-5 and IL- 13 may also be secreted) and are associated with "help" for humoral immunity. Th 17 cells, more recently described, characteristically secrete IL- 17 (A through F), and may enhance innate immune responses to a pathogen, by recruitment and activation of professional phagocytes. Depending on disease pathology, adjuvants may be chosen to prefer a ThI response (e.g., antigen-specific cytolytic cells) versus a Th2 response (e.g., antigen-specific antibodies) or a ThI 7 response.

[0032] Unmethylated CpG dinucleotides or-similar-motifs are known to activate B lymphocytes and macrophages (see, U.S. Pat. No. 6,218,371). Other forms of bacterial DNA can be used as adjuvants. Bacterial DNA is among a class of structures which have patterns allowing the immune system to recognize their pathogenic origins to stimulate the innate immune response leading to adaptive immune responses. These structures are called pathogen-associated molecular patterns (PAMP) and include lipopolysaccharides, teichoic acids, unmethylated CpG motifs, double-stranded RNA, and mannins.

[0033] Most ADP-ribosylating exotoxins are organized as A:B heterodimers with a B subunit containing the receptor binding activity and an A subunit containing the ADP- ribosyltransferase activity. Exemplary exotoxins include cholera toxin (CT), E. coli heat-labile enterotoxin (LT), diphtheria toxin, Pseudomonas exotoxin A (ETA), pertussis toxin (PT), C. botulinum toxin C2, C. botulinum toxin C3, C. limosum exoenzyme, B. cereus exoenzyme, Pseudomonas exotoxin S, S. aureus EDIN, and B. sphaericus toxin. Alternatively, mutant bARE, for example containing mutations of the trypsin cleavage site (e.g., Dickenson et al., Infect Immun, 63:1617-1623, 1995) or mutations affecting ADP-ribosylation (e.g., Douce et al., Infect Immun, 65:28221-282218, 1997) may be used. Exotoxin subunit-containing fragments may be used, e.g., CTA, CTB, LTA or LTB. In general, toxins can be chemically inactivated to form toxoids which are less toxic but remain immunogenic.

[0034] An adjuvant may be chemically conjugated to other antigens including, for example, carbohydrates, polypeptides, glycolipids, and glycoprotein antigens. Chemical conjugation with toxins, their subunits, or toxoids with these antigens would be expected to enhance the immune response to these antigens. To overcome the problem of the toxicity of the toxins (e.g., diphtheria toxin is known to be so toxic that one molecule can kill a cell) and to overcome the problems of working with such potent toxins as tetanus, several workers have taken a recombinant approach to producing genetically-produced toxoids. This is based on inactivating the catalytic activity of the ADP-ribosyl transferase by genetic deletion. These toxins retain the binding capabilities, but lack the toxicity, of the natural toxins. Additionally, several techniques exist to chemically modify toxins and can address the same problem. These techniques could be important for certain applications, especially pediatric applications, in which ingested toxins might possibly elicit adverse reactions.

[0035] Undesirable properties or harmful side effects (e.g., allergic or hypersensitive reaction; atopy, contact dermatitis, or eczema; systemic toxicity) may be reduced by modification without destroying its effectiveness in immunization. Modification may involve, for example, removal of a reversible chemical modification (e.g., proteolysis) or encapsulation in a coating which reversibly isolates one or more components of the formulation from the immune system. For example, one or more components of the formulation may be encapsulated in a particle for delivery (e.g., microspheres, nanoparticles). Phagocytosis of a particle may, by itself, enhance activation of an antigen presenting cell by upregulating expression of MHC Class I and/or Class II molecules and/or costimulatory molecules (e.g., CD40, B7 family members like is CD80 and CD86). Alternative methods of upregulating such molecules by activating an antigen presenting cell are also known to the skilled artisan.

[0036] An adjuvant may be used that creates causes an antigen or allergen to be slowly released in the body thus prolonging the exposure of immune cells to the antigen or allergen, i.e., one that creates a depot effect. This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in-oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLϊquide, Paris, France); MF-59; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle- forming agent; IDEC Pharmaceuticals Corporation, San Diego, Calif.).

[0037] An adjuvant that both creates a depot effect and stimulates the immune system may be used. This class of adjuvants includes but is not limited to ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsion containing MPL and QS21; SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

[0038] In some embodiments certain bacterial protein toxins and their derivatives or subcomponents are utilized as adjuvants. For example, one can use cholera toxin (CT), Escherichia coli heat-labile toxin (LT), pertussis toxin, shiga toxin, anthrax toxin, pseudomonas exotoxin A, or nontoxic derivatives of such toxins. See Mrsny et al., Drug Discovery Today 7:247-258, 2002. Pneumolysin, a toxin from the pneumococcus itself, may likewise act as an adjuvant. Derivatives can be rendered nontoxic by, for example, mutating the nucleic acid sequence that encodes them; some amino acid substitutions are known to reduce toxicity while retaining adjuvant action {e.g., LT(Rl 02G) or LT(S63K)). Alternatively, one can use only the inherently nontoxic subunits of the toxins responsible for cell binding (e.g., "B subunits" such as CT-B and LT-B). See Holmgren et al., Vaccine JJ.: 1179-1184, 1993). Such adjuvants can simply be combined or admixed with the antigens to be parenterally, e.g., intramuscularly, administered. Alternatively, protein adjuvants can be conjugated to (a broad term that encompasses any coupling or association) the antigen (i.e., to CWPS or a portion, fragment, or derivative thereof). For example, CWPS and a protein adjuvant can be chemically coupled as described by Szu et al. (supra). In addition to the proteinaceous adjuvants listed above, one can use a non-proteinaceious adjuvant (e.g., the adjuvant can be a liposome, a lipid-based composition such as RHINOV AX® (an adjuvant formulation based on caprylic-capric glycerides dissolved in polysorbate 20 and water, Lyfjathroun, Reykjavik, Iceland), or a glycolipid such as derivitives of the lipid A component of Gram-negative bacterial endotoxin). [0039] In addition to antigen and adjuvant, the formulation may comprise a vehicle. For example, the formulation may comprise an Aquaphor, Freund, Ribi, or Syntex emulsion; water- in-oil emulsions (e.g., aqueous creams, ISA-720), oil-in-water emulsions (e.g., oily creams, ISA-51, MF59), microemulsions, anhydrous lipids and oil-in-water emulsions, other types of emulsions; gels, fats, waxes, oil, silicones, and humectants (e.g., glycerol).

[0040] Carrier Proteins: In addition to (or as an alternative to) adjuvants, the CWPS- containing compositions of the invention can include heterologous carrier proteins that are chemically coupled to the antigen. These include proteins that may or may not lack adjuvant activity when admixed but that, when coupled, enhance the presentation of the CWPS-containing compositions to antigen-processing cells, particularly useful in immunologically immature subjects. For example, CWPS (or a portion, fragment, or derivative thereof) can be conjugated to the outer membrane protein complex of a Gram-negative bacterium such as Neisseria meningitidis (or proteins within those complexes), to various bacterial toxins and toxoids {e.g., diphtheria or tetanus toxins or their respective conventional toxoids or genetically detoxified toxins referred to as cross-reacting material, e.g., CRM 197), or haemocyanins (some of these materials are mentioned elsewhere herein).

[0041] The effect of the linkage to carriers may be additive to that of the above-described adjuvants. For example, a carrier protein can be conjugated to CWPS and administered with (e.g., admixed with) one of the adjuvants described herein.

[0042] Formulations, routes and methods of administration: A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, intravenous, intraarterial, intraperioneal, intradermal, transdermal, or subcutaneous administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0043] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [0044] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0045] The dose of CWPS (or any portion, fragment, or derivative thereof) will be a dose that is safe and effective {i.e., able to generate a protective immune response in a patient upon the completion of the treatment protocol) and appropriate for a particular type of formulation. Suitable dosages of antigens are determined by those of ordinary skill in the art by routine methods, and the dosage can range from micrograms to milligrams. For example, an average adult human can be given 1 μg - 10 mg of CWPS (or a portion, fragment, or derivative thereof) {e.g., 1 μg - 1 mg; 1 μg - 200 μg). The recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0046] These doses can be administered on one or several {e.g., two, three, or four successive occasions) and can be varied appropriated based on the patient's age, body weight and other known criteria used to determine dosages of various drugs and antigens. The antibody response in an individual can be monitored by assaying for antibody titer or bactericidal activity and boosted if necessary to enhance the response.

[0047] Compositions containing anti-CWPS antibodies can be similarly formulated and administered to confer passive immunity, as known in the art.

[0048] Patients and Conditions Amenable to Treatment: The patient can be any animal susceptible to pneumococcal infection. For example, the patient can be a mammal, such as a human, a domesticated animal (e.g., a dog or cat; or a farm animal such as a horse, pig, or cow), a rodent, or a nonhuman primate. Unless specifically noted, the "patient" may also be referred to herein as a "host" or "subject."

[0049] The compositions and methods of the invention can be used alone or in combination with existing or experimental conjugated vaccines.

[0050] The compositions of the invention can be administered to patients of any age, including patients whose immune systems are not fully mature (e.g., children under the age of two). In that event, conjugated antigens may be administered at, for example, about two months of age, and conjugated or unconjugated antigens can be administered subsequently (at, for example, about four, six, 12, and/or 18 months of age. CWPS-based compositions can also be administered to elderly patients (e.g., patients over the age of 65). While otherwise healthy patients may also be treated, the ability to mount an immune response against the immunogen need not be perfect for the CWPS-based compositions described here to confer some degree of protection against pneumococcus. Physicians, veterinarians, and others who routinely care for patients will be able to determine whether a patient is apparently in good enough health to consider administering a CWPS-based composition.

[0051] The CWPS-based compositions and methods of the present invention are also useful as components of multivalent vaccines, which are capable of eliciting an immune response against a plurality of infectious agents.

[0052] The invention is illustrated further by the following non-limiting examples, which demonstrate that intraparenteral, and in particular intramuscular, administration of CWPS, with cholera toxin (CT) as an adjuvant, exhibits a significant and impressive protection against pneumococcal infection.

[0053] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein,

EXAMPLES

[0054] In the past, parenteral injection of CWPS (two doses of 2 μg CWPS intraperitoneally, without adjuvant), gave no protection compared with saline. The question was whether larger doses, given with adjuvant, could be protective. Mice were thus immunized with CWPS (50 μg) + CT (1 μg) or CT alone, intramuscularly (in the calf muscle), twice at a one week interval. Four weeks after the second immunization, mice were challenged intranasally with a strain of serotype 6B. Density of nasopharyngeal colonization was determined one week later by serial dilutions of tracheal washes on BAP+Gent. The density of colonization in mice that received CWPS+CT intramuscularly was significantly lower than control mice (geometric mean density of colonization 1365 cfu/ml of tracheal wash vs. 83939 cfu/ml, respectively, P=O-OOl 5), as shown in Figure 2.

Claims

[0055] What is claimed is:

1. A method for inhibiting pneumococcus bacterial colonization and reducing the ability of the pneumococcus bacteria to cause disease in a subject, the method comprising parenteral administration to the subject a composition comprising a therapeutically effective amount of a teichoic acid-containing cell wall polysaccharide (CWPS) preparation of pneumococcus in a pharmaceutically acceptable carrier.

2. The method of claim 1 , wherein the disease is pneumonia.

3. The method of claim 1, wherein the disease is otitis media.

4. The method of claim 1 , wherein the disease is meningitis.

5. The method of claim 1 , wherein the disease is bacteremia.

6. A method for inducing an immune response against pneumococcus in a subject, the method comprising parenteral administration to the subject of a composition comprising a teichoic acid-containing cell wall polysaccharide (CWPS) preparation of pneumococcus in a pharmaceutically acceptable carrier, wherein the CWPS elicits an immune response in the subject.

7. The method of claim 6, wherein the immune response is CD4⁺ T cell mediated.

8. The method of claim 1 or 6, wherein the teichoic acid-containing CWPS preparation further comprises an adjuvant.

9. The method of claim 1 or 6, wherein the CWPS preparation includes fragments of the pneumococcal cell wall peptidoglycan.

10. The method of claim 1 or 6, wherein the CWPS preparation includes a glycolipid endgroup such as described for lipoteichoic acid, Forssman or F antigen.

1. The method of claim 1 or 6 wherein the teichoic acid-containing CWPS preparation comprises a repeating unit of:

, wherein R=H or COCH₃.