FIELD OF INVENTION
The present invention relates to novel improved multivalent pneumococcal vaccines and their methods of preparation. The novel vaccines of the invention may be further used for preventing and treating infections caused by Streptococcus pneumoniae.
BACKGROUND OF THE INVENTION
Streptococcus pneumoniae, or pneumococcus, is gram-positive, lancet-shaped cocci, alpha-hemolytic, bile soluble aerotolerant anaerobe and a member of the genus Streptococcus. Streptococcus pneumoniae is a normal inhabitant of the human upper respiratory tract. It can cause pneumonia, usually of the lobar type, paranasal sinusitis and otitis media, or meningitis, which is usually secondary to one of the former infections. It also causes osteomyelitis, septic arthritis, endocarditis, peritonitis, cellulitis and brain abscesses. Streptococcus pneumoniae is currently the leading cause of invasive bacterial disease in children and the elderly. Streptococcus pneumoniae is known in medical microbiology as pneumococcus, referring to its morphology and its consistent involvement in pneumococcal pneumonia.
The ability of the pneumococcus to resist the major mechanism of clearance of the organism from the bloodstream (i.e. opsonophagocytosis) requires expression of the major virulence factor of the organism, which is a polysaccharide capsule. Pneumococcal capsular polysaccharides (PS) are responsible for its anti-phagocytic properties and inhibition of adherence to host cells, which is a critical step in carriage and possibly later aspects in the pathogenesis of disease. Hence, the capsule of S. pneumoniae has long been recognized as the major virulence factor. Ninety different pneumococci serotypes have been identified and each serotype corresponds to a different chemical composition of the capsule. The capsule is currently used as an antigen in pneumococcal vaccines. Pneumococcal capsular polysaccharide vaccines have been licensed since 1977. The 23-valent unconjugated vaccine (Merck' PNEUMOVAX® 23) is designed to provide coverage against -90% of the most
frequently reported isolates. This vaccine does not, however induces immune memory, hence is not effective in children below 2 years of age. Advances in the conjugation technology has led to conjugation of the polysaccharides to carrier proteins leading to a T-cell response, enabling its use in younger children (Wyeth's 7-valent Prevnar® and 13-valent Prevnar 13®, GSK's Synflorix ).Multivalent conjugate vaccines and combination vaccine formulations have been introduced to permit protection against multiple pathogen serotypes within a simple immunization schedule. While the combination of purified capsular polysaccharide vaccines has not altered the immunogenicity of the individual polysaccharides, a reduced immunogenicity of individual conjugate vaccines when presented in multivalent formulations has been observed. The reduced immunogenicity of conjugate vaccines in combined formulations has been attributed largely to the epitopic suppression phenomenon, and in some cases, to non-epitope specific suppression.
In 1929, Avery and coworkers showed that covalent binding of capsular polysaccharides to proteins increases the immunogenicity of the polysaccharides. This comprises linkage of capsular polysaccharides to a protein carrier, either by covalent binding or through reactive groups. The difference in the immune response towards pure PS vaccines is the observed switch to a TD response promoted by the protein. This leads to the induction of memory B cells and an improved B cell response. In addition, it leads to a better immune response early in childhood. This is explained by the stepwise maturation of the human immune system with the earlier maturation of the TD response compared to anti-polysaccharide antibodies (TI-2 response). Unfortunately, linkage of polysaccharides to proteins is restricted: too much carrier antigen may impair the antibody response to the polysaccharides by antigen competition or carrier-mediated epitope suppression. As a result when adding conjugated vaccines to existing immunization schedules, the issue of carrier-induced epitopic suppression (or "carrier suppression", as it is generally known) must be addressed, particularly suppression arising from carrier priming. "Carrier suppression" is the phenomenon whereby pre-immunization of an animal with a carrier protein prevents it from later eliciting an immune response against a new antigenic epitope that is presented on that carrier.
By now it is well recognized and established that various components in vaccines are not necessarily independent and interaction is possible. Negative interactions are known as immunological interferences and positive interactions are known as enhancement. It is not an easy task to develop a multivalent conjugate vaccine, because there is a risk of immune suppression leading to a suboptimal response to the conjugated polysaccharide due to the presence of too much of a particular carrier protein in a multi-conjugate vaccine.
For a protein to be an acceptable carrier in a conjugate vaccine it must be non-toxic and it must have epitopes suitable for its interaction with T cells. A variety of proteins, including bacterial pili, outer membrane proteins (OMPs) and excreted toxins of pathogenic bacteria, preferably in toxoid form, have been employed as carriers for carbohydrate antigens, including pneumococcal polysaccharides. Most popular as carrier proteins are tetanus and diphtheria toxoids, which are readily available and accepted for human use. However, the use of detoxified bacterial toxins as carrier proteins has some disadvantages. The process of chemically detoxifying produces lot¬to-lot variations. Thus, physical and chemical properties of the toxin can be substantially modified, which can affect the conjugation efficiency. Conjugation of these proteins with large amounts of saccharide may further affect protein conformational features and inactivate T- and/or B-cell epitopes. This might limit the amount of saccharide to be coupled to the protein, since a precondition of the conjugation is to maintain the T-cell activating properties of the carrier protein. Bacterial toxins offer advantages over their corresponding toxoids if cytotoxic effects can be reduced by the conjugation itself. Alternative carrier proteins have been developed, such as CRM197, a nontoxic analog of diphtheria toxoid. These proteins have the same advantages as native toxins—light or heavy loading of saccharide is possible without influencing the carrier characteristics. Although diphtheria and tetanus toxin-derivatized proteins have proven to be successful carrier proteins, both in animal and human studies, such problems as hypersensitivity or suppression of anti-carbohydrate response caused by the pre-existence of anti-carrier antibodies may still be a matter of concern, especially when a broad range of saccharides is analyzed. These
negative effects could become more evident when conjugated carrier proteins are tested in polyvalent or combination vaccine formulations.
It may be necessary to develop and use multiple carrier proteins as an approach to reduce interference when more than one conjugate vaccine is used. Further the selection of a carrier protein and its specific amounts, for a polysaccharide based vaccine will require a balance and fine tuning between the necessity to use a carrier working in all patients (broad MHC recognition), the induction of high levels of anti-polysaccharide antibody responses and low antibody response against the carrier.
Fattom et al., Vaccine 17 (1999) 126-133 investigated the phenomenon of interference in combined polysaccharide-conjugate vaccines in animals. This article stated that interference is due to competition between capsular polysaccharides (CPs) bound to homologous carrier proteins for a limited number of carrier-specific primed helper T cells. Such competition would result in a diminished T cell help and consequently in a reduced immune response to one or more polysaccharide components of the combined vaccine. The data presented in this manuscript supports the suggestion that, in addition to epitopic suppression induced by pre-existing carrier protein antibodies, the epitopic load at the site of injection might contribute to the severity of the suppression. This article finally recommends that epitopic suppression may be abrogated by choosing a conjugate methodology that blocks B-cell epitopes on the carrier protein or by diversification of the carrier protein used for these vaccines. The use of different or diverse carrier proteins in a single multivalent pneumococcal conjugate vaccine is reported in Australian patent 748716 (granted from W098/51339), with multiple carriers being used in order to avoid carrier suppression. The authors predicted that there is a maximum load of a carrier protein that can be tolerated in a multivalent conjugate vaccine without giving rise to negative interference. In Olander et al. Vaccine (2001) 20:336-341, it was reported that pneumococcal conjugate vaccines including mixed carrier proteins elicited, in parallel to the anti-pneumococcus response, unintentional booster responses to the carriers. Further Peeters, C. in "Effect of carrier priming on immunogenicity of saccharide-protein conjugate vaccines" Infect. Immun. (1991) 59:3504-3510, in abstract states that "prior exposure to the carrier protein can
either enhance or suppress antibody response to polysaccharides administered in saccharide-protein conjugates". The conjugates used in this study were tetanus toxoid or the CRM197 mutant as the carrier protein. The effect of the carrier-protein dosage on the humoral response to the protein itself has also proven to be complex. In human infants the significance and the difficulty of optimizing the carrier protein amounts, especially TT has been highlighted in Dagan et al, Infect. Immun, (Sept. 2004), p. 5383-5391, this study reported that in infants who simultaneously received DT, TT, whole-cell pertussis vaccine (DTwP), TT-conjugated Hib vaccine (polyribosylribitol phosphate tetanus) toxoid [PRP-T]), and a 4-valent TT-conjugated PCV, a reduced response to Hib and TT was observed, and the magnitude of the reduced response depended on the total dose of the TT. This study further tested the co-administration of aP based vaccines with 11-valent vaccine (PncD/Tll) containing seven polysaccharides conjugated to TT and four polysaccharides (those judged to need the largest carrier amounts) conjugated to DT. When aP was present in the concomitantly administered vaccine, a reduction in the primary immunogenicity of the pneumococcal polysaccharides conjugated to TT was observed in the above study.
The very first multicarrier protein based pneumococcal vaccine (DT/TT) reported by Aventis never made it to the market, hence there is no commercial success evident from Aventis's approach. The first vaccine of Wyeth that was launched in the market uses a single carrier namely CRM-197. However GSK has criticized this approach saying that they have found that CRM can have a negative effect on the immune response to certain antigens, which are herein termed sensitive antigens. GSK has developed and launched a pneumococcal polysaccharide protein D-conjugate vaccine (Synflorix ®) that contains ten capsular polysaccharide serotypes from the bacterium Streptococcus pneumoniae, eight of which are conjugated to a non-lipidated cell-surface lipoprotein (protein D) of non-typeable Haemophilus influenzae and two of which are conjugated to either tetanus or diphtheria toxoid. However protein D can be an extremely costly antigen and is cannot be easily prepared.
The final results of making a multivalent pneumococcal vaccine comprising different combination and amounts of carrier proteins cannot be predicted. Prior art still suggests
the uncertainty associated with multiple carriers and multiple conjugates. Very little is known and not much can be predicted regarding the optimum composition and concentration of carriers and polysaccharides for such combination vaccines. It is clear that there is still a need for developing multivalent pneumococcal conjugate vaccines which overcomes the disadvantages of the prior art vaccines. Ideally the carrier protein(s) used should induce strong helper effect to a conjugated B-cell epitope (e.g. polysaccharide) without inducing an antibody response against itself. The use of multiple epitopes, which are immunogenic in the context of most major histocompatibility complex class II molecules, is the key towards this goal. Further the novel vaccines should be effective in the presence of other antigens like wP/aP based vaccines. Present inventors have identified a unique balance in a unique combination and ratios of such epitopes. When designing a vaccine the desire is to create something which is clinically effective, safe, non-reactogenic and cost effective. The present inventors have strived to achieve these qualities in the novel vaccines of the present invention. The compositions resulting from the claimed strategies of the present invention reduces the risk of negative interference phenomenon between the polysaccharide conjugates, enhances the immunogenic response to the conjugated polysaccharides and is safe for use in humans.
SUMMARY OF THE INVENTION
The present invention provides generally a multivalent immunogenic composition comprising 10 or more polysaccharides conjugated to 2 or more different protein conjugates, wherein each of the conjugates contains a capsular polysaccharide from a different serotype of Streptococcus pneumonia conjugated to 2 or more different proteins, together with a physiologically acceptable vehicle. Optionally, an adjuvant, such as an aluminum-based adjuvant, is included in the formulation.
The present invention also provides a method of inducing an immune response to a Streptococcus pneumoniae capsular polysaccharide conjugate, comprising administering to an animal or mammal an immunologically effective amount of any of
the immunogenic compositions or vaccines of the present invention.
The present invention specifically relates to an improved Streptococcus pneumonia vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that serotypes 5, 7F and 14 are always conjugated to TT. In some of the embodiments it may be preferred that 5, 7F, 14 and 19F are always conjugated to TT
In some embodiments the present invention relates to an improved Streptococcus pneumonia vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that serotypes 5, 7F and 14 are always conjugated to TT, wherein in some embodiments serotypes, 1 and 4 are conjugated to the same carrier protein.
The present invention also relates to an improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum
albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that serotype 18C is never conjugated to DT.
In some embodiments the present invention further relates to an improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that in some preferred embodiments serotype 19F and 23 F are always conjugated to different carrier proteins.
The present invention further provides that any of the immunogenic compositions administered is a single 0.5 to 1 mL dose formulated to contain: 1 to 6 ug of serotypes 1 and 18C, 1 to 3 µg of serotypes 4, 9V and 19F, about 1 µg of serotypes 5, 7F& 14, 1 to 10 ug of serotype 6B, 2 to 10 µg of serotype 23F; with total TT, DT and CRM-197 content in conjugates always <15 µg, < 30 µg and <30 µg respectively; optionally with adjuvants, buffer and other excipients. In some embodiments the formulation may contain 1 to 6 µg of serotype 1, 1 to 3 µg of serotype 4 & 19F, about 1 µg of serotype 5, 7F& 14, 4 to 10 µg of serotype 6B, 1 to 2.5 µg of serotype 9V, 2 to 5 µg of serotype 18C, 2 to 6 µg of serotype 23F; approximately 1 of 11 µg of TT, 0 to 26 µg of DT and 0 to 21 µg of CRM 197; optionally with adjuvants, buffer and other excipients.
DESCRIPTION OF THE INVENTION
When developing novel conjugate pneumococcal vaccines, it must be borne in mind that a dose-response relationship exists between carrier, saccharide and host that affects the immunological mechanisms impacting on the function of conjugate vaccines. This
relationship may (a) reduce vaccine effectiveness due to antigenic competition caused by an excess number of serotypes in the vaccine (b) induce immune tolerance/ suppression of epitopes associated with carrier protein following repeated immunizations with conjugate vaccines and (c) affect carrier priming as a result of prior or co-administration of unconjugated carrier proteins that are often part of other childhood vaccines. It would be very difficult to apply the success of prior pneumococcal vaccines in designing a new improved vaccine. When each selected polysaccharide is conjugated to different carrier proteins in different amounts, and amounts it is clear that they would result in an immune response which would be conceivably different and distinct from the prior known formulations.
Optimizing the valency of conjugate vaccines is not easy, because the protein component of multivalent vaccines probably competes for a limited number of carrier-specific T cells. A large amount of a specific type of carrier protein may lead to high T cell-specific epitopic load; carrier mediated suppression of the antibody response, especially after repeated injections, and thus become a limiting factor for addition of further conjugates. In addition the amount of carrier protein might influence the immunogenicity of other conjugate vaccines sharing the same carrier when simultaneously administered. This appeared with simultaneously given Haemophilus influenzae type b (Hib) and pneumococcal tetanus-conjugated vaccines (Dagan et al, Infec Immun (1998):66:2093-8). Anti Hib PS response was suppressed with increasing doses of tetanus toxoid in the pneumococcal vaccine. Furthermore increasing the tetanus toxoid content seemed to be associated with low priming capacity of the pneumococcal conjugate vaccine (Ahman et al, Vaccine (1999); 17:2726-32). For these reasons it is desirable to optimize the carrier protein content. The use of a unique combination of mixed carrier, in specific amounts and ratios can reduce the load of an individual protein and might reduce interference in immunogenicity.
The present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more carrier proteins, wherein the vaccine comprises serotypes 5, 7F and 14 always conjugated to TT. In some of the embodiments it may be preferred that 5, 7F, 14 and 19F are always conjugated to TT
The present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, preferably 3 different carrier proteins, wherein the vaccine comprises serotypes 5, 7F and 14 always conjugated to TT. In some of the embodiments it may be preferred that 5, 7F, 14 and 19F are always conjugated to TT
In certain embodiments the present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, preferably 3 different carrier proteins, wherein the vaccine comprises serotypes 5, 7F and 14 always conjugated to TT, wherein is some embodiments serotypes 1 and 4 are always conjugated to the same carrier protein. In some of the embodiments it may be preferred that 5, 7F, 14 and 19F are always conjugated to TT
The present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, preferably 3 different carrier proteins, wherein serotype 18C is never conjugated to DT.
In some embodiments the present invention provides an improved Streptococcus pneumoniae vaccine comprising 10 or more capsular saccharides from different S. pneumoniae serotypes conjugated to 2 or more different carrier proteins, preferably 3 different carrier proteins, wherein the selected embodiments serotypes 19F and 23F are always conjugated to different carriers proteins.
In some embodiments the present invention provides an improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes, conjugated to at least 3 or more carrier proteins wherein serotypes 5, 7F and 14 are conjugated to TT and remaining serotypes are conjugated to 1 or 2 different secondary carrier proteins, and wherein the secondary carrier proteins are different from the first carrier protein. In some of the embodiments it may be preferred that 5, 7F, 14 and 19F are always conjugated to TT
In preferred embodiments the present invention provides an improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes, conjugated to at least 2 or more carrier proteins wherein the ratio of serotypes 5, 7F & 14 is 1:1:1.
In some embodiments the present invention provides an improved Streptococcus pneumonia vaccine comprising 7 or more polysaccharides from different S. pneumonia serotypes, conjugated to 2- 3 carrier proteins selected from one or more of TT, DT and CRM197, wherein the total quantity of TT is below 15 µg, the total quantity of DT is below 30 ug and the total quantity of CRM-197 is below 30 ug, with the proviso that TT is never 0 µg. In certain embodiments the total quantity of TT may be below 11 µg, the total quantity of DT may be between 0 to 26 µg and the total quantity of CRM-197 may be between 0 to 21 µg, with the proviso that TT is never 0 µg.
In some embodiments the present invention provides an improved Streptococcus pneumonia vaccine comprising 7 or more polysaccharides from different S. pneumonia serotypes, conjugated to 2- 3 carrier proteins selected from one or more of TT, DT and CRM197, wherein the total quantity of TT is below 15 µg, the total quantity of DT is below 30 µg and the total quantity of CRM-197 is below 30 µg, with the proviso that when DT is 0 µg, TT is less than 10 µg. In certain embodiments the total quantity of TT may be below 11 µg, the total quantity of DT may be between 0- 26 µg and the total quantity of CRM-197 may be between 0 to 21 ug, with the proviso that when DT is 0 ug, TT is about 7 to 8 µg.
In some embodiments the present invention provides an improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes, conjugated to 2- 3 carrier proteins selected from one or more of TT, DT and CRM197, wherein the total quantity of TT is below 15 µg, the total quantity of DT is below 30 ug and the total quantity of CRM-197 is below 30 µg, with the proviso that when CRM197 is 0 µg, TT is less than 13 µg. In certain embodiments the total quantity of TT may be below 11 ug, the total quantity of DT may be between 0- 26 ug and the total quantity of CRM-197 may be between 0 to 21 µg, with the proviso that when CRM197 is 0 µg, TT is about 8 to 11 µg.
The present invention also provides specific examples wherein 10 serotypes of S. pneumoniae 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F are used with specific carrier proteins DT, TT, and CRM in specific amounts for preparation of immunogenic composition or vaccine of the present invention.
An improved Streptococcus pneumoniae vaccine comprising 7 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 3 or more carrier proteins wherein the specific conjugates are selected from the examples provided below.
Polysaccharide
Typically the Streptococcus pneumoniae vaccine of the present invention will comprise capsular saccharide antigens wherein the saccharides are derived from at least ten serotypes of S. pneumoniae. The number of S. pneumoniae capsular saccharides can range from 10 different serotypes to 23 different serotypes. In one embodiment there are 10, 11, 12, 13, 14 or 15 different serotypes.
In one embodiment the multivalent pneumococcal vaccine of the invention will be selected from the following serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, although it is appreciated that one or two other serotypes could be substituted depending on the age of the recipient receiving the vaccine and the geographical location where the vaccine will be administered. For example, a 10-valent vaccine may comprise polysaccharides from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F.
Carrier protein
The vaccine compositions according to the present invention in general comprises at least two carrier proteins, herein designated carrier protein 1 and carrier protein 2, respectively. It is also highly preferred within the invention that the vaccine composition may comprise more than two carrier proteins, specifically 3 carrier proteins, third carrier designated as carrier protein 3.
Carrier proteins can be directed linked to the polysaccharide or can be linked to the saccharide antigen by means of a linkers like hexanediamine or adipic dihydrazide.
It is most preferred that carrier protein 1 is different from carrier protein 2 and carrier protein 3 is different from carrier proteins 1 and 2.
Carrier proteins 1, 2 & 3 may independently be any suitable carrier protein known to the skilled person. Thus they may independently be selected from the group consisting of bacterially derived proteins, virally derived proteins, lipopeptides, members of the polyhistidine triad family of proteins and fusions thereof, keyhole limpet hemocyanin, PPD (purified protein derivative of Tuberculin), pneumococcal pneumolysin (PLY), including detoxified PLY for example dPLY-GMBS or dPLY-formol, OMPC (meningococcal outer membrane protein - usually extracted from N. meningitidis serogroup), PorB (from N. meningitidis), PD (Haemophilus influenzae protein D), heat shock protein, Plasmodium flaciparum pfg27, lactate dehydrogenase peptide, gpl 20 of HIV, pertussis proteins, cytokines, lymphokines, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens, such as N19 protein, pneumococcal surface protein, iron uptake proteins, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin, ovalbumin, immunoglobulins and hormones, such as insulin and immune stimulating peptides such as P2 and/ or P30 from Tetanus Toxin or the synthetic PADRE epitope (Pan DR. Epitope).
Bacterially derived proteins may be any protein, which is naturally produced by a bacterium or fragments and variants thereof, wherein fragments comprises at least 10, such as at least 20, for example at least 30 consecutive amino acids of the naturally produced bacterial protein and wherein variants are proteins sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95% sequence identity with a naturally produced bacterial protein. Bacterially derived proteins are preferably bacterial toxins or variants of bacterial toxins, for example such variants which are not or are less toxic to mammals, such as human beings. Variants of bacterial toxins, which are inactivated either by genetic mutation, chemical treatment or by conjugation, are also referred to as toxoids herein. Bacterial toxins may for example be tetanus toxin and/or diphtheria toxin and/or Pseudomonas aeruginosa exotoxin A or toxin A or B of
C. difficile. Variants of bacterial toxins useful as carrier proteins within the scope of the present invention includes bacterial toxoids, such as tetanus toxoid, fragment C of tetanus toxoid, diphtheria toxoid (such as diphtheria toxoid as described in US patent 4,496,538, for example in Example III) , pertussis toxoid, bacterial cytolysins or pneumolysin. Mutations of pneumolysin (Ply) have been described which lower the toxicity of pneumolysin (WO 90/06951, WO 99/03884). Useful variants of diphtheria toxin which lower its toxicity are known and include for example CRM197 and other mutants described in US 4,709,017, US 5,843,711, US 5,601,827, and US 5,917,017, as well as CRM176, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations reported in literature.
In a most preferred embodiment carrier proteins 1, 2 and 3 may independently be selected from the group consisting of tetanus toxoid (TT), diphtheria toxoid (DT), and diphtheria toxin mutant CRM 197.
Carrier proteins may be prepared by any suitable means, for example they may be purified from a natural source by conventional techniques or they may be recombinantly produced. Methods for preparation of recombinant proteins are well known to the skilled person and are for example described in Molecular Cloning: A Laboratory Manual (Third Edition) by Sambrook and Russell. Briefly, a nucleic acid is introduced into a desirable host cell; said nucleic acid comprising a first nucleic acid sequence encoding the carrier protein is operably linked to a second nucleic acid sequence directing expression of the first nucleic acid sequence in said host cell. For example, CRM197 is produced by C. diphtheriae infected by the nontoxigenic phase ß197tox-created by nitrosoguanidine mutagenesis of the toxigenic corynephage b (Uchida et al, Nature New Biology (1971) 233; 8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs from it by a single base change in the structural gene. This leads to a glycine to glutamine change of amino acid at position 52 which makes fragment A unable to bind NAD and therefore renders it non-toxic (Pappenheimer, Ann Rev, Biochem. (1977) 46; 69-94, Rappuoli, Applied and Environmental Microbiology (September 1983) p 560-564).
Carrier protein expressed by the host cell may then be purified by conventional means.
Conjugation method
The prime consideration during the conjugation process is to achieve covalent linkage between polysaccharide and the carrier protein without bringing about any significant changes to the structures of the individual components.
The saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known coupling technique. The conjugation method may rely on activation of the saccharide with l-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide may thus be coupled directly or via a spacer (linker) group to an amino group on the carrier protein. Preferably, the cyanate ester (optionally made by CDAP chemistry) is coupled with hexane diamine or ADH and the amino-derivatised saccharide is conjugated to the carrier protein using carbodiimide (e.g. ED AC or EDC) chemistry via a carboxyl group on the protein carrier.
Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S—NHS, EDC, TSTU.
The conjugates can also be prepared by direct reductive amination methods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods are described in EP-O-161-188, EP-208375 and EP-0-477508.
A further method involves the coupling of a cyanogen bromide (or CDAP) activated saccharide derivatised with adipic acid dihydrazide (ADH) to the protein carrier by Carbodiimide condensation (Chu C. et al Infect. Immunity, 1983 245 256), for example using ED AC. Any suitable carbodiimide may be used as long as it is capable of conjugating saccharides and proteins in an aqueous medium. In one embodiment the carbodiimide may be EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) [also known as EDC] or it may be a carbodiimide other than.ED AC.
Various other conjugation techniques are known in the art. Conjugates can be prepared by direct reductive amination methods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat. No. 4,673,574 (Anderson).
The immunogenic compositions of the invention may additionally be combined with DTPa or DTPw vaccine (for instance one containing DT, TT, and either a whole cell pertussis (Pw) vaccine or an acellular pertussis (Pa) vaccine (comprising for instance pertussis toxoid, FHA, pertactin, and, optionally agglutinogins 2 and 3). Such combinations may also comprise a vaccine against hepatitis B (for instance it may comprise hepatitis B surface antigen [HepB], optionally adsorbed onto aluminium phosphate). In one embodiment the immunogenic composition of the invention comprises Hib, MenA and MenC saccharide conjugates, or Hib and MenC saccharide conjugates, or Hib, MenC and MenY saccharide conjugates, or MenA, MenC, MenW and MenY saccharide conjugates, wherein at least one, two or all the saccharide conjugates are made according the method of the invention.
Optionally, the immunogenic composition or vaccine of the invention contains an amount of an adjuvant sufficient to enhance the immune response to the immunogen. Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs)
Example 1 Example 2
(Table Removed)

WE CLAIM:
1. An improved Streptococcus pneumonia vaccine comprising either of (a) 7 or more (b) 10 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that serotypes 5, 7F and 14 are always conjugated to TT.
2. An improved Streptococcus pneumonia vaccine comprising either of (a) 7 or more (b) 10 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD), with the proviso that serotype 18C is never conjugated to DT.
3. An improved Streptococcus pneumonia vaccine comprising either of (a) 7 or more (b) 10 or more polysaccharides from different S. pneumoniae serotypes conjugated to at least 2 or more carrier proteins selected from a group comprising diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein
derivative of tuberculin (PPD), with the proviso that serotype 19F and 23F are always conjugated to different carrier proteins.
4. An improved Streptococcus pneumonia vaccine comprising either of (a) 7 or more (b) 10 or more polysaccharides from different S. pneumonia serotypes conjugated to 2- 3 carrier proteins selected from one or more of TT, DT and CRM197, wherein the total quantity of TT is below 15 µg, the total quantity of DT is below 30 µg and the total quantity of CRM-197 is below 30 µg.
5. An improved Streptococcus pneumonia vaccine according to claim 1 wherein the ratio of serotypes 5, 7F and 14 is 1:1:1.
6. An improved Streptococcus pneumonia vaccine according to claim 1 wherein serotype 19F is also always conjugated to TT.
7. An improved Streptococcus pneumonia vaccine according to claims 1 or 5 wherein serotypes 1 and 4 are conjugated to same carrier protein.
8. A method of inducing an immune response to Streptococcus pneumonia, by administrating a vaccine as claimed in claims 1-7 to an animal or mammal.
9. Formulation of a vaccine according to claims 1-6, wherein it contains 1 to 6 µg of serotypes 1 and 18C, 1 to 3 µg of serotypes 4, 9V and 19F, about 1 µg of serotypes 5, 7F and 14, 1 to 10 µg of serotype 6B, 2 to 10 µg of serotype 23F; with total TT, DT and CRM-197 content in conjugates always less than 15 µg, 30 ug, and 30 µg respectively; optionally with adjuvants, buffer and other excipients.
10. Formulation of a vaccine according to claims 1-6, wherein it contains 1 to 6 µg of serotype 1, 1 to 3 µg of serotype 4 & 19F, about 1 µg of serotype 5, 7F and 14, 4 to 10 µg of serotype 6B, 1 to 2.5 µg of serotype 9V, 2 to 5 µg of serotype 18C, 2 to 6 µg of serotype 23F; with about 1 to 11 µg of TT, 0 to 26 µg of DT
and 0 to 21 µg of CRM197; optionally with adjuvants, buffer and other excipients.