Pneumococcal fusion protein vaccine

Abstract

The present invention relates to a technique for preventing and / or treating pneumococcal infections.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 730,199, filed on September 12, 2018, the content of which is hereby incorporated herein by reference in its entirety. Background of the Invention

[0003] Streptococcus pneumoniae remains a major cause of serious diseases, including bacteremia, sepsis, meningitis, and pneumonia in children and adults worldwide. Incidence and mortality rates are high in infants, young children, the elderly, and subjects with certain underlying medical conditions.

[0004] Streptococcus pneumoniae is a Gram - positive encapsulated coccus that colonizes the nasopharynx in approximately 5 - 10% of healthy adults and 20 - 40% of healthy children. Normal colonization becomes infectious when S. pneumoniae is carried to the eustachian tube, sinuses, lungs, bloodstream, meninges, joint space, bones, and abdominal cavity. S. pneumoniae infection is the most common cause of bacteremia, pneumonia, meningitis, sinusitis, and acute otitis media [CDC, 2010].

[0005] Pneumococcal disease can be either invasive or non - invasive. The most common form of non - invasive disease, non - bacteremic pneumococcal pneumonia, remains one of the most common causes of pneumonia hospitalization. Invasive pneumococcal disease (IPD) is defined as S. pneumoniae isolated from normally sterile sites (e.g., cerebrospinal fluid, blood, joint fluid, pleural fluid, or peritoneal fluid). The highest incidence of IPD occurs at the age extremes, i.e., in elderly adults and young children less than 2 years of age. In the United States, before the first pneumococcal vaccine became available, S. pneumoniae caused approximately 17,000 cases of invasive disease annually in children less than 5 years of age, including 700 cases of meningitis and 200 deaths [CDC, 2000]. The highest incidence and mortality rates have been reported in developing countries, but the disease burden is also substantial in industrialized countries.

[0006] S. pneumoniae has several virulence factors that enable the organism to evade the immune system. Examples include the polysaccharide capsule that prevents phagocytosis by host immune cells, proteases that inhibit complement - mediated opsonization, and proteins that cause lysis of host cells. Among the polysaccharide capsules, the presence of complex polysaccharides forms the basis for the differentiation of pneumococci into different serotypes. To date, nearly 100 S. pneumoniae serotypes have been identified.

[0007] Two vaccines against S. pneumoniae are currently available in the United States: pneumococcal conjugate vaccine (PCV13) or ) and pneumococcal polysaccharide vaccine (PPSV23 or ). PCV13 does not confer protection against most known pneumococcal serotypes. Although PPSV23 includes polysaccharide components of more pneumococcal serotypes than PCV13, the immune response it induces is neither long-lasting nor memory-based upon subsequent challenge. PPSV23 protects adults and the elderly from invasive pneumococcal disease; however, no consistent effect has been observed in pneumonia prevention [Gruber et al., 2008].

[0008] Accordingly, there is a medical need for a vaccine that provides T cell-dependent immunity against a broad range of pneumococcal serotypes. Summary of the Invention

[0009] The present disclosure addresses the lack of suitable techniques for preventing and / or treating pneumococcal infections. In particular, the present disclosure addresses the challenge of providing a vaccine with sufficient immunogenicity to protect against invasive pneumococcal disease and pneumonia. The techniques described herein can induce T cell and B cell responses and / or provide immunity against a broad range of pneumococcal serotypes, including one or more serotypes not included in commercially available vaccines such as PCV13 or PPSV23.

[0010] In some embodiments, when administered to a subject, the fusion proteins described herein can induce a higher Th17 response, at least 25% or more higher than the Th17 response induced by the individual antigenic components of the fusion protein, including, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or more. In some embodiments, when administered to a subject, the fusion proteins described herein can induce a higher Th17 response, at least 1.1-fold or more higher than the Th17 response induced by the individual antigenic components of the fusion protein, including, for example, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold or more.

[0011] In some embodiments, when administered to a subject, the fusion proteins described herein can induce an immune response against one or more representative non-vaccine pneumococcal serotypes that are not included in commercially available vaccines such as PCV13 or PPSV23. In some embodiments, when administered to a subject, the fusion proteins described herein can induce an immune response against one or more non-vaccine pneumococcal serotypes selected from the group consisting of 6B, 16F, 15A, and 35B. Brief Description of the Drawings

[0012] When combined with the attachedFigure 1 Upon reading, the teachings of the present invention described herein will be more fully understood based on the following description of various illustrative embodiments. It should be understood that the accompanying drawings described below are for illustrative purposes only and are not intended to limit the scope of the teachings of the present invention in any way.

[0013] Figure 1 is a schematic diagram of an exemplary CP1 fusion protein. Such an exemplary CP1 fusion protein comprises a biotin-binding protein such as truncated rhizavidin (e.g., amino acids 45-179 of wild-type rhizavidin (designated as Rhavi)), a first linker (e.g., the GGGGSSS linker), the SP1500 polypeptide (e.g., amino acids 27-278 of Streptococcus pneumoniae protein SP1500), a second linker (e.g., the amino acid sequence or linker AAA), and the SP0785 polypeptide (e.g., amino acids 33-399 of Streptococcus pneumoniae protein SP0785). In some embodiments, the CP1 fusion protein may further comprise a His tag. For the GGGGSSS linker, the SSS amino acid sequence may be from the Sac I site on the PET21 / 24b plasmid, where the GGGG amino acid sequence is added to produce a flexible linker with minimal steric hindrance. Alternatively, the GGGGSSS linker may be synthesized. The AAA amino acid sequence may be from the Not I site on the PET21 / 24b plasmid or may be synthesized.

[0014] Figure 2 Illustrates the immune response to the SP1500 and SP0785 polypeptide components of the exemplary fusion protein CP1. The left panel shows the Th17 response to the SP1500 polypeptide and the SP0785 polypeptide of the exemplary fusion protein CP1. The Th17 response is shown as the geometric mean concentration of IL-17A secreted in the culture medium after stimulation of peripheral blood samples from mice immunized with either cholera toxin alone or with the SP1500 or SP0785 polypeptide adjuvanted with cholera toxin. Each point on the graph represents the secreted IL-17A of one mouse. The right panel shows the protection from Streptococcus pneumoniae colonization after immunization with either cholera toxin alone or with the SP1500 or SP0785 polypeptide adjuvanted with cholera toxin and intranasal challenge with Streptococcus pneumoniae. Each point on the graph represents the Streptococcus pneumoniae CFU per nasal wash of one mouse. The horizontal bars in both graphs represent the geometric mean of the secreted IL-17A (left panel) and the geometric mean of the CFU per nasal wash (right panel) for each group, respectively. The data were statistically analyzed by the Mann-Whitney U test. CT: cholera toxin; CFU: colony-forming unit; ELISA: enzyme-linked immunosorbent assay; IL-17A: interleukin 17A. **p < 0.01; ***p < 0.001.

[0015] Figure 3 Describe the immune response to the exemplary fusion protein CP1. The left panel shows the Th17 response to the exemplary fusion protein CP1. The Th17 response is shown as the geometric mean concentration of IL-17 secreted in the culture medium after stimulation of peripheral blood samples from mice immunized with truncated rhizobium avidin (Rhavi) or CP1 (both adjuvanted with cholera toxin). Each point on the graph represents the secreted IL-17A from one mouse. The right panel shows protection from Streptococcus pneumoniae colonization after immunization with truncated rhizobium avidin (Rhavi), CP1, or heat-killed (inactivated) whole-cell pneumococcus (WCC) (adjuvanted with cholera toxin) and intranasal challenge with S. pneumoniae. Each point on the graph represents the S. pneumoniae CFU per nasal wash from one mouse. The horizontal bars in both graphs represent the geometric mean of the secreted IL-17A for each group (left panel) and the geometric mean of the CFU per nasal wash (right panel), respectively. The data were statistically analyzed by the Mann-Whitney U test. CT: cholera toxin; CFU: colony-forming unit; IL-17A: interleukin 17A; rhavi: truncated rhizobium avidin; (amino acids 45 - 179 of full-length rhizobium avidin). ***p < 0.001.

[0016] Figure 4 Demonstrate the presence of functional antibodies against a representative S. pneumoniae serotype (e.g., serotype 6B) in CP1 immune sera. Streptococcus pneumoniae serotype 6B was incubated at various dilutions in a modified opsonophagocytic assay (COPA) with heat-inactivated pre-immune (P0) and immune (P3) sera from each of two rabbits (87 and 88) immunized with CP1 adjuvanted with aluminum phosphate. After overnight incubation, the colony-forming units (CFU) were counted on blood agar plates for each dilution and serum combination. The presence of functional antibodies was indicated by killing of S. pneumoniae, i.e., a reduction in CFU after incubation with immune sera. Each vertical bar on the graph represents the CFU / ml for each sample of the designated CP1 serum and dilution at the designated time point (bottom of the graph).

[0017] Figure 5Demonstrate the presence of functional antibodies against representative Streptococcus pneumoniae serotypes (e.g., serotype 15A) in CP1 immune sera. In a modified opsonophagocytic assay (COPA), Streptococcus pneumoniae serotype 15A was incubated with heat-inactivated pre-immune (P0) and immune (P3) sera from each of two rabbits (87 and 88) immunized with CP1 adjuvanted with aluminum phosphate at a 1 / 2 dilution. After overnight incubation, colony-forming units (CFU) of each serum were counted on blood agar plates. The presence of functional antibodies was indicated by killing of Streptococcus pneumoniae, i.e., a decrease in CFU after incubation with immune sera. Each vertical bar on the graph represents CFU / ml of each sample of the designated CP1 serum at the designated time point (bottom of the figure).

[0018] Figure 6 Demonstrate the presence of functional antibodies against representative Streptococcus pneumoniae serotypes (e.g., serotype 35B) in CP1 immune sera. In a modified opsonophagocytic assay (COPA), Streptococcus pneumoniae serotype 35B was incubated with heat-inactivated pre-immune (P0) and immune (P3) sera from each of two rabbits (87 and 88) immunized with CP1 adjuvanted with aluminum phosphate at a 1 / 2 dilution. After overnight incubation, colony-forming units (CFU) of each serum were counted on blood agar plates. The presence of functional antibodies was indicated by killing of Streptococcus pneumoniae, i.e., a decrease in CFU after incubation with immune sera. Each vertical bar on the graph represents CFU / ml of each sample of the designated CP1 serum at the designated time point (bottom of the figure).

[0019] Figure 7 Demonstrate the presence of functional antibodies against representative Streptococcus pneumoniae serotypes. In a modified opsonophagocytic assay (COPA), Streptococcus pneumoniae serotypes 6B (Group A), 16F (Group D), 15A (Group B), and 35B (Group C) were incubated with heat-inactivated pre-immune (P0) and immune (P3) sera from rabbits (87, 88, and 1762) immunized with CP1 adjuvanted with aluminum phosphate at various dilutions. The presence of functional antibodies was demonstrated by killing of Streptococcus pneumoniae. Results are expressed as the percentage of killing activity relative to incubation with matched pre-immune (P0) sera, i.e., the percentage decrease in Streptococcus pneumoniae colony-forming units (CFU). Each vertical bar in Figures A - D represents the percentage of killing activity against the designated Streptococcus pneumoniae serotype (top of each figure) observed at the designated dilution of the designated CP1 serum (bottom of each figure).

[0020] Figure 8Describe the immune response (e.g., Th17 response) to the exemplary fusion protein CP1 compared to the SP1500 or SP0785 polypeptides. Mice were immunized with CP1, SP1500, or SP0785 polypeptides adjuvanted with cholera toxin, or with Rhavi protein (control) adjuvanted with cholera toxin. The Th17 response is shown as the geometric mean concentration of IL-17A secreted in the culture medium after stimulation of peripheral blood samples from immunized mice with purified SP0785 polypeptide (Figure A), purified SP1500 polypeptide (Figure B), or killed (inactivated) pneumococcal whole cells (WCV; Figure C). Each point on the graph represents the IL-17A secreted by one mouse. The horizontal bar represents the geometric mean of the IL-17A secreted by each group. CT: Cholera toxin.

[0021] Figure 9 Describe the immune response (e.g., Th17 response) to the exemplary fusion protein CP1 compared to the fusion protein SP0785-linker (SSSGG)-SP1500-linker (SSVDKL)-PdT. Mice were immunized with CP1 or SP0785-linker (SSSGG)-SP1500-linker (SSVDKL)-PdT adjuvanted with cholera toxin, or with Rhavi protein (control) adjuvanted with cholera toxin. The Th17 response is shown as the geometric mean concentration of IL-17A secreted in the culture medium after stimulation of peripheral blood samples from immunized mice with purified SP0785 polypeptide (Figure A) or purified SP1500 polypeptide (Figure B). Each point on the graph represents the IL-17A secreted by one mouse. The horizontal bar represents the geometric mean of the IL-17A secreted by each group. CT: Cholera toxin.

[0022] Figure 10 Describe the immune response (e.g., Th17 response) to the exemplary fusion protein CP1 compared to a mixture (unconjugated) of SP0785, SP1500, and Rhavi polypeptides. Mice were immunized with CP1 or a mixture (unconjugated) of SP0785, SP1500, and Rhavi polypeptides adjuvanted with cholera toxin, or with Rhavi protein (control) adjuvanted with cholera toxin. The Th17 response is shown as the geometric mean concentration of IL-17A secreted in the culture medium after stimulation of peripheral blood samples from immunized mice with purified SP0785 polypeptide (Figure A), purified SP1500 polypeptide (Figure B), or killed (inactivated) pneumococcal whole cells (WCV; Figure C). Each point on the graph represents the IL-17A secreted by one mouse. The horizontal bar represents the geometric mean of the IL-17A secreted by each group. CT: Cholera toxin; mixture: A mixture (unconjugated) of SP0785, SP1500, and Rhavi polypeptides.

[0023] Figure 11 Describe the hemolytic activity of the exemplary fusion protein CP1 and the fusion protein SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT (PdT fusion) against sheep red blood cells. Sheep red blood cells were incubated with the positive control protein pneumolysin (Ply), pneumolysoid PdT, CP1, or the fusion protein SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT at the concentrations indicated on the x-axis. The hemolytic activity measured by the OD 420 of the supernatant is plotted on the y-axis.

[0024] Certain definitions

[0025] In this application, unless otherwise apparent from the context, (i) the term "a" can be understood to mean "at least one / species"; (ii) the term "or" can be understood to mean "and / or"; (iii) the terms "comprising" and "including" can be understood to cover the specifically recited components or steps, whether presented by themselves or in conjunction with one or more additional components or steps; and (iv) the terms "about" and "approximately" can be understood to allow for the standard deviation as understood by a person of ordinary skill in the art; and (v) when ranges are provided, endpoints are included.

[0026] About: The term "about" when used herein in reference to a value means a value similar in the context of the value being referred to. Generally, a person of ordinary skill in the art familiar with the context should understand the degree of relevant variation covered by "about" in that context. For example, in some embodiments, the term "about" can cover a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the value being referred to.

[0027] Administration: As used herein, the term "administration" generally refers to the administration of a composition to a subject or system to effect the delivery of the composition or an agent included in the composition. One of ordinary skill in the art will recognize the various routes that can be used for administration to a subject such as, for example, a human. By way of example, in some embodiments, administration can be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration can be bronchial (e.g., by bronchial instillation), buccal, cutaneous (which can be or include, for example, one or more of topical to the dermis, intradermal, intercutaneous, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intracameral, within a particular organ (e.g., intrahepatic), transmucosal, transnasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by endotracheal instillation), transvaginal, transvitreal, etc. In some embodiments, administration can involve only a single administration. In some embodiments, administration can involve a fixed number of administrations. In some embodiments, administration can involve administration as intermittent (e.g., multiple doses spaced in time) and / or periodic (e.g., separate doses separated by a constant time period) dosing. In some embodiments, administration can involve continuous administration (e.g., infusion) for at least a selected time period.

[0028] Agent: Generally, as used herein, the term "agent" can be used to refer to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. In appropriate circumstances, as will be apparent to one of skill in the art from the context, the term can be used to refer to an entity that is or includes a cell or organism or a part, extract, or component thereof. Alternatively or additionally, as will be apparent from the context, the term can be used to refer to a natural product, in that it is found in and / or obtained from nature. In some cases, in addition, as will be apparent from the context, the term can be used to refer to one or more artificial entities, in that it is designed, engineered, and / or produced by an artificial act and / or is not found in nature. In some embodiments, an agent can be utilized in isolated or pure form; in some embodiments, an agent can be utilized in crude form. In some embodiments, potential agents can be provided in a collection or library form, e.g., a form that can be screened to identify or characterize the active agents therein. In some cases, the term "agent" can refer to a compound or entity that is or includes a polymer; in some cases, the term can refer to a compound or entity that includes one or more polymeric moieties. In some embodiments, the term "agent" can refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or is substantially free of one or more particular polymeric moieties. In some embodiments, the term can refer to a compound or entity that lacks or is substantially free of any polymeric moieties.

[0029] Amino acid: In its broadest sense, the term "amino acid" as used herein refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, via the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structural formula H 2 N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any one of the twenty standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from natural sources. In some embodiments, an amino acid, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, may contain a structural modification compared to the above general structural formula. For example, in some embodiments, compared to the general structural formula, an amino acid may be modified by methylation, amidation, acetylation, polyethylene glycolylation, glycosylation, phosphorylation, and / or substitution (e.g., substitution of an amino group, a carboxyl group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid compared to a polypeptide containing the unmodified amino acid that is otherwise identical. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing the unmodified amino acid that is otherwise identical. As will be apparent from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

[0030] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes the typical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, a complete antibody as it occurs in nature is an approximately 150 kDa tetrameric agent composed of two identical heavy chain polypeptides (each approximately 50 kDa) and two identical light chain polypeptides (each approximately 25 kDa) that associate with one another to form a structure commonly referred to as a “Y-shaped” structure. Each heavy chain is composed of at least four domains (each approximately 110 amino acids in length)—an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH1, CH2, and a carboxyl-terminal CH3 (located at the base of the stem of the Y). A short region called the “switch” links the heavy chain variable region and the constant region. The “hinge” links the CH2 domain and the CH3 domain to the remainder of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides in the complete antibody to one another. Each light chain is composed of two domains—an amino-terminal variable (VL) domain, followed by a carboxyl-terminal constant (CL) domain, which are spaced apart from one another by another “switch”. The complete antibody tetramer is composed of two heavy chain-light chain dimers, where the heavy chain and the light chain are connected to one another by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions to one another such that the dimers are connected to one another and form the tetramer. Naturally occurring antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed by two β-sheets (e.g., 3-strand, 4-strand, or 5-strand folds) that are packed against one another in a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops called “complementary determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form β-sheets that provide a structural framework for the domain and bring the CDR loop regions from both the heavy chain and the light chain together in three-dimensional space such that they create a single hypervariable antigen-binding site located at the tip of the Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also binds to receptors on effector cells including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity of the Fc region for Fc receptors and / or other binding properties can be modulated via glycosylation or other modifications. In some embodiments, the antibodies produced and / or utilized according to the present invention include an Fc domain that is glycosylated, including an Fc domain having such glycosylation that is modified or engineered. For the purposes of the present invention, in some embodiments, any polypeptide or polypeptide complex that includes sufficient immunoglobulin domain sequences as found in a natural antibody can be referred to and / or used as an “antibody”, whether such polypeptide is produced naturally (e.g., by reacting an organism with an antigen) or by recombinant engineering, chemical synthesis, or other artificial systems or methods.In some embodiments, the antibody is a polyclonal antibody; in some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody has a constant region sequence characteristic of a murine, rabbit, primate, or human antibody. In some embodiments, as is known in the art, antibody sequence elements are humanized, primatized, chimeric, etc. In addition, as used herein, the term "antibody" in suitable embodiments (unless otherwise stated or apparent from the context) can refer to any of the constructs or formats known or developed in the art for exploiting the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, the antibodies utilized according to the present invention are in a format selected from, but not limited to, the following: intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid-like antibodies; masking antibodies (e.g., ); small modular immunopharmaceuticals ( S mall M odular I mmuno P harmaceuticals; "SMIPs TM "); single-chain or tandem bifunctional antibodies VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; miniproteins; and In some embodiments, the antibody may lack covalent modifications (such as glycan linkages) that it would have in its native state. In some embodiments, the antibody may contain covalent modifications (such as glycan, payload [e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.], or other side groups [e.g., polyethylene glycol, etc.] linkages).

[0031] Antigen: As used herein, the term "antigen" refers to (i) an agent that induces an immune response; and / or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or binds to an antibody. In some embodiments, the antigen induces a humoral response (e.g., including the production of antigen-specific antibodies); in some embodiments, the antigen induces a cellular response (e.g., involving T cells, the receptors of which specifically interact with the antigen). In some embodiments, the antigen induces both a humoral response and a cellular response. In some embodiments, the antigen binds to an antibody and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or include any chemical entity such as, for example, small molecules, nucleic acids, polypeptides, carbohydrates, lipids, polymers (in some embodiments other than biopolymers (e.g., other than nucleic acid or amino acid polymers)), etc. In some embodiments, the antigen is or comprises a polypeptide. In some embodiments, the antigen is or comprises a polysaccharide. One of ordinary skill in the art will appreciate that, generally, an antigen can be provided in isolated or pure form, or alternatively can be provided in crude form (e.g., together with other materials, such as in an extract such as a cell extract or other relatively crude preparation of a source containing the antigen). In some embodiments, the antigen utilized according to the present invention is provided in crude form. In some embodiments, the antigen is a recombinant antigen. In some embodiments, the antigen is a polypeptide or polysaccharide that, upon administration to a subject, induces a specific and / or clinically relevant immune response against the polypeptide or polysaccharide. In some embodiments, the antigen is selected to induce a specific and / or clinically relevant immune response against the polypeptide or polysaccharide.

[0032] associated with: When the term "associated with" is used herein, two entities are "associated with" each other if the presence, level, and / or form of one entity is related to the presence, level, and / or form of the other entity. In some embodiments, two or more entities are physically "associated with" each other if they interact directly or indirectly such that they are physically close to and / or remain physically close to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other. In some embodiments, two or more entities that are physically associated with each other are not covalently linked, but rather are non-covalently associated, for example, by means of affinity interactions, electrostatic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0033] Binding: It should be understood that as used herein, the term "binding" generally refers to non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction by means of physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated under any of a variety of circumstances, including when the interacting entities or moieties are studied in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or when in a biological system or cell).

[0034] Carrier protein: As used herein, the term "carrier protein" refers to a protein or peptide that is conjugated, complexed, or otherwise associated with a hapten (e.g., a small peptide or lipid) or a less immunogenic antigen (e.g., a polysaccharide), and that induces or enhances an immune response against such conjugated, complexed, or otherwise associated hapten (e.g., a small peptide or lipid) or less immunogenic antigen (e.g., a polysaccharide). In some embodiments, such an immune response is or includes a response against the hapten conjugated, complexed, or otherwise associated with such carrier protein. In some embodiments, such an immune response is or includes a response against both the carrier protein and the hapten or less immunogenic antigen conjugated, complexed, or otherwise associated with such carrier protein. In some embodiments, no significant immune response against the carrier protein itself occurs. In some embodiments, an immune response against the carrier protein can be detected; in some such embodiments, the immune response against such carrier protein is strong. In some embodiments, the carrier protein is conjugated, complexed, or otherwise associated with one or more other molecules.

[0035] Colonization: As used herein, the term "colonization" generally refers to the ability of a microorganism to grow at a target site or surface. By way of example, the term "colonization" refers to the ability of a microorganism (e.g., a bacterium) to grow at an anatomical site of a host (e.g., a mucosa, the gastrointestinal tract, a site of injury, an organ, etc.).

[0036] Combination Therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, these regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any dose of the second regimen); in some embodiments, these agents are administered in an overlapping dosing regimen. In some embodiments, administering a "combination therapy" may involve administering one or more agents or treatment regimens to a subject who is receiving treatment with other agents or treatment regimens in combination form. For clarity, combination therapy does not require that the individual agents be administered together (or even necessarily simultaneously) in a single composition, but in some embodiments, two or more agents or their active moieties may be administered together in a combined composition or even in the form of a combined compound (e.g., as part of a single chemical complex or covalent entity).

[0037] Derivative: The term "derivative" or its grammatical equivalents as used herein refers to a structural analogue of a reference substance. That is, a "derivative" is a substance that exhibits significant structural similarity to the reference substance, e.g., sharing a core or common structure, but also differs in certain discrete ways. Such a substance shall be termed "derived from" the said reference substance. In some embodiments, a derivative is a substance that can be generated from the reference substance by chemical manipulation. In some embodiments, a derivative is a substance that can be generated via a synthetic method that performs substantially similar to the method by which the reference substance was generated (e.g., sharing multiple steps therewith).

[0038] Domain: The term "domain" as used herein refers to a segment or portion of an entity. In some embodiments, a "domain" is associated with specific structural and / or functional characteristics of the entity such that when the domain is physically separated from the remainder of its parent entity, it substantially or fully retains the specific structural and / or functional characteristics. Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from the (parent) entity and attached to a different (recipient) entity, substantially retains and / or confers upon the recipient entity one or more of the structural and / or functional characteristics that characterize it in the parent entity. In some embodiments, a domain is a segment or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a segment of a polypeptide; in some such embodiments, a domain is characterized by specific structural elements (e.g., a specific amino acid sequence or sequence motif, α-helical properties, β-sheet properties, coiled-coil properties, random coil properties, etc.) and / or by specific functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0039] Dosage form or unit dosage form: Those skilled in the art will understand that the term "dosage form" can be used to refer to a physically discrete unit of an active agent (such as a therapeutic agent or a diagnostic agent) for administration to a subject. Generally, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is a unit dose amount (or an integral part thereof) suitable for administration according to a dosing regimen that has been determined to be associated with a desired or beneficial outcome when administered to a relevant population (i.e., in a therapeutic dosing regimen). Those of ordinary skill in the art understand that the total amount of a therapeutic composition or therapeutic agent administered to a particular subject is determined by one or more attending physicians and may involve the administration of multiple dosage forms.

[0040] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can be used to refer to a collection (usually more than one) of unit doses administered separately to a subject, typically spaced apart by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, the dosing regimen comprises multiple doses, each of which is spaced apart in time from the other doses. In some embodiments, the individual doses are spaced apart from each other by periods of the same length; in some embodiments, the dosing regimen comprises multiple doses and at least two different periods of time separating the individual doses. In some embodiments, all administrations within the dosing regimen have the same unit dose. In some embodiments, the different administrations within the dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose at a first dosing amount, followed by one or more additional doses at a second dosing amount different from the first dosing amount. In some embodiments, the dosing regimen comprises a first dose at a first dosing amount, followed by one or more additional doses at a second dosing amount the same as the first dosing amount. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0041] Fragment: A "fragment" of a material or entity as described herein has a structure that includes discrete portions of the whole but lacks one or more portions found in the whole. In some embodiments, the fragment consists of such discrete portions. In some embodiments, the fragment includes discrete portions of the whole that share one or more functional features found in the whole. In some embodiments, the fragment consists of such discrete portions. In some embodiments, the fragment consists of or comprises the following: characteristic structural elements or portions found in the whole. In some embodiments, a fragment of a polymer, such as a polypeptide or polysaccharide, comprises or consists of the following: at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of the following: at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% monomer units (e.g., residues) as found in the whole polymer. In some embodiments, the whole material or entity may be referred to as the "parent" of the whole.

[0042] Homology: As used herein, the term "homology" refers to the overall relatedness between polymeric molecules such as, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "homologous" to each other if their sequences have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. In some embodiments, polymeric molecules are considered to be "homologous" to each other if their sequences have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity (e.g., containing residues with related chemical properties at corresponding positions). By way of example, as is well known to those of ordinary skill in the art, certain amino acids are commonly classified as "hydrophobic" or "hydrophilic" amino acids and / or classified as having "polar" or "nonpolar" side chains, similar to each other. Substitution of one amino acid for another of the same type is often considered a "homologous" substitution.

[0043] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules such as, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "substantially identical" to each other if their sequences have at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity. By way of example, the percent identity between two nucleic acid or polypeptide sequences can be calculated by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or substantially 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules have identity at that position. The percent identity between two sequences varies with the number of aligned positions shared by the sequences, taking into account the number of gaps and the length of each gap needed to optimally align the two sequences. Sequence comparison and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. By way of example, the algorithm of Meyers and Miller, 1989 incorporated into the ALIGN program (version 2.0) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, nucleic acid sequence comparison using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix.

[0044] Improve, increase, inhibit, or decrease: As used herein, the terms “improve,” “increase,” “inhibit,” “decrease,” or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, the appropriate reference measurement may be or include a measurement in a particular system (e.g., in a single subject) that is otherwise comparable in the absence (e.g., before and / or after) of the presence of a particular agent or treatment, or in the presence of an appropriate comparable reference agent. In some embodiments, the appropriate reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of the relevant agent or treatment.

[0045] Immunologically effective amount or immunologically effective dose: As used herein, an “immunologically effective amount” or “immunologically effective dose” refers to an amount of an antigenic or immunogenic substance, such as an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition, which, when administered to a subject as a single dose or as part of a series of doses, is sufficient to enhance the subject's own immune response to a subsequent exposure to a pathogen. In some embodiments, the pathogen is Streptococcus pneumoniae. In some embodiments, the immune response is against one or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against two or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against nine or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against thirteen or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against fifteen or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against twenty-three or more different serotypes of Streptococcus pneumoniae. In some embodiments, the immune response is against twenty-four or more different serotypes of Streptococcus pneumoniae. The immunologically effective amount can vary based on the subject to be treated, the type of subject, the degree of immune response desired, etc. In some embodiments, the immunologically effective amount is sufficient to treat or protect a subject suffering from or at risk of suffering from a disease. In some embodiments, the immunologically effective amount refers to a non-toxic but sufficient amount that can be an amount that treats, attenuates, or prevents an infection and / or disease (e.g., a bacterial infection, a Streptococcus pneumoniae infection, bacterial colonization, pneumococcal colonization, complications associated with a bacterial infection, complications associated with a pneumococcal infection, etc.) in any subject. In some embodiments, the immunologically effective amount is sufficient to induce an immunoprotective response after administration to a subject.

[0046] Immune protective response or protective response: As used herein, "immune protective response" or "protective response" refers to an immune response that mediates immunological memory induced by an antigen or immunogen. In some embodiments, the immune protective response is induced by administering to a subject a substance such as an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, or pharmaceutical composition. In some embodiments, immune protection involves one or more of the following: active immune surveillance, a more rapid and effective post-immune activation response compared to the response observed in an untreated subject, efficient clearance of the activator or pathogen, followed by a rapid resolution of inflammation. In some embodiments, the immune protective response is an adaptive immune response. In some embodiments, the immune protective response is sufficient to protect the immunized subject from a toxigenic infection (e.g., Streptococcus pneumoniae infection) caused by one or more specific pathogens against which the vaccine is directed.

[0047] Immunization: As used herein, "immunization" or its grammatical equivalents refer to the process of inducing an immune response against an infectious organism or agent ("active immunization") in a subject, or alternatively, providing to the subject components of the immune system against an infectious organism or agent ("passive immunization"). In some embodiments, immunization involves administering to a subject one or more antigens, immunogens, immunogenic complexes, vaccines, immune molecules (e.g., antibodies), immune sera, immune cells (e.g., T cells or B cells), or pharmaceutical compositions. In some embodiments, immunization is performed by administering to the subject an immunologically effective amount of a substance such as an antigen, immunogen, immunogenic complex, immunogenic composition, vaccine, immune molecule (e.g., antibody), immune serum, immune cell (e.g., T cells or B cells), or pharmaceutical composition. In some embodiments, immunization elicits an immune protective response in the subject. In some embodiments, active immunization is performed by administering to the subject an antigenic or immunogenic substance such as an antigen, immunogen, immunogenic complex, vaccine, or pharmaceutical composition. In some embodiments, passive immunization is performed by administering to the subject components of the immune system such as immune molecules (e.g., antibodies), immune sera, or immune cells (e.g., T cells or B cells).

[0048] Isolated: As used herein, the term "isolated" or its grammatical equivalents means that a substance and / or entity has (1) been separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) been designed, produced, prepared, and / or manufactured by man. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of the other components with which it was initially associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as would be understood by one of ordinary skill in the art, a substance can still be considered "isolated" or even "pure" after having been combined with certain other components such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in these embodiments, the percentage of separation or purity of the substance is calculated without including these carriers or excipients. In some embodiments, by way of just one example, a biopolymer such as a polypeptide or polysaccharide that occurs in nature is considered "isolated" when: a) it is not associated with some or all of the components that accompany it in its native state in nature due to its derivative origin or source; b) it is substantially free of other polypeptides or nucleic acids of the same species as those that produce it in nature; c) it is expressed or otherwise associated with components from a cell or other expression system that does not have the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide or polysaccharide that is chemically synthesized or synthesized in a cell system different from the cell system that produces it in nature is considered an "isolated" polypeptide or polysaccharide. Alternatively or additionally, in some embodiments, a polypeptide or polysaccharide that has been subjected to one or more purification techniques can be considered an "isolated" polypeptide or polysaccharide to the extent that it has been separated from a) the components with which it is associated in nature; and / or b) the other components with which it was associated when initially produced.

[0049] Linker: As used herein, the term "linker" is used to refer to an entity that connects two or more components to form a multi-component agent. For example, one of ordinary skill in the art will appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between these domains that connect them to each other. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general formula S1-L-S2, where S1 and S2 can be the same or different and represent two domains that are associated with each other through the linker (L). In some embodiments, the length of the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids. In some embodiments, the linker is characterized by its tendency not to adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide. A variety of different linker elements that can be suitably used in engineering polypeptides (such as fusion polypeptides) are known in the art (Holliger et al., 1993; Poljak, 1994).

[0050] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a therapeutically effective unit dosage suitable for administration in a treatment regimen that, when administered to a relevant population, has a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including solid or liquid forms suitable for: oral administration, such as drenching (aqueous or non-aqueous solutions or suspensions), tablets (e.g., tablets targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, a sterile solution or suspension or sustained release formulation; topical administration, such as in the form of a cream, ointment, or controlled release patch or spray for application to the skin, lungs, or mouth; intravaginal or rectal, such as in the form of a pessary, cream, or foam; sublingual; ophthalmic; transdermal; or nasal, pulmonary, and to other mucosal surfaces.

[0051] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applied to a carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other components of the composition and not harmful to its recipient.

[0052] Polysaccharide: As used herein, the term "polysaccharide" refers to a polymeric carbohydrate molecule composed of long chains of monosaccharide units linked together by glycosidic, phosphodiester, or other bonds and yielding the constituent monosaccharides or oligosaccharides upon hydrolysis. The structure of polysaccharides ranges from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen; structural polysaccharides such as cellulose and chitin and microbial polysaccharides; and antigenic polysaccharides found in microorganisms including but not limited to capsular polysaccharide (CPS), O-polysaccharide (OPS), core O-polysaccharide (COPS), and lipopolysaccharide (LPS).

[0053] Polypeptide: As used herein, the term "polypeptide" generally has its meaning recognized in the art as a polymer of at least three amino acids linked to each other, for example, by peptide bonds. One of ordinary skill in the art should understand that the term "polypeptide" is intended to be of sufficient generality to cover not only polypeptides having the complete sequences listed herein but also polypeptides representing functional fragments of these complete polypeptides (i.e., fragments that retain at least one activity). In addition, one of ordinary skill in the art should understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, the related term "polypeptide" as used herein encompasses any polypeptide that retains activity and shares at least about 30 - 40% (often greater than about 50%, 60%, 70%, or 80%) overall sequence identity with another polypeptide of the same class and further generally includes at least one region having much higher identity (often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions) that generally encompasses at least 3 - 4 and often up to 20 or more amino acids. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0054] Prevention: As used herein in connection with a disease, disorder, and / or medical condition, the term "prevent / prevention" refers to reducing the risk of developing a disease, disorder, and / or condition, and / or delaying onset, and / or reducing the frequency and / or severity of one or more features or symptoms of a particular disease, disorder, or condition. In some embodiments, prevention is evaluated on a population basis such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population predisposed to the disease, disorder, or condition. In some embodiments, prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

[0055] Protein: As used herein, the term "protein" encompasses polypeptides. A protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. One of ordinary skill in the art will appreciate that a "protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. One of ordinary skill in the art will appreciate that a protein can sometimes include more than one polypeptide chain linked, for example, by one or more disulfide bonds or associated by other means. A polypeptide can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein can comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide that is less than about 100 amino acids in length, less than about 50 amino acids in length, less than 20 amino acids in length, or less than 10 amino acids in length. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0056] Recombinant: As used herein, the term "recombinant" is intended to mean a polypeptide that is designed, engineered, prepared, expressed, produced, manufactured, and / or isolated by recombinant means, such as, for example, a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from a transgenic animal or otherwise engineered to express one or more genes or a genetic component encoding and / or directing the expression of a polypeptide or one or more of its components, parts, elements, or domains (e.g., a mouse, rabbit, sheep, fish, etc.); and / or a polypeptide prepared, expressed, produced, or isolated by any other means involving splicing selected nucleic acid sequence elements or ligating selected nucleic acid sequence elements to each other, chemically synthesizing the selected sequence elements, and / or otherwise generating a nucleic acid encoding and / or directing the expression of a polypeptide or one or more of its components, parts, elements, or domains. In some embodiments, one or more of these selected sequence elements are found in nature. In some embodiments, one or more of these selected sequence elements are designed by computer simulation. In some embodiments, one or more of these selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of a known sequence element, such as a natural or synthetic source from, for example, the germline of a target source organism (e.g., human, mouse, etc.).

[0057] Reference: As used herein, the term "reference" describes a standard or control relative to which a comparison is being made. For example, in some embodiments, a target agent, animal, subject, population, sample, sequence, or value is compared to a reference or control agent, animal, subject, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or assayed substantially contemporaneously with the target test or assay. In some embodiments, the reference or control is a historical reference or control optionally implemented in a tangible medium. Generally, as will be understood by those of skill in the art, the reference or control is assayed or characterized under conditions or circumstances comparable to those being evaluated. Those of skill in the art will appreciate when there is sufficient similarity to justify reliance on and / or comparison with a particular potential reference or control.

[0058] Response: As used herein, "response" to treatment can refer to any beneficial change in a subject's disorder that is caused by or associated with the treatment. Such changes can include stabilization of the disorder (e.g., prevention of worsening that would occur in the absence of treatment), improvement of disorder symptoms, and / or improvement in the prospects for a cure of the disorder, among others. It can refer to subject response or tumor response. Subject or tumor response can be measured according to a wide variety of criteria including clinical and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, positron emission tomography, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, the presence or level of biomarkers in samples obtained from the subject, cytology, and / or histology. Accurate response criteria can be selected in any suitable manner, provided that when comparing groups of subjects and / or tumors, the groups to be compared are evaluated based on the same or comparable criteria for measuring response rates. Those of ordinary skill in the art will be able to select appropriate criteria.

[0059] Risk: As will be understood in context, the "risk" of a disease, condition, and / or disorder refers to the likelihood that a particular subject will develop the disease, condition, and / or disorder. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%. In some embodiments, the risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of disease, condition, disorder, and / or event. In some embodiments, the reference sample or group of reference samples is from a subject comparable to a particular subject. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0060] Serotype: As used herein, the term "serotype", also known as serovar, refers to the different variations within a bacterial or viral species or in the immune cells of different subjects. These microorganisms, viruses, or cells are grouped together based on their cell surface antigens, allowing for epidemiological classification at the organism to subspecies level. A group of serovars with common antigens may be referred to as a serogroup or sometimes as a serotype.

[0061] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments a prenatal human form). In some embodiments, the subject has a related disease, disorder, or condition. In some embodiments, the subject is predisposed to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics that are characterized by a predisposition to or risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject to whom a diagnosis and / or therapy has been administered and / or has been administered.

[0062] Predisposed: A subject who is "predisposed" to a disease, disorder, or condition is at risk of developing the disease, disorder, or condition. In some embodiments, a subject who is predisposed to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, a subject who is predisposed to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, a subject who is predisposed to a disease, disorder, or condition is a subject who has been exposed to conditions associated with developing the disease, disorder, or condition. In some embodiments, the risk of developing the disease, disorder, and / or condition is a population-based risk (e.g., a family member of a subject with the disease, disorder, or condition).

[0063] Symptom reduction: As used herein, "symptom reduction" occurs when the degree (e.g., intensity, severity, etc.) and / or frequency of one or more symptoms of a particular disease, disorder, or condition is reduced, e.g., to a statistically and / or clinically significant or relevant level. For clarity, delaying the onset of a particular symptom is considered a form of reduced symptom frequency.

[0064] Treatment: As used herein, the term "treatment" (and "treat / treating") refers to the administration of any therapy that partially or fully relieves, ameliorates, alleviates, inhibits one or more symptoms, features, and / or causes of a particular disease, disorder, and / or affliction, delays its onset, reduces its severity, and / or decreases its incidence. In some embodiments, such treatment may be in a subject who does not exhibit signs of the relevant disease, disorder, and / or affliction and / or a subject who exhibits only early signs of the disease, disorder, and / or affliction. Alternatively or additionally, such treatment may be in a subject who exhibits one or more established signs of the relevant disease, disorder, and / or affliction. In some embodiments, the treatment may be in a subject who has been diagnosed with the relevant disease, disorder, and / or affliction. In some embodiments, the treatment may be in a subject who is known to have one or more predisposing factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or affliction.

[0065] Vaccination: As used herein, the term "vaccination" refers to, for example, the administration of a composition designed to generate an immune response to a pathogen. For the purposes of the present invention, vaccination may be administered before, during, and / or after exposure to the pathogen and in some embodiments shortly before, during, and / or after exposure to the pathogen. In some embodiments, vaccination includes multiple administrations of a vaccination composition that are appropriately spaced in time. In some embodiments, vaccination elicits immunization. Detailed Description

[0066] The present disclosure generally relates to novel immunogenic fusion proteins of Streptococcus pneumoniae that can be used, for example, to induce and / or increase an immune protective response or reduce pneumococcal colonization in subjects at risk of or suffering from pneumococcal infection.

[0067] Two pneumococcal vaccines are currently available in the United States. PCV13, a 13-valent conjugate vaccine, has been approved for the prevention of invasive pneumococcal disease (IPD) caused by the 13 serotypes contained in the vaccine in children and for the prevention of pneumonia and IPD in adults. In this vaccine, the covalent conjugation of the sugars of the 13 pneumococcal serotypes to the CRM197 protein produces glycoprotein conjugates that are capable of inducing a T cell-dependent immune response against one or more of the 13 pneumococcal serotypes represented by the sugars. [PREVNAR 13 prescribing information, 2017]. Although the incidence of pneumococcal infections with multi-drug resistant serotypes contained in PCV13 appears to have decreased after the approval of this vaccine, note that the incidence of multi-drug resistant serotypes 35B, 23A, 23B, and 15B infections has increased, and these serotypes are just a few of the more than 84 known pneumococcal serotypes not included in PCV13. In addition, PCV13 has been reported to have marginal activity against serotype 3 because of its prevalence remaining in the population [Richter et al., 2014].

[0068] The second vaccine, PPSV23, is a 23-valent polysaccharide vaccine and is designated for the prevention of pneumococcal disease in adults aged greater than 50 years or in individuals aged greater than 2 years who are at increased risk of pneumococcal disease. It consists of purified capsular polysaccharides from 23 pneumococcal serotypes. Although this vaccine has the potential to provide protection against more serotypes compared to PCV13, it does not provide protection against the emerging serotypes 35B, 23A, and 23B. Additionally, PPSV23 elicits a T cell-independent polysaccharide immune response that stimulates mature B-lymphocytes rather than T-lymphocytes. Thus, this vaccine induces an immune response that is neither long-lasting nor has memory upon subsequent challenge. PPSV23 is not effective against colonization. Additionally, polysaccharide vaccines are not used in infants and children less than 2 years of age because these children respond poorly to T cell-independent antigens [PNEUMOVAX 23 prescribing information, 2017; CDC, 2010]. Data indicate that PPSV23 can protect adults and the elderly from IPD; however, no consistent effect has been observed in pneumonia prevention [Gruber et al., 2008].

[0069] The presently disclosed novel immunogenic proteins represent a substantial advance over the currently available options for immunizing patients against pneumococcal infections. Such immunogenic proteins can be used, for example, to induce and / or increase an immune protective response or to reduce pneumococcal colonization in a subject (e.g., a subject at risk of or suffering from a pneumococcal infection).

[0070] Fusion protein

[0071] The present disclosure describes novel immunogenic fusion proteins of Streptococcus pneumoniae. In WO2014 / 124228, the inventors demonstrated that pneumococcal antigens SP0785 and SP1500 elicited strong IL-17 recall responses in re-stimulated human PBMCs and splenocytes of mice exposed to pneumococcus, respectively. Immunization of mice with SP0785 + cholera toxin adjuvant or SP1500 + cholera toxin adjuvant resulted in a significant reduction (about 100-fold) in pneumococcal colonization. Immunization with a fusion of SP0785 with pneumolysin PdT or a fusion of SP0785 with pneumolysin PdT further conjugated with polysaccharide of Salmonella typhi protected 80% of the mice from sepsis in a lethal challenge with live Streptococcus pneumoniae. Immunization with fusions of SP0785, SP1500, and pneumolysin PdT further conjugated with polysaccharide of Salmonella typhi also resulted in a significant reduction (about 10-fold) in pneumococcal colonization.

[0072] The fusion proteins described and / or utilized herein provide improved immunogenicity and IL-17 responses to protein stimulation, as well as further reduction of Streptococcus pneumoniae colonization and protection against invasive disease.

[0073] The fusion proteins comprise one, two, or more polypeptides that elicit (e.g., predominantly elicit) a T cell response or both a T cell and a B cell response. In some embodiments, the fusion protein comprises one or more polypeptides listed in Table 1. In some embodiments, the fusion protein comprises two polypeptides listed in Table 1. In some embodiments, the fusion protein comprises three polypeptides listed in Table 1. In some embodiments, the fusion protein comprises one or more polypeptides encoded by one or more genes listed in Table 1. In some embodiments, the fusion protein comprises two polypeptides encoded by two or more genes listed in Table 1. In some embodiments, the fusion protein comprises three polypeptides encoded by three genes listed in Table 1.

[0074] Table 1. Exemplary polypeptide components of the fusion proteins

[0075]

[0076]

[0077] In some embodiments, the fusion protein comprises one or more antigenic polypeptides of Streptococcus pneumoniae having an amino acid sequence comprising any one of SEQ ID NOs: 3-8, or an antigenic fragment thereof. In some embodiments, the fusion protein comprises two antigenic polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 3-8, or an antigenic fragment thereof. In some embodiments, the fusion protein comprises (i) two antigenic polypeptides having an amino acid sequence comprising any one of SEQ ID NOs: 3-8, or an antigenic fragment thereof, and (ii) a biotin-binding moiety comprising SEQ ID NO: 1 or 2, or a biotin-binding fragment thereof. In some such embodiments, at least one antigenic polypeptide is or comprises the SP0785 polypeptide (e.g., SEQ ID NOs: 3-5). In some such embodiments, at least one antigenic polypeptide is or comprises the SP1500 polypeptide (e.g., SEQ ID NOs: 6-8).

[0078] In some embodiments, the fusion protein comprises one or more polypeptides homologous to the Streptococcus pneumoniae polypeptides listed in Table 1, e.g., the SP0785 polypeptide or the SP1500 polypeptide isolated from different serotypes of Streptococcus pneumoniae. Individual serotypes of Streptococcus pneumoniae contain many mutations relative to each other, some of which result in different protein sequences between different serotypes. One of ordinary skill in the art can readily substitute the amino acid sequence or a portion thereof with a homologous amino acid sequence from a different Streptococcus pneumoniae serotype. In some embodiments, the antigenic polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to the polypeptide listed in Table 1 or an antigenic fragment thereof. Serotype variation can be used to design such variants of the polypeptides listed in Table 1.

[0079] In some embodiments, the fusion proteins described herein comprise one or more fragments of the polypeptides listed in Table 1, e.g., a biotin-binding fragment of avidin, an antigenic fragment of the SP0785 polypeptide with or without a signal sequence, or an antigenic fragment of the SP1500 polypeptide with or without a signal sequence. In some embodiments, the fusion proteins described herein comprise truncated mutants that are close in size to the polypeptides listed in Table 1. For example, they may lack up to one, two, three, four, five, ten, or twenty amino acids (referring to the constituent polypeptides in the fusion protein) from one or both termini. In some embodiments, the fragment is a truncated fragment of any one of SEQ ID NOs: 1-8, lacking 1-5, 1-10, or 1-20 amino acid residues from the N-terminus, C-terminus, or both of any one of SEQ ID NOs: 1-8. In some embodiments, the fragment is a truncated fragment of any one of SEQ ID NOs: 1-8, lacking 1-10 amino acid residues from the N-terminus, C-terminus, or both of any one of SEQ ID NOs: 1-8. For example, the fragment may lack 10 amino acid residues at the N-terminus and C-terminus of any one of SEQ ID NOs: 1-8, resulting in a protein lacking 20 amino acid residues. Internal deletions are also contemplated, such as internal deletions of 1-10, 11-20, 21-30, or 31-40 amino acids.

[0080] In some embodiments, the fusion protein comprises an N-terminal polypeptide and a C-terminal polypeptide. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide are antigenic polypeptides, e.g., polypeptides having an amino acid sequence comprising one or more of SEQ ID NOs: 3-8, or antigenic fragments or variants thereof. In some embodiments, one or both of the N-terminal polypeptide and the C-terminal polypeptide are biotin-binding moieties, e.g., polypeptides having an amino acid sequence comprising SEQ ID NO: 1 or 2, or biotin-binding fragments thereof. In some embodiments, one of the N-terminal polypeptide or the C-terminal polypeptide is a biotin-binding moiety, e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO: 1 or 2, or a biotin-binding fragment thereof, and the other terminal polypeptide is an antigenic polypeptide, e.g., a polypeptide having an amino acid sequence comprising one or more of SEQ ID NOs: 3-8, or an antigenic fragment or variant thereof.

[0081] In some embodiments, the N-terminal polypeptide and the C-terminal polypeptide bind directly to each other. In some embodiments, the N-terminal polypeptide and the C-terminal polypeptide are linked via a linker peptide. When a linker is present, its length and / or amino acids can be adjusted to obtain a more flexible, semi-rigid or rigid linker. Exemplary flexible peptide linkers are shown as SEQ ID NOs: 37-40. The length of the linker can generally be 1-40, such as 3-10 or 10-30, and in particular 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In some embodiments, the fusion protein comprises one linker. In some embodiments, the fusion protein comprises two linkers. In some embodiments, one or both linkers are selected from SEQ ID NO: 37 (GGGGSSS) and SEQ ID NO: 38 (AAA). In some embodiments, the fusion protein comprises SEQ ID NO: 37 (GGGGSSS) and SEQ ID NO: 38 (AAA). In some embodiments, the fusion protein comprises the amino acid sequence AAA residues from the Not I restriction site. In some embodiments, the fusion protein comprises the linker of SEQ ID NO: 37 (GGGGSSS) and the amino acid sequence AAA residues from the Not I restriction site.

[0082] Exemplary fusion proteins are shown in Table 2.

[0083] Table 2. Exemplary fusion proteins

[0084]

[0085]

[0086] In some embodiments, the present disclosure provides fusion proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the fusion proteins listed in Table 2. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs: 17-26. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO: 23. In some embodiments, the fusion protein is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to CP1.

[0087] In some embodiments, the fusion proteins described herein comprise antigenic fragments of the fusion proteins shown in Table 2. In some embodiments, the fusion protein is or comprises an antigenic fragment of any one of SEQ ID NOs: 17-26. For example, the fusion protein may lack up to one, two, three, four, five, ten, or twenty amino acids from the N-terminus, C-terminus, or both of any one of SEQ ID NOs: 17-26. In some embodiments, the same number of residues are removed from the N-terminus and C-terminus, while in other embodiments, the number of residues removed from the N-terminus is different compared to the C-terminus. In some embodiments, the fusion protein is or comprises an antigenic fragment of SEQ ID NO: 23. In some embodiments, the fusion protein is or comprises an antigenic fragment of CP1.

[0088] In some embodiments, the fusion proteins described herein comprise a biotin-binding moiety. In some embodiments, the fusion protein comprises a biotin-binding moiety and one or more polypeptide antigens. In some embodiments, the fusion protein comprises a biotin-binding moiety and two or more polypeptide antigens. As used herein, "biotin-binding moiety" refers to a biotin-binding polypeptide or protein, a biotin-binding fragment thereof, or a biotin-binding domain thereof. In some embodiments, the biotin-binding moiety of the fusion protein comprises streptavidin or a biotin-binding fragment thereof, as further described in WO 2012 / 155053, the contents of which are incorporated herein by reference in their entirety.

[0089] In some embodiments, the fusion proteins described herein comprise a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:1 (rhodococcal avidin), or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:2 (amino acids 45-179 of rhodococcal avidin, designated Rhavi), or a biotin-binding fragment thereof. In some embodiments, the fusion protein comprises a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:4 (amino acids 33-399 of Streptococcus pneumoniae SP0785 polypeptide), or an antigenic fragment thereof. In some embodiments, the fusion protein comprises a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:7 (amino acids 27-278 of Streptococcus pneumoniae SP1500 polypeptide), or an antigenic fragment thereof.

[0090] In some embodiments, the fusion proteins described herein comprise: (a) a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:1 (rhodococcal avidin), or a biotin-binding fragment thereof; (b) a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:4 (amino acids 33-399 of the Streptococcus pneumoniae SP0785 polypeptide), or an antigenic fragment thereof; and (c) a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:7 (amino acids 27-278 of the Streptococcus pneumoniae SP1500 polypeptide), or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises the amino acid sequence AAA residues from the Not I restriction site. In some embodiments, the fusion protein comprises the linker of SEQ ID NO:37 (GGGGSSS) and the amino acid sequence AAA residues from the Not I restriction site.

[0091] In some embodiments, the fusion proteins described herein comprise: (a) a biotin-binding moiety that is or comprises a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:2 (amino acids 45-179 of rhizobium avidin, designated Rhavi), or a biotin-binding fragment thereof; (b) a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:4 (amino acids 33-399 of Streptococcus pneumoniae SP0785 polypeptide), or an antigenic fragment thereof; and (c) a polypeptide that comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO:5 (amino acids 27-278 of Streptococcus pneumoniae SP1500 polypeptide), or an antigenic fragment thereof. In some embodiments, the fusion protein further comprises one or more linkers. In some embodiments, the one or more linkers are selected from SEQ ID NO:37 (GGGGSSS) and SEQ ID NO:38 (AAA). In some embodiments, the fusion protein comprises the amino acid sequence AAA residues from the Not I restriction site. In some embodiments, the fusion protein comprises the linker of SEQ ID NO:37 (GGGGSSS) and the amino acid sequence AAA residues from the Not I restriction site. In some embodiments, the fusion proteins described herein comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence SEQ ID NO:23. In some embodiments, the fusion protein comprises the amino acid sequence SEQ ID NO:23. In some embodiments, the fusion protein consists of the amino acid sequence SEQ ID NO:23 (CP1).

[0092] In some embodiments, the fusion proteins described herein include variants or fragments of the polypeptides listed in Table 1. In some embodiments, the fusion proteins described herein include polypeptides encoded by variants or fragments of the genes listed in Table 1. In some embodiments, the fragments contained in the fusion proteins described herein are of a size approaching that of the full-length polypeptide or the polypeptides listed in Table 1. For example, they may lack up to one, two, three, four, five, ten, twenty, or thirty amino acids from one or both termini. In some embodiments, the fragment has a length of 25 - 50 amino acids, or a length of 50 - 100, or 100 - 150, or 150 - 200, or 200 - 250, or 250 - 300, or 300 - 350 amino acids. In some embodiments, the fragment is produced by processing or partial processing of the signal sequence by an expression host such as Escherichia coli, an insect cell line (e.g., baculovirus expression system), or a mammalian (e.g., human or Chinese hamster ovary) cell line. The above-mentioned fragments or their sub-fragments (e.g., fragments of 8 - 50, 8 - 30, or 8 - 20 amino acid residues) preferably have one of the following biological activities, such as increasing the amount of released IL-17 by at least 1.5-fold or 2-fold or more (e.g., as an absolute measure or relative to a control protein).

[0093] The DNA and protein sequences of each gene and polypeptide can be identified by searching for the locus tag in the Streptococcus pneumoniae TIGR4 genome in a publicly available database (e.g., EntrezGene) (on the NCBI NIH website on the World Wide Web, www.ncbi.nlm.nih.gov / sites / entrez?db=gene), and the indicated sequences are also included within the scope of the present disclosure.

[0094] Certain polypeptides of Table 1, their variants, and additional exemplary polypeptides and linkers that make up the various embodiments of the fusion proteins are described in more detail below.

[0095] The SP0785 polypeptide (e.g., SEQ ID NO: 3 - 5) and its variants

[0096] SP0785 is a conserved hypothetical Streptococcus pneumoniae protein described in WO 2014 / 124228. In some embodiments, the SP0785 polypeptide is an efflux transporter that is conserved across S. pneumoniae strains. In some embodiments, the SP0785 polypeptide is or comprises a full-length SP0785 polypeptide. For example, in some embodiments, the full-length SP0785 polypeptide has 399 amino acids (38 kDa) and is represented by the amino acid sequence shown in SEQ ID NO:3. Amino acids 1-32 of SEQ ID NO:3 are predicted to be the signal sequence and transmembrane domain of the SP0785 polypeptide (amino acids 1-32 of the full-length protein). In some embodiments, the fusion protein comprises the SP0785 polypeptide of S. pneumoniae. In some embodiments, the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367 or 399 contiguous amino acids of the SP0785 polypeptide.

[0097] In some embodiments, the SP0785 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367 or 399 contiguous amino acids of the sequence shown in SEQ ID NO:3 [full-length]. In some embodiments, the SP0785 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 367 or 399 contiguous amino acids of the sequence shown in SEQ ID NO:3 [full-length].

[0098] In some embodiments, the SP0785 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 367 consecutive amino acids of the sequence shown in SEQ ID NO:4 [minus the signal sequence and transmembrane domain]. In some embodiments, the SP0785 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 367 consecutive amino acids of the sequence shown in SEQ ID NO:4 [minus the signal sequence and transmembrane domain].

[0099] Sequence variations occur at the protein level between different Streptococcus pneumoniae serotypes, and the consensus sequence of a combination of SP785 sequences from different Streptococcus pneumoniae serotypes is provided as SEQ ID NO:5. Thus, in some embodiments, the fusion protein comprises a polypeptide having an amino acid sequence comprising or consisting of SEQ ID NO:5 or an antigenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NO:3 or 4). In some embodiments, the SP0785 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 367 consecutive amino acids of the sequence [consensus] shown in SEQ ID NO:5. In some embodiments, the SP0785 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, or 367 consecutive amino acids of the sequence [consensus] shown in SEQ ID NO:5.

[0100] An exemplary nucleotide sequence encoding the SP0785 polypeptide is provided herein as SEQ ID NO:11.

[0101] The SP1500 polypeptide (e.g., SEQ ID NO:6 - 8) and variants thereof

[0102] The SP1500 is described in WO 2014 / 124228. In some embodiments, the SP1500 polypeptide is an amino acid ABC transporter and an amino acid-binding polypeptide that is conserved across Streptococcus pneumoniae strains. In some embodiments, the SP1500 polypeptide is or comprises the full-length SP1500 polypeptide. For example, in some embodiments, the full-length SP1500 polypeptide has 278 amino acids (28 kDa) and is represented by the amino acid sequence as set forth in SEQ ID NO:6. Amino acids 1-26 of SEQ ID NO:6 are predicted to be the signal sequence of the SP1500 polypeptide (amino acids 1-26 of the full-length protein). In some embodiments, the fusion protein comprises the SP1500 polypeptide of Streptococcus pneumoniae. In some embodiments, the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 252 or 278 contiguous amino acids of the SP1500 polypeptide.

[0103] In some embodiments, the SP1500 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 252 or 278 contiguous amino acids of the sequence [full-length] as set forth in SEQ ID NO:6. In some embodiments, the SP1500 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 252 or 278 contiguous amino acids of the sequence [full-length] as set forth in SEQ ID NO:6.

[0104] In some embodiments, the SP1500 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO:7 [minus the signal sequence]. In some embodiments, the SP1500 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO:7 [minus the signal sequence].

[0105] Sequence variation occurs at the protein level between different Streptococcus pneumoniae serotypes, and the consensus sequence of the combination of SP1500 sequences from different Streptococcus pneumoniae serotypes is provided as SEQ ID NO:8. In some embodiments, the fusion protein comprises a polypeptide having an amino acid sequence comprising or consisting of SEQ ID NO:8 or an antigenic fragment thereof (e.g., in place of a polypeptide having an amino acid sequence comprising one of SEQ ID NO:6 or 7). In some embodiments, the SP1500 polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO:8 [consensus]. In some embodiments, the SP1500 polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 252 consecutive amino acids of the sequence shown in SEQ ID NO:8 [consensus].

[0106] Exemplary nucleotide sequences encoding the SP1500 polypeptide are provided herein as SEQ ID NO:14.

[0107] Streptavidin

[0108] In some embodiments, the fusion proteins described herein are components of non-covalent multiple antigen presentation system (MAPS) immunogenic complexes. In some embodiments, the MAPS complex utilizes the high affinity (dissociation constant [K D ≈10 -15 M) non-covalent binding between biotin or a biotin derivative and streptavidin, where streptavidin is a biotin-binding protein with no significant predicted homology to human proteins.

[0109] Streptavidin is a naturally occurring dimeric protein in the streptavidin family and was first discovered in the symbiotic bacterium Rhizobium etli of Phaseolus vulgaris. Streptavidin has only 22% amino acid identity with avidin, a protein common in eggs, but the amino acid residues related to biotin binding are highly conserved [Helppolainen et al., 2007]. In some embodiments, the nucleotide sequence of streptavidin is shown as SEQ ID NO:9. In some embodiments, the amino acid sequence of streptavidin is shown as SEQ ID NO:1. Amino acids 1-44 of SEQ ID NO:1 are predicted to be the signal sequence of streptavidin (amino acids 1-44 of the full-length protein). In some embodiments, the fusion protein comprises streptavidin. In some embodiments, the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 179 consecutive amino acids of the streptavidin polypeptide.

[0110] In some embodiments, the streptavidin polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 179 consecutive amino acids of the sequence shown in SEQ ID NO:1 [full length]. In some embodiments, the streptavidin polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 179 consecutive amino acids of the sequence shown in SEQ ID NO:1 [full length].

[0111] In some embodiments, the streptavidin polypeptide of the fusion protein comprises at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 135 consecutive amino acids of the sequence shown in SEQ ID NO:2 [minus signal sequence]. In some embodiments, the streptavidin polypeptide of the fusion protein comprises an amino acid sequence having at least 60% or higher (including, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 or 135 consecutive amino acids of the sequence shown in SEQ ID NO:2 [minus signal sequence].

[0112] In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 50 - 179 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 55 - 179 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 60 - 179 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 65 - 179 of SEQ ID NO:1.

[0113] In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 45 - 175 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 45 - 171 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 45 - 167 of SEQ ID NO:1. In some embodiments, the streptavidin polypeptide of the fusion protein is or comprises amino acids 45 - 163 of SEQ ID NO:1.

[0114] Linker or spacer

[0115] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is or comprises one or more amino acids. In some embodiments, the fusion protein comprises an antigen polypeptide linked to a biotin - binding moiety via a linker. In some embodiments, the fusion protein comprises a first antigen polypeptide, a second antigen polypeptide, a biotin - binding moiety, and at least one linker. In some embodiments, the first antigen polypeptide and the second antigen polypeptide are linked by a linker. In some embodiments, the first antigen polypeptide or the second antigen polypeptide is linked to the biotin - binding moiety by a linker. In some embodiments, the first antigen polypeptide and the second antigen polypeptide are linked by a first linker; and the first antigen polypeptide or the second antigen polypeptide is linked to the biotin - binding moiety by a second linker.

[0116] In some embodiments, the linker inserts a structure between two protein moieties. In some embodiments, the structure is or comprises an α - helix. In some embodiments, the structure is or comprises a β - strand. In some embodiments, the structure is or comprises a coil / turn. In some embodiments, the structure is or comprises a loop. In some embodiments, the linker reduces the steric hindrance between the two protein moieties linked by the linker. In some embodiments, the linker reduces the unfavorable interactions between the two protein moieties linked by the linker. In some embodiments, the linker comprises a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline, and / or alanine residues. In some embodiments, the linker is hydrophilic. In some embodiments, the linker is hydrophobic. In some embodiments, the linker increases the stability of the fusion protein comprising the linker.

[0117] In some embodiments, the linker does not interfere with the folding of the antigen polypeptide to which it is attached. In some embodiments, the linker does not interfere with the antigenicity of the antigen polypeptide to which it is attached. In some embodiments, the linker does not reduce the antigenicity of the antigen polypeptide to which it is attached. In some embodiments, the linker does not eliminate the antigenicity of the antigen polypeptide to which it is attached. In some embodiments, the effect of the linker is determined by comparing the polypeptide with the polypeptide attached to the linker.

[0118] In some embodiments, the linker does not interfere with the folding of the biotin-binding moiety to which it is attached. In some embodiments, the linker does not interfere with the biotin-binding ability of the biotin-binding moiety to which it is attached. In some embodiments, the linker does not reduce the biotin-binding ability of the biotin-binding moiety to which it is attached. In some embodiments, the linker does not eliminate the biotin-binding ability of the biotin-binding moiety to which it is attached. In some embodiments, the effect of the linker is determined by comparing the biotin-binding moiety with the biotin-binding moiety attached to the linker.

[0119] In some embodiments, the linker is not antigenic. In some embodiments, the linker does not elicit a T cell response. In some embodiments, the linker does not elicit a B cell response. In some embodiments, the linker does not induce a T cell or B cell response.

[0120] In some embodiments, the linker comprises two or more amino acids. In some embodiments, the length of the linker can be 3 - 100, 5 - 100, 10 - 100, 20 - 100, 30 - 100, 40 - 100, 50 - 100, 60 - 100, 70 - 100, 80 - 100, 90 - 100, 5 - 55, 10 - 50, 10 - 45, 10 - 40, 10 - 35, 10 - 30, 10 - 25, 10 - 20, 10 - 15, 3 - 10, 3 - 9, 3 - 8, 3 - 7, 3 - 6, 3 - 5, 3 - 4 or 2 - 3 amino acids. In some embodiments, the linker comprises 10 - 100, 10 - 90, 10 - 80, 10 - 70, 10 - 60, 10 - 50, 10 - 40, 10 - 30, 10 - 20, 10 - 15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 amino acids. In some embodiments, the linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids.

[0121] In some embodiments, the linker is a flexible linker. Flexible linkers can be used to connect domains that require a degree of movement or interaction and can include small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules and thus reduce unfavorable interactions between the linker and the protein moiety. In some embodiments, the linker comprises small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. In some embodiments, the linker is a Gly-Ser linker.

[0122] In some embodiments, the linker is or comprises the amino acid sequence GGGGS (SEQ ID NO:37). In some embodiments, the linker is or comprises (GGGGS) n (SEQ ID NO:39), where n represents the number of repeating GGGGS units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the polypeptide linker can have as or comprise GGGGSGGGGSGGGGS (SEQ ID NO:41) (i.e., (GGGGS) 3 ) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:42) (i.e., (GGGGS) 6 ) as its amino acid sequence. In some embodiments, the linker comprises one or more of Gly, Ser, Thr, Ala, Lys, and Glu. In some embodiments, the linker is or comprises KESGSVSSEQLAQFRSLD (SEQ ID NO:43). In some embodiments, the linker is or comprises EGKSSGSGSESKST (SEQ ID NO:44). In some embodiments, the linker is or comprises (Gly) n (SEQ IDNO:45), where n represents the number of repeating Gly residues and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises GGG. In some embodiments, the linker is or comprises (Gly) 6 (SEQ ID NO:40). In some embodiments, the linker is or comprises (Gly) 8(SEQ ID NO:46). In some embodiments, the linker is or comprises GSAGSAAGSGEF (SEQ ID NO:47). In some embodiments, the linker is or comprises the amino acid sequence of AAA (SEQ ID NO:38).

[0123] In some embodiments, the linker is a rigid linker. Rigid linkers are useful for maintaining a fixed distance between domains and maintaining their independent functions. Rigid linkers may also be useful when the spatial separation of domains is critical for maintaining the stability or biological activity of one or more components in the fusion. In some embodiments, the linker is or comprises (EAAAK) n (SEQ ID NO:48), where n represents the number of repeated EAAAK units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises A(EAAAK) n A, (SEQ ID NO:49), where n represents the number of repeated EAAAK units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises A(EAAAK) n A, where n represents the number of repeated EAAAK units and is 2, 3, 4 or 5. In some embodiments, the linker is or comprises A(EAAAK) 4 ALEA(EAAAK) 4 A(SEQ ID NO:50). In some embodiments, the linker is or comprises [A(EAAAK) n A] m (SEQ ID NO:51), where n is 2, 3 or 4, and m is 1 or 2. In some embodiments, the linker is or comprises AEAAAKEAAAKA (SEQ ID NO:52).

[0124] In some embodiments, the linker is or comprises (X-Pro) n (SEQ ID NO:53), where X represents any amino acid, where n represents the number of repeated X-Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises (Ala-Pro) n(SEQ ID NO:54), where n represents the number of repeated Ala - Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises (Ala - Pro) n , where n represents the number of repeated Ala - Pro units and is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0125] In some embodiments, the linker is or comprises (Lys - Pro) n (SEQ ID NO:55), where n represents the number of repeated Lys - Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises (Glu - Pro) n (SEQ ID NO:56), where n represents the number of repeated Glu - Pro units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. In some embodiments, the linker is or comprises (Ala - Pro) 7 (SEQ ID NO:57).

[0126] In some embodiments, the linker is or comprises GAPGGGGGAAAAAGGGGGGAP (GAG linker, SEQ ID NO:58). In some embodiments, the linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (GAG2 linker, SEQ ID NO:59). In some embodiments, the linker is or comprises GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGG GGAP (GAG3 linker, SEQ ID NO:60).

[0127] Suitable linkers or spacers also include those having an amino acid sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology or identity to the above - exemplified linkers.

[0128] Additional linkers applicable to some embodiments are visible in U.S. Patent Publication No. 2012 / 0232021, filed Mar. 2, 2012, and [Chen, 2013], the disclosures of which are incorporated herein by reference in their entireties.

[0129] Tagged fusion protein

[0130] In some embodiments, the fusion proteins described herein may contain tags. The tags can be N-terminal or C-terminal. For example, a tag can be added to a polypeptide (either via addition or modification on the coding DNA sequence) to facilitate purification, detection, solubility, or to confer other desired properties to the protein. In some embodiments, the tag can be a peptide, oligopeptide, or polypeptide that can be used for affinity purification. In some embodiments, the tag is, comprises, or is derived from one or more of the following: polyhistidine (His), glutathione S-transferase (GST), tandem affinity purification (TAP), FLAG, myc, human influenza hemagglutinin (HA), maltose binding protein (MBP), vesicular stomatitis virus glycoprotein (VSV-G), thioredoxin, V5, avidin, streptavidin, biotin carboxyl carrier protein (BCCP), calmodulin, Nus, S-tag, lipoprotein D, and galactosidase. In some embodiments, the His tag is or comprises the amino acid sequence of H n where n is an integer between 2 and 10. Exemplary His tags include HHHHHH (SEQ ID NO:15) and MSYYHHHHHH (SEQ ID NO:16). In other embodiments, the fusion protein does not contain a tag such as a protein purification tag and is purified by methods that do not rely on affinity for a purification tag. In some embodiments, the fusion protein contains no more than 1, 2, 3, 4, 5, 10, or 20 additional amino acids at one or both ends of the polypeptide of Table 1 or the fusion protein of Table 2.

[0131] In some embodiments, the fusion proteins described herein may contain a membrane translocation sequence (MTS) to facilitate introduction of the fusion protein into mammalian cells and subsequent stimulation of a cell-mediated immune response. Exemplary membrane translocation sequences include the hydrophobic region in the signal sequence of Kaposi fibroblast growth factor, the MTS of synuclein, the third helix of the Antennapedia homeodomain, SN50, the integrin 3h region, HIV Tat, pAntp, PR-39, abaecin, apidaecin, Bac5, Bac7, the Plasmodium berghei (P.berghei) CS protein, and those MTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680, and 6,248,558.

[0132] Nucleic acid

[0133] In some embodiments, the present disclosure provides nucleic acids encoding one or more of the polypeptides and / or fusion proteins described herein, such as DNA, RNA, or analogs thereof. The underlying DNA sequences of the polypeptides described herein can be modified in a manner that does not affect the sequence of the protein product, and such sequences are included in the present invention. In some embodiments, the DNA sequences can be codon-optimized to improve expression in a host such as a bacterial cell line (e.g., Escherichia coli), an insect cell line (e.g., using a baculovirus expression system), or a mammalian (e.g., human or Chinese hamster ovary) cell line.

[0134] In some embodiments, the present disclosure provides nucleic acids having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% identity to the nucleic acid sequences provided in Table 1, Table 2, or variants or portions thereof, such as DNA, RNA, or analogs thereof. In some embodiments, the nucleic acid has a length of 600 - 2000, 800 - 1800, 1000 - 1600, or 1200 - 1400 nucleotides. In some embodiments, the nucleic acid has a length of 600 - 1600, 800 - 1800, 1000 - 2000, 2000 - 3000, or 3000 - 4000 nucleotides. In some embodiments, the nucleic acid can be used to recombinantly produce the polypeptides or fusion proteins of Table 1 or Table 2 or antigenic fragments thereof. In some embodiments, the nucleic acid can be used as a vaccine.

[0135] The nucleic acid sequences encoding the SP0785 variants (SEQ ID NO: 3 and 4) are provided as SEQ ID NO: 11 and 12. The nucleic acid sequences encoding the SP1500 variants (SEQ ID NO: 6 and 7) are provided as SEQ ID NO: 13 and 14. The nucleic acid sequences encoding different fusion proteins (SEQ ID NO: 17 - 26) are provided as SEQ ID NO: 27 - 36. In all cases, due to the degeneracy of the genetic code, other DNA sequences (including multiple codon-optimized sequences) can be envisioned by those of ordinary skill in the art to encode these polypeptides and fusion proteins.

[0136] Nucleic acids encoding the polypeptides or fusion proteins or fragments thereof of Table 1 or Table 2 can be cloned into any of a variety of expression vectors under the control of a variety of regulatory elements and fused with other target sequences. Methods for cloning nucleic acids are routine and conventional in the art. For general references describing the molecular biology methods mentioned in this application (such as isolating, cloning, modifying, labeling, manipulating, sequencing, and otherwise processing or analyzing nucleic acids and / or proteins), see, for example, Sambrook et al., 1989; Ausubel et al., 1995; Davis et al., 1986; Hames et al., 1985; Dracopoli et al., 2018; and Coligan et al., 2018.

[0137] Use of the fusion protein

[0138] In some embodiments, the fusion proteins described herein do not have hemolytic activity or have minimal hemolytic activity. For example, in some embodiments, the hemolytic activity of the fusion proteins described herein can be determined by turbidimetry (OD 420 ) by measuring the protein concentration that lyses 50% of red blood cells after incubating different dilutions of the fusion protein with red blood cells (e.g., sheep red blood cells). In some such embodiments, the hemolytic activity of the fusion proteins described herein can be characterized in that, for a given protein concentration, OD 420 is less than 0.4 or lower, including, for example, less than 0.3, less than 0.25, less than 0.2 or lower.

[0139] In some embodiments, the polypeptides and fusion proteins of Streptococcus pneumoniae described herein, and fragments and variants thereof, are immunogenic. These polypeptides and fusion proteins can be immunogenic in mammals such as mice, rats, guinea pigs, or humans. An antigenic polypeptide or fusion protein is generally one that is capable of eliciting a significant immune response in an assay or in a subject. The immune response can be innate, humoral, cell-mediated, or mucosal (combining elements of innate, humoral, and cell-mediated immunity). For example, the antigenic polypeptide or fusion protein can increase the amount of IL-17 produced by T cells. Alternatively or additionally, the antigenic polypeptide or fusion protein can (i) induce the production of antibodies (e.g., neutralizing antibodies) that bind to the polypeptide and / or the whole bacterium, (ii) induce Th17 immunity, (iii) activate a CD4+ T cell response, e.g., by increasing the number of CD4+ T cells and / or increasing the localization of CD4+ T cells at the site of infection or reinfection, (iv) activate a CD8+ T cell response, e.g., by increasing the number of CD8+ T cells and / or increasing the localization of CD8+ T cells at the site of infection or reinfection, (v) activate CD4+ and CD8+ responses, (vi) activate CD4- / CD8- immunity, (vii) induce Th1 immunity, (viii) induce antimicrobial peptides, (ix) activate innate immunity, or any combination of the foregoing. In some embodiments, the antigenic polypeptide or fusion protein elicits the production of a detectable amount of antibodies specific for the antigen.

[0140] In some embodiments, the fusion proteins described herein are antigens or have antigenic properties. In some embodiments, the fusion proteins described herein are carrier proteins or have carrier properties. In some embodiments, the fusion proteins described herein are both antigens and carrier proteins. In some embodiments, the fusion proteins described herein have both carrier properties and antigenic properties simultaneously.

[0141] In some embodiments, the fusion proteins described herein are antigens of an immunogenic complex (e.g., a multiple antigen presentation system (MAPS) complex as described in WO2012 / 155007, the entire content of which is incorporated herein by reference for the purposes indicated herein). In some embodiments, the fusion proteins described herein are carrier proteins of an immunogenic complex. In some embodiments, the fusion proteins described herein are both carrier proteins and antigens of an immunogenic complex.

[0142] In some embodiments, the polypeptides of the fusion proteins described herein have less than 20%, 30%, 40%, 50%, 60%, or 70% identity with human self-antigens and / or gut commensal bacteria (such as certain Bacteroides, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Escherichia, and Lactobacillus species). Examples of human self-antigens include insulin, proliferating cell nuclear antigen, cytochrome P450, and myelin basic protein.

[0143] The polypeptides contained in the fusion proteins described herein may comprise one or more immunogenic portions and one or more non-immunogenic portions. Immunogenic portions can be identified by various methods, including protein microarrays, ELISPOT / ELISA techniques, and / or specific assays of different deletion mutants (such as fragments) of the polypeptide in question. Immunogenic portions can also be identified by computer algorithms. Some such algorithms, such as EpiMatrix (produced by EpiVax), use computational matrix methods. Other computational tools for identifying antigenic epitopes include PEPVAC (vaccine based on promiscuous epitopes, hosted on the World Wide Web by the Dana Farber Cancer Institute at immunax.dfci.harvard.edu / PEPVAC), MHCPred (which uses partial least squares method and is hosted on the World Wide Web by the Jenner Institute at www.jenner.ac.uk / MHCPred), and the Immune Epitope Database algorithm at tools.immuneepitope.org on the World Wide Web. The antigenic fragments of the polypeptides described herein contain at least one immunogenic portion, as measured experimentally or identified by an algorithm (e.g., the SYFPEITHI algorithm, available at www.syfpeithi.de).

[0144] Table 3 and Table 4 present representative predicted epitopes of SP0785 and SP1500, respectively.

[0145] Table 3. Top 20 of 353 total predicted MHC II binding sites (HLA-DRB1*0101) for an exemplary SP0785 polypeptide

[0146]

[0147]

[0148] Table 4. The top 20 out of 239 total predicted MHC II binding sites (HLA-DRB1*0101) of exemplary SP1500 polypeptides

[0149]

[0150]

[0151] Immunogenic and vaccine compositions

[0152] The present disclosure also provides an immunogenic composition (e.g., a vaccine composition) of one or more of the fusion proteins described herein, or an immunogenic composition (e.g., a vaccine composition) comprising one or more of the fusion proteins described herein. In some embodiments, the immunogenic composition comprises one or more fusion proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the fusion proteins listed in Table 2. In some embodiments, the immunogenic composition comprises a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs: 17-26. In some embodiments, the immunogenic composition comprises a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO: 23. In some embodiments, the immunogenic composition comprises a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity to CP1 (e.g., as described in Figure 1 ).

[0153] In some embodiments, the immunogenic composition may also comprise portions of the fusion proteins described herein, such as internal deletion mutants, truncated mutants, and fragments. In some embodiments, the portions of the fusion proteins are immunogenic. The immunogenicity of a portion of a fusion protein is readily determined using the same assays used to determine the immunogenicity of the full-length fusion protein. In some embodiments, the portion of the fusion protein has substantially the same immunogenicity as the full-length fusion protein. In some embodiments, the immunogenicity is not less than 10%, 20%, 30%, 40%, or 50% of the immunogenicity of the fusion protein of Table 2.

[0154] Multicomponent immunogenic and vaccine compositions

[0155] In some embodiments, the immunogenic compositions (e.g., vaccine compositions) described herein include the fusion proteins described herein and one or more additional, or two or more known Streptococcus pneumoniae antigens. In some cases, the known Streptococcus pneumoniae antigens are primarily antibody targets. In some cases, the known Streptococcus pneumoniae antigens are polysaccharides. In some cases, the known Streptococcus pneumoniae antigens can protect against Streptococcus pneumoniae colonization, or prevent Streptococcus pneumoniae infections such as sepsis, pneumonia, meningitis, otitis media, sinusitis, or infections in other sites or organs caused by Streptococcus pneumoniae.

[0156] A class of suitable Streptococcus pneumoniae antigens that are well recognized in the art are pneumococcal surface protein A (PspA) and derivatives of PspA. Derivatives of PspA include proline-rich segments with non-proline blocks (PR+NPB, also known as PRN, and further described in Daniels et al., 2010), and related constructs that contain all or fragments of the proline-rich region of PspA (e.g., regions that contain one or more of the sequences PAPAP, PKP, PKEPEQ, and PEKP and optionally include non-proline blocks). In some embodiments, the fragment or variant of PspA comprises a proline-rich segment having a non-proline block and 10, 20, 30, 40, or more additional amino acids of the PspA sequence. Peptides containing NPB are particularly immunogenic, indicating that NPB may be an important epitope.

[0157] Another class of suitable Streptococcus pneumoniae antigens that are recognized in the art is pneumolysin-like. Pneumolysin-like is homologous to the Streptococcus pneumoniae protein pneumolysin (PLY or Ply), but has reduced toxicity compared to pneumolysin. Pneumolysin-like can be a naturally occurring or engineered derivative of pneumolysin. In some embodiments, pneumolysin-like has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to pneumolysin. In some embodiments, in an assay for one or both of hemolytic activity on red blood cells and polymorphonuclear leukocyte inhibition, pneumolysin-like exhibits less than 1 / 2, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 200, 1 / 500 or 1 / 1000 the toxicity of pneumolysin. Both assays are described in Saunders et al., 1989. Exemplary pneumolysin-likes include PdT, a triple mutant further described in Berry et al., 1995; Pd-A and Pd-B, further described in Paton et al., 1991; rPd2 and rPd3, further described in Ferreira et al., 2006; Ply8, MPLY and L460D, further described in, for example, US 2009 / 0285846 and L. Mitchell, 2011; or variants thereof. In some embodiments, pneumolysin has a mutation in the catalytic center, such as at or near amino acid 428 or 433.

[0158] Other suitable Streptococcus pneumoniae antigens include choline-binding protein A (CbpA) and its derivatives (Ogunniyi et al., 2001); pneumococcal surface adhesin A (PsaA); caseinolytic protease; sortase A (SrtA); pilus 1 RrgA adhesin; PpmA; PrtA; PavA; LytA; Stk-PR; PcsB; RrgB and its derivatives. CpbA derivatives include the constructs described in WO 2012 / 134975. Such constructs can comprise one or more copies of the R2 domain, R21 and / or R22 subdomains of CpbA, or active variants and fragments thereof, or any combination thereof. Such constructs can also comprise pneumolysin-like.

[0159] In some embodiments, an immunogenic composition (e.g., a vaccine composition) comprises one or more of the fusion proteins described herein in a mixture with one or more polypeptides from Table 1 or antigenic fragments or variants thereof. In some embodiments, the mixture comprises full-length polypeptides and fragments that are produced by processing or partial processing of a signal sequence by an expression host such as Escherichia coli, an insect cell line (e.g., a baculovirus expression system), or a mammalian cell line (e.g., human or Chinese hamster ovary).

[0160] In some embodiments, in the absence of any other antigen, the immunogenic composition comprises one or more fusion proteins of any one of SEQ ID NOs: 17-26. In some embodiments, in the absence of any other antigen, the immunogenic composition comprises the fusion protein of SEQ ID NO: 23. In some embodiments, in the absence of other antigens, the immunogenic composition comprises a combination of one or more fusion proteins of any one of SEQ ID NOs: 17-26 and one or more additional proteins of any one of SEQ ID NOs: 1-8. In some embodiments, in the absence of any other antigen, the immunogenic composition comprises a combination of the fusion protein of SEQ ID NO: 23 and one or more additional proteins of any one of SEQ ID NOs: 1-8.

[0161] In some embodiments, the fusion proteins described herein can be conjugated to Streptococcus pneumoniae polysaccharides. In some embodiments, the fusion proteins described herein can be non-covalently complexed with Streptococcus pneumoniae polysaccharides. Streptococcus pneumoniae polysaccharides can be described, for example, in U.S. Patent No. 5,623,057, U.S. Patent No. 5,371,197, or PCT / US2011 / 023526. Non-covalent complexes can be those of, for example, multiple antigen presenting systems (MAPS), as described in PCT / US2012 / 037412, PCT / US2012 / 037541, and Zhang et al., 2013.

[0162] In some embodiments, the fusion proteins described herein are covalently bound to another molecule. For example, this can increase the half-life, solubility, bioavailability, or immunogenicity of the fusion protein. Molecules that can be covalently bound to the fusion protein include carbohydrates, biotin, poly(ethylene glycol) (PEG), polysialic acid, N-propionylated polysialic acid, nucleic acids, polysaccharides, and PLGA. There are many different types of PEG, with molecular weights ranging from less than 300 g / mol to more than 10,000,000 g / mol. The PEG chains can be linear, branched, or have a comb or star geometry. In some embodiments, the fusion protein is covalently bound to a moiety that stimulates the immune system. An example of such a moiety is a lipid moiety. In some cases, the lipid moiety is recognized by Toll-like receptors (TLRs) such as TLR-2 or TLR-4 and activates the innate immune system.

[0163] In some embodiments, the fusion proteins described herein are mixed with one or more other components using known methods to form a multi-component immunogenic composition. In some embodiments, the fusion proteins described herein are nanoencapsulated with one or more other components using known methods. In some embodiments, the fusion proteins described herein are molded into nanoparticles or microparticles with one or more other components using known methods. In some embodiments, the fusion proteins described herein are conjugated via covalent bonds with one or more other components to form a multi-component immunogenic composition. In some embodiments, the fusion proteins described herein are non-covalently linked with one or more other components using known methods to form a multi-component immunogenic composition. Other methods of combining the fusion protein and one or more other components are described, for example, in PCT / US2012 / 37412 and PCT / US2009 / 44956.

[0164] Nucleic acid-based immunogenic compositions and vaccines

[0165] The present disclosure also provides an immunogenic composition (e.g., a vaccine composition) comprising one or more nucleic acids encoding a fusion protein described herein, or an immunogenic composition (e.g., a vaccine composition) comprising one or more nucleic acids encoding a fusion protein described herein. In some embodiments, the immunogenic composition comprises one or more nucleic acids encoding a fusion protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to the fusion proteins listed in Table 2. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs: 17-26. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to SEQ ID NO: 23. In some embodiments, the immunogenic composition comprises a nucleic acid encoding a fusion protein that is or comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to CP1.

[0166] In some embodiments, the immunogenic composition comprises one or more nucleic acids having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to any one of SEQ ID NOs: 27-36. In some embodiments, the immunogenic composition comprises a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO: 33. In all cases, due to the degeneracy of the genetic code, other DNA sequences (including multiple codon-optimized sequences) can encode such fusion proteins. In some embodiments, these nucleic acids are expressed in the immunized individual, resulting in the production of the encoded Streptococcus pneumoniae fusion protein, and the Streptococcus pneumoniae fusion protein so produced has an immunostimulatory or immunoprotective effect in the immunized individual.

[0167] Such nucleic acid-containing immunostimulatory compositions can comprise, for example, an origin of replication and / or a promoter driving the expression of one or more nucleic acids encoding one or more fusion proteins of SEQ ID NOs: 27-36. Such compositions can also comprise a bacterial plasmid vector into which is inserted a promoter (sometimes a strong viral promoter), one or more nucleic acids encoding one or more fusion proteins of SEQ ID NOs: 17-26, and a polyadenylation / transcription termination sequence. In some cases, the nucleic acid is DNA. In some cases, the nucleic acid is RNA.

[0168] Use of immunogenic and vaccine compositions

[0169] In some embodiments, an immunogenic composition or vaccine comprising one or more of the fusion proteins described herein is characterized by an increase in one or more opsonization potentials or immune responses against one or more of the fusion proteins relative to a predetermined level, as measured by ELISA and / or by a functional antibody assay. In some embodiments, the one or more opsonization potentials or immune responses against one or more of the fusion proteins are increased by at least 30% or more relative to a predetermined level, including, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, as measured by ELISA and / or by a functional antibody assay. In some embodiments, the one or more opsonization potentials or immune responses against one or more of the fusion proteins are increased by at least 1-fold, 2-fold, 3-fold, 4-fold or 5-fold relative to a predetermined level, as measured by ELISA and / or by a functional antibody assay. In some embodiments, the predetermined level is a pre-immunization level (e.g., the level observed when a subject is not immunized or is immunized in the absence of one or more of the fusion proteins described herein).

[0170] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising one or more of the fusion proteins described herein induces an immune response against Streptococcus pneumoniae. In some embodiments, after administration to a subject, the immunogenic composition or vaccine induces an immune response against one or more Streptococcus pneumoniae serotypes. In some embodiments, after administration to a subject, the immunogenic composition or vaccine induces a protective immune response against one or more Streptococcus pneumoniae serotypes. In some embodiments, the immune response is an antibody response or a B cell response. In some embodiments, the immune response is a T cell response. In some embodiments, the immune response is an innate immune response. In some embodiments, the immune response is a CD4+ T cell response, including a Th1, Th2, or Th17 response, or a CD8+ T cell response, or a CD4+ and CD8+ T cell response, or a CD4- / CD8- T cell response. In some embodiments, the immune response is an antibody response or a B cell response and a T cell response. In some embodiments, the immune response is an antibody response or a B cell response, a T cell response, and an innate immune response.

[0171] In some embodiments, an immunogenic composition or vaccine comprising one or more of the fusion proteins described herein can be used for the prophylactic and / or therapeutic treatment of Streptococcus pneumoniae. Accordingly, the present disclosure provides a method of immunizing a subject afflicted with or susceptible to Streptococcus pneumoniae infection, the method comprising administering an immunologically effective amount of any immunogenic composition or vaccine that comprises one or more of the fusion proteins described herein. The subject to be immunized can be male or female and can be an infant, child, adolescent, or adult. In some embodiments, the subject being treated is human. In other embodiments, the subject is a non-human animal.

[0172] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising the fusion protein described herein treats or prevents Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents bacteremia caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents sepsis caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents organ damage caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents meningitis caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents pneumonia caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents otitis media caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine treats or prevents sinusitis caused by Streptococcus pneumoniae infection.

[0173] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising the fusion protein described herein inhibits Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits bacteremia caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits sepsis caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits organ damage caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits meningitis caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits pneumonia caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits otitis media caused by Streptococcus pneumoniae infection or reduces its incidence. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits sinusitis caused by Streptococcus pneumoniae infection or reduces its incidence.

[0174] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising the fusion protein described herein reduces the severity of Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of bacteremia caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of sepsis caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of organ damage caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of meningitis caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of pneumonia caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of otitis media caused by Streptococcus pneumoniae infection. In some embodiments, after administration to a subject, the immunogenic composition or vaccine reduces the severity of sinusitis caused by Streptococcus pneumoniae infection.

[0175] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising the fusion protein described herein inhibits the transmission of Streptococcus pneumoniae from the subject to another subject. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits the colonization of Streptococcus pneumoniae in the subject. In some embodiments, after administration to a subject, the immunogenic composition or vaccine inhibits the colonization of Streptococcus pneumoniae in the nasopharynx of the subject.

[0176] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein induces an immune response in the subject against Streptococcus pneumoniae at a level higher than that of a control composition. In some embodiments, after administration to a subject, the immunogenic composition or vaccine induces an immune response against one or more Streptococcus pneumoniae serotypes at a level higher than that of a control composition. In some embodiments, the higher level is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% of the control composition.

[0177] In some embodiments, after administration to a subject, an immunogenic composition or vaccine comprising a fusion protein described herein induces an immune response that helps prevent the establishment of Streptococcus pneumoniae at a level higher than that of a control composition. In some embodiments, the immunogenic composition or vaccine prevents colonization at a level higher than that of a control composition. In some embodiments, the immunogenic composition or vaccine inhibits Streptococcus pneumoniae infection in non-colonized or non-infected subjects at a level higher than that of a control composition. In some embodiments, the immunogenic composition or vaccine reduces the duration of Streptococcus pneumoniae colonization in subjects that are already colonized at a level higher than that of a control composition. In some embodiments, the higher level is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% of the control composition.

[0178] Antibody composition

[0179] Some embodiments provide an antibody composition comprising antibodies generated in a mammal immunized with an immunogenic composition or vaccine comprising a fusion protein described herein. In some embodiments, the antibodies comprise at least one antibody selected from the group consisting of monoclonal antibodies (mAbs) and anti-idiotypic antibodies. In some embodiments, the antibody composition comprises an isolated gamma globulin fraction. In some embodiments, the antibody composition comprises polyclonal antibodies. In some embodiments, the antibody composition is administered to a subject.

[0180] Vaccine preparation

[0181] The optimal amount of the components of a particular vaccine comprising a fusion protein described herein can be determined by standard studies involving observing an appropriate immune response in a subject. After an initial immunization, the subject may receive one or more booster immunizations that are sufficiently spaced in time.

[0182] An immunogenic composition or vaccine comprising a fusion protein described herein and / or its formulation can be formulated in unit dosage form for ease of administration and uniformity of dosage. The therapeutically effective dosage level specific to any particular subject or organism can depend on various factors including: the severity or degree of risk of the infection; the activity of the particular vaccine or vaccine composition employed; other characteristics of the particular vaccine or vaccine composition employed; the age, body weight, general health, sex of the subject, the diet of the subject, the pharmacokinetic profile of the subject, the time of administration (e.g., with respect to other subject activities such as eating, sleeping, receiving other medications including the administration of other vaccines), the route of administration, the excretion rate of the particular vaccine or vaccine composition employed; vaccines combined or co-administered with the vaccine composition employed; and similar factors well known in the medical arts.

[0183] An immunogenic composition or vaccine comprising a fusion protein described herein for use according to the present disclosure can be formulated into a composition (e.g., a pharmaceutical composition) according to known techniques. Vaccine preparation is generally described in Vaccine Design (Powell and Newman, 1995). By way of example, an immunizing amount of the vaccine product can be formulated with one or more pharmaceutically suitable carrier materials, organic or inorganic, liquid or solid.

[0184] Generally, pharmaceutically acceptable carriers include solvents, dispersion media, and the like that are compatible with the administration of the drug. By way of example, and depending on the judgment of the formulator (Martin, 1975), materials that can serve as pharmaceutically acceptable carriers include, but are not limited to: sugars, such as lactose, glucose, dextrose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; polyols, such as glycerol, propylene glycol, and liquid polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, and preservatives, and antioxidants may also be present in the composition.

[0185] Vaccines can be formulated by combining one or more of the fusion proteins described herein with a carrier and / or other optional components by any available means, including, for example, conventional mixing, granulation, dissolution, lyophilization, or similar processes.

[0186] Vaccines containing one or more of the fusion proteins described herein can be lyophilized until they are to be used, at which time they are reconstituted temporarily with a diluent. In some embodiments, the vaccine components or composition are lyophilized in the presence of one or more other components (such as adjuvants) and reconstituted temporarily with a saline solution. Alternatively, the individual components or sets of components can be lyophilized and / or stored separately (such as in a vaccination kit), the components are reconstituted, and mixed or administered separately to a subject before use.

[0187] Lyophilization can produce a more stable composition (such as by preventing or reducing polysaccharide antigen degradation). Lyophilization of vaccines or vaccine components is well known in the art. Generally, liquid vaccines or vaccine components are often lyophilized in the presence of an anti-caking agent (such as a sugar, such as sucrose or lactose). In some embodiments, the anti-caking agent is present, for example, at an initial concentration of 10 - 200 mg / ml. Lyophilization typically occurs in a series of steps, such as a cycle starting at -69°C, gradually adjusted to -24°C over 3 h, then held at this temperature for 18 h, then gradually adjusted to -16°C over 1 h, then held at this temperature for 6 h, then gradually adjusted to +34°C over 3 h, and finally held at this temperature for more than 9 h.

[0188] In some embodiments, the vaccine comprising the fusion protein described herein is a liquid. In some embodiments, the liquid is a reconstituted lyophylate. In some embodiments, the pH of the vaccine is about 5, about 6, about 7, or about 8. In some embodiments, the pH of the vaccine is between about 5 and about 7.5. In some embodiments, the pH of the vaccine is between 5 and 7.5. In some embodiments, the pH of the vaccine is between about 5.3 and about 6.3. In some embodiments, the pH of the vaccine is between 5.3 and 6.3. In some embodiments, the pH of the vaccine is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0189] The vaccines or vaccine components used according to the present disclosure can be incorporated into liposomes, cochleates, biodegradable polymers (such as polylactide, polyglycolide, and polylactide-co-glycolide), or immunostimulating complexes (ISCOMs).

[0190] In certain cases, it may be desirable to prolong the action or release of the vaccines used according to the present invention, for example, by slowing the absorption of one or more vaccine components. This delay in absorption can be achieved, for example, by using a liquid suspension of crystalline or amorphous materials with poor water solubility. Then, the absorption rate of the product depends on its dissolution rate, which in turn depends on size and form. Alternatively or additionally, delayed absorption can be achieved by dissolving or suspending one or more vaccine components in an oil vehicle. Injectable depot forms can also be employed to delay absorption. These depot forms can be prepared by shaping the microcapsule matrix of one or more vaccine components into a biodegradable polymer network. Depending on the ratio of the polymer to the vaccine component and the nature of the specific polymer employed, the release rate can be controlled.

[0191] Examples of biodegradable polymers that can be employed according to the present disclosure include, for example, poly(orthoesters) and poly(anhydrides). A specific exemplary polymer is polylactide-polyglycolide.

[0192] Depot injectable formulations can also be prepared by coating the product with liposomes or microemulsions that are compatible with body tissues.

[0193] Polymeric delivery systems can also be used in non-depot formulations including, for example, oral formulations. For example, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used in oral formulations. Polysaccharide antigens or conjugates can be formulated with these polymers, for example, to prepare particles, microparticles, extrudates, solid dispersions, mixtures, or other combinations to facilitate the preparation of suitable formulations (such as oral formulations).

[0194] Vaccines comprising one or more of the fusion proteins described herein according to the present disclosure include immunogenic compositions and may additionally include one or more additional active agents (i.e., agents that exert a biological effect - non-inert components). For example, one or more adjuvants are often included in vaccine preparation. It should be understood that these additional agents can be formulated with one or more other vaccine components or can be kept separate and combined at the time of or near the time of administration. In some embodiments, these additional components can be administered separately from some or all of the other vaccine components within an appropriate time window for achieving the relevant effect.

[0195] Adjuvant

[0196] Vaccine formulations and immunogenic compositions comprising the fusion proteins described herein can include adjuvants. Generally, an adjuvant is an agent that enhances the immune response against an antigen. Adjuvants can be broadly classified into two categories based on their main mechanism of action: vaccine delivery systems and immunostimulatory adjuvants (see, for example, Singh et al., 2003). In most vaccine formulations, adjuvants provide signals to the immune system such that it generates a response against the antigen, and the antigen is required to drive the specificity of the response against the pathogen. Vaccine delivery systems are often particulate formulations such as emulsions, microparticles, immunostimulating complexes (ISCOMs), nanoparticles (which can be, for example, particles and / or matrices), and liposomes. In contrast, immunostimulatory adjuvants sometimes originate from or are derived from pathogens and can represent pathogen-associated molecular patterns (PAMPs) that activate cells of the innate immune system, such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), or CpG-containing DNA.

[0197] Alternatively, adjuvants can be classified as organic adjuvants and inorganic adjuvants. Inorganic adjuvants include alum salts such as aluminum phosphate, amorphous hydroxy aluminum phosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants contain organic molecules including polymers. Non-limiting examples of organic adjuvants include cholera toxin / toxoid, other enterotoxins / toxoids, or labile toxins / toxoids of Gram-negative bacteria, interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (such as interferon γ), granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0198] Adjuvants can also be classified according to the responses they induce. In some embodiments, the adjuvant induces the generation, proliferation, or activation of Th1 cells or Th2 cells. In other embodiments, the adjuvant induces the generation, proliferation, or activation of B cells. In still other embodiments, the adjuvant induces the activation of antigen-presenting cells. These categories are not mutually exclusive; in some cases, the adjuvant activates more than one type of cell.

[0199] In some embodiments, the adjuvant induces the generation, proliferation, or activation of Th17 cells. The adjuvant can promote the secretion of IL-17 by CD4+ T cells or CD8+ T cells. In some embodiments, the adjuvant that induces the generation, proliferation, or activation of Th17 cells is an adjuvant that produces at least a 2-fold and in some cases a 10-fold experimental sample to control ratio in the following assay. In the assay, the experimenter compares the levels of IL-17 secreted by two cell populations: (1) cells from animals immunized with the adjuvant and a polypeptide known to induce Th17 generation, proliferation, or activation, and (2) cells from animals treated with the adjuvant and an irrelevant (control) polypeptide. The adjuvant that induces the generation, proliferation, or activation of Th17 cells can cause the cells of population (1) to produce more than 2-fold or more than 10-fold more IL-17 than the cells of population (2). IL-17 can be measured, for example, by ELISA or ELISPOT. Certain toxins such as cholera toxin and labile toxin (produced by enterotoxigenic Escherichia coli (E. coli) or ETEC) activate the Th17 response. Thus, in some embodiments, the adjuvant is a toxin or a toxoid. Cholera toxin has been successfully used in a mouse model to induce protective immunity in combination with certain polypeptides from Table 1. One form of the labile toxin is produced by Intercell. Mutant derivatives of the labile toxin (toxoid) that are active as adjuvants but have significantly lower toxicity can also be used. Exemplary detoxified mutant derivatives of the labile toxin include mutants lacking ADP-ribosyltransferase activity. Specific detoxified mutant derivatives of the labile toxin include LTK7 (Douce et al., 1995) and LTK63 (Williams et al., 2004), LT-G192 (Douce et al., 1999), and LTR72 (Giuliani et al., 1998).

[0200] In some embodiments, the adjuvant comprises VLP (virus-like particles). One such adjuvant platform, the alphavirus replicon, uses alphavirus to induce the activation of Th17 cells and is produced by Alphavax. In some embodiments of the alphavirus replicon system, the alphavirus can be engineered to express a target antigen, a target cytokine (e.g., IL-17 or a cytokine that stimulates IL-17 production), or both, and can be produced in a helper cell line. More detailed information can be found in U.S. Patent Nos. 5,643,576 and 6,783,939. In some embodiments, a vaccine formulation is administered to a subject in combination with a nucleic acid encoding a cytokine.

[0201] Certain classes of adjuvants activate toll-like receptors (TLRs) to activate the Th17 response. TLRs are well-known proteins that can be present on the leukocyte membrane and recognize foreign antigens, including microbial antigens. Administration of a known TLR ligand along with a target antigen, such as in the form of a fusion protein, can promote the development of an immune response specific to the target antigen. An exemplary adjuvant that activates TLRs contains monophosphoryl lipid A (MPL). Traditionally, MPL has been produced in the form of detoxified lipopolysaccharide (LPS) endotoxin obtained from Gram-negative bacteria such as Salmonella minnesota. In particular, sequential acid and base hydrolysis of LPS yields the immunologically active lipid A moiety, which is MPL, and lacks glycosyl groups and all but one of the phosphates present in LPS. A variety of synthetic TLR agonists, particularly TLR-4 agonists, are disclosed in Evans et al., 2003. Similar to MPL adjuvants, these synthetic compounds activate the innate immune system via TLRs. Another type of TLR agonist is a synthetic phospholipid dimer such as E6020 (Ishizaka et al., 2007). Various TLR agonists, including TLR-4 agonists, have been produced and / or sold by, for example, the Infectious Disease Research Institute (IRDI), Corixa, Esai, Avanti Polar Lipids, Inc., and Sigma Aldrich. Another exemplary adjuvant that activates TLRs contains a mixture of MPL, trehalose dicorynomycolate (TDM), and dimethyldioctadecylammonium bromide (DDA). Another TLR-activating adjuvant is R848 (resiquimod).

[0202] In some embodiments, the adjuvant is or comprises saponin. Generally, saponins are triterpene glycosides, such as those isolated from the bark of the Quillaja saponaria tree. Saponin extracts from biological sources can be further fractionated (e.g., by chromatography) to isolate the fractions of the extract that have optimal adjuvant activity and acceptable toxicity. Typical fractions of the extract from the Quillaja saponaria tree used as adjuvants are designated fraction A and C.

[0203] In some embodiments, adjuvant combinations are used. Three exemplary adjuvant combinations are MPL and alum, E6020 and alum, and MPL and ISCOM.

[0204] Adjuvants can bind covalently or non-covalently to an antigen. In some embodiments, an adjuvant can comprise a protein that induces an inflammatory response via activation of an antigen-presenting cell (APC). In some embodiments, one or more of these proteins can be recombinantly fused to a selected antigen such that the resulting fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately enhances antigen presentation to T cells and initiation of a T cell response (see, e.g., Wu et al., 2005).

[0205] In some embodiments, an immunogenic composition or vaccine comprising a fusion protein described herein is formulated and / or administered in combination with an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, and aluminum hydroxyphosphate. In some embodiments, the adjuvant comprises aluminum phosphate. In some embodiments, the adjuvant is aluminum phosphate.

[0206] Typically, the same adjuvant or adjuvant mixture is present in each dose of the vaccine. Optionally, however, the adjuvant can be administered with the first dose of the vaccine and not with subsequent doses (i.e., booster injections) of the vaccine. Alternatively, a strong adjuvant can be administered with the first dose of the vaccine, and a weaker adjuvant or a lower dose of the strong adjuvant can be administered with subsequent doses of the vaccine. The adjuvant can be administered before, simultaneously with, or after administration of the antigen to the subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are suitable for human subjects, non-human animals, or both.

[0207] Vaccines used in accordance with the present disclosure can include other antimicrobial therapies or be administered concurrently with antimicrobial therapies. By way of example, these vaccines can include or be administered with one or more agents that kill pathogens or arrest pathogen growth. These agents include, for example, penicillin, vancomycin, erythromycin, azithromycin, and clarithromycin, cefotaxime, ceftriaxone, levoflaxin, gatifloxacin.

[0208] Alternatively or additionally, vaccines used in accordance with the present invention can include or be administered with one or more other vaccines or therapies. By way of example, one or more non-pneumococcal antigens can be included in or administered with the vaccine.

[0209] Additional Components and Excipients

[0210] In addition to the fusion proteins described herein and the adjuvants described above, the vaccine formulation or immunogenic composition may further comprise one or more additional components.

[0211] In some embodiments, the vaccine formulation or immunogenic composition may comprise one or more stabilizers such as, for example, sugars (such as sucrose, glucose or fructose), phosphates (such as disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or sodium dihydrogen phosphate), glutamates (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine and their alkyl esters), inosine or sodium borate.

[0212] In some embodiments, the vaccine formulation or immunogenic composition comprises one or more buffering agents such as, for example, a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, the vaccine formulation may be administered in a saline such as, for example, phosphate buffered saline (PBS) or distilled water.

[0213] In some embodiments, the vaccine formulation or immunogenic composition comprises one or more surfactants such as, but not limited to, for example, polysorbate 80 (TWEEN 80), polysorbate 20 (TWEEN 20), polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (TRITON X-100), and a polymer of 4-(1,1,3,3-tetramethylbutyl)phenol with formaldehyde and ethylene oxide (TYLOXAPOL). The surfactant may be an ionic surfactant or a non-ionic surfactant.

[0214] In some embodiments, the vaccine formulation or immunogenic composition comprises one or more salts such as, for example, sodium chloride, ammonium chloride, calcium chloride or potassium chloride.

[0215] In some embodiments, a preservative is included in the vaccine or immunogenic composition. In other embodiments, no preservative is used. Preservatives are most commonly used in multi-dose vaccine vials and are less commonly required for single-dose vaccine vials. In some embodiments, the preservative is 2-phenoxyethanol, methylparaben and propylparaben, benzyl alcohol and / or sorbic acid.

[0216] Administration method

[0217] In some embodiments, an immunogenic composition or vaccine comprising the fusion proteins described herein is administered to a subject at risk of developing pneumococcal disease, such as an infant, young child, adolescent, or older adult. In some embodiments, the immunogenic composition or vaccine is administered to a subject at increased risk of developing pneumococcal disease, such as an immunocompromised subject, a subject with sickle cell disease or other hemoglobinopathies, congenital or acquired asplenia, splenic dysfunction, chronic renal failure or nephrotic syndrome, diseases associated with treatment with immunosuppressive drugs or radiation therapy including malignancies, leukemia, lymphoma, Hodgkin's disease, or solid organ transplantation, congenital or acquired immunodeficiency, HIV infection, cerebrospinal fluid leak, cochlear implant, chronic heart disease, chronic lung disease, diabetes, alcoholism, chronic liver disease, smoking, asthma, systemic malignancy, multiple myeloma, or solid organ transplantation. It should be understood that a subject may be considered at risk of developing a disease in the absence of any symptoms of the disease being diagnosed. For example, a subject will be considered at risk of developing a disease if the subject is known to be or is intended to be in a situation with a relatively high risk of infection.

[0218] Any effective route of administration can be utilized, such as oral, nasal, enteral, parenteral, intramuscular or intravenous, subcutaneous, transdermal, intradermal, rectal, vaginal, topical, ophthalmic, pulmonary, or by contact. In some embodiments, the immunogenic composition or vaccine can be injected (e.g., via intramuscular, intraperitoneal, intradermal, and / or subcutaneous routes); or delivered via mucosal delivery (e.g., to the oral / digestive tract, respiratory tract, and / or genitourinary tract). In some cases, intranasal administration may be particularly suitable for, for example, treating pneumonia or otitis media (since nasopharyngeal carriage of pneumococcus can be more effectively prevented, thus alleviating the infection at its earliest stage). In some embodiments, it may be necessary to administer different doses of the immunogenic composition or vaccine by different routes; in some embodiments, it may be necessary to administer different components of a dose by different routes.

[0219] In some embodiments, a pharmaceutical composition (e.g., an immunogenic composition or vaccine) is administered intradermally. The conventional technique for intradermal injection, the "Mantoux procedure," includes the steps of cleaning the skin and then stretching it with one hand, and inserting the needle at an angle between 10 - 15° with the bevel of a narrow gauge needle (26 - 31 gauge) facing upward. Once the bevel of the needle is inserted, lower the barrel of the needle and further advance it while providing slight pressure to lift it under the skin. Then, inject the liquid very slowly, thereby forming a bleb or bulge on the skin surface, and then slowly withdraw the needle.

[0220] Devices have been described that are specifically designed to administer a liquid agent into or onto the skin, such as those described in WO 99 / 34850 and EP 1092444, and also such as the jet injection devices described in WO 01 / 13977; U.S. Patent No. 5,480,381, U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S. Patent No. 5,704,911, U.S. Patent No. 5,383,851, U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Patent No. 5,312,335, U.S. Patent No. 5,503,627, U.S. Patent No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 4,940,460, WO 97 / 37705 and WO 97 / 13537. Other methods of intradermal administration of an immunogenic composition or vaccine can include a conventional syringe and needle, or a device designed for ballistic delivery of a solid vaccine (WO 99 / 27961), or a transdermal patch (WO 97 / 48440; WO 98 / 28037); or a device for administration to the skin surface (transdermal or percutaneous delivery WO 98 / 20734; WO 98 / 28037).

[0221] As described above, a pharmaceutical composition (such as an immunogenic composition or vaccine) can be administered in a single dose or in multiple doses. It should be understood that administration can be a single "dose" as long as all relevant components are administered to the subject within a time window; each component does not have to be present in a single composition. For example, the administration of two different immunogenic compositions or vaccines within a period of less than 24 hours is considered a single dose. Just to give one example, immunogenic compositions or vaccines having different antigenic components can be administered in separate compositions but as part of a single dose. As noted above, these separate compositions can be administered via different routes or via the same route. Alternatively or additionally, in embodiments where the immunogenic composition or vaccine is combined with an additional type of active agent, the immunogenic composition or vaccine can be administered via one route, and the second active agent can be administered via the same route or via a different route.

[0222] Administer the pharmaceutical composition (e.g., an immunogenic composition or a vaccine) in these amounts and for these durations as required to achieve the desired result. In some embodiments of the invention, the immunogenic composition or vaccine comprises an immunologically effective amount of at least an immunogenic composition. The precise amount required to achieve an immunologically effective amount can vary depending on the immunogenic composition and, among different subjects, depending on the type, age and general condition of the subject, stage of the disease, particular drug mixture, its mode of administration, etc.

[0223] Select the amount of the fusion protein described herein in each dose of the pharmaceutical composition (e.g., an immunogenic composition or a vaccine) to allow the vaccine to induce an appropriate immunoprotective response without significant adverse side effects when administered as described herein.

[0224] In some embodiments, the pharmaceutical composition comprising the fusion protein described herein induces a Th1 and / or Th17 cell response after administration to a subject. In some embodiments, the pharmaceutical composition induces an opsonin / bactericidal response against Streptococcus pneumoniae after administration to a subject. In some embodiments, the pharmaceutical composition comprising the fusion protein disclosed herein reduces the rate of mucosal surface spread of Streptococcus pneumoniae and / or reduces the mucosal surface colonization of Streptococcus pneumoniae after administration to a subject. In some embodiments, the pharmaceutical composition reduces the rate of nasopharyngeal or pulmonary spread of Streptococcus pneumoniae and / or reduces the colonization of Streptococcus pneumoniae after nasopharyngeal or pulmonary spread.

[0225] Some embodiments provide methods of immunizing a subject against Streptococcus pneumoniae infection, the method comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising the fusion protein described herein. Some embodiments provide methods of immunizing a subject against Streptococcus pneumoniae infection, the method comprising administering to the subject an immunologically effective amount of a vaccine composition comprising the fusion protein described herein. Some embodiments provide methods of immunizing a subject against Streptococcus pneumoniae infection, the method comprising administering to the subject an immunologically effective amount of a pharmaceutical composition comprising the fusion protein described herein.

[0226] Combination prophylaxis or combination therapy

[0227] In some embodiments, the immunogenic composition or vaccine comprising the fusion protein described herein can be administered in combination with another agent. In some embodiments, the agent is or comprises PCV13. In some embodiments, the agent is or comprises PPSV23. In some embodiments, the agent is or comprises an antibiotic.

[0228] Administration

[0229] In some embodiments, administration of an immunogenic composition or vaccine comprising a fusion protein described herein may involve single-dose delivery. In some embodiments, administration may involve an initial dose followed by one or more additional vaccination doses that are sufficiently spaced apart. A vaccination schedule is a program for administering one or more specified pneumococcal vaccines at one or more specified ages of a subject by one or more specified routes of administration.

[0230] The present disclosure provides a vaccination method involving administering at least one dose of a vaccine to an infant subject. In some embodiments, the infant subject is 18 months of age or younger. In some embodiments, the infant subject is 12 months of age or younger. In some embodiments, the infant subject has previously received one or more doses of a conjugated pneumococcal polysaccharide vaccine; in other embodiments, the infant subject has not received a pneumococcal vaccine. In some embodiments, the infant subject has previously been infected with Streptococcus pneumoniae or exposed to Streptococcus pneumoniae infection.

[0231] The present disclosure provides a vaccination method involving administering at least one dose of a vaccine to a toddler subject. In some embodiments, the toddler subject is 5 years of age or younger. In some embodiments, the toddler subject is 4 years of age or younger. In some embodiments, the toddler subject has previously received one or more doses of a conjugated pneumococcal polysaccharide vaccine; in other embodiments, the toddler subject has not received a pneumococcal vaccine. In some embodiments, the toddler subject has previously been infected with Streptococcus pneumoniae or exposed to Streptococcus pneumoniae infection.

[0232] The present disclosure provides a vaccination method involving administering at least one dose of a vaccine to an adolescent subject. In some embodiments, the adolescent subject is 18 years of age or younger. In some embodiments, the adolescent subject is 15 years of age or younger. In some embodiments, the adolescent subject has previously received one or more doses of a conjugated pneumococcal polysaccharide vaccine; in other embodiments, the adolescent subject has not received a pneumococcal vaccine. In some embodiments, the adolescent subject has previously been infected with Streptococcus pneumoniae or exposed to Streptococcus pneumoniae infection.

[0233] The present disclosure provides a vaccination method involving administering at least one dose of a vaccine to an adult subject. In some embodiments, the adult subject is greater than about 50 years of age. In some embodiments, the adult subject is greater than about 65 years of age. In some embodiments, the adult subject has previously received one or more doses of a conjugated pneumococcal polysaccharide vaccine; in other embodiments, the adult subject has not received a pneumococcal vaccine. In some embodiments, the adult subject has previously been infected with Streptococcus pneumoniae or exposed to Streptococcus pneumoniae infection.

[0234] An immunization schedule of the present disclosure is provided to induce an immune response (e.g., an immunoprotective response) in a subject that is sufficient to reduce at least one measure selected from the group consisting of the incidence, prevalence, frequency, and / or severity of at least one infection, disease, or disorder, and / or at least one surrogate marker of the infection, disease, or disorder, in a population and / or subgroup of subjects. A supplemental immunization schedule is an immunization schedule that has such an effect relative to the standard schedule that it supplements. The supplemental schedule may require additional administration and / or supra-immunogenic doses of the immunogenic compositions or vaccines disclosed herein as seen in the standard schedule, or may require administration of the immunogenic compositions or vaccines in a non-standard portion of the schedule. The complete immunization schedule of the invention may include both the standard schedule and the supplemental schedule. For illustrative purposes, exemplary sample immunization schedules are provided. A detailed description of the methods for assessing the immunogenic responses discussed herein allows for modification of the sample immunization schedules without undue experimentation.

[0235] In one embodiment of the present disclosure, the first pneumococcal vaccine administration is typically performed when the subject is older than about 2 weeks, older than about 5 weeks, older than about 1 year, older than about 2 years, older than about 15 years, or older than about 18 years.

[0236] In some embodiments of the present disclosure, the first pneumococcal vaccine administration is performed when the subject is older than about 50 years, older than about 55 years, older than about 60 years, older than about 65 years, or older than about 70 years.

[0237] In some embodiments of the present disclosure, a single vaccine administration is employed. It is possible that the objectives of the present invention can provide a single administration, particularly when one or more of the vaccine polypeptides, polysaccharides, and / or conjugates or combinations thereof used are potent, and in such a case, a single-dose schedule is sufficient to induce a sustained immunoprotective response.

[0238] In some embodiments, it is desirable to administer two or more doses of the vaccine for higher immunoprotective efficacy and coverage. Thus, in some embodiments, the number of doses is at least two, at least three, or more doses. There is no fixed maximum number of doses; however, good clinical practice is to not immunize more frequently than necessary to achieve the desired effect.

[0239] Without being bound by theory, the first dose of the vaccine administered according to the present disclosure can be considered a "prime" dose. In some embodiments, more than one dose is included in the immunization schedule. In such a context, subsequent doses can be considered "boost" doses.

[0240] The priming dose can be administered to an untreated subject (a subject who has never received a conjugated polysaccharide vaccine before). In some embodiments, the priming dose can be administered to a subject who has previously received a conjugated polysaccharide vaccine at least five or more years before administering the initial vaccine dose according to the present invention. In other embodiments, the priming dose can be administered to a subject who has previously received a conjugated polysaccharide vaccine at least twenty or more years before administering the priming vaccine according to the present invention.

[0241] When the vaccination schedule requires two or more separate doses, the interval between doses is considered. The interval between two consecutive doses can be the same throughout the vaccination schedule, or it can vary with the age of the subject. In the vaccination schedule of the present invention, once the first vaccine dose has been administered, there is a first interval before the subsequent dose is administered. The first interval is generally at least about 2 weeks, 1 month, 6 weeks, 2 months, 3 months, 6 months, 9 months, 12 months or longer. In the case where more than one subsequent dose is administered, a second (or greater) interval can be set between these subsequent doses. In some embodiments, all intervals between subsequent doses have the same length; in other embodiments, the length of the second interval can vary. In some embodiments, the interval between subsequent doses can be at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months or at least about 2 years. In some embodiments, the interval between doses can be at most 3 years, at most about 4 years or at most about 5 years or 10 years or longer. In some embodiments, the interval between subsequent doses can decrease with the age of the subject.

[0242] Those skilled in the art will appreciate that there are various possible combinations and sub - combinations of the timing of the first administration, the shortest interval, the longest interval, and the total number of administrations (absolutely or within the stated period), and all of these combinations and sub - combinations are to be considered within the expectations of the inventors, but are not explicitly listed here.

[0243] Assay for determining immune response

[0244] In some embodiments, methods for assessing the immunogenicity of a pharmaceutical composition, immunogenic composition, or vaccine comprising a fusion protein described herein include using one or more in vitro bioassays (including B cell and T cell responses such as antibody levels according to ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, Th1 / Th17 cell responses, cytokine level measurements, and functional antibody levels measured by, e.g., OPK, serum bactericidal killing (SBA), agglutination, motility, cytotoxicity, or adhesion); and in vivo assays in animal models of pneumococcal disease (e.g., pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization) to evaluate, measure, and / or compare immune responses. Parameters of the in vivo assays include bacterial clearance from mucosal surfaces or the bloodstream, alleviation or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of nasopharyngeal colonization, reduction in mortality, and passive and active protection following challenge with a pneumococcal pathogen that is the target of the immunogenic composition. In some embodiments, the immune response is compared to a control composition.

[0245] In some embodiments, methods for assessing the potency of a pharmaceutical composition, immunogenic composition, or vaccine comprising a fusion protein described herein include using one or more in vitro bioassays (including B cell and T cell responses such as antibody levels according to ELISA, multiplex ELISA, MSD, Luminex, flow cytometry, Th1 / Th17 cell responses, cytokine level measurements, and functional antibody levels measured by, e.g., OPK, serum bactericidal killing (SBA), internalization, active neutralization, agglutination, motility, cytotoxicity, or adhesion); and in vivo assays in animal models of pneumococcal disease (e.g., pneumonia, bacteremia, meningitis, sepsis, otitis media, nasopharyngeal colonization) to evaluate, measure, and / or compare immune responses. Parameters include bacterial clearance or reduction from mucosal surfaces or the bloodstream, alleviation or prevention of bacteremia, meningitis, sepsis, or otitis media, reduction or prevention of nasopharyngeal colonization, reduction in mortality, and passive and active protection following challenge with a pneumococcal pathogen that is the target of the immunogenic composition. In some embodiments, the immune response is compared to a control composition.

[0246] Generally, it may be necessary to evaluate the humoral response, cellular response, and / or interactions between the two. In the case of evaluating the humoral response, the antibody titers and / or types (e.g., total IgG, IgG1, IgG2, IgM, IgA, etc.) of specific pathogen antigens (e.g., polypeptides or polysaccharides, serotype-specific or conserved across two or more serotypes) can be measured, for example, before and / or after administration of an initial or booster dose of the vaccine (and / or compared to antibody levels in the absence of antigenic stimulation). The cellular response can be evaluated by monitoring the response to the antigen, such as a delayed hypersensitivity reaction. The cellular response can also be directly measured by assessing the response of peripheral blood mononuclear cells (PBMCs) monocytes to the target antigen stimulation. Precursors and memory B cell populations can be evaluated in an enzyme-linked immunospot (ELISpot) assay against specific pathogen antigens.

[0247] The RIA method detects specific antibodies (e.g., Schiffiman et al., 1980) by incubating serum with radiolabeled polysaccharide or polypeptide in suspension. The antigen - antibody complex is then precipitated with ammonium sulfate, and the counts per minute (cpm) of the radiolabeled pellet are measured.

[0248] In the ELISA assay, specific antibodies from the serum of vaccinated subjects are quantified by incubating with antigen (e.g., polypeptide or polysaccharide, serotype-specific or conserved across two or more serotypes) that has been adsorbed to a solid support (e.g., Koskela and Leinonen (1981); Kojima et al., 1990; Concepcion and Frasch, 2001). Enzyme-conjugated secondary detection antibodies are used to detect the bound antibodies. ELISA also allows for isotype and subclass analysis of the immune response (i.e., IgM vs. IgG, or IgG1 vs. IgG2) by using isotype or subclass-specific secondary antibodies and can be adapted to evaluate the avidity of the antibodies (Anttila et al., 1998; Romero-Steiner et al., 2005). Multiplex assays (e.g., Luminex) facilitate the simultaneous detection of antibodies against multiple antigens. The antigens are conjugated to spectrally distinct microspheres, which are mixed and incubated with the serum. Antibodies that bind to the antigens on the coated microspheres are detected using secondary antibodies (e.g., R-phycoerythrin-conjugated goat anti-human IgG).

[0249] The method used to evaluate functional antibodies in serum is the opsonophagocytic assay (OPA), which quantifies only the antibodies that can opsonize bacteria and cause bacterial uptake and killing. The standard assay utilizes human phagocytic effector cells, a complement source, bacteria, and diluted serum. The assay reading is the serum endpoint titer at which ≥50% killing is present compared to bacteria incubated only with complement and human cells (Romero-Steiner et al., 1997). This killing OPA can also be multiplexed by using target strains of pathogens carrying different antibiotic resistance markers (Kim et al., 2003). Another type of multiplex opsonin assay is the non-killing assay, in which the uptake of fluorescently stained encapsulated pathogens or fluorescent microspheres conjugated to antigens from the target pathogen by phagocytic effector cells in the presence of diluted serum plus a complement source is evaluated by FC (Martinez et al., 1999). The opsonin activity of serum antibodies plus complement can also be evaluated by measuring the oxidative response of phagocytic human effector cells to the ingested pathogens (Munro et al 1985; Ojo-Amaize et al., 1995).

[0250] Certain in vivo model systems can be used to evaluate the protection provided by serum antibodies induced by immunogenic compositions or vaccines comprising the fusion proteins described herein. In such a passive protection system, mice or rats are challenged with a pathogen plus diluted serum, and the endpoint titer or mortality of the serum providing defense against pneumonia, bacteremia, organ, or tissue colonization is determined (Stack et al., 1998; Saeland et al., 2000).

[0251] In some embodiments, the efficacy of immunization can be determined by measuring the level of one or more cytokines, which is carried out by stimulating T cells from a subject after immunization. The level of one or more cytokines can be compared with the level of one or more cytokines in the same subject before immunization. An increase in the level of one or more cytokines (e.g., 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more increase compared to the cytokine level before immunization) will indicate an increased response to the immunogenic composition or vaccine. In some embodiments, one or more cytokines are selected from GM-CSP; IL-1α; IL-1β; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-10; IL-12; IL-17A; IL-17F or other members of the IL-17 family; IL-22; IL-23; IFN-α; IFN-β; IFN-γ; MIP-1α; MIP-1β; TGF-β; TNFα or TNF-β. In a non-limiting example, the efficacy of immunization can be determined by measuring the level of IL-17 (especially IL-17A), which is carried out by stimulating T cells from a subject after immunization. The IL-17 level can be compared with the IL-17 level in the same subject before immunization. An increase in the level of IL-17 (e.g., IL-17A) (e.g., 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or more increase) will indicate an increased response to the immunogenic composition or vaccine.

[0252] In some embodiments, neutrophils can be assayed for pneumococcal killing in the presence of T cells or antibodies from a patient. An increase in pneumococcal killing (e.g., a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or greater increase) will indicate an increased response to the immunogenic composition or vaccine. For example, Th17 cell activation can be measured, where an increase in Th17 cell activation (e.g., a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or greater increase) is associated with an increased response to the immunogenic composition or vaccine. In another non-limiting example, Th1 cell activation can be measured, where an increase in Th1 cell activation (e.g., a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or greater increase) is associated with an increased response to the immunogenic composition or vaccine. The level of antibodies specific for the immunogenic composition or vaccine can also be measured, where an increase in the level of specific antibodies (e.g., a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold or greater increase) is associated with an increase in efficacy. In some embodiments, two or more of these assays are used. For example, the level of IL-17 and the level of immunogenic composition-specific or vaccine-specific antibodies can be measured. Alternatively, epidemiological markers can be tracked, such as the incidence, severity, or duration of Streptococcus pneumoniae infection in vaccinated subjects compared to unvaccinated subjects.

[0253] The efficacy of an immunogenic composition or vaccine can also be assayed in various model systems such as, for example, a murine challenge model. For example, the murine BALB / c or C57BL / 6 strains can be used. After administering the test immunogenic composition or vaccine (in single or multiple doses) to a subject, the experimenter administers a challenge dose of Streptococcus pneumoniae. In some cases, the challenge dose administered intranasally is sufficient to cause Streptococcus pneumoniae colonization (especially nasal colonization) in unvaccinated animals, and in some cases, the challenge dose administered via inhalation is sufficient to cause sepsis and high lethality in unvaccinated animals. In some cases, the challenge dose administered intraperitoneally is sufficient to cause sepsis and high lethality in unvaccinated animals. In some cases, the challenge dose administered intravenously is sufficient to cause sepsis and high lethality in unvaccinated animals. Then, the reduction in colonization or the decrease in lethality in the vaccinated animals can be measured.

[0254] Certain in vivo model systems can be used to evaluate the protection provided by serum antibodies induced by the vaccines of the invention. In these passive protection systems, mice or rats are challenged with the pathogen plus diluted serum, and the endpoint titer of the serum providing defense against bacteremia, organ, or tissue colonization or the mortality rate is determined (Stack et al., 1998; Saeland et al., 2000).

[0255] Example

[0256] Example 1: Induction of Th17 response in mice mediated by the SP0785 and SP1500 components of fusion protein CP1 and Defense against pneumococcal nasal colonization

[0257] Purpose:

[0258] This example compared the ability of the individual Streptococcus pneumoniae proteins SP0785 and SP1500 and the exemplary fusion protein CP1 to stimulate a Th17 response and protect mice from nasal colonization by S. pneumoniae after intranasal immunization with the adjuvant cholera toxin (CT). The exemplary fusion protein CP1 is a fusion protein comprising truncated rhizobial avidin (amino acids [45 - 179], designated Rhavi), the SP0785 polypeptide, and the SP1500 polypeptide. In some embodiments, the fusion protein CP1 is or comprises Rhavi-linker(GGGGSSS)-SP1500-linker(AAA)-SP0785.

[0259] Overview:

[0260] 1. As demonstrated by interleukin 17A (IL-17A) secretion after intranasal immunization with the adjuvant cholera toxin (CT), the S. pneumoniae proteins SP0785 and SP1500, as well as the fusion protein CP1, can each generate a robust Th17 response in mice.

[0261] 2. Intranasal immunization of mice with SP1500 or SP0785 and CT, or CP1 and CT, respectively, significantly reduced nasal colonization by S. pneumoniae.

[0262] Materials and Methods:

[0263] Recombinant protein production

[0264] His-tagged recombinant proteins were expressed in Escherichia coli and purified using Ni-nitrilotriacetic acid affinity chromatography. A second purification was performed using size exclusion chromatography with a Superdex 200 column. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad).

[0265] Formulation

[0266] Cholera toxin (CT) was used as an adjuvant for intranasal immunization to facilitate induction of a T cell response to the individual proteins. Prior to administration, the proteins or pneumococcal whole cell vaccine (amounts shown in Table 5) were mixed with 1 μg CT in a saline solution to a final volume of 20 μl per dose.

[0267] Intranasal mouse immunization protocol

[0268] For intranasal immunization with protein adjuvanted with CT and chloroform-inactivated pneumococcal whole cell vaccine (WCC), C57BL / 6 mice (groups of n = 10) received two immunizations at 1-week intervals. Peripheral blood samples were collected 3 weeks after the last immunization for ex vivo IL-17A stimulation in the presence of the appropriate antigen as a stimulant.

[0269] Table 5. Intranasal immunization study groups in mice

[0270]

[0271] Abbreviations: CP1: fusion protein 1 (Rhavi-linker (GGGGSSS)-SP1500-linker (AAA)-SP0785); Rhavi: truncated rhizobial avidin, amino acids [45-179]; WCC: pneumococcal whole cell vaccine (chloroform-inactivated); CT: cholera toxin

[0272] Note: All recombinant proteins carry His tags.

[0273] IL-17A induction and measurement in whole blood

[0274] Ex vivo stimulation of peripheral blood samples collected 3 weeks after the last intranasal immunization was performed in round-bottom 96-well plates. All stimulants were diluted in stimulation medium (DMEM F-12; 10% FBS, 50 μM 2-mercaptoethanol, 10 μg / ml ciprofloxacin) to a final concentration of 10 μg / ml. In each well, 25 μl of heparinized blood was added to 225 μl of stimulation medium containing the designated stimulant, followed by incubation at 37 °C and 5% CO 2 for 6 days. Supernatants were collected after centrifugation and analyzed for IL-17A using an ELISA kit (R&D systems).

[0275] Measurement of nasopharyngeal Streptococcus pneumoniae infection and colonization

[0276] One to two weeks after blood collection, mice were challenged intranasally with 10 7 CFU of Streptococcus pneumoniae serotype 6B (strain 603). On day 7 after infection, nasopharyngeal washes were performed on euthanized mice. After growth on blood agar plates, the pneumococcal CFU in each nasal wash was counted.

[0277] Statistical analysis

[0278] Statistical analysis was performed using PRISM (GraphPad software). Mann-Whitney U test was used to analyze all data regarding IL-17A concentration and nasopharyngeal colonization density. The geometric mean concentration of IL-17A for each group was calculated, and the geometric mean density of colonization for each group was calculated.

[0279] Results and Discussion:

[0280] IL-17A response and colonization reduction after intranasal immunization with SP1500, SP0785 or CP1

[0281] As Figure 2 shown in the left panel, intranasal immunization with SP0785 or SP1500 adjuvanted with CT elicited a strong antigen-specific Th17 response compared to immunization with CT alone, as demonstrated by increased IL-17A production after in vitro stimulation of peripheral blood with purified SP0785 or SP1500. The increased secretion of IL-17A was associated with a corresponding statistically significant decrease in Streptococcus pneumoniae CFU recovered from nasopharyngeal washings on day 7 after challenge of immunized mice ( Figure 2 , right panel).

[0282] As Figure 3 shown in the left panel, after intranasal immunization with CT, when the Rhavi protein was compared to the protein fusion of Rhavi-linker (GGGGSSS)-SP1500-linker (AAA)-SP0785 (CP1), CP1 retained the antigen-specific induction of IL-17A. When these mice were challenged with Streptococcus pneumoniae and compared to intranasal immunization with pneumococcal whole cell vaccine, as measured by pneumococcal CFU, Rhavi alone had no protective effect, while the protective effect of CP1 was comparable to that of killed (inactivated) pneumococcal whole cells ( Figure 3 , right panel).

[0283] Example 2: Bactericidal activity of antiserum against fusion protein CP1

[0284] Materials and Methods:

[0285] Growth of bacteria

[0286] Streptococcus pneumoniae strains 6B, 15A, 16F, and 35B were inoculated into 10 mL Todd Hewitt Broth (THB) cultures with yeast extract. The cultures were incubated at 37 °C in 5% CO 2 for 4 - 7 hours until OD 600Reached 0.5 - 0.8 (logarithmic mid - phase). Bacteria were harvested by spinning at 3,000 g for 7 minutes at 4°C, and the bacterial pellet was resuspended in 10 mL of THB containing 10% glycerol and stored at -80°C. Colony - forming unit (CFU) estimates were determined by serially diluting the frozen stock on trypticase soy agar with 5% sheep blood (Becton, Dickenson, and Company) and incubating at 37°C and 5% CO 2 for 18 to 24 hours.

[0287] Complement opsonophagocytosis assay (COPA)

[0288] The frozen stock of Streptococcus pneumoniae was thawed and resuspended at 2×10 5 CFU / ml in assay buffer (Hank's buffered saline with 10% heat - inactivated FBS). 10 μl of the bacterial suspension was added to each well of a 96 - well plate, followed by 20 μl of heat - inactivated rabbit serum diluted in assay buffer for testing in the assay. The bacteria and rabbit serum were incubated with shaking at room temperature for 30 minutes. 10 μl of young rabbit complement (Pel - Freeze Biologicals) was added to each well, followed by incubation with shaking at room temperature for 30 minutes. HL60 cells (ATCC) were washed with assay buffer and resuspended to 1×10 7 cells / ml. 40 μl of the HL60 suspension (HL60 to bacteria ratio of 200:1) was added to each well, followed by incubation with shaking at 37°C and 5% CO 2 for 1 hour. The plate was transferred to ice and incubated for 20 minutes. Then each sample (undiluted, 1 / 5 and 1 / 25 dilutions in water) was plated on 5% blood agar plates. After incubation overnight at 37°C and 5% CO 2 , the CFUs of each sample and dilution were counted.

[0289] Rabbit serum

[0290] New Zealand white rabbits (n = 3) were immunized with 100 μg per dose of Rhavi - linker (GGGGSSS) - SP1500 - linker (AAA) - SP0785 (CP1, His - tagged) and 0.625 mg of elemental aluminum from AlPO 4 for a total of three doses at two - week intervals between each immunization (rabbits 87, 88, and 1762). Serum was collected before immunization (P0) and two weeks after the third immunization (P3) and stored at -80°C.

[0291] Results and Discussion:

[0292] Opsonophagocytic activity of antibodies against CP1 against Streptococcus pneumoniae

[0293] An improved opsonophagocytic assay (COPA) was established to study protein antibody-mediated killing of Streptococcus pneumoniae. Sera from two rabbits immunized with CP1 (P3, from rabbits 87 and 88) were assayed compared to pre-immune sera (P0). Incubation with P3 immune sera resulted in a decrease in CFU ( Figure 4 ), or alternatively, an increase in the percentage of killing activity ( Figure 7 , Figure A), compared to incubation with pre-immune sera (P0) against Streptococcus pneumoniae serotype 6B (the type incorporated into the commercially available Prevnar 13 vaccine) at all dilutions tested for both rabbits. The killing activity of the immune sera was dependent on HL60 cells and active complement (data not shown). The same sera were assayed against two serotypes not incorporated into the commercially available vaccine, namely Streptococcus pneumoniae serotype 15A ( Figure 5 and Figure 7 , Figure B: 1 / 2 dilution) and serotype 35B ( Figure 6 and Figure 7 , Figure C: 1 / 2 dilution). Sera from a third rabbit immunized with CP1 (P3, from rabbit 1762) were assayed compared to pre-immune sera (P0) against Streptococcus pneumoniae serotype 16F (another serotype not incorporated into the commercially available vaccine ( Figure 7 , Figure D: 1 / 2, 1 / 6, 1 / 18, 1 / 54 dilutions)). For all rabbits and non-vaccine serotypes tested, incubation with P3 immune sera resulted in a decrease in CFU or alternatively showed an increase in the percentage of killing activity compared to incubation with pre-immune sera (P0). The killing activity of the immune sera was dependent on HL60 cells and active complement (data not shown).

[0294] Example 3: Comparison of induction of Th17 response in mice after immunization with a mixture of SP0785, SP1500 and truncated streptavidin Rhavi (unconjugated) or with fusion protein CP1 or SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT Recombinant protein production

[0295] Materials and Methods:

[0296] Formulation

[0297] Recombinant proteins with histidine tags were expressed in Escherichia coli and purified using Ni-nitrilotriacetic acid affinity chromatography. A second purification was performed using size exclusion chromatography with a Superdex 200 column. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad).

[0298] Intranasal mouse immunization protocol

[0299] Cholera toxin (CT) was used as an adjuvant for intranasal immunization to facilitate the induction of T cell responses to individual proteins. Before administration, the protein (in the amounts shown in Table 6) was mixed with 1 μg CT in a saline solution with a final volume of 20 μl per dose.

[0300] IL-17A induction and measurement in whole blood

[0301] For intranasal immunization with CT-adjuvanted protein, C57BL / 6 mice (groups of n = 15) received 2 immunizations at 1-week intervals. Peripheral blood samples were collected 3 weeks after the last immunization for ex vivo IL-17A stimulation in the presence of the appropriate antigen as a stimulant.

[0302] Table 6. Mouse intranasal immunization study groups

[0303]

[0304] Abbreviations: CP1: fusion protein 1 (Rhavi-linker (GGGGSSS)-SP1500-linker (AAA)-SP0785); Rhavi: truncated rhizobial avidin, amino acids [45 - 179]; CT: cholera toxin

[0305] Note: All recombinant proteins carried His tags.

[0306] Statistical analysis

[0307] Ex vivo stimulation of peripheral blood samples collected 3 weeks after the last intranasal immunization was performed in 96-well round-bottom plates. All stimulants (purified protein or heat-killed (inactivated) pneumococcal whole cells) were diluted in stimulation medium (DMEM F-12; 10% FBS, 50 μM 2-mercaptoethanol, 10 μg / ml ciprofloxacin) to a final concentration of 10 μg / ml. In each well, 25 μl of heparinized blood was added to 225 μl of stimulation medium containing the designated stimulant, followed by incubation at 37 °C and 5% CO 2 2 for 6 days. Supernatants were collected after centrifugation and analyzed for IL-17A using an ELISA kit (R&D systems).

[0308] IL-17A response after intranasal immunization with a mixture of SP1500, SP0785 and Rhavi protein or with CP1 or SP0785-linker(SSSGG)-SP1500-

[0309] Statistical analysis was performed using PRISM (GraphPad software). All data on IL-17A concentration were analyzed using the Mann-Whitney U test. The geometric mean concentration of IL-17A for each group was calculated.

[0310] Results and Discussion:

[0311] linker(SSVDKL)-PdT Figure 8

[0312] As Figure 9 shown, intranasal immunization with CT-adjuvanted CP1 induced a stronger antigen-specific Th17 response compared to immunization with CT-adjuvanted SP0785 or SP1500 or CT alone (control), with or without CT. The Th17 response was indicated by increased IL-17A production after in vitro stimulation of the peripheral blood of immunized mice with purified SP0785 (Figure A), purified SP1500 (Figure B), or heat-killed (inactivated) pneumococcal whole cells (WCV; Figure C).

[0313] As Figure 10 shown, intranasal immunization with CT-adjuvanted CP1 also induced a stronger antigen-specific Th17 response compared to immunization with CT-adjuvanted fusion protein SP0785-linker (SSSGG)-SP1500-linker (SSVDKL)-PdT or CT alone (control). The Th17 response was indicated by increased IL-17A production after in vitro stimulation of the peripheral blood of immunized mice with purified SP0785 (Figure A) or purified SP1500 (Figure B).

[0314] As Example 4: Comparison of hemolytic activities of CP1 and SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT shown, intranasal immunization with CT-adjuvanted CP1 induced a stronger antigen-specific Th17 response compared to immunization with a combination (mixture) of CT-adjuvanted SP0785, SP1500, and Rhavi or CT alone (control). The Th17 response was indicated by increased IL-17A production after in vitro stimulation of the peripheral blood of immunized mice with purified SP0785 (Figure A), purified SP1500 (Figure B), or heat-killed (inactivated) pneumococcal whole cells (WCV; Figure C).

[0315] Recombinant protein production Determination of hemolytic activity of fusion protein Materials and Methods:

[0316] Figure 11

[0317] His-tagged recombinant fusion proteins CP1 and SP0785-linker (SSSGG)-SP1500-linker (SSVDKL)-PdT were expressed in Escherichia coli and purified using Ni-nitrilotriacetic acid affinity chromatography. A second purification was performed using size exclusion chromatography with a Superdex 200 column. Protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Bio-Rad).

[0318] ​

[0319] The assay was adapted from Benton et al., 1997. The assay buffer contained 10 mM dithiothreitol, 0.1% bovine serum albumin in PBS pH 7.4, and 2% sheep red blood cells. The sheep red blood cells were prepared as follows: Add 200 μl of sheep blood + 1 ml of PBS pH 7.4, mix well, centrifuge, and wash 3 times at 8,000 rpm for 30 sec each; finally, resuspend the blood cells in 10 ml of ice-cold PBS and place on ice until use. The assay was performed as follows: Dilute pneumolysin standard (Ply), pneumolysin-like PdT, and the fusion protein to test at the designated concentrations in 100 μl / well on the plate (CP1 and SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT), then add 50 μl of 2% sheep red blood cells to all wells. Incubate the plate at 37 °C for 30 minutes. After incubation, centrifuge the plate at 2,000 rpm for 5 minutes at room temperature, and transfer 100 μl of the supernatant to an empty 96-well plate to measure the absorbance 420 at OD. [Benton, K.A., J.C. Paton and D.E. Briles. 1997. Differences in virulence for mice among Streptococcus pneumoniae strains of capsular types 2, 3, 4, 5, and 6 are not attributable to differences in pneumolysin production. Infect Immun. 65:1237-44.]

[0320] Results and Discussion:

[0321] As ​ shown, incubation of sheep red blood cells with the fusion protein CP1 did not result in hemolysis at any of the tested concentrations. Incubation with the fusion protein SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT at concentrations higher than 1 mg / ml and 0.5 mg / ml, respectively, or with pneumolysin-like PdT alone, resulted in almost complete hemolysis of sheep red blood cells. These results suggest that the SP0785 and SP1500 portions of the two fusion proteins do not contribute to hemolytic activity. Instead, the hemolytic activity of the fusion protein SP0785-linker(SSSGG)-SP1500-linker(SSVDKL)-PdT can be attributed to pneumolysin-like PdT

[0322] Sequence

[0323] SEQ ID NO:1, Rhizobium avidin protein, full length [amino acids 1-179]:

[0324]

[0325] SEQ ID NO:2, truncated Rhizobium avidin protein, designated as Rhavi [amino acids 45-179]:

[0326]

[0327] SEQ ID NO:3, SP0785 protein, full length [amino acids 1-399], strain TIGR4:

[0328]

[0329] SEQ ID NO:4, SP0785 protein lacking the signal sequence [amino acids 33-399]:

[0330] Note: One T394A mismatch with the SP0785 NCBI sequences ABJ54007.1 and YP816180

[0331]

[0332] SEQ ID NO:5, consensus SP0785 protein [amino acids 1-399]:

[0333]

[0334]

[0335]

[0336]

[0337] SEQ ID NO:6, SP1500 protein, full length [amino acids 1-278], strain TIGR4:

[0338]

[0339] SEQ ID NO:7, SP1500 protein lacking the signal sequence [amino acids 27-278]:

[0340]

[0341] SEQ ID NO:8, consensus SP1500 protein [amino acids 1-278]:

[0342]

[0343]

[0344]

[0345]

[0346] SEQ ID NO:9, the streptavidin gene encoding full-length streptavidin of rhizobia:

[0347]

[0348] SEQ ID NO:10, the streptavidin gene encoding truncated streptavidin (designated as Rhavi [amino acids 45-179]) of rhizobia:

[0349]

[0350] SEQ ID NO:11, the SP0785 gene encoding full-length SP0785 protein [amino acids 1-399], strain TIGR4:

[0351]

[0352]

[0353] SEQ ID NO:12, the SP0785 gene encoding SP0785 protein lacking a signal sequence [amino acids 33-399]:

[0354]

[0355] SEQ ID NO:13, the SP1500 gene encoding full-length SP1500 protein [amino acids 1-278], strain TIGR4:

[0356]

[0357] SEQ ID NO:14, the SP1500 gene encoding SP1500 protein lacking a signal sequence [amino acids 27-278]:

[0358]

[0359] SEQ ID NO:15, His tag 1:

[0360]

[0361] SEQ ID NO:16, His tag 2:

[0362]

[0363] SEQ ID NO:17, Fusion protein SP1500 - SP0785:

[0364]

[0365] SEQ ID NO:18, Fusion protein SP0785 - SP1500:

[0366]

[0367] SEQ ID NO:19, Fusion protein Rhavi - SP1500 - SP0785:

[0368]

[0369] SEQ ID NO:20, Fusion protein Rhavi - SP0785 - SP1500:

[0370]

[0371] SEQ ID NO:21, Fusion protein SP1500 - SP0785 - Rhavi:

[0372]

[0373] SEQ ID NO:22, Fusion protein SP0785 - SP1500 - Rhavi:

[0374]

[0375] SEQ ID NO:23, Fusion protein CP1, Rhavi - linker(GGGGSSS) - SP1500 - linker(AAA) - SP0785:

[0376]

[0377] SEQ ID NO:24, Fusion protein Rhavi - GGGGSSS - SP0785 - AAA - SP1500:

[0378]

[0379]

[0380] SEQ ID NO:25, Fusion protein SP1500 - GGGGSSS - SP0785 - AAA - Rhavi:

[0381]

[0382] SEQ ID NO: 26, Fusion protein SP0785 - linker (GGGGSSS) - SP1500 - linker (AAA) - Rhavi:

[0383]

[0384]

[0385] SEQ ID NO: 27, Codon - optimized nucleic acid sequence encoding the fusion protein SP1500 - SP0785

[0386]

[0387] SEQ ID NO: 28, Codon - optimized nucleic acid sequence encoding the fusion protein SP0785 - SP1500

[0388]

[0389] SEQ ID NO: 29, Codon - optimized nucleic acid sequence encoding the fusion protein Rhavi - SP1500 - SP0785:

[0390] SEQ ID NO: 30, Codon - optimized nucleic acid sequence encoding the fusion protein Rhavi - SP0785 - SP1500:

[0391]

[0392] SEQ ID NO: 31, Codon - optimized nucleic acid sequence encoding the fusion protein SP1500 - SP0785 - Rhavi:

[0393]

[0394] SEQ ID NO: 32, Codon - optimized nucleic acid sequence encoding the fusion protein SP0785 - SP1500 - Rhavi:

[0395]

[0396] SEQ ID NO: 33, Codon - optimized nucleic acid sequence encoding the fusion protein CP1 Rhavi - linker (GGGGSSS) - SP1500 - linker (AAA) - SP0785:

[0397]

[0398] SEQ ID NO:34, Codon-optimized nucleic acid sequence encoding the fusion protein Rhavi-linker(GGGGSSS)-SP0785-linker(AAA)-SP1500:

[0399]

[0400]

[0401] SEQ ID NO:35, Codon-optimized nucleic acid sequence encoding the fusion protein SP1500-linker(GGGGSSS)-SP0785-linker(AAA)-Rhavi:

[0402]

[0403] SEQ ID NO:36, Codon-optimized nucleic acid sequence encoding the fusion protein SP0785-linker(GGGGSSS)-SP1500-linker(AAA)-Rhavi:

[0404]

[0405] SEQ ID NO:37, Linker sequence [7 amino acids]:

[0406] GGGGSSS

[0407] SEQ ID NO:38, Linker sequence [3 amino acids]:

[0408] AAA

[0409] SEQ ID NO:39, Linker sequence [repeated 5-amino acid sequence]:

[0410] (GGGGS) n

[0411] SEQ ID NO:40, Linker sequence [6 amino acids]:

[0412] GGGGGG

[0413] SEQ ID NO:41, Linker sequence [15 amino acids]:

[0414] GGGGSGGGGSGGGGS

[0415] SEQ ID NO:42, Linker sequence [30 amino acids]:

[0416] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

[0417] SEQ ID NO:43, Linker sequence [18 amino acids]:

[0418] KESGSVSSEQLAQFRSLD

[0419] SEQ ID NO:44, Linker sequence [14 amino acids]:

[0420] EGKSSGSGSESKST

[0421] SEQ ID NO:45, Linker sequence:

[0422] (Gly) n

[0423] SEQ ID NO:46, Linker sequence [8 amino acids]:

[0424] GGGGGGGG

[0425] SEQ ID NO:47, Linker sequence [12 amino acids]:

[0426] GSAGSAAGSGEF

[0427] SEQ ID NO:48, Linker sequence [repeated sequence of 5 amino acids]:

[0428] (EAAAK) n

[0429] SEQ ID NO:49, Linker sequence:

[0430] A(EAAAK) n A

[0431] SEQ ID NO:50, Linker sequence:

[0432] A(EAAAK) 4 ALEA(EAAAK) 4 A

[0433] SEQ ID NO:51, Linker sequence:

[0434] [A(EAAAK) n A] m

[0435] SEQ ID NO:52, Linker sequence [12 amino acids]:

[0436] AEAAAKEAAAKA

[0437] SEQ ID NO:53, Linker sequence [repeated sequence of 2 amino acids]:

[0438] (XP) n

[0439] SEQ ID NO:54, Adapter sequence:

[0440] (AP) n

[0441] SEQ ID NO:55, Adapter sequence:

[0442] (KP) n

[0443] SEQ ID NO:56, Adapter sequence:

[0444] (QP) n

[0445] SEQ ID NO:57, Adapter sequence [14 amino acids]:

[0446] APAPAPAPAPAPAP

[0447] SEQ ID NO:58, GAG adapter sequence [21 amino acids]:

[0448] GAPGGGGGAAAAAGGGGGGAP

[0449] SEQ ID NO:59, GAG2 adapter sequence [39 amino acids]:

[0450] GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP

[0451] SEQ ID NO:60, GAG3 adapter sequence [57 amino acids]:

[0452] GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP

[0453] References

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[0501] WO2012 / 155007

[0502] WO 2012 / 155053

[0503] US 2009 / 0285846

[0504] 等同物

[0505] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the invention is not intended to be limited to the above description, but rather is set forth in the appended claims.

Claims

1. A fusion protein, which sequentially comprises from the N-terminus to the C-terminus: (i) A biotin-binding portion, wherein the biotin-binding portion consists of the amino acid sequence of SEQ ID NO:2; (ii) An SP1500 polypeptide, wherein the SP1500 polypeptide consists of the amino acid sequence of SEQ ID NO:7; and (iii) An SP0785 polypeptide, wherein the SP0785 polypeptide consists of the amino acid sequence of SEQ ID NO:

4.

2. The fusion protein according to claim 1, which further comprises: (iv) A first linker located between the biotin-binding portion and the SP1500 polypeptide and / or a second linker located between the SP1500 polypeptide and the SP0785 polypeptide.

3. The fusion protein according to claim 2, wherein the first linker consists of the amino acid sequence GGGGSSS.

4. The fusion protein according to claim 2, wherein the second linker consists of the amino acid sequence AAA.

5. The fusion protein according to any one of claims 1 to 4, wherein the fusion protein consists of the amino acid sequence of SEQ ID NO:

23.

6. A nucleic acid, which comprises a nucleotide sequence encoding the fusion protein according to any one of claims 1 - 4, wherein the nucleotide sequence comprises: (i) A first part encoding the biotin-binding portion; (ii) A second part encoding the SP1500 polypeptide; and (iii) A third part encoding the SP0785 polypeptide.

7. The nucleic acid according to claim 6, wherein the first part of the nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

10.

8. The nucleic acid according to claim 6 or 7, wherein the second part of the nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

14.

9. The nucleic acid according to claim 6 or 7, wherein the third part of the nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:

12.

10. The nucleic acid according to claim 6, wherein the nucleotide sequence encoding the fusion protein consists of the nucleotide sequence of SEQ ID NO:

33.

11. An expression vector, which comprises the nucleic acid according to any one of claims 6 - 10.

12. The expression vector according to claim 11, which further comprises a promoter nucleotide sequence operably linked to the nucleic acid.

13. The expression vector according to claim 11 or 12, which further comprises a polyadenylation sequence and / or a transcription termination sequence.

14. A cell, which comprises the nucleic acid according to any one of claims 6 - 10 or the expression vector according to any one of claims 11 - 13.

15. The cell according to claim 14, wherein the cell is an expression host cell.

16. The cell according to claim 15, wherein the expression host cell is selected from the group consisting of: bacterial cell lines, insect cell lines, and mammalian cell lines.

17. The cell according to claim 16, wherein the bacterial cell line is an Escherichia coli cell line.

18. The cell according to claim 16, wherein the insect cell line is a baculovirus expression system.

19. The cell according to claim 16, wherein the mammalian cell line is a human cell line or a Chinese hamster ovary (CHO) cell line.

20. The cell according to any one of claims 14 - 19, wherein the sequence of the nucleic acid according to any one of claims 6 - 10 is codon-optimized to improve expression in the cell.

21. A method for producing the fusion protein according to any one of claims 1 - 5, the method comprising the steps of: introducing the nucleic acid according to any one of claims 6 - 10 or the expression vector according to any one of claims 11 - 13 into an expression host cell.

22. A method for producing the fusion protein according to any one of claims 1 - 5, the method comprising the steps of: providing or obtaining the cell according to any one of claims 14 - 20; and isolating the fusion protein from the cell.

23. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 - 5.

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