Vaccine composition against Streptococcus pneumoniae infections

Abstract

Immunogenic compositions are provided that include at least two antigenic determinants, the antigenic determinants being derived from at least two proteins selected from ABC-T, PavA and ZmpB of Streptococcus pneumoniae bacteria, the immunogenic compositions including one or more genetic constructs encoding the at least two antigenic determinants, the antigenic determinants being derived from at least two proteins selected from ABC-T, PavA and ZmpB of Streptococcus pneumoniae bacteria, the immunogenic compositions including at least one antigenic determinant and one or more genetic constructs encoding at least one different antigenic determinant, the antigenic determinants being derived from at least two proteins selected from ABC-T, PavA and ZmpB of Streptococcus pneumoniae bacteria. Vaccines, methods of treating or preventing or immunizing against Streptococcus pneumoniae bacterial infection, and kits including the immunogenic compositions are also provided.

Description

[Technical field]

[0001] The present invention relates to immunogenic compositions comprising Streptococcus pneumoniae proteins, new vaccines effective against Streptococcus pneumoniae bacteria, the invention also includes methods of treating or preventing Streptococcus pneumoniae bacterial infections, and methods of immunizing against Streptococcus pneumoniae bacterial infections. Further included within the scope of the present invention are nucleic acids encoding Streptococcus pneumoniae proteins for inclusion in DNA / RNA-based vaccinations. [Background technology]

[0002] Streptococcus pneumoniae is a nasopharyngeal commensal bacterium that normally invades sterile sites and causes pneumonia, bloodstream infections, and meningitis, and can also cause noninvasive disease such as otitis media. Although the population structure and evolutionary genetics of pneumococci are well defined, it is unclear whether pneumococci that cause invasive disease are genetically distinct from those that do not. Kulohoma et al. (Infection and Immunity;2015,83,10,4165-4896) reported the whole genome sequences of 140 isolates of S. pneumoniae taken from bloodstream infections and meningitis and found that these isolates shared a core of 1,427 genes and did not differ in their disease manifestations and core genome or accessory gene content. Therefore, the presence / absence of a gene alone does not explain the invasive behavior of pneumococci, but instead genetic variations such as nucleotide polymorphisms or Unitigs may be contributing factors.

[0003] From the prior art, it is known that there are more than 100 pneumococcal serotypes, each of which produces a biochemically distinct capsular polysaccharide (CPS) and differs in its propensity to cause invasive disease. Serotype 1 is one of the most common causes of invasive pulmonary disease (IPD), accounting for 11.7% of cases in Africa globally, and in contrast to other serotypes, serotype 1 has been associated with outbreaks in closed communities and fatal meningitis outbreaks in West Africa. The high burden of serotype 1 in IPD highlights the need for an effective vaccine against this serotype. Cornick et al; (Vaccine, 2017, 35(6), 972-980) showed the prevalence of seven protein vaccine candidates (CbpA, PcpA, PhtD, PspA, SP0148, SP1912, SP2108) among 445 serotype 1 pneumococci from 26 different countries across four continents. CbpA (76%), PspA (68%), PhtD (28%), and PcpA (11%) were not universally encoded in the study population and would not provide complete coverage for serotype 1. For example, PcpA was widely present in cases of European ancestry (82%) but rarely present in cases of African ancestry (2%). They proposed that a multivalent vaccine incorporating CbpA, PcpA, PhtD, and PspA could be predicted to provide coverage for 86% of the world's population and advocated further investigation of protein vaccines further incorporating single variants of SP0148, SP1912, and / or SP2108 from Streptococcus pneumoniae TIGR4.

[0004] Pneumococcal vaccines protect against serious and potentially fatal pneumococcal infections in high-risk individuals such as infants under 2 years of age, adults and children over 65 years of age, and immunocompromised individuals with certain long-term health conditions such as heart and kidney conditions. Currently, two types of pneumococcal vaccines are in common use in the UK, and are given differentially to infants and those over 65 years of age, depending on age. Prevenar13® (also known as PCV-13) includes the Pneumococcal Polysaccharide Conjugate Vaccine (PCV), which provides protection against 13 serotypes of Streptococcus pneumoniae bacteria and is used to vaccinate children under 2 years of age, while the Pneumococcal Polysaccharide Vaccine PPV-23 (also known as Pneumovax), which protects against 23 serotypes, is given to people over 65 years of age. The problem with this vaccine is that it is not particularly effective in small doses. In fact, both vaccines are thought to be only 50-70% effective in preventing pneumococcal disease. Furthermore, a major limitation of PCV is that it only induces protective antibodies against serotypes included in the vaccine formulation, and as a result, non-vaccine serotypes may increase in frequency in IPD and carriage after PCV introduction, as observed after the introduction of PCV7 in the United States (Weinberger et al; Lancet, 2011, 378; 1968-1973).

[0005] Prerequisites for an effective pneumococcal vaccine are conserved expression and minimal variation of the target antigens in the pneumococcal population, and the ability to induce a robust human immune response.

[0006] A further need is to provide a pneumococcal vaccine that is effective across all ages, particularly newborns and infants.

[0007] Demonstrated herein is an effective pneumococcal vaccine with a strongly suggested ability to induce a robust human immune response, which vaccination will be effective across all ages, particularly neonates, infants, the elderly, and immunocompromised individuals.

[0008] More than 100 different strains of S. pneumoniae are a major, worldwide cause of morbidity and mortality in children and adults. Commercially available pneumococcal vaccines such as Prevenar-13 (children) and Pneumovax-23 (adults) are widely used, but they use strain-specific carbohydrate antigens, which lead to high production costs (the most expensive vaccine in the world, pre-Covid pandemic) and dramatically limit their scope of application. The present invention was developed in a completely different way to existing pneumococcal vaccines: instead of targeting the outer sugar molecules of the bacteria, which differ from strain to strain, and therefore limit the scope of protection, the present invention was developed with the intention of targeting a highly conserved protein present in all 100 strains of pneumococcus. Our studies have shown that the immunological composition of the present invention is able to induce a comparable or better protective immune response against pneumococcal strains covered by Pfizer's PCV-13 vaccine as well as against strains that are not covered. The combination of broad coverage and low production costs distinguishes the immunological composition of the present invention from all existing and developmental vaccines against pneumococcus. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Kulohoma et al(Infection and Immunity;2015,83,10,4165-4896) [Non-Patent Document 2] Cornick et al;(Vaccine,2017,35(6),972-980) [Non-Patent Document 3] Weinberger et al;Lancet,2011,378;1968-1973 Summary of the Invention

[0010] According to a first aspect of the present invention there is provided an immunogenic composition comprising at least two antigenic determinants derived from at least two proteins selected from ABC-T, PavA and ZmpB of the bacterium Streptococcus pneumoniae.

[0011] According to a second aspect of the present invention there is provided an immunogenic composition comprising one or more genetic constructs encoding at least two antigenic determinants derived from at least two proteins selected from ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

[0012] According to a third aspect of the present invention there is provided an immunogenic composition comprising at least one antigenic determinant and one or more genetic constructs encoding at least one different antigenic determinant, the antigenic determinant being derived from at least two proteins selected from ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

[0013] The present invention also provides a vaccine comprising an immunogenic composition according to the first, second or third aspect of the invention.

[0014] The present invention further provides a method of treating, preventing, diagnosing or screening for a Streptococcus pneumoniae bacterial infection in an individual, comprising administering to an individual in need thereof an immunological composition of the first, second or third aspect of the invention or a vaccine of the invention in an amount sufficient to treat, prevent, diagnose or screen for a Streptococcus pneumoniae bacterial infection.

[0015] The present invention further provides a method of immunizing against Streptococcus pneumoniae bacteria-initiated infection in an individual comprising administering to the individual in need thereof an effective amount of the immunological composition of the first, second or third aspect of the invention, or the vaccine of the invention.

[0016] The present invention is based on the finding that immunization with a combination of antigenic determinants derived from any two of the proteins ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium provides a good immunogenic response. The antigenic determinants may be used directly or administered in the form of a genetic construct encoding the antigenic determinant, or a mixture of antigenic determinants and genetic constructs encoding different antigenic determinants may also be used. Thus, the immunogenic composition of the present invention may comprise at least two antigenic determinants, or one or more genetic constructs encoding at least two antigenic determinants, or at least one genetic construct encoding at least one antigenic determinant and at least one different antigenic determinant.

[0017] In a preferred embodiment of the invention, the immunogenic composition is based on the provision of antigenic determinants from each of the proteins ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium, which may be provided in any combination of antigenic determinants and genetic constructs encoding one or more antigenic determinants (for example there may be three antigenic determinants, one genetic construct encoding two antigenic determinants and a third antigenic determinant, one or more genetic constructs encoding one antigenic determinant and a second and a third antigenic determinant, or one or more genetic constructs encoding all three antigenic determinants).

[0018] In embodiments of the invention in which two or more antigenic determinants are encoded by genetic constructs, each different antigenic determinant may be encoded by a different genetic construct, or two or more different antigenic determinants may be encoded by a single genetic construct. For example, in an immunogenic composition of the invention that includes one or more genetic constructs encoding antigenic determinants from each of the proteins ABC-T, PavA, and ZmpB of the bacterium Streptococcus pneumoniae, each antigenic determinant may be encoded by a separate genetic construct, or two antigenic determinants may be encoded by one genetic construct and the other by a second genetic construct, or all three antigenic determinants may be encoded by a single genetic construct.

[0019] Genetic constructs suitable for use in the present invention include any nucleic acid-based structure suitable for expressing an antigenic determinant when administered to a recipient. Suitable genetic constructs include DNA constructs (e.g., DNA plasmids or DNA vaccines), RNA constructs (e.g., RNA vaccines or messenger RNA), bacterial-based constructs and viral-based constructs (such as inactivated or attenuated viral constructs).

[0020] DNA vaccines contain DNA that codes for a specific protein (antigen) from a pathogen. The DNA is injected into the body and taken up by cells, whose normal metabolic processes synthesize the protein based on the genetic code in the plasmid.

[0021] RNA vaccines or mRNA (messenger RNA) vaccines are a type of vaccine that uses artificial copies of natural chemicals to generate an immune response. Vaccines involve transfecting synthetic RNA molecules into human cells to stimulate an adaptive immune response, and typically the mRNA molecules are coated with a drug delivery vehicle, usually a PEGylated lipid nanoparticle.

[0022] The invention also includes the possibility of using DNA / RNA using delivery systems such as attenuated live viruses, including but not limited to retroviruses, adenoviruses, herpes simplex viruses, vaccinia viruses or liposome-based delivery systems, e.g. emulsions, microparticles, immune stimulating complexes (ISCOMs) and liposomes.

[0023] The immunogenic composition of the present invention may be for human use in human medicine. Preferably, the composition is for administration to a subject. Preferably, the subject is a human. In one embodiment, the subject is an adult, such as an elderly adult over 65 years of age; an infant under 1 year of age; a child between 1 and 2 years of age; a child between 2 and 5 years of age; or an immunocompromised individual of any age.

[0024] In one embodiment of the invention, the antigenic determinant from ABC-T, if present, is 30-50 kDa, ideally about 40 kDa, the antigenic determinant from PavA, if present, is 55-70 kDa, ideally about 63 kDa, and the antigenic determinant from ZmpB, if present, is 125-155 kDa, ideally about 148 kDa. These sequences encompass surface exposed fragments of the proteins which are believed to contain immune epitopes in adjacent regions.

[0025] In one embodiment of an immunogenic composition of the invention comprising an antigenic determinant derived from ABC-T, or a genetic construct encoding an antigenic determinant derived from ABC-T, the antigenic determinant comprises or consists of the protein sequence of SEQ ID NO: 1, or an immunologically effective fragment thereof, or an immunologically effective analogue of that sequence or fragment.

[0026] In one embodiment of an immunogenic composition of the invention comprising an antigenic determinant derived from PavA, or a genetic construct encoding an antigenic determinant derived from PavA, the antigenic determinant comprises or consists of the protein sequence of SEQ ID NO:2, or an immunologically effective fragment thereof, or an immunologically effective analogue of that sequence or fragment.

[0027] In one embodiment of an immunogenic composition of the invention comprising an antigenic determinant derived from ZmpB, or a genetic construct encoding an antigenic determinant derived from ZmpB, the antigenic determinant comprises or consists of the protein sequence of SEQ ID NO: 3, or an immunologically effective fragment thereof, or an immunologically effective analogue of that sequence or fragment.

[0028] Preferably, the immunogenic composition of the invention further comprises an immunostimulant, more preferably the immunostimulant is an adjuvant as described below.

[0029] Preferably, the composition is selected from the group consisting of pneumococcal surface protein A (PspA), pneumococcal choline binding protein A (PcpA), secreted 45-kDa protein Usp45-hydrolase (PcsB), serine / threonine protein kinase (StkP), peptide permease enzyme, manganese ABC transporter (PsaA), pneumolysin D (PlyD), pneumolysin toxoid (dPly), choline binding protein A (CbpA) and D (CbpD), histidine triad protein D (PhtD), histidine triad protein E (PhtE), histidine triad protein F (HtF), histidine triad protein G (HtF), histidine triad protein H (HtF), histidine triad protein I ... and a further set of one or more antigenic determinants from Streptococcus pneumoniae bacteria, which may be encapsulated or conjugated, selected from the group including histidine triad protein A (PhtA), histidine triad protein B (PhtB); pneumococcal iron uptake protein A (PiuA), protein cell wall segregation (PcsB), pneumococcal serine-threonine kinase (StkP) and analogs, plasmin and fibronectin binding protein A (PfbB), SP0148, SP1912 and SP2108, SP0785, SP1500, SP2216.

[0030] According to a further aspect of the invention there is provided an immunogenic composition as described herein in the form of a kit, e.g., sealed in a suitable container which protects the contents from the external environment. Such a kit may include instructions for use.

[0031] Preferably, the immunogenic composition is hermetically packaged in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of the composition. In one embodiment, the composition is supplied as a liquid, suspension, tablet or spray. In another embodiment, the composition is supplied as a dry sterile lyophilized powder or water-free concentrate in a hermetically sealed container, which can be reconstituted, for example with water or saline, to obtain the appropriate concentration for administration to a subject.

[0032] When the vaccines of the invention are administered systemically, for example, by subcutaneous or intramuscular injection, needle and syringe, or needle-free administration devices, for example, but not limited to, inhalation or intranasal delivery can be used. The vaccine formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0033] Although the immunogenic compositions and vaccines of the invention are generally provided as a single composition, i.e. a single composition containing all the necessary components, it will be understood that the invention also relates to compositions and vaccines provided as multiple compositions, for example a first composition comprising one or more antigenic determinants and / or a genetic construct encoding one or more antigenic determinants, and a second composition comprising a genetic construct encoding a different one of the specific antigenic determinants and / or a different one of the specific antigenic determinants, or a first composition comprising one or more specific antigenic determinants and / or a genetic construct encoding one or more antigenic determinants of the specific antigenic determinants, and a second composition comprising one or more adjuvants with or without a further one of the antigenic determinants and / or a genetic construct encoding a further one of the antigenic determinants. In this case, the multiple compositions may be administered simultaneously or sequentially. Accordingly, the invention further provides a method of treating or preventing a Streptococcus pneumoniae bacterial infection in an individual, or a method of immunizing against a Streptococcus pneumoniae bacterial infection in an individual, comprising administering a sufficient amount of at least one antigenic determinant derived from a protein selected from ABC-T, PavA and ZmpB of a Streptococcus pneumoniae bacterium, and simultaneously or subsequently administering a sufficient amount of at least one antigenic determinant derived from a protein selected from a different one of ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

[0034] The present invention further provides an immunological composition according to the invention or a vaccine according to the invention for use in the treatment or prevention of a Streptococcus pneumoniae bacterial infection in an individual, and / or for immunizing against and / or diagnosing or screening for a Streptococcus pneumoniae bacterial infection in an individual.

[0035] The invention further provides a kit comprising an immunogenic composition according to the invention or a vaccine according to the invention, and means for administering the immunogenic composition or vaccine to an individual. The kit of the invention may include additional components selected from one or more of a carrier and a means for resuspending the lyophilized composition or vaccine.

[0036] It will be understood that preferred features according to one aspect of the invention apply mutatis mutandis to all aspects of the invention.

[0037] Embodiments of the invention are further described below with reference to the accompanying drawings. [Brief description of the drawings]

[0038] [Figure 1] Figure 1 shows SEQ ID NO: 1, a truncated fragment of the Pneumococcal ATP-binding cassette transporter permease (ABC-T) protein sequence. Gene ID: 13695552, Gene Symbol: HMPREF1038_02239, Protein WP_000834690.1. [Diagram 2] FIG. 2 shows the Pneumococcal adherence and virulence factor A (PavA) protein sequence SEQ ID NO:2. [Diagram 3] FIG. 3 shows SEQ ID NO:3, a truncated fragment of the Pneumococcal zinc metalloprotease B (ZmpB) protein sequence. [Figure 4]Figure 1 shows immune binding activity of human sera from children, n=13, community acquired patients (CAP): n=5, elderly patients: n=3, healthy volunteers: n=5, and adults with HIV: n=5. Mouse sera from naive wild type mice were used as negative control (control serum) to show the absence of binding. Plates were coated with either PavA alone, ABCT alone, ZmpB alone, or all three proteins combined (PAZ). [Diagram 5] Figure 1: Validation of the role of three pneumococcal antigens, ABC-T, PavA and ZmpB, in CNS invasion. Survival numbers were determined at various time points (days) post-infection in brain tissue from a meningitis mouse model. [Figure 6] Figure 6 shows clinical scores in mice immunized with a combination of three antigenic determinants from ABC-T, PavA and ZmpB, either alone or in combination with alum or CpG-chitosan. Figure 6A is neonatal and Figure 6B is adult. Abbreviations: Ag: mixture of ABC-T, PavA and ZmpB antigens; PCV-13: pneumococcal conjugate vaccine (PCV-13 / Prevenar-13). [Figure 7A] FIG. 1 shows the results of an antibody dose response immunization study. [Figure 7B] FIG. 1 shows the results of cell-mediated immune responses upon vaccination with vaccine formulations. [Figure 7C] FIG. 1 shows that antibody-mediated immunity results from vaccination with a vaccine formulation that enhances disease protection compared to conventional alum adjuvant alone. [Figure 7D] FIG. 1 shows that cell-mediated immunity results from vaccination with a vaccine formulation that enhances disease protection compared to conventional alum adjuvant alone. [Figure 8]Figure 8 shows efficacy of vaccine and adjuvant formulations for commercial Prevenar-13® (PCV-13) in models of pneumococcal sepsis and pneumonia in adult and neonatal mice. Kaplan-Meier survival curves are shown upon challenge with pneumococci in adult (Figure 8A) and neonatal (Figure 8B) mice. Viable bacterial counts in lung tissue and blood of neonatal mice are shown in Figures 8C and 8D, respectively. Abbreviations: Ag: antigen (combination of three antigenic determinants derived from ABC-T, PavA and ZmpB); PrPV: composition of the invention as described herein. [Figure 9] FIG. 9B shows that the vaccine formulation of the present invention provides better immune protection efficacy against non-PCV13-responsive serotypes (i.e., serotypes 8, 11A and 33F) compared to PCV-13, as measured by survival rate (FIG. 9A) and clinical pain score (FIG. 9B). [Figure 10] FIG. 1 shows that the vaccine formulations of the invention activate / stimulate CD4+ T cell responses in ex vivo human peripheral blood mononuclear cells (PBMCs). [Figure 11] FIG. 1 shows that the vaccine formulation of the present invention activates plasma B cells in human PBMCs. [Figure 12] Figure 12 shows results from a passive transfer experiment: Serum from mice immunized with a vaccine formulation of the invention confers passive immunity to naive mice, as demonstrated by mouse survival time (Figure 12A) and colony forming units (CFU) in blood and lungs (Figure 12B). [Figure 13] 13A-B show results from passive transfer experiments of human PBMCs: Concentrated supernatants from human PBMC cultures stimulated with a vaccine formulation of the invention confer passive immunity to naive mice, as demonstrated by mouse survival time (FIG. 13A) and colony forming units (CFU) in blood and lungs (FIG. 13B). [Figure 14] Figure 1 shows the results of an opsonophagocytic killing assay from mice. Immunization with a vaccine formulation of the invention (PrPv) promotes in vitro opsonization of pneumococci and outcompetes Prevenar-13 (PCV-13). [Figure 15] FIG. 15A shows that vaccination with the mixed protein formulation of the present invention exhibits enhanced immunogenicity compared to single protein formulations adjuvanted with CpG chitosan; FIG. 15A shows antibody dose responses for antigen-specific IgG1 and IgG2a, and FIG. 15B shows cell-mediated immune responses upon vaccination. [Figure 16] Vaccination with the mixed protein formulation of the present invention enhances disease protection compared to single proteins adjuvanted with CpG chitosan; Figure 16A shows survival curves of adult mice and Figure 16B shows colony forming units (CFU) in blood and lungs. [Figure 17] 17A shows that the protein combinations (ABC-T+PavA+ZmpB) and (ABC-T+ZmpB) provided 100% protection (n=5 mice per group), and the protein combinations (PavA+ABC-T) and (PavA+ZmpB) provided 60% protection, while 80% of mice receiving only vehicle solution succumbed to infection within 46 hours of dosing. (17A). Also shown are viable counts (CFU) measured in blood (17B) and lung tissue (17C). All formulations also contained CpG-chitosan. [Figure 18] Figure 1 shows the results of nasal colonization (1st prime and 2nd booster) by Streptococcus pneumoniae after immunization with antigen only (green), CpG-chitosan only (blue) and antigen mixed with CpG-chitosan (red) for 0, 1, 3, 9 and 15 days post-immunization, as shown in panels A, B, C, D and E, respectively. Abbreviations: CFU: colony forming units; NP: nasopharyngeal; pi: post-infection. [Figure 19] Figure 1 shows results from ex vivo immune cell expansion (CD3+CD4+ cells harvested from mesenteric lymph nodes) obtained in mice immunized with a mixed protein formulation of the invention and subsequently colonized with S. pneumoniae.Staining panels are shown: CD4-FITC; CD3-APC; Ki67-PEcy7. [Figure 20]Figure 20 shows the induction of IL-17A positive memory T cells (CD62L+CD44+) in lung tissues of mice immunized with vehicle, CpG-c chitosan alone, or the composition of the present invention (PrPV), i.e., ABC-T+PavA+ZmpB+CpG-chitosan. Representative flow cytometry plots are shown in Figure 20A, and group comparisons are shown in Figure 20B. Abbreviations: MFI, mean fluorescence intensity. [Figure 21] FIG. 1 shows the protective efficacy of immunizing mice with the vaccine antigen formulation of the present invention containing a β-glucan-based adjuvant different from CpG-chitosan. [Figure 22] FIG. 2 shows SEQ ID NO:4, the mRNA sequence of a truncated fragment of the Pneumococcal ATP-binding cassette transporter permease (ABC-T) that matches the amino acid sequence shown in FIG. 1 (SEQ ID NO:1). [Figure 23] FIG. 3 shows SEQ ID NO:5, the mRNA sequence of pneumococcal adherence and virulence factor A (PavA), which corresponds to the amino acid sequence shown in FIG. 2 (SEQ ID NO:2). [Figure 24] FIG. 4 shows SEQ ID NO:3, the mRNA sequence of a truncated fragment of Pneumococcal zinc metalloprotease B (ZmpB) that matches the amino sequence shown in FIG. 3 (SEQ ID NO:3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The term "immunogenic composition" refers to any composition capable of eliciting an immune response in the human or other animal body, which may include, for example, the production of antibodies, a T cell and / or a B cell mediated immune response, or any other such response to the introduction of a vaccine into the body that confers protection against future disease or infection.

[0040] References herein to a "vaccine" refer to a biological preparation, as defined above, suitable for being placed in the human or animal body to elicit an immune response.

[0041] As used herein, the terms "treat" and "treatment" refer to any and all uses of treating a condition or symptom, preventing the establishment of a condition or disease, or preventing, hindering, slowing, ameliorating or reversing the progression of a condition or disease or other undesirable symptoms.

[0042] The term "vaccine protein antigen" as used herein refers to an antigen, whether it is a protein-containing cell fragment, a purified protein, a synthetic peptide, or whether its nature consists of amino acids alone or in combination with other biological molecules such as carbohydrates or lipids derived from the infectious organism against which the vaccine is intended to protect. The purpose of including a vaccine protein antigen is to induce specific and protective immunity against infectious diseases caused by the organism from which the vaccine protein is derived.

[0043] References herein to an "antigenic determinant" refer to a part of a protein antigen against which antibodies, B cells or T cells can be directed, and are synonymous with "epitope."

[0044] Reference herein to an antigenic determinant "derived from" a particular protein refers to a peptide that corresponds to all or a part of the particular protein, or is an analog of all or a part of the protein, and has an antigenic effect similar to that of the protein. For example, an antigenic determinant derived from the protein ABC-T of Streptococcus pneumoniae bacteria may comprise or consist of the entire sequence of the ABC-T protein, or may comprise or consist of any fragment of the protein, so long as it exhibits at least one antigenic effect exhibited by the ABC-T protein. Alternatively, an antigenic determinant derived from the protein ABC-T of Streptococcus pneumoniae bacteria may comprise or consist of an analog of the entire sequence of the ABC-T protein, or a fragment of an analog of the protein, so long as it exhibits at least one antigenic effect exhibited by the ABC-T protein. The same is true for antigenic determinants derived from the proteins PavA and ZmpB of Streptococcus pneumoniae bacteria. Examples of suitable antigenic determinants derived from the proteins ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium comprise or consist of the protein sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, or an immunologically effective fragment thereof, or an immunologically effective analogue of said sequences or fragments thereof. In a particular embodiment of the invention, the antigenic determinant derived from ABC-T is between 30 and 50 kDa, and / or the antigenic determinant derived from PavA is between 55 and 70 kDa, and / or the antigenic determinant derived from ZmpB is between 125 and 155 kDa.

[0045] The proteins shown in Figures 1, 2 and 3 / SEQ ID NOs: 1, 2 and 3 may include additional amino acids that are not part of the native sequence but are included to aid in recombinant production and / or purification of the protein, so long as they do not affect the immunological properties of the antigen. Examples of suitable additional amino acids include polyhistidine tags such as (HHHHHH). In certain embodiments of the invention, the truncated fragments of ABC-T shown in Figure 1 and / or the sequences of PavA shown in Figure 2 and / or the truncated fragments of ZmpB shown in Figure 3 may include a terminal polyhistidine tag HHHHHH. When looking at or calculating percent identity / homology, the poly-histidine tag may not be part of the calculation.

[0046] Reference herein to an "analog" of a particular protein refers to a protein having sufficient identity / homology (the terms are used interchangeably herein) to the protein to exhibit at least one antigenic effect exhibited by the protein.

[0047] To determine the identity / homology percentage of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap.

[0048] In one embodiment, the antigenic determinants that are analogs of ABC-T, PavA and ZmpB of Streptococcus pneumoniae bacteria used in the present invention have an amino acid sequence that is sufficiently identical or substantially identical to the amino acid sequence of SEQ ID NO: 1, 2 or 3, or a fragment thereof. The term "sufficiently identical" or "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or a minimum number of identical or equivalent (e.g., having similar side chains) amino acid residues with a second amino acid sequence such that the first and second amino acid sequences have a common structural domain or a common functional activity. For example, amino acid sequences that contain a common structural domain that has at least about 60% or 65% identity, suitably 75% identity, more suitably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as being sufficiently identical or substantially identical.

[0049] As used herein, an "immunologically effective analog or portion" of a protein includes a fragment of the protein that is involved in an interaction that elicits an immunological response. Typically, an immunologically effective analog or portion of a protein includes a domain or motif that has at least one activity of the protein, for example, an immunologically effective analog or portion may retain one or more immunogenic portions. A polypeptide has S. pneumoniae immunological effectiveness as defined herein if it has one, two, and preferably more of the following properties: (1) when expressed during the course of S. pneumoniae infection, it can promote or mediate the attachment of S. pneumoniae to cells; (2) it has an enzymatic activity, structure, or regulatory function characteristic of an S. pneumoniae protein; (3) or the gene encoding it can rescue a lethal mutation in an S. pneumoniae gene; (4) it contributes to the immune evasion properties of the bacterium. A polypeptide has immunological effectiveness if it is an antagonist, agonist, or superagonist of a polypeptide having one of the above properties.

[0050] Immunologically effective "fragments" or "analogs" are those that have an in vivo or in vitro activity characteristic of the S. pneumoniae polypeptides of the invention, particularly proteins having the sequences of SEQ ID NOs: 1-3, or other naturally occurring S. pneumoniae polypeptides, such as one or more of the biological activities described herein. Particularly preferred are fragments that exist in vivo, such as fragments resulting from post-transcriptional processing, or from translation of alternatively spliced ​​RNA. Fragments include those that are expressed in native or endogenous cells, as well as those made in expression systems, such as CHO cells. Because peptides such as S. pneumoniae polypeptides often exhibit a range of physiological properties, which can be attributed to different portions of the molecule, useful S. pneumoniae fragments or S. pneumoniae analogs are those that exhibit biological activity in any biological assay for S. pneumoniae activity.

[0051] Reference herein to a "genetic construct encoding an antigenic determinant" refers to any nucleic acid structure suitable for expressing an antigenic determinant when administered to a recipient, including DNA constructs (e.g., DNA plasmids), RNA constructs (e.g., messenger RNA), bacterial-based constructs, and viral-based constructs (such as inactivated or live-attenuated viral constructs). The genetic construct may encode an antigenic determinant that corresponds to the complete sequence of a native protein, or may encode an immunologically effective fragment or an immunologically effective analog of the native sequence or a fragment thereof.

[0052] In embodiments of the invention that include a genetic construct encoding a protein comprising a fragment of ABC-T corresponding to the sequence of SEQ ID NO:1, the genetic construct may comprise a nucleic acid sequence (DNA or RNA) corresponding to SEQ ID NO:4 or an analogue thereof (i.e., the RNA sequence shown in Figure 22, although an equivalent DNA sequence is also an embodiment of the invention).

[0053] In an embodiment of the invention comprising a genetic construct encoding a protein, including the protein PavA, corresponding to the sequence of SEQ ID NO:2, the genetic construct may comprise a nucleic acid sequence (DNA or RNA) corresponding to SEQ ID NO:5 or an analogue thereof (i.e. the RNA sequence shown in Figure 23, although an equivalent DNA sequence is also an embodiment of the invention).

[0054] In an embodiment of the invention comprising a genetic construct encoding a protein comprising a fragment of ZmpB corresponding to the sequence of SEQ ID NO:3, the genetic construct may comprise a nucleic acid sequence (DNA or RNA) corresponding to SEQ ID NO:6 or an analogue thereof (i.e., the RNA sequence shown in Figure 24, although an equivalent DNA sequence is also an embodiment of the invention).

[0055] References herein to "immunostimulating" refer to immunostimulants and immunostimulants that stimulate the immune system by inducing activation or increasing the activity of any of their components.

[0056] Reference herein to an "adjuvant" is intended to include a pharmacological or immunological agent that modifies the effect of another agent, in this example, a vaccine composition of the invention. Adjuvants used in the present invention act to enhance or increase the magnitude and / or durability of the immune response of the vaccine composition, which may also result in a reduction in the amount of antigen required to induce protective and long-lasting immunity.

[0057] Proteins, protein fragments and genetic constructs used in vaccine compositions are often conjugated or mixed with immunostimulatory or immunostimulatory substances / adjuvants. The incorporation of adjuvants into vaccine formulations aims to enhance, accelerate and prolong the specific immune response to vaccine antigens. The advantages of adjuvants include enhancing the immunogenicity of weaker antigens, reducing the amount of antigen required for successful immunization and reducing the frequency of booster immunizations to achieve a sufficient level of protective immunity. Optionally, adjuvants can also be used to optimize the desired immune response, for example with respect to immunoglobulin classes and induction of cytotoxic or helper T-lymphocyte responses.

[0058] In one embodiment, the immunogenic composition of the invention is directed to one or more substances selected from substances having a stimulatory effect on cytosolic pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), nucleic acid-sensing receptors such as retinoic acid-inducible gene I (RIG-I), or nutrient sensors such as mTOR and GCN2; any stress, cell death and tissue damage pathways that result in the release of damage-associated molecular patterns (DAMPs) such as the following pathways: MyD88 / TLR-signaling pathway, cGAS-stimulator of interferon genes (STING) pathway, NF-κB pathway, nucleic acid, etc. (necrosis / necrotizing pathways). The therapeutic agent may comprise one or more substances selected from: substances acting on at least one of the following signaling pathways: uric acid, ATP and proteins that activate the innate immune system, any signaling pathway that results in immune cell recruitment or epigenetic changes that maintain the innate immune system in an alert state, i.e., memory-like state, for extended periods of time; substances having the property of inducing inflammasome activation resulting from cell differentiation towards CD4+Th2 (interleukin-4), Th1 (interferon-gamma)- or Th17 (IL17A)-mediated immunity; immune enhancers; polysaccharide-based substances acting on IL-1β, CLR and TNF-α signaling pathways; delivery systems and mucosal adjuvants.

[0059] Additionally or alternatively, the immunogenic compositions of the invention may be comprised of bacterial proteins and / or polysaccharide substances such as capsules and analogs thereof, toxins / toxoids and analogs thereof, TLR ligands and analogs thereof, agonists such as Pam3CSK4, Pam2CSK4, MPLA (an LPS derivative), CpG (a short, single-stranded synthetic DNA molecule containing an unmethylated cytosine triphosphate deoxynucleotide "C" followed by a guanine triphosphate deoxynucleotide "G", where "p" refers to a phosphodiester bond between consecutive nucleotides, although some ODNs have a modified phosphorothioate (PS) backbone instead), P Microparticles and nanoparticles made of lipid-based scaffolds co-formulated with any other immunostimulant such as olyI:C, CpG motifs, chitosan or β-glucan, flagellin, poly(lactic-co-glycolic acid) or PLGA-poly-L-lysine / poly-gamma-glutamic acid (PLGA-PLL / gamma PGA), or extracellular vesicles (EVs) such as monophosphoryl lipid A (MPLA), polyethylene glycol (PEG), oligomannose, poly(I:C), outer membrane vesicles (OVMs); water-in-oil, oil-in-water and thermoreversible oil-in-water emulsions, e.g. Montanide ISA-51, Montanide ISA-720 and analogues thereof, alum or aluminum salt adjuvants, liposomes, virosomes, archaeosomes, outer membrane vesicles, niosomes, saponins and immune stimulating complexes (ISCOMs), polymeric particles, cytokines including pro-inflammatory cytokines such as IFN-γ, IL-1, IL-2, IL-4, IL-12, IL-17A / F, GM-CSF and MPI, virus-like particles (VLPs), detoxified variants of bacterial components such as LPS, e.g. monoclonal antibodies, Sulfolyl lipid A (MPL), muramyl dipeptide (MDP lipophilic and hydrophobic), QS21, PLGA, CT, liposome-based cationic adjuvant formulations such as CAF01-09 (composed of Span80, polyoxyethylene cetyl-stearyl ether, mannitol, squalene) and cholesterol, phosphatidylcholine and phosphatidylserine, or analogs composed of α-Gal ceramide, NOD-like receptor protein 3 (NLRP3) inflammasome,Apoptosis-associated speck-like protein containing CARD (ASC), NLR family CARD domain-containing protein 4 (NLRC4) inflammasome, Absent in melanoma 2 (AIM2) inflammasome, dsRNA:poly(I:C), poly-IC:LC, monophosphoryl lipid A (MPL), LPS, flagellin, imidazoquinolines: imiquimod (R837), resiquimod (848), CpG oligodeoxynucleotides (ODN), muramyl dipeptide (MDP), saponin (QS-21), polysaccharide capsules and analogs thereof, toxins / toxoids and analogs thereof, unmethylated DNA (CpG) and analogs thereof, cytokines such as IL-2, IL-12, TNF-α and conjugates. The antibodies may include one or more substances selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokines such as RANTES (regulated upon activation, expressed and secreted by normal T cells), macrophage inflammatory protein (MIP)-1α, costimulatory or adhesion molecules such as CD80, lymphocyte function-associated antigen-3 and polyarginine tails, polysaccharide-based substances with properties similar to those presented by chitosan, such as CpG-delta inulin, cochleates, virus-like particles (VLPs), microparticles such as virosomes, PLA (polylactic acid), PLG (poly[lactide-co-glycolide]), cholera toxin (CT) derivatives, and heat-labile enterotoxins (LTK3 and LTR72).

[0060] In certain embodiments, the immunological composition of the invention may comprise one or more substances selected from TLR agonists, agents capable of inducing a CD4+ T cell-mediated immune response (especially with a Th1 and / or Th17 differentiation profile), oil-in-water emulsions, water-in-oil emulsions, agents acting on the MyD88 / TLR signaling pathway, agents acting on the cGAS stimulator of interferon genes (STING) pathway, and particulate polysaccharide substances. In a particularly preferred embodiment of the immunological composition of the invention, the adjuvant comprises a combination of a TLR agonist and an agent capable of acting on the MyD88 / TLR signaling pathway or the cGAS stimulator of interferon genes (STING) pathway, for example, a combination of CpG with chitosan and / or β-glucan.

[0061] Freeze-drying of vaccines is well known in the art. Typically, liquid vaccines are freeze-dried in the presence of a clotting inhibitor such as a sugar, e.g., sucrose or lactose (present at an initial concentration of 10-200 mg / ml). Freeze-drying usually requires several steps, for example a cycle starts at -69°C, gradually adjusts the temperature to -24°C over 3 hours, then maintains the temperature for 18 hours, then gradually adjusts to -16°C, then 1°C, then maintains the temperature for 6 hours, then adjusts to +34°C over 3 hours, and finally maintains for 9 hours.

[0062] Lyophilization of the formulation results in a more stable formulation (e.g., degradation of the polysaccharide antigen is prevented). This process is also surprisingly responsible for higher antibody titers against pneumococcal polysaccharides. This was found in particular for PS6B conjugates and 3DMPL adjuvants (preferably without aluminum-based adjuvants) and pneumococcal proteins selected from the group consisting of PhtA, PhtB, PhtD, PhtE, SpsA, LytB, LytC, LytA, Spl25, SpiO1, Spl28, Spl30 and Spi33.

[0063] In some embodiments of the invention, the immunogenic composition or vaccine may be lyophilized.

[0064] In another aspect, the invention encompasses a vector comprising a nucleic acid encoding a S. pneumoniae polypeptide or a S. pneumoniae polypeptide variant described herein; a host cell transfected with the vector; and a method for producing a recombinant S. pneumoniae polypeptide or a S. pneumoniae polypeptide variant comprising: culturing the cell, e.g., in cell culture medium, and isolating the S. pneumoniae polypeptide or S. pneumoniae polypeptide variant, e.g., from the cell or cell culture medium.

[0065] Methods for producing antibodies in a host animal are also provided. The methods of the invention comprise immunizing an animal with at least one immunogenic component derived from S. pneumoniae, the immunogenic component comprising an immunogenic composition or vaccine of the invention or a sequence-conservative or function-conservative variant thereof, or a polypeptide contained within any ORF comprising the complete protein coding sequence of which any of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 forms a part, or a polypeptide sequence contained within any of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 forms a part. Host animals include any warm-blooded animal, including but not limited to mammals and birds. Such antibodies are useful as reagents for immunoassays to assess the abundance and distribution of S. pneumoniae specific antigens.

[0066] Delivery systems that have been investigated to achieve more efficient peptide vaccine design include nanoscale size (<1000 nm) materials such as virus-like particles (VLPs), outer membrane vesicles (OMVs), liposomes, immune stimulating complexes (ISCOMs), polymers, and non-degradable nanospheres, which have been highlighted as potential delivery vehicles for vaccine antigens that can stabilize vaccine antigens and act as adjuvants. Furthermore, they offer the ability to design vaccines containing multiple protein antigenic fragments that specifically target immune cells, resulting in more effective uptake by antigen-presenting cells.

[0067] Alternative delivery systems may include biodegradable materials, such as natural polymeric compounds such as starch, alginate and cellulose, biosynthetic materials such as poly-β-hydroxybutyrate (PHB), and copolymers such as polylactic acid (PLA), polyurethane, polylactic-co-glycolic acid (PLGA) and polymethylmethacrylate resin (PMMA), which have attracted much attention in vaccine research due to their biocompatibility, biodegradability and often low toxicity, which can protect antigens from degradation, increase antigen stability, provide sustained release, resulting in enhanced overall immunogenicity. These are also promising adjuvants / delivery systems for the novel pneumococcal vaccine of the present invention.

[0068] Further delivery systems include, but are not limited to, probiotics, such as Lactobacilli and Bifidobacteria species; viral vector systems such as Modified Vaccinia Virus Ankara (MVA) and various other viruses including poxviruses and adenoviruses; and bacteriophages such as phage T4.

[0069] It will be understood that the vaccine of the present invention can be used with other existing vaccines / conjugate vaccines. The vaccine of the present invention can be used as a carrier protein to improve currently available formulations, for example, but not limited to, Prevenar contains carrier protein CRM197. Thus, CRM197 can be replaced by one or more or all of the proteins / polypeptides of the present invention.

[0070] The compositions described herein can be administered alone or in combination with other treatments, either simultaneously or sequentially. Administration can be repeated at intervals of daily, twice weekly, weekly or monthly. The treatment schedule for each individual subject can depend on factors such as the route of administration and the severity of the condition being treated.

[0071] The present invention also encompasses combination vaccines that provide protection against a variety of pathogens. Currently, many pediatric vaccines are administered as combination vaccines to reduce the number of injections a child must receive. Thus, pediatric vaccines containing antigens from other pathogens can be formulated with the vaccine of the present invention. For example, the vaccine of the present invention can be formulated (or administered alone but simultaneously) with the well-known "trivalent" combination vaccines containing diphtheria toxoid (DT), tetanus toxoid (TT), pertussis components [typically detoxified filamentous hemagglutinin (FHA), which may contain pertactin (PRN) and / or agritomom, 1+2], such as the commercially available INFANRIXDTPa™ vaccine (SmithKline Beacham Biologicals) containing DT, TT, PT, FHA and PRN antigens, or with pertussis components whole cell, such as the vaccine marketed by SmithKline Beecham Biologicals SA as Tritanrix™. Combination vaccines may also contain additional antigens such as Hepatitis B virus surface antigen (HBsAg), poliovirus antigens (e.g., inactivated trivalent poliovirus - IPV), Moraxella catarrhalis outer membrane proteins, atypical Haemophilus influenzae proteins, and N. meningitidis B outer membrane proteins.

[0072] The composition may be administered in one or more doses, followed by one or more further "booster" doses administered days, weeks or years later. For example, when the composition is administered to a child, the first dose may be given at age 1-12 years, and the booster dose at age 16 years. For adolescents who receive their first dose at age 13-15 years, a booster dose may be given at age 16-18 years. For individuals who may not have received a pneumococcal vaccine in childhood, a prime and / or booster dose of the immunogenic composition or vaccine of the invention may be administered or co-administered with a seasonal vaccine, such as a seasonal influenza / influenza vaccine. The immunogenic composition or vaccine of the invention may also be administered as a prime or booster in adults over 65 years of age who have received one or two doses of PPSV-23 (Pneumovax) at one or two year intervals. The injections may contain the same dose of active ingredient, or may contain different doses. Preferably, the doses are administered by injection, although needle-free administration is also within the scope of the invention.

[0073] The immunogenic composition or vaccine may be administered to children, adolescents, or adults. "Children" generally includes infants up to the age of 2 years. In some countries, infants are generally administered two doses, with a third "booster" dose administered in the second year of life, while in other countries only two doses may be administered, and in other countries four doses may be used.

[0074] The specific dosage level and frequency of administration for any particular patient may vary and depend on a variety of factors, including the age, weight, general health, sex, diet, and mode of administration of the individual being treated. Typically, the appropriate dosage can be determined by a physician. The composition can be administered as a dose of 0.00001 μg / Kg body weight to 5 mg / Kg body weight, preferably 0.0001 μg / Kg to 5 mg / Kg, preferably 0.001 g / Kg to 1 mg / Kg, preferably 0.01 g / Kg to 500 g / Kg, preferably 0.02 μg / Kg to 300 g / Kg body weight.

[0075] Currently, there are two licensed pneumococcal vaccines targeting both capsular polysaccharides: (a) a 23-valent pneumococcal polysaccharide-based vaccine (PPV-23, marketed under the trade name Pneumovax); and (b) 7-, 10-, and 13-valent pneumococcal conjugate vaccines (PCV-7, -10, -13, marketed under the trade name Prevenar). However, PPV-23 is poorly immunogenic in children under 2 years of age and does not generate immune memory responses or herd immunity. Conjugation of pneumococcal polysaccharides to carrier proteins converted polysaccharide-based vaccines from T-cell-independent to T-cell-dependent antigens and enhanced their immunogenicity. The conjugate vaccines induced efficient immune memory and associated herd immunity.

[0076] Other suitable S. pneumoniae vaccines that can be used in conjunction with the vaccines of the present invention include whole cell S. pneumoniae vaccines, which are inactivated cell preparations of a non-encapsulated strain of S. pneumoniae in which the autolysin (lytA) gene has been deleted and the pneumolysin (ply) gene has been replaced with pdT. Additionally, other suitable candidates include pneumococcal surface protein A (PspA), pneumococcal choline binding protein A (PcpA), secreted 45-KDa protein Usp45-hydrolase (PcsB), serine / threonine protein kinase (StkP), peptide permease enzyme, manganese ABC transporter (PsaA), pneumolysin D (PlyD), pneumolysin toxoid (dPly), choline binding proteins A (CbpA) and D (CbpD), histidine triad protein D (PhtD). , histidine triad protein E (PhtE), histidine triad protein A (PhtA), histidine triad protein B (PhtB); pneumococcal iron uptake protein A (PiuA), protein cell wall segregation (PcsB), pneumococcal serine-threonine kinase (StkP) and analogs, plasmin and fibronectin binding protein A (PfbB), SP0148, SP1912 and SP2108, SP0785, SP1500, SP2216.

[0077] The vaccine compositions of the invention may be administered by any conventional route, including oral (e.g., tablets or capsules), nasal (e.g., sprays), inhalation, topical (e.g., creams or lotions) and injection. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or intradermal. The vaccine compositions of the invention are administered in an effective amount. An "effective amount" is that amount of vaccine composition which alone, or together with further doses, produces the desired response. In the case of preventing pneumococcal disease, the desired response is to provide protection upon challenge by an infectious agent.

[0078] The compositions described herein may be formulated using pharma- ceutically acceptable carriers, excipients, buffers, stabilizers, or diluents, or other substances known to those skilled in the art. Suitable pharma-ceutically acceptable carriers, excipients, or diluents are described, for example, in (Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company

[1990] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press

[2000] ). The exact nature of the carrier or other substances depends on the route of administration.

[0079] The composition may be formulated in a form suitable for the intended mode of administration, for example as a powder, spray, tablet, solution or suspension, optionally with a suitable carrier, excipient or diluent (or a combination thereof). For parenteral administration, the composition may be in the form of a sterile aqueous solution, which may contain other substances, such as salts or buffers. Those skilled in the art are well able to prepare suitable solutions, for example, using isotonic vehicles, such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Optionally, phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycosyltransferase. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, including lysine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents, e.g., EDTA; sugars such as sucrose, mannitol, lactose, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or buffers such as non-ionic surfactants, e.g., TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0080] "Therapeutically effective amount" means a sufficient amount of a composition to show benefit to a subject, including, but not limited to, inducing / increasing an immune response to Streptococcus pneumoniae bacteria in a subject and / or reducing the severity or duration of disease in a subject. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g., dosage, etc., is within the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated.

[0081] An immune response is induced or increased if there is a detectable difference in immune response indicators measured before and after administration of a particular composition, including, but not limited to, antibody titers or specificities detected by assays such as enzyme-linked immunosorbent assay (ELISA), bactericidal assays, flow cytometry, immunoprecipitation, Outterlony immunodiffusion, binding detection assays such as spot, Western blot or antigen array, cytotoxicity assays, ELISpot, ex vivo peripheral blood monocytic and lymphocyte stimulation assays, mouse peritonitis and challenge models, etc.

[0082] In some embodiments, a therapeutically effective amount of a vaccine formulation or composition described herein is an amount sufficient to generate antigen-specific antibodies (e.g., anti-Streptococcus pneumoniae bacteria). In some embodiments, a therapeutically effective amount is sufficient to provide seroprotection in a subject, i.e., to generate sufficient antigen-specific antibodies to prevent / protect against infection. In some embodiments, seroprotection is conferred to at least 60%, 70%, 80%, 90%, or at least 95% of vaccinated subjects. In some embodiments, an effective amount of an immunogenic composition or vaccine of the invention is sufficient to seroconvert a subject at least 50% of the time. In some embodiments, a therapeutically effective amount is sufficient to seroconvert a subject at least 60%, 70%, 80%, 90%, or at least 95% of the time. Whether a subject has seroconverted can be assessed by any method known in the art, such as obtaining a serum or peripheral blood sample from the subject and performing an assay to detect anti-Streptococcus pneumoniae antibodies or S. pneumoniae-induced lymphocyte responses. In some embodiments, a subject is seroconverted when a serum sample from the subject contains an amount of anti-Streptococcus pneumoniae antibodies or lymphocyte-mediated response above a threshold or predetermined baseline.

[0083] In vitro studies bacterial isolates Streptococcus pneumoniae serotype-2, lab-adapted strain D39 (NCTC7466) was obtained from the National Collection of Type Culture (London, UK). All other pneumococcal strains used in this study, e.g., hypervirulent S. pneumoniae serotype 1, were clinical isolates obtained from the biobank of isolates stored at Liverpool Royal Hospital (RLBUHT) or the Malawi Wellcome Trust Centre, Blantyre, Malawi. Pneumococcus identification was confirmed by optochin susceptibility testing, and serotypes were determined using standard Quellung reactions with pneumococcal capsule-specific antisera (Statens Serum Institute, Copenhagen, Denmark). All isolates were stored in the laboratory at -80°C on Protect™ cryobeads (Pro-Diagnotics Lab, Inc.) and subcultured on 5% horse blood agar plates incubated at 37°C for 18 h under anaerobic conditions. Multilocus sequence typing (MLST) was also performed on all isolates.

[0084] Tissue samples for bacterial viability counts Viable bacterial counts in blood and lungs were determined at the indicated intervals after pneumococcal infection. Tissue samples were disrupted using an IKA T10 homogenizer. Viable bacterial counts in lung cell suspensions and blood samples were determined by serial dilutions in sterile PBS and plating on blood agar containing 5% (v / v) defibrinated horse blood (Oxoid) and 10ug / ml gentamicin (sigma). Once dry, plates were incubated overnight at 37°C in 5% (v / v) CO2 and bacterial colonies were counted the following day.

[0085] Mutagenesis of Streptococcus pneumoniae.

[0086] Generation of competent S. pneumoniae cells and subsequent transformation were performed using the complete transformation medium (C+Y medium, pH=6.8) method (Lacks and Hotchkiss, 1960). Single deletion mutants were constructed by replacing the open reading frame (ORF) with the aphA3 cassette (conferring kanamycin resistance) and then selecting kanamycin-resistant recombinants on blood agar-based medium supplemented with kanamycin. The recombination was verified by Sanger sequencing.

[0087] Recombinant protein production and adsorption to CpG-chitosan.

[0088] Each vaccine candidate Escherichia Coli optimized synthetic gene was obtained from GeneArt (Thermo Fisher Scientific) and included a truncated fragment of ABC-T (SEQ ID NO:1) corresponding to the sequence shown in Figure 1, the complete sequence of PavA (SEQ ID NO:2) corresponding to the sequence shown in Figure 2, and a truncated fragment of ZmpB (SEQ ID NO:3) corresponding to the sequence shown in Figure 3.

[0089] For each antigen, 6xHis-tagged C-terminal recombinant pneumococcal proteins were cloned into E. coli BL21(DE3) cultured in Terrific Broth (Thermo Fisher Scientific). ABC-T is a large multi-membrane spanning protein: the sequence from Met435 to Gly790 (SEQ ID NO:1, approximately 40 kDa) was amplified and subcloned as a T7 promoter C-term 6xHis-tagged recombinant entity. PavA was expressed in its full length, i.e., Met1 to Ser551 (SEQ ID NO:2, approximately 63 kDa), as a T7 promoter C-term 6xHis-tagged recombinant protein. The largest extracellular domain of ZmpB, i.e., Met140 to Ala1876 (SEQ ID NO:3, approximately 148 kDa), was amplified and subcloned as a T7 promoter C-term 6xHis-tagged truncated protein. All 6xHis-tagged proteins were recovered from an IMAC Nickel (Bio-Rad laboratories) column and passed four times through a Pierce™ high capacity endotoxin removal spin column (Thermo Scientific Fisher) to ensure residual endotoxin levels were less than 0.05 units / ml. The expressed proteins were then combined with chitosan (CS) polymer (Protosan, Novomatrix) as single protein or combined protein formulations (i.e., formulations containing a pair of proteins or all three proteins), i.e., 1-10 μg of antigen per vaccine formulation. Pneumococcal antigen-adsorbed CS nanoparticles were formed upon stirring in a sterile phosphate buffered saline aqueous solution containing 100 μg / ml of pneumococcal antigen (1-10 ug of antigen per vaccine formulation, either as single or combined formulations). The CS nanoparticles were then mixed with CpG (unmethylated cytosine-phosphorothioate-guanine oligonucleotide) or CpG ODN1826 (group 2) supplied by Integrated DNA Technologies Inc.

[0090] A mixture of the three antigens corresponding to SEQ ID NOs: 1, 2 and 3, in the absence of any adjuvant, is an immunological composition of the invention, and is referred to herein as the "antigens."

[0091] The above compositions comprising a mixture of two of the three antigens corresponding to SEQ ID NOs: 1, 2 and 3 in combination with CpG and adsorbed to chitosan microparticles are immunological compositions of the present invention and are referred to herein by reference to their respective antigenic determinants, i.e. "ABC-T+ZmpB", "PavA+ZmpB" and "PavA+ABC-T".

[0092] The above composition comprising a mixture of three antigens corresponding to SEQ ID NOs: 1, 2 and 3 combined with CpG and absorbed onto chitosan microparticles is a preferred immunological composition of the present invention and is referred to herein as "PrPV" (protein-based pneumococcal vaccine).

[0093] ABC-T was found to be a large, multiple transmembrane domain and highly insoluble / toxic in E. coli. The largest ectodomain was expressed according to a design based on TMHMM server analysis. PavA, full length protein was expressed. ZmpB was determined to be very large, multiple functional domains as identified by NCBI domain homology search. Trial expression of fragments of the protein was done to see which largest portion gave the best expression.

[0094] Opsonophagocytosis assay (OPK).

[0095] The purpose of this assay is to reproducibly measure the opsonization capacity of test serum samples against Streptococcus pneumoniae using a method adapted from a published standardized CDC method (Steiner et al Clinical and Diagnostic Laboratory Immunology 4:415:1997). This assay mimics the in vitro conditions that are the primary in vivo mechanism for the elimination of invading Streptococcus pneumoniae followed by phagocytosis and then killing of the microorganism. "Phagocytosis" is the process by which a cell absorbs material and encapsulates it in a cytoplasmic vacuole (phagosome). Pneumococci are killed when engulfed by healthy mammalian phagocytes. "Opsonization" is the process that facilitates phagocytosis by depositing opsonins, such as antibodies and complement, on the antigen.

[0096] Many opsonophagocytosis assays have been described in the literature. A standardized CDC method has been tested in many laboratories (Steiner et al., ICAAC, Sept. 16-20, 2000, Toronto). This test was then modified in SB to provide a basis for comparison with other laboratories using commonly available reagents and controls and expressing results as serum titers (dilutions) that promote the killing of 50% of viable pneumococci, a unit commonly used for this type of test. Moreover, this modified test has been shown to be able to generate results that correspond well with four other laboratories (Steiner et al., ICAAC, Sept. 16-20, 2000, Toronto).

[0097] OPK was performed to test the ability of vaccinated mouse antisera to opsonize and mediate killing of live pneumococci. A reference opsonophagocytosis assay described elsewhere (Bangert et al, 2012 JID) was used with minor modifications. Reaction mixtures were prepared consisting of heat-inactivated serum obtained from vaccinated versus non-vaccinated mice, differentiated HL-60 cells or freshly isolated human neutrophils, pneumococci, and baby rabbit complement. Control reactions lacking all components except complement and / or antibodies, effector cells, or pneumococci were run in parallel and incubated at 37°C with shaking. Aliquots were removed before and after incubation, serially diluted in sterile PBS, and samples were plated on blood agar plates for CFU counting. A total of 5x10 4 5x10 DMF-differentiated HL-60 cells 2 The wells were incubated with opsonized S. pneumoniae and complement for 45 min at 37°C / 5% CO2. Viable colony counts were performed after an incubation period of approximately 18 h under anaerobic conditions at 37°C. IVIG at a final dilution of 1:16 was used as a source of pathogen-specific antibodies for opsonization. Wells containing non-opsonized S. pneumoniae and heat-inactivated complement were used as controls.

[0098] Ex vivo studies PBMC isolation and ex vivo stimulation assay PBMCs were isolated from whole blood by Ficoll-Paque Plus (GE Healthcare Bio-Sciences). Briefly, whole blood was collected in heparin tubes and diluted with an equal volume of DPBS in a 50 ml Falcon tube. A volume of 30 ml of diluted blood was layered on top of the Ficoll and centrifuged at 400 g for 30 min at room temperature. Mononuclear cells (MNCs) form a defined cell layer at the interface between the plasma and Ficoll. The top layer (plasma) was carefully removed and the MNC layer was then collected in a new 50 ml Falcon tube. The MNCs were washed by refilling with DPBS and centrifuged at 300 g for 10 min. The supernatant was discarded and the cell pellet was resuspended in complete RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin and 200 mM L-glutamine. Intracellular cytokine (IFN-γ and IL-17A) assays were performed using PBMCs to examine their antigen-specific and T cell responses to the three pneumococcal antigens. Briefly, PBMCs (1x10 6 PBMCs (1000 / well) were freshly cultured in RP10 (RPMI-1640 containing 10% FBS, 1 mM GlutaMAX-I supplement, 55 μM 2-mercaptoethanol, 50 U / mL penicillin, 50 μg / mL streptomycin, and 10 mM HEPES) for 72 h (37° C., 5% CO2) in the presence of pneumococcal antigens adsorbed to CpG-chitosan. Brefeldin A (BFA; 5 μg / mL; Sigma-Aldrich) was included for the last 14 h. PBMCs were then fixed, permeabilized, and labeled with cell surface marker-specific antibodies (anti-CD3 FITC, anti-CD4 PerCP Cy5.5, and anti-CD8 APC; all from BD Biosciences) and anti-IFN-PE and IL-17A-APC (Invitrogen Corporation) to detect intracellular cytokines. Proliferative responses were assessed using Ki67 labeling.

[0099] In vivo mouse studies Mouse nasopharyngeal carriage model.

[0100] S. pneumoniae was streaked onto blood agar and grown overnight at 37°C, 5% CO2. S. pneumoniae was identified by the presence of an α-hemolytic zone around each colony and an inhibition zone around an optotin disk. A sweep of colonies was inoculated into brain heart infusion (BHI) broth and grown statically overnight at 37°C. The following day, 750 μl of the overnight growth was subcultured into BHI containing 20% ​​(v / v) fetal calf serum (FCS) and grown statically for 4–6 h until mid-log phase growth (OD500 0.8), at which point the broth was divided into 500 μl aliquots and stored at -80°C in BHI broth with FCS until use for up to 1 month. Mice were anesthetized with 2.5% isofluorane in oxygen and aliquoted for a total of 10 6 CFU / 10ul of bacterial suspension was administered intranasally using a micropipette. At the designated time points, animals were euthanized in a CO2 chamber and various tissues were dissected.

[0101] Nasopharyngeal-to-brain meningitis model Mice were anesthetized with a mixture of O2 and isofluorane and 10 8 Mice were infected intranasally at 10 CFU / 10 ul. Mice were scored periodically for clinical signs of disease and culled at predetermined times post infection by cervical dislocation, and tissues were aseptically removed for determination of viable bacterial counts. Blood samples were taken by cardiac puncture.

[0102] Mouse challenge tests in pneumonia and sepsis models Pneumococcal isolates were streaked onto 5% blood agar plates. Single colonies were grown overnight in BHI and then subcultured in BHI + 20% serum for storage at -80°C. Inocula were administered to mice in sterile PBS vehicle solution at various densities: for the pneumonia model, mice were anesthetized and 10 6 In the sepsis model, mice were infected intranasally with 10 6 CFU / 100 μl / mouse were administered intravenously. All experiments were performed over a 72 hour monitoring period.

[0103] Immunization protocol.

[0104] The immunogenicity of the trivalent protein-based vaccine + CpG chitosan formulation (PrPV) of the present invention was evaluated in C57BL / 6 mice (1 week old infants vs. 6 week old adults) after intranasal immunization. A volume of 10 μl of the solution was administered intramuscularly on days 7, 14 and 21 to groups of n=10 infant mice / group. Adult mice were immunized in the same manner at weeks 6 (prime), 8 (boost 1) and 10 (boost 2). Control groups received either PBS or free antigen solution, i.e., antigen solution without adjuvant. In preliminary experiments, the optimal vaccine dose (ranging from 1-10 μg / animal) was determined by measuring the antibody response 2 weeks after the third vaccination. In addition to a negative / placebo control group, the vaccine formulation was also evaluated against the currently licensed vaccine PCV-13 (trade name Prevenar13®).

[0105] Passive transition studies.

[0106] Adult mice immunized with either vehicle or a vaccine PrPV formulation containing ABC-T+PavA+ZmpB adsorbed to CpG-chitosan were selected by cardiac puncture and serum (100-150 μl / mouse) was transferred intravenously to naive animals. Four hours after transfer, animals were challenged with S. pneumoniae either intranasally (pneumoniae model) or intravenously (sepsis model). Mouse survival and tissue viability were recorded.

[0107] Measurement of immune response.

[0108] To characterize anti-pneumococcal adaptive immunity, antigen-specific serum antibody responses (total IgG, antigen-specific IgG1, IgG2a) were determined using a commercially available sandwich ELISA assay (R&D systems). Cell-mediated immunity was also determined by measuring pneumococcal antigen-specific CD3+CD4+ T cell responses: single cell suspensions were prepared from mesenteric lymph nodes and restimulated ex vivo in the presence or absence of pneumococcal antigens (ABC-T+PavA+ZpmB). Cytokine responses were determined using intracellular staining kits for Th1 / Th2 and Th17 panels (BD Biosciences), with a special focus on IFN-γ / IL-4 and IL-17.

[0109] Comparative genomic analysis One hundred and forty pneumococcal isolates (n=70) from Malawian adults and children with meningitis to bloodstream infection (bacteremia; n=70) without clinical meningitis were subjected to Illumina sequencing. Mapped genes were clustered and divided into three categories: orthologous genes shared by all isolates (core genes), orthologous genes shared between two or more isolates (accessory genes), and genes unique to one isolate. A total of 3,164 orthologous gene clusters were discovered: 45% (1,428 core genes) were present in all of the tested isolates, 1,612 (51%) were present in two or more isolates, and the remaining 124 genes (4%) were not grouped within a cluster. Unexpectedly, no differences were found between the core genome or accessory gene content of the 140 isolates. To compare the evolutionary history of gene gain / loss events (unpublished), an approach built on stochastic mixing (GLOOME) was employed. Here, a small subset of meningitis-specific genes was identified, namely pneumococcal adherence virulence factor A (PavA, gene ID: 12889101); ATP-binding cassette / transporter permease (ABC-T, gene ID: 13695552); and zinc metalloprotease B (ZmpB, NCBI gene ID: 7680834). These genes were of interest based on their low sequence homology to human proteins, their documented functions, and their surface expression patterns. We then extended the genomic analysis to a dataset consisting of more than 25,0000 pneumococcal isolates. In line with the goal of designing a vaccine formulation that provides global coverage across ages, we evaluated a set of isolates comprising 44 different pneumococcal serotypes originating from both developed and developing countries, spanning six continents (Asia, Africa, North and South America, Europe, and Australia) and 36 different countries.

[0110] Due to their important role in pneumococcal virulence, it was expected that the three pneumococcal proteins might also be the most variable in sequence. For example, this has been demonstrated in the case of pneumolysin, where 21 different allelic variants have been identified so far. Therefore, the degree of gene sequence variability (allelic variants) was determined for each identified candidate gene, their genetic relationships were assessed, prevalence at the overall level, serotype-specific associations were determined, and the most common variant forms were selected.

[0111] statistical analysis For comparison of multiple groups, the statistical significance of the endpoints was assessed by one-way ANOVA followed by Tukey's multiple comparison post-hoc test if the data were normally distributed. If the data were not normally distributed, the statistical significance of the endpoints was assessed by Kruskal-Wallis test followed by Dunn's multiple comparison post-hoc test. For comparison of two groups, an unpaired two-tailed Student's t-test was used. Data are shown in bar graphs as mean ± SEM. Pneumococcal challenge survival curves were analyzed using the log-rank (Mantel-Cox) test. Results with p-values ​​less than 0.05 were considered significant (*p-value < 0.05, **p-value < 0.01, ***p < 0.001, ****p < 0.0001). Statistical analysis was performed using Graph Pad Prism.

[0112] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including but not limited to" and are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0113] It should be understood that any property, integer, feature, compound, chemical moiety or group described in connection with a particular aspect, embodiment or example of the invention is applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. All of the features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel, or any novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings), or any novel, or any novel combination of steps of any method or process so disclosed.

[0114] The reader's attention is directed to all articles and documents related to this application that are filed contemporaneously with or prior to this application and that are open to public inspection herewith, and the contents of all such articles and documents are incorporated herein by reference. The following examples are illustrative and are not intended to limit the scope of the invention.

[0115] [Example 1] Levels of specific IgG antibodies against ZmpB, AbcT and PavA were determined by enzyme immunoassay in sera obtained from n=12 children (2-7 years old), n=12 healthy adults (18-45 years old), n=12 adult patients with pneumococcal hospital-acquired pneumonia (CAP), n=12 adults with HIV, and n=12 elderly patients aged 65-75 years. High-binding microtiter plates (Corning) were coated with 5ug / ml total protein (either as single AbcT, PavA, ZmpB or in combination of AbcT+PavA+ZmpB) in carbonate-bicarbonate buffered saline (50mM, pH 9.2) and incubated overnight at 4°C. Blocking was performed with 1% w / v BSA in PBS. Sera were serially diluted 1:20 to 1:40,960 in 0.5% BSA w / v in PBS. The secondary antibody used was alkaline phosphatase-conjugated monoclonal anti-human IgG (Sigma, A2064-1ML). An OD of ≥ 0·04 (2 standard deviations of control serum) for all measurements was considered positive. Samples with undetectable anti-ZmpB or anti-AbcT / PavA were assigned a value corresponding to half the detection limit. Protein-specific IgG results are given semiquantitatively based on OD values ​​(OD units at 405 nm). These were calculated from the mean OD readings of triplicate samples after subtracting the OD readings of serum negative control wells.

[0116] Figure 4 shows the binding activity of human serum antibodies taken from healthy adult volunteers or individuals susceptible to pneumococcal invasive disease, i.e. children, elderly and patients with HIV. The results suggest that three proteins described herein, i.e. ABC-T, PavA and ZmpB, contain antibody binding regions (or B cell epitopes) recognized by human antibodies, thus supporting their use as potential vaccine candidates.

[0117] [Example 2] Figure 5 shows results from the validation of ubiquitously expressed conserved pneumococcal antigens in a meningitis mouse model. S. pneumoniae in 10 μl of sterile saline was applied nasally or injected into the cisterna magna. 100 μL of brain tissue homogenate was transferred to wells on a 96-well plate and 10-fold serial dilutions were performed in sterile PBS. Aliquots of 60 μL were spotted onto blood agar plates containing 10 μg / ml gentamicin. Bacterial loads (CFU) from tissue homogenates were enumerated from infected animals in a preclinical meningitis model at days 0, 1, 3, 7, 10 and 14 post-infection. These results indicate that the virulence of S. pneumoniae, i.e., its ability to spread and invade host tissues, is significantly attenuated when expression of ABC-T, PavA and ZmpB proteins is null. These findings strongly support the use of these proteins as vaccine candidates.

[0118] [Example 3] Mice were immunized with a mixture of three antigenic determinants corresponding to SEQ ID NOs: 1, 2 and 3 of the present invention, alone (Ag) or in combination with alum (Alum+Ag) or CpG-chitosan (CpG-chitosan+Ag) / PrPV using a prime-boost regimen, and 5.10 5 CFU / mouse were challenged intranasally in 50 μl. Mice were monitored for the onset of disease signs in (FIG. 6A) adult and (FIG. 6B) neonatal C57BL / 6 mice, and clinical pain scores were recorded (n=10 / group).

[0119] Antibody dose response-immunization studies were performed with a mixture of various amounts of three antigenic determinants (Ags) corresponding to SEQ ID NOs: 1, 2 and 3 of the present invention, each in combination with a fixed amount of CpG-chitosan. Antigen-specific antibody (Spe-IgG1, Spe-IgG2a) levels in antisera were measured from mice (n=10) vaccinated with prime+booster doses of recombinant protein antigens (Ags) adsorbed to CpG-chitosan at 0.1, 1.0 and 10 μg antigen / mouse. Antisera from PBS / vehicle-vaccinated animals were included as controls. Results are shown as mean±SEM (Figure 7A). Cell-mediated immune responses were determined using mesenteric lymph nodes harvested 35 days after immunization upon ex vivo restimulation with PavA+ABC-T+ZmpB (500 μg / mL) for 72 hours. Enzyme-linked immunosorbent assays (ELISA) were performed to measure IL-17 and IFN-γ cytokine production. (Figure 7B).

[0120] Mice were vaccinated with PBS (vehicle), adjuvant alone (alum or CpG-chitosan), or adjuvant + antigen (alum + 1.0 μg Ag; CpG-chitosan + 1.0 μg Ag). At 35 days post-vaccination, mice were vaccinated with 5x10 5 CFU of a highly virulent pneumococcal serotype 1 clinical isolate was challenged intranasally and selected at the onset of disease symptoms. Vaccine antigen-specific responses in mesenteric lymph nodes harvested 35 days after immunization were determined by ex vivo restimulation with PavA+ABC-T+ZmpB (500 μg / mL) for 72 hours. Enzyme-linked immunosorbent assays (ELISAs) were performed to measure IL-17 (Th1) and IL-4 (Th2) cytokine production. Mean ± SEM. *p<0.05; **: p<0.01 Kruskal-Wallis and Dunn post-hoc test vs. vehicle (Figures 7C and 7D). Vaccination with PrPV formulations enhances disease protection compared to conventional alum adjuvant.

[0121] [Example 4] The protective efficacy of PrPV was compared against PCV-13 in models of pneumococcal sepsis and pneumonia in adult (FIG. 8A) and neonatal (FIG. 8B) mice. Mice were immunized with recombinant ZmpB, ABC-T, and PavA in combination with Alhydrogel ("Alum+Ag") and challenged with 5x10 S. pneumoniae. 5 CFU(in). Animals were monitored for survival for 7 days after challenge. The number of surviving mice is plotted as a percentage of the total (n=10 mice / group). Figure 8C shows bacterial density in the lungs of individual newborn mice 3 days after challenge. Blood was collected by cardiac puncture and spotted on blood agar plates for viable CFU counts (Figure 8D). For all experiments, naïve mice and mice immunized with adjuvant only served as negative controls.

[0122] In further experiments, mice were immunized with recombinant ZmpB, ABC-T and PavA corresponding to SEQ ID NOs: 1, 2 and 3 in combination with CpG-chitosan (PrPV) followed by immunization with 1x10 S. pneumoniae 6 CFU were challenged in. Animal survival was monitored up to 5 days after challenge. The number of surviving mice was plotted as a percentage of the total (n=10 mice / group). Non-vaccine pneumococcal serotypes, such as 8, 11A and 33F, which have been reported as emerging serotypes in England and Wales, were tested to evaluate the breadth of protective scope of the vaccine formulation. PCV-13 was used as a comparison (n=10 mice / group). Mouse survival (Figure 9A) and clinical pain scores were monitored (Figure 9B). These results indicate that the PrPV formulation of the present invention provides better immune protection against non-vaccine serotypes compared to the commercial vaccine PCV-13 (Prevenar-13).

[0123] [Example 5] Isolated human PBMCs were stimulated with PCV-13 (1:50) and PrPV for 72 h, respectively, and T cell responses were examined by intracellular staining. + CD4 +Gating out T cells and Ki67 in the rectangular gate with percentages shown at the top + Shown to represent proliferating T cells. IFNγ + CD4 + Th1 cells (B) and IL-17A + CD4 + The percentage of Th17 cells (C) is shown in the upper right quadrant of the dot plot. Data from two individual donors are summarized in a bar graph. MC: medium control. The results showed that PrPV inhibited CD4 + It has been shown that it activates T cell responses. In a similar experiment, isolated human PBMCs were stimulated with PCV-13 (1:50), chitosan_CpG and PrPV for 7 days, respectively, and plasma B cell activation was subsequently detected. Memory B cells (CD19 + CD27 + IgD - ) plasma B cells (CD27 high CD38 high The percentage of PrPV-activated plasma B cells in human PBMCs (gated as PrPV+) is shown in the contour plot below (Figure 11). Data are representative of two individual donors. These results show that PrPV activates plasma B cells in human PBMCs.

[0124] [Example 6] Adult mice (n=10 / group) immunized with either vehicle or PrPV formulations (containing ABC-T / PavA / ZmpB corresponding to SEQ ID NOs: 1, 2, and 3 adsorbed to CpG-chitosan) were selected by cardiac puncture and serum (100-150 μl / mouse) was transferred intravenously to naive animals. Four hours after transfer, animals were challenged intravenously with a disease-inducing dose of S. pneumoniae (sepsis model). Mouse survival times (Figure 12A) and viable CFU counts in blood and lungs (Figure 12B) were recorded. These results show that serum from PrPV-immunized mice confers passive immunity in naive mice.

[0125] Human PBMCs from healthy volunteers were isolated from whole blood by Ficoll-Paque Plus using density gradient separation and cultured for 7-8 hours in the presence of PrPV compositions. Supernatants were collected, concentrated using Vivaspin concentrators (10K MWCO), and administered intravenously to adult naive mice. Four hours after transfer, animals (n=5 / group) were challenged with S. pneumoniae either intranasally (pneumoniae model) or intravenously (sepsis model). Mouse survival times (Figure 13A) and viable CFU counts in blood and lungs (Figure 13B) were recorded. These results indicate that concentrated supernatants from PrPV-stimulated human PBMC cultures confer passive immunity in naive mice.

[0126] [Example 7] Adult mice (n=10 / group) immunized with either vehicle or PrPV formulations were selected by cardiac puncture and serum (100-150 μl / mouse, dilution 1:16) was collected for use in a functional opsonophagocytic killing assay (OPK assay). OPK was performed to test the ability of vaccinated mouse antisera to opsonize and mediate killing of live pneumococci. A reference opsonophagocytosis assay was used, whereby reaction mixtures were prepared consisting of heat-inactivated serum obtained from vaccinated versus non-vaccinated mice, differentiated HL-60 cells or freshly isolated human neutrophils, pneumococci, and infant rabbit complement. Control reactions lacking all components except complement and / or antibodies, effector cells or pneumococci were run in parallel and incubated at 37 °C with shaking. The results show that serum from PrPV-immunized mice promotes in vitro opsonization of pneumococci and outcompetes the licensed PCV-13 vaccine (Figure 14).

[0127] [Example 8] The antibody dose response of single proteins combined with CpG-chitosan against PrPV compositions was studied. Antigen-specific antibody (Spe-IgG1, Spe-IgG2a) levels were measured in antisera from mice (n=10) vaccinated with single proteins or PrPV compositions (labeled "CpG-chitosan+Mix") at a prime+booster dose of 1.0 μg / mouse. Results are shown as mean ± SEM (Figure 15A). Cell-mediated immune responses upon vaccination with single vs. mixed protein formulations were also studied. Vaccine antigen-specific responses in mesenteric lymph nodes harvested 35 days after immunization were determined by ex vivo restimulation with PavA, ABC-T, or ZmpB (500 μg / mL) for 72 hours. Enzyme-linked immunosorbent assays (ELISAs) were performed to measure IL-17A (Th17) and IFN-γ (Th1) cytokine production (Figure 15B). These results indicate that vaccination with the mixed protein formulation displays enhanced immunogenicity compared to the single protein-adjuvanted CpG formulation.

[0128] Adult mice (n=10 / group) were vaccinated with PBS (vehicle), single antigens + CpG-chitosan or PrPV. 35 days after vaccination, mice were vaccinated with 5x10 5 CFU of a highly virulent pneumococcal serotype 1 clinical isolate was challenged intranasally and selected at the onset of disease symptoms. Figure 16A shows the survival curves. Viable bacterial counts in blood and lungs were determined at preselected intervals after intranasal infection (Figure 16B). Tissue samples were homogenized using an IKA T10 homogenizer. Viable bacterial counts in lung cell suspensions and blood samples were determined by serial dilution in sterile PBS and plating on blood agar containing 5% (v / v) defibrinated horse blood (Oxoid) and 10ug / ml gentamicin (sigma). Once dry, plates were incubated overnight at 37°C in 5% (v / v) CO2 and bacterial colony counts were assessed the following day. These results indicate that vaccination with the combined protein formulation of the present invention provides enhanced disease protection compared to single protein adjuvanted formulations.

[0129] Adult mice (n=5 / group) were vaccinated with PBS (vehicle) or adjuvant + antigen (CpG-chitosan + 1.0 μg Ag in paired protein preparations). The paired protein preparations were proteins corresponding to SEQ ID NOs: 1, 2 and 3 (PavA + ABC-T or ABC-T + ZpmB or PavA + ZmpB). At 35 days post vaccination, mice were vaccinated with 5x10 5 CFU of a highly virulent pneumococcal serotype 1 clinical isolate was challenged intranasally and selected at the onset of disease symptoms. Figure 17A shows the survival curves. Viable counts in blood (Figure 17B) and lungs (Figure 17C) were determined at preselected intervals after intranasal infection. Tissue samples were homogenized using an IKA T10 homogenizer. Viable counts in lung cell suspensions and blood samples were determined by serial dilutions in sterile PBS and plating on blood agar containing 5% (v / v) defibrinated horse blood (Oxoid) and 10ug / ml gentamicin (sigma). Once dry, plates were incubated overnight at 37°C in 5% (v / v) CO2 and bacterial colony counts were assessed the following day. These results indicate that vaccination with paired protein formulations induces a protective effect in the following order: PavA+ZmpB, PavA+ABC-T, ABC-T+ZmpB.

[0130] [Example 9] The effect of the immunological composition of the invention (PrPV) on pneumococcal colonization and shedding was evaluated in C57BL / 6 mice after intramuscular immunization. A volume of 10 μl of the solution was administered intramuscularly to n=10 suckling mice / group on postnatal days 7, 14 and 21. Control groups received either adjuvant alone or free antigen solution, i.e., antigen solution without adjuvant. Two weeks after the second booster, the inoculum was administered at 10 μl. 5Mice were challenged intranasally with pneumococcal CFU / 10 μl / mouse in sterile PBS vehicle. Bacterial density (by tissue homogenization and nose tap) was determined over a 15-day monitoring period ( FIG. 18 ). Clearance of pneumococci from the human nasopharynx can have potentially harmful consequences. Thus, a rational approach is to develop a vaccine that allows pneumococci to persist in the nasopharynx but prevents dissemination to body sites that result in infection. The PrPV composition of the present invention provides protection against pneumococcal disease but does not affect pneumococcal ecology in the mouse nasopharynx.

[0131] [Example 10] The immunological response induced by PrPV in mice was evaluated by flow cytometry. A volume of 10 μl of PrPV solution was administered intramuscularly to n=10 suckling mice / group on postnatal days 7, 14 and 21. Further groups of mice received either adjuvant alone ("CpG-chitosan") or vehicle control solution. Two weeks after the second booster, mice were administered 2.10 μl of PrPV in 50 μl / mouse. 5 CFU pneumococcus serotype 1. Immune cell proliferation measured in mesenteric lymph nodes 3 days after pneumococcal infection is shown in Figure 19. The results show that the composition of the invention (PrPV) is potent in inducing a proliferative response (measured by the percentage of CD3+CD4+Ki-67 positive cells) in lymphoid structures of PrPV-immunized mice.

[0132] [Example 11] Adult mice (n=5 / group) were vaccinated with PBS (vehicle), CpG-chitosan adjuvant alone ("CpG-chitosan"), or PrPV (i.e., CpG-chitosan + 1.0 μg SEQ ID NO:1, 1.0 μg SEQ ID NO:2 + 1.0 μg SEQ ID NO:3 / mouse). 35 days after vaccination, mice were vaccinated with 5x10 5Highly virulent pneumococcal serotype 1 clinical isolates of CFU were nasally challenged and sorted after 24 hours. The vaccine antigen-specific responses in the lung single cell suspensions collected at the experimental endpoint were determined by ex vivo restimulation with PavA, ABC-T or ZmpB (500 μg / mL) for 24 hours. Briefly, the lungs of the mice were dissected and transferred to small tubes containing Hank's balanced salt solution (HBSS). The remaining tracheal tissue was carefully removed, and the lung tissue was cut into small pieces using a Petri dish. A digestion solution containing 300 U / ml of collagenase and 75 μg / ml of DNase I was added to the lung pieces (2 ml / mouse) and incubated at 37 °C for 40 minutes to 1 hour. The digested tissue was passed through a cell strainer, centrifuged, and the pellet was resuspended in 5 ml of 1x RBC lysis buffer (incubation for 4 minutes), the cells were counted, and seeded at a density of 4x10 6 cells / ml. The induced single cell suspensions were stained with anti-CD62L, anti-CD44 or anti-IL-17A antibodies conjugated to their respective fluorophores and analyzed by flow cytometry. Figure 20A shows representative dot plots of each group, namely naive, antigen, CpG-chitosan (adjuvant alone) and PrPV, and the upper right quadrant shows the percentage of CD62L+CD44+IL17A positive cells. Figure 20B shows that the percentage of CD62L+CD44+IL17A positive cells in PrPV-immunized mice is significantly higher than that of all other groups (**<p<0.01;***p<0.001). These results suggest that vaccination with PrPV elicits significantly higher IL-17A-mediated protective immunity compared to the vehicle or adjuvant-only groups.

[0133] [Example 12] The protective efficacy of the antigens of the invention was tested when co-formulated with β-glucan (β1,3 / 1,6-D-glucan, IRI1501, isolated from Saccharomyces cerevisiae). Adult mice were vaccinated with PBS (vehicle), adjuvant alone (beta-glucan-based formulation supplemented or not with CpG), or adjuvant + a mixture of ZmpB and PavA antigens corresponding to SEQ ID NOs: 3 and 2 (beta-glucan-based adjuvant + 1.0 μg Ag supplemented or not with CpG). 35 days after vaccination, mice were vaccinated with 5x10 5 CFU of a highly virulent pneumococcal serotype 1 clinical isolate were challenged intranasally and selected at the onset of disease symptoms. Survival curves (Figure 21A) and viable numbers (CFU) were determined in blood (Figure 21B) and lung tissue (Figure 21C). A protective efficacy of 40% was obtained in mice administered either (antigen alone) or (antigen + CpG + β-glucan), suggesting that β-glucan-based adjuvanted formulations do not interfere with the protective efficacy conferred by the antigens of the invention and may be developed into potent pneumococcal vaccines upon further optimization, e.g., dose, immunization schedule, and / or reformulation with other immunostimulatory compounds.

Claims

1. 1. An immunogenic composition comprising at least two antigenic determinants, said antigenic determinants being derived from at least two proteins selected from ABC-T, PavA and ZmpB of the bacterium Streptococcus pneumoniae.

2. 1. An immunogenic composition comprising one or more genetic constructs encoding at least two antigenic determinants, said antigenic determinants being derived from at least two proteins selected from ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

3. 1. An immunogenic composition comprising at least one antigenic determinant and one or more genetic constructs encoding at least one different antigenic determinant, said antigenic determinant being derived from at least two proteins selected from ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

4. The immunogenic composition according to any one of claims 1 to 3, wherein said antigenic determinants are derived from all three proteins ABC-T, PavA and ZmpB of the Streptococcus pneumoniae bacterium.

5. said ABC-T derived antigenic determinant comprises or consists of the protein sequence of SEQ ID NO:1, or an immunologically effective fragment thereof, or an immunologically effective analogue of said sequence or fragment; and / or said PavA-derived antigenic determinant comprises or consists of the protein sequence of SEQ ID NO:2, or an immunologically effective fragment thereof, or an immunologically effective analogue of said sequence or fragment; and / or The ZmpB-derived antigenic determinant comprises or consists of the protein sequence of SEQ ID NO: 3, or an immunologically effective fragment thereof, or an immunologically effective analogue of said sequence or fragment. The immunogenic composition according to any one of claims 1 to 4.

6. The immunogenic composition according to any one of claims 1 to 5, further comprising an immunostimulant, preferably said immunostimulant being an adjuvant.

7. The adjuvant is selected from the group consisting of substances that have a stimulatory effect on cytosolic pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), nucleic acid-sensing receptors such as C-type lectin receptors (CLRs), retinoic acid-inducible gene I (RIG-I), or nutrient sensors such as mTOR and GCN2; and substances that stimulate the following pathways: MyD88 / TLR-signaling pathway, cGAS-stimulator of interferon genes (STING) pathway, NF-κB pathway, any stress, cell death and tissue damage pathways that result in the release of damage-associated molecular patterns (DAMPs) such as nucleic acids, uric acid, ATP and proteins that activate the innate immune system (necrosis / necrosis pathway).

7. The immunogenic composition of claim 6, comprising one or more substances selected from: substances acting on at least one of the signaling pathways leading to immune cell recruitment or epigenetic changes that maintain the innate immune system in an alert state, i.e. memory-like state, for extended periods of time; substances having the property of inducing inflammasome activation resulting from cell differentiation towards CD4+ Th2 (interleukin-4), Th1 (interferon-gamma)- or Th17 (IL17A) mediated immunity; immune enhancers; polysaccharide-based substances acting on IL-1β, CLR and TNF-α signaling pathways); delivery systems and mucosal adjuvants.

8. The adjuvant may be any of the following: bacterial protein and / or polysaccharide substances and analogs thereof, such as capsules, toxins / toxoids and analogs thereof, TLR ligands and analogs thereof, agonists such as Pam3CSK4, Pam2CSK4, MPLA (LPS derivatives), CpG, PolyI:C, CpG motifs; microparticles and nanoparticles such as chitosan or β-glucan, flagellin, poly(lactic-co-glycolic acid) or PLGA-poly-l-lysine / poly-gamma-glutamic acid (PLGA-PLL / gamma PGA), or microparticles and nanoparticles made of lipid-based scaffolds co-formulated with any other immunostimulants such as monophosphoryl lipid A (MPLA), polyethylene glycol (PEG), oligomannose, poly(I:C), extracellular vesicles (EV) such as outer membrane vesicles (OVM); Montanide; oil-based encapsulation or emulsion-based systems, including water-in-oil, oil-in-water and thermoreversible oil-in-water emulsions such as ISA-51, Montanide ISA-720 and analogues thereof; alum or aluminum salt adjuvants; liposomes, virosomes, archaeosomes, outer membrane vesicles, niosomes, saponins and immune stimulating complexes (ISCOMs), polymeric particles; cytokines, including pro-inflammatory cytokines such as IFN-γ, IL-1, IL-2, IL-4, IL-12, IL-17A / F, GM-CSF and MPI; virus-like particles (VLPs); bacterial components such as detoxified variants of LPS such as monophosphoryl lipid A (MPL); muramyl dipeptides ( MDP lipophilic and hydrophobic), QS21, PLGA, CT, liposome-based cationic adjuvant formulations such as CAF01-09 (composed of Span 80, polyoxyethylene cetyl-stearyl ether, mannitol, squalene) and analogs composed of cholesterol, phosphatidylcholine and phosphatidylserine, or α-Gal ceramide, NOD-like receptor protein 3 (NLRP3) inflammasome, apoptosis-associated speck-like protein (ASC) containing CARD, NLR family CARD domain-containing protein 4 (NLRC4) inflammasome, Absent in melanoma2 (AIM2) inflammasome, dsRNA:poly(I:C), poly-IC:LC, monophosphoryl lipid A (MPL), LPS, flagellin, imidazoquinolines: imiquimod (R837), resiquimod (848), bacterial molecules or derivatives including CpG oligodeoxynucleotides (ODN), muramyl dipeptide (MDP), saponin (QS-21), polysaccharide capsules and analogues thereof, toxins / toxoids and analogues thereof, unmethylated DNA (CpG) and analogues thereof, cytokines such as IL-2, IL-12, TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF), RANTES (Regulated upon Activation and Positive Responses), 8. The immunogenic composition of claim 6 or 7, comprising one or more substances selected from: inflammatory cytokines such as macrophage inflammatory protein (MIP)-1α, co-stimulatory or adhesion molecules such as CD80, lymphocyte function associated antigen-3 and polyarginine tails, polysaccharide-based substances with properties similar to those presented by chitosan, e.g. CpG-delta inulin, cochleates, virus-like particles (VLPs), microparticles such as virosomes, PLA (polylactic acid), PLG (poly[lactide-coglycolide]), cholera toxin (CT) derivatives, and heat-labile enterotoxins (LTK3 and LTR72).

9. 9. The immunogenic composition according to any one of claims 6 to 8, wherein the adjuvant comprises one or more substances selected from a TLR agonist, an agent capable of inducing a CD4+ T cell mediated immune response (in particular with a Th1 and / or Th17 differentiation profile), an oil-in-water emulsion, a water-in-oil emulsion, an agent acting on the MyD88 / TLR signalling pathway, an agent acting on the cGAS-Stimulator of Interferon Genes (STING) pathway and a particulate polysaccharide substance, preferably wherein the adjuvant comprises a combination of a TLR agonist and an agent capable of acting on the MyD88 / TLR signalling pathway or the cGAS-Stimulator of Interferon Genes (STING) pathway, e.g. a combination of CpG with chitosan and / or beta-glucan.

10. Pneumococcal surface protein A (PspA), pneumococcal choline-binding protein A (PcpA), secreted 45-kDa protein Usp45-hydrolase (PcsB), serine / threonine protein kinase (StkP), peptide permease enzyme, manganese ABC transporter (PsaA), pneumolysin D (PlyD), pneumolysin toxoid (dPly), choline-binding protein A (CbpA) and D (CbpD), histidine triad protein D (PhtD), histidine triad protein E (PhtE), histidine triad protein A (PhtA) 10. The method according to claim 1, further comprising one or more further antigenic determinants from encapsulated or conjugated Streptococcus pneumoniae bacteria selected from the group comprising: histidine triad protein B (PhtB); pneumococcal serine-threonine uptake protein A (PiuA), protein cell wall segregation (PcsB), pneumococcal serine-threonine kinase (StkP) and analogs, plasmin and fibronectin binding protein A (PfbB), SP0148, SP1912 and SP2108, SP0785, SP1500 and SP2216. An immunogenic composition of.

11. The immunogenic composition of any one of claims 1 to 10, which is a liquid, suspension, tablet or spray, or is lyophilized.

12. The immunogenic composition according to any one of claims 1 to 11, delivered by a group comprising virus-like particles (VLPs), outer membrane vesicles (OMVs), liposomes, immune stimulating complexes (ISCOMs), polymeric, non-degradable, biodegradable substances, natural polymeric compounds, starches, alginates, celluloses, biosynthetic substances, poly beta-hydroxybutyrate (PHB), copolymers, polylactic acid (PLA), polyurethanes, poly(lactic-co-glycolic acid) (PLGA), polymethyl methacrylate resins (PMMA), probiotics, Lactobacilli and Bifidobacteria species; viral vector systems; modified vaccinia virus Ankara (MVA), poxviruses, adenoviruses and bacteriophages.

13. A vaccine comprising the immunogenic composition according to any one of claims 1 to 5.

14. A vaccine according to claim 13, comprising one or more of the features according to any one of claims 6 to 12.

15. Use of an immunological composition according to any one of claims 1 to 12 or a vaccine according to claim 13 or 14 in the manufacture of a medicament for a method of treating or preventing a Streptococcus pneumoniae bacterial infection.

16. Use of an immunological composition according to any one of claims 1 to 12 or a vaccine according to claim 13 or 14 in the manufacture of a medicament for a method of immunizing against a Streptococcus pneumoniae bacterial infection.

17. 17. The use according to claim 15 or 16, wherein said immunological composition or vaccine is administered orally, nasally, by inhalation or by injection.

18. 18. The use according to claim 17, wherein the administration is by a route selected from the group comprising intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, intradermal, intranasal and inhalation.

19. 15. An immunological composition according to any one of claims 1 to 12 or a vaccine according to claim 13 or claim 14 for use in the treatment or prevention of a Streptococcus pneumoniae bacterial infection in an individual, and / or for immunizing against a Streptococcus pneumoniae bacterial infection in an individual, and / or for diagnosing or screening for a Streptococcus pneumoniae bacterial infection in an individual.

20. A kit comprising an immunogenic composition according to any one of claims 1 to 12 or a vaccine according to claim 13 or claim 14, and means for administering said immunogenic composition or vaccine to an individual.

21. 1. Use of at least one antigenic determinant derived from a protein selected from ABC-T, PavA and ZmpB of a Streptococcus pneumoniae bacterium and at least one antigenic determinant derived from a protein selected from a different one of ABC-T, PavA and ZmpB of a Streptococcus pneumoniae bacterium in the manufacture of a medicament for a method of treating or preventing a Streptococcus pneumoniae bacterial infection in an individual, or a method of immunizing against a Streptococcus pneumoniae bacterial infection in an individual, the method comprising administering a sufficient amount of the at least one antigenic determinant derived from a protein selected from ABC-T, PavA and ZmpB of a Streptococcus pneumoniae bacterium and simultaneously or subsequently administering a sufficient amount of the at least one antigenic determinant derived from a protein selected from a different one of ABC-T, PavA and ZmpB of a Streptococcus pneumoniae bacterium.

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