Pneumococcal vaccine

Abstract

The present invention relates to immunogenic compositions and vaccine compositions comprising the antigens AliA or immunogenic fragments thereof and PnrA and immunogenic fragments thereof from Streptococcus pneumoniae and uses thereof in the treatment and / or prevention of infections by S. pneumoniae.

Description

[0001] PNEUMOCOCCAL VACCINE

[0002] Technical field

[0003] The present invention relates to immunogenic compositions and vaccine compositions comprising the antigens AliA and PnrA from Streptococcus pneumoniae and uses thereof in the treatment and / or prevention of infections by 5. pneumoniae.

[0004] Background

[0005] Pneumococcal disease is a major public health concern globally and contributes significantly to clinical disease burden and economic burden worldwide due to its high rate of global morbidity and mortality. Pneumococcal disease often afflicts young children and older adults, as well as the immunocompromised population. The incidence rate of pneumococcal disease is estimated to be higher in children <2 years and adults >65 years and the World Health Organization (WHO) estimates that about a million children succumb to this disease annually, with more than 300,000 being under 5 years of age. Immunization is the most effective public health strategy to combat pneumococcal disease and several vaccine formulations have been developed in this regard. However, there is a large need to improve vaccines against pneumococcal disease.

[0006] Pneumococcal disease refers to symptomatic infections caused by the bacterium Streptococcus pneumoniae, commonly referred to as pneumococci. Pneumococci are Grampositive bacteria that are encapsulated with a polysaccharide capsule, which is an important virulence factor. Colonization of the human nasopharynx by 5. pneumoniae is rather common and serves as the primary reservoir for transmission, which takes place person to person by contact with respiratory secretions (mucosal or saliva) from patients as well as healthy carriers. Carriage refers to the asymptomatic presence of pneumococci in the nasopharynx. Although most carriers do not show symptoms, it can cause local inflammation. Carriage is essential for the subsequent development of pneumococcal disease. The term invasive pneumococcal disease (IPD) refers to more severe and invasive pneumococcal infections, such as bacteremia, sepsis, meningitis and osteomyelitis, in which the bacterium can be isolated from normally sterile sites. The incubation period is uncertain but assumed to be around 1-3 days. Serious pneumococcal infections include pneumonia, meningitis and febrile bacteremia, while less serious and common manifestations are otitis media, sinusitis and bronchitis. Pneumonia is the most common manifestation of pneumococcal disease, whereas bacterial spread within the respiratory tract may result in middle-ear infection, sinusitis or recurrent bronchitis. Pneumococcal resistance to antimicrobials is a serious and rapidly increasing problem worldwide.

[0007] Pneumococci are Gram-positive bacteria that are encapsulated with a polysaccharide capsule, which is an important virulence factor. More than 100 different capsular serotypes of the pathogen have been identified worldwide. These serotypes have been defined based on differences in their capsular polysaccharide composition. The distribution of these serotypes has been seen to vary with time and geographic location. Pneumococcal serotype distribution varies over time, by age, clinical manifestation, vaccination background and geography.

[0008] Antibodies to capsular polysaccharide antigens of 5. pneumoniae provide serotype-specific protection against pneumococcal infections. Pneumococcal vaccines are aimed to cover the serotypes associated with severe pneumococcal disease, however the vaccines available today only protect against a total of 24 of the at least 100 known serotypes. There is a need to address remaining unmet medical need for coverage of pneumococcal disease caused by serotypes not found in vaccines available per today as well as potential for serotype replacement over time. Serotype replacement refers to the phenomenon where the prevalence of non-vaccine serotypes increases or new serotypes appear and spread in a population after the introduction of a vaccine that targets specific serotypes. Hence, there is a need for immunogenic compositions covering pathogenic serotypes and medical uses that can be used to induce a high immune response in humans against a majority of 5. pneumoniae serotypes.

[0009] Thus, there is a large medical need to provide vaccine compositions which provide efficient protection from infection, as well as spreading of infection, by more serotypes of 5. pneumoniae. Summary of the Invention

[0010] It is an object of the present disclosure to provide an immunogenic composition and / or a vaccine composition, which overcomes the problems associated with the prior art. In particular, it is an object of the present disclosure to provide an immunogenic composition and / or a vaccine composition which is useful in the treatment and / or prevention of infection by Streptococcus pneumoniae, in particular in the treatment and / or prevention of infection by 5. pneumoniae of several different serotypes, including serotypes which are not covered by existing vaccines.

[0011] It is furthermore an object of the present invention to provide an immunogenic composition and / or a vaccine composition that also prevents spreading of 5. pneumoniae in the population.

[0012] These and other objects which are evident to the skilled person from the present disclosure are met by different aspects of the invention as claimed and as generally disclosed herein.

[0013] The present inventors have identified a combination of antigens from 5. pneumoniae, namely AliA and PnrA, which is particularly suitable in an immunogenic and / or vaccine composition. As is shown in the appended Examples, the specific combination of antigens is able to elicit both mucosal and systemic antibody responses, elicits cellular immunity, and results in decreased bacterial load in the nose and improved survival in subjects administered the composition. Furthermore, the present inventors have surprisingly found that said combination is expected to be useful in the treatment and / or prevention of infection by 5. pneumoniae of several different serotypes, including at least one serotype which is not covered by the presently available vaccines.

[0014] Thus, in the first aspect of the present disclosure there is provided an immunogenic composition capable of eliciting an immune response against 5. pneumoniae in a subject when administered to said subject, said immunogenic composition comprising a first component and a second component, wherein a) said first component is at least one component selected from the group consisting of a nucleic acid encoding a PnrA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the PnrA protein of 5. pneumoniae; a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and b) said second component is at least one component selected from the group consisting of a nucleic acid encoding an Al i A protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae; a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, wherein said PnrA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:1 and said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2 or SEQ ID NO:152, such as wherein said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2.

[0015] In one embodiment there is provided an immunogenic composition capable of eliciting an immune response, such as a protective immune response, against 5. pneumoniae in a subject when administered to said subject, said immunogenic composition comprising a first component and a second component, wherein a) said first component is at least one component selected from the group consisting of a nucleic acid encoding a PnrA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the PnrA protein ofS. pneumoniae; a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein ofS. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system or a nucleic acid encoding at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a nucleic acid encoding an AliA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae; a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein ofS. pneumoniae, and wherein said second component further comprises at least one coupling moiety of a coupling system or a nucleic acid encoding at least one coupling moiety of a coupling system; wherein said PnrA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:1 and said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2 or SEQ ID NO:152; wherein the coupling system comprises at least one coupling moiety and at least one complimentary moiety and allows for the formation of at least one isopeptide bond between said at least one coupling moiety and at least one complementary moiety, and wherein the coupling system is derived from pilus proteins from Gram positive bacteria, which proteins are capable of spontaneously forming one or more isopeptide bonds.

[0016] For clarity, it is to be understood that in embodiments wherein the first and / or second component is a polypeptide, each components further comprises at least one coupling moiety of a coupling system in the form of amino acid sequence. In embodiments wherein the first and / or second component is a nucleic acid, each component further comprises a nucleic acid sequence encoding at least one coupling moiety of a coupling system.

[0017] In one embodiment, there is provided the immunogenic composition as defined herein wherein a) said first component is at least one component selected from the group consisting of a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, and wherein said second component further comprises at least one coupling moiety of a coupling system. In one embodiment, there is provided the immunogenic composition as defined herein wherein a) said first component is a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system; and b) said second component is a polypeptide corresponding to the AliA protein of 5. pneumoniae; and wherein said second component further comprises at least one coupling moiety of a coupling system. It will be understood that said least one coupling moiety of a coupling system comprised in the first and / or second component may be a polypeptide or peptide. Thus, in one embodiment said first component further comprises a polypeptide or peptide corresponding at least one coupling moiety of a coupling system. In one embodiment said second component further comprises a polypeptide or peptide corresponding at least one coupling moiety of a coupling system.

[0018] The present inventors have surprisingly found that the combination of antigens as defined by said first component and said second component not only provides several desired immunological responses, which are considered important for achieving a therapeutic effect, but also as show in Example 9 that said composition is expected to achieve said therapeutic effect for a large number of serotypes of 5. pneumoniae as explained herein.

[0019] The term "immune response" refers to the ability to induce a humoral and / or cell mediated immune response, preferably but not only in vivo. A humoral immune response comprises a B-cell mediated antibody response. A cell mediated immune response comprises a T-cell mediated immune response, including but not limited to CD4+ T-cells and CD8+ T-cells. The ability of an antigen to elicit immune responses is called immunogenicity, which can be humoral and / or cell-mediated immune responses. An immune response of the present invention is preferably an immune response against 5. pneumoniae, in particular against several serotypes of 5. pneumoniae. Said immune response may be a protective immune response. The term "immunogenic", as used herein, refers to the properties of an immunogen, which is an entity capable of eliciting a humoral and / or cell-mediated immune response. As used herein, the term "fragment" of a protein, such as an immunogenic fragment, is meant to refer to a portion of the amino acid sequence of the full-length polypeptide. In the context of the present disclosure, "an immunogenic fragment" as defined in any one of the herein discussed embodiments, refers to a fragment of an immunogenic protein which retains the same or a similar degree of immunogenicity as the immunogenicity of said protein. For example, an immunogenic fragment of an immunogenic protein in the present context may be of a length corresponding to 50% or more, such as 60% or more, such as 70% or more, such as 80% or more, such as 90% or more, such as 95% or more of said full length immunogenic protein. The "subject" as used herein relates to an animal, such as a mammal, which can be, for instance, a mouse, rat, guinea pig, hamster, rabbit, dog, cat, or primate. The subject may also be a human. Thus, in one embodiment, said subject is a human subject.

[0020] The term "PnrA" when used herein relates to the surface-exposed nucleoside-binding protein which is part of an ABC transporter system of 5. pneumoniae. SEQ ID NO:1 depicts an exemplarily amino acid sequence of said PnrA of 5. pneumoniae. However, the term "PnrA" also encompasses PnrA polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO:1. The term "AliA" when used herein relates to the ATP-binding cassette transporter which is found in 5. pneumoniae and which is involved in nutrient uptake. SEQ ID NO:2 and SEQ ID NO:152 depict exemplarily amino acid sequence of said AliA of 5. pneumoniae. The term "AliA" also encompasses AliA polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:152. As further discussed below, the skilled person is aware that a certain degree of mismatch between two amino acid sequences has no significant bearing on the structure and function of a protein comprising any of the two amino acid sequences. It will be appreciated that such degree of mismatch is reasonable and may be introduced into the herein disclosed exemplary amino acid sequences without any impairment of the structure and function of proteins encoded by these exemplary amino acid sequences. Thus, in one embodiment, the PnrA protein of 5. pneumoniae comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and any amino acid sequence having at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:1. In one particular embodiment, said PnrA protein of 5. pneumoniae comprises or consists of an amino acid sequence according to SEQ ID NO:1. In one embodiment, said AliA protein of 5. pneumoniae comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:152 and any amino acid sequence having at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:2 or SEQ ID NO:152. In one embodiment, said AliA protein of 5. pneumoniae comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and any amino acid sequence having at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:2. In one embodiment, said AliA protein of 5. pneumoniae comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:152 and any amino acid sequence having at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:152. In one particular embodiment, said AliA protein of 5. pneumoniae comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:152, such as wherein said AliA protein of 5. pneumoniae comprises or consists of an amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:152. Herein the term "protein" refers to a molecule comprising a polymer of amino acids linked together by peptide bonds. Said term is not meant herein to refer to a specific length of the molecule and is interchangeably used with the term "polypeptide". A polypeptide comprises an amino acid sequence, and, thus, sometimes a polypeptide comprising an amino acid sequence is referred to herein as a "polypeptide comprising a polypeptide sequence". Thus, herein the term "polypeptide sequence" is interchangeably used with the term "amino acid sequence". The term "amino acid" or "aa" herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogues refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g. homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogues have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. As used herein the term "sequence identity" or "% identity" refers to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. For purposes of the present invention, the sequence identity between two amino acid sequences or between two nucleotide sequences is determined using the NCBI BLAST program version 2.3.0 (Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402). Sequence identity of two amino acid sequences can be determined with blastp set at the following parameters: Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence = 11, Extension = 1; Compositional adjustments: Conditional compositional score matrix adjustment. The term "% identity" may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as% identity. As the skilled person will realize, the properties of a polypeptide, such as the immunogenicity of the polypeptides of the present disclosure, may be dependent on the sequence structure of the polypeptide and the presence and accessibility of immunogenic regions within said polypeptide. It is therefore possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the disclosure encompasses uses of nucleic acids encoding modified variants of the immunogenic polypeptide as described herein, which are such that the immunogenic characteristics are retained. For example, it is possible that one or several amino acid residues belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc.) could be exchanged for another amino acid residue from the same functional group. It is also possible, that one or several amino acid residues are exchanged for one or several amino acid residues that belong to a different functional group, provided that the resulting polypeptide retains its immunogenic properties. It will be understood the above reasoning is equally valid in the context of other proteins and components, such as moieties of the coupling system as disclosed herein and autotransporters as disclosed herein according to the first aspect and related aspects, and will not be repeated for the mere sake of brevity.

[0021] The present inventors envision, that the advantageous properties achieved by the immunogenic and / or vaccine composition as disclosed herein are achieved independent of the form in which said first component and said second component as defined herein are administered. Thus, it is envisioned that substantially the same beneficial effects will be achieved by administration of protein or polypeptide immunogenic and / or vaccine composition as by administration of a nucleic acid immunogenic and / or vaccine composition, such as an RNA vaccine or a DNA immunogenic and / or vaccine composition, or alternatively by a combination composition comprising both protein or polypeptide components and nucleic acid components.

[0022] Thus, in one embodiment, there is provided an immunogenic composition as defined herein, wherein said first component is selected from the group consisting of a nucleic acid encoding the PnrA protein of 5. pneumoniae and a nucleic acid encoding an immunogenic fragment of the PnrA protein of 5. pneumoniae.

[0023] In one embodiment, there is provided an immunogenic composition as defined herein, wherein said second component is selected from the group consisting of a nucleic acid encoding the AliA protein of 5. pneumoniae and a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae. In one embodiment, there is provided an immunogenic composition as defined herein, wherein said first component is selected from the group consisting of a nucleic acid encoding the PnrA protein of 5. pneumoniae and a nucleic acid encoding an immunogenic fragment of the PnrA protein of 5. pneumoniae, and wherein said second component is selected from the group consisting of a nucleic acid encoding the AliA protein of 5. pneumoniae and a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae.

[0024] Herein, the terms "polynucleotide", "nucleotide sequence", "nucleic acid" or "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides which are usually linked from one deoxyribose or ribose to another. The term "polynucleotide" preferably includes single and double stranded forms of DNA or RNA. A nucleic acid molecule of this invention may include both sense and antisense strands of RNA (containing ribonucleotides), cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[0025] In one embodiment, said nucleic acid as defined in a) and / or b) is selected from the group consisting of DNA and RNA. In one embodiment, nucleic acid as defined in a) and / or b) is DNA. As used herein, the term "DNA" refers to a deoxyribonucleic acid that carries genetic information for one or more proteins in the context of the present disclosure. Generally, such a DNA encodes a polypeptide and is transcribed into a messenger RNA (mRNA) which in turn is translated into the encoded protein in the target cell. The term "codon-optimized" or "codon-optimization" in the context of a nucleic acid (RNA or DNA) sequence as used herein refers to a nucleic acid sequence designed for improved codon composition of a recombinant gene, such as a transgene, based on various criteria without altering the amino acid sequence of the recombinant gene. This is possible because most amino acids are encoded by more than one codon. In the context of the present disclosure, a codon- optimized nucleic acid sequence is optimized for improved translation in a cell, such as a human cell, into which the codon-optimized nucleic acid sequence has been delivered.

[0026] In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3, any codon-optimized variant thereof or any nucleic acid having at least 70% identity thereto and / or wherein the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:4, any codon-optimized variant thereof or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3 or any nucleic acid having at least 70% identity thereto and / or wherein the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:4 or any nucleic acid having at least 70% identity thereto.

[0027] In one embodiment, the nucleic acid as defined in a) and / or b) is DNA, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3, any codon-optimized variant thereof, or any nucleic acid having at least 70% identity thereto and / or the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153, any codon optimized variant thereof, or any nucleic acid having at least 70% identity thereto, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3 or any codon-optimized variant thereof and / or the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153 or any codon-optimized variant thereof, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3 and / or the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153.

[0028] In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3, any codon-optimized variant thereof, or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3 or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence according to SEQ ID NO:3 any codon-optimized variant thereof.

[0029] In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:4, any codon optimized variant thereof or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:4 or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:4 or any codon-optimized variant thereof.

[0030] In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153, any codon-optimized variant thereof or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153 or any nucleic acid having at least 70% identity thereto. In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence according to SEQ ID NO:153 or any codon-optimized variant thereof.

[0031] In one embodiment, the nucleic acid as defined in a) comprises or consists of a nucleic acid sequence according to SEQ ID NO:3, any codon-optimized variant thereof or any sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:3. In one embodiment, the nucleic acid as defined in b) comprises or consists of a nucleic acid sequence according to SEQ ID NO:4, any codon-optimized variant thereof or any sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:4. In one embodiment, the nucleic acid as defined in b) comprises or consists of a nucleic acid sequence according to SEQ ID NO:153, any codon-optimized variant thereof or any sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:153. As explained above, codon-optimization may be advantageous to facilitate protein translation. In one embodiment, each of said codon-optimized variant of SEQ ID NO:3, 4 or 153, independently exhibits at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:3, 4 or 153, respectively.

[0032] In one embodiment said nucleic acid as defined in a) and / or b) is inserted into a plasmid or a vector. In one embodiment, said nucleic acids as defined in a) and b) are inserted into different plasmids or vectors. In one embodiment, said nucleic acids as defined in a) and b) are inserted into the same plasmid or vector. Said plasmid or vector may allow for the expression of said polypeptide as defined in a) and b) as one polypeptide chain, for example as a fusion polypeptide. In one embodiment, said nucleic acids as defined in a) and b) are inserted into a bicistronic plasmid or vector. Such bicistronic plamid or vector may allow for the simultaneous expression of said polypeptide as defined in a) and b) separately from the same RNA transcript.

[0033] Said nucleic acid as defined in a) and / or b) may be inserted into a vector, such as a viral vector, such as said viral vector is selected from a group consisting of a retroviral vector, an alphaviral vector, a poxviral vector, cytamegaloviral vector, a lentiviral vector, an adenoviral vector and adeno-associated viral vector, such as a group consisting of an adenoviral vector and an adeno associated viral vector. The skilled person is aware of other suitable ways to administer or deliver DNA, for example naked DNA, such as but not limited to physical methods, for example injection, electroporation, gene gun or sonoporation, or chemical methods, for example lipoplexes, polymersomes, polyplexes, dendrimers, as well as via attachment to inorganic nanoparticles, assembly of the DNA itself into virus mimicking particles. The skilled person will appreciate that the nucleic acids as defined in a) and / or b) herein, may be prepared for such administration or delivery.

[0034] In one embodiment, the nucleic acid as defined in a) and / or b) is RNA. In one particular embodiment, the RNA is a messenger RNA (mRNA). Said RNA may be obtained by an RNA manufacturing method, such as by in vitro transcription. As used herein, the term "mRNA" refers to a messenger ribonucleic acid. Generally, such an mRNA encodes a polypeptide and is translated into the protein it encodes in the target cell.

[0035] As appreciated by those skilled in the art, codon optimality is one feature that contributes greatly to mRNA stability. Stable mRNAs are enriched in codons designated optimal, whereas unstable mRNAs contain predominately non-optimal codons. Substitution of optimal codons with synonymous, non-optimal codons results in dramatic mRNA destabilization, while the converse substitution significantly increases stability. Codon optimality impacts ribosome translocation, connecting the processes of translation elongation and decay through codon optimality (Presnyak et al, Cell, 160(6):llll-24 (2015)). It is envisioned that codon optimized versions of the herein disclosed mRNAs are useful in the immunogenic composition as disclosed herein.

[0036] In one embodiment, the nucleic acid as defined in a) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, a fragment thereof encoding an immunogenic polypeptide fragment, any nucleic acid having at least 70% identity to SEQ ID NO:5 or to said fragment thereof, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, any nucleic acid having at least 70% identity to thereto, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:5, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as the group consisting of SEQ ID NO:5 and any codon-optimized and / or nucleoside modified version thereof. In one embodiment, the nucleic acid as defined in a) is a codon-optimized and / or nucleoside modified version of SEQ ID NO:5. In one embodiment, wherein the nucleic acid is RNA, the nucleic acid as defined in a) comprises a nucleic acid sequence selected from a group consisting of any nucleic acid sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:5 and any codon-optimized version thereof.

[0037] In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:154, a fragment of SEQ ID NO:6 encoding an immunogenic polypeptide fragment, a fragment of SEQ ID NO:154 encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as the group consisting of SEQ ID NO:6 and any codon-optimized and / or nucleoside modified version thereof or such as the group consisting of SEQ ID NO:154 and any codon-optimized and / or nucleoside modified version thereof. In one embodiment, the nucleic acid as defined in b) comprises a nucleic, acid sequence selected from the group consisting of SEQ ID NO:6, a fragment thereof encoding an immunogenic polypeptide fragment, any nucleic acid having at least 70% identity to SEQ ID NO:6 or to said fragment thereof, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, any nucleic acid having at least 70% identity to thereto, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in a) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as the group consisting of SEQ ID NO:6 and any codon-optimized and / or nucleoside modified version thereof. In one embodiment, wherein the nucleic acid is RNA, the nucleic acid as defined in b) comprises a nucleic acid sequence selected from a group consisting of any nucleic acid sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:6 and any codon-optimized version thereof.

[0038] In one embodiment, the nucleic acid as defined in b) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:154, a fragment thereof encoding an immunogenic polypeptide fragment, any nucleic acid having at least 70% identity to SEQ ID NO:154 or to said fragment thereof, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in b) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:154, any nucleic acid having at least 70% identity to thereto, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as wherein the nucleic acid as defined in b) comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:154, a fragment thereof encoding an immunogenic polypeptide fragment, and any codon-optimized and / or nucleoside modified version thereof, such as the group consisting of SEQ ID NO:154 and any codon-optimized and / or nucleoside modified version thereof. In one embodiment, the nucleic acid as defined in b) is selected from the group consisting of a codon-optimized and / or nucleoside modified version of SEQ ID NO:6 and a codon-optimized and / or nucleoside modified version of SEQ ID NO:154, such as wherein the nucleic acid as defined in b) is a codon-optimized and / or nucleoside modified version of SEQ ID NO:6 or a codon-optimized and / or nucleoside modified version of SEQ ID NO:154. In one embodiment, wherein the nucleic acid is RNA, the nucleic acid as defined in b) comprises a nucleic acid sequence selected from a group consisting of any nucleic acid sequence having at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO:154 and any codon-optimized version hereof.

[0039] The skilled person is familiar with the concept of codon optimization and knows that it refers to experimental approaches designed to improve the codon composition of a recombinant nucleic acid based on various criteria without altering the amino acid sequence encoded by the nucleic acid. In the present context, the term "codon-optimized" refers to nucleic acid sequences comprising modified codons compared to the native naturally occurring sequences. As appreciated by those skilled in the art, one example of codon-optimization involves altering a protein-encoding nucleic acid sequence derived from one organism for optimal expression in another, such as altering of a protein-encoding nucleic acid sequence derived from a prokaryote for optimal expression in a mammal, such as altering of a proteinencoding nucleic acid sequence derived from S. pneumoniae for optimal expression in humans. The skilled person is aware of that several different approaches and algorithms for codon optimization are known in the art.

[0040] As appreciated by those skilled in art, one or more nucleoside(s) may be replaced by other naturally modified nucleosides or by synthetic nucleoside analogues during RNA, such as mRNA, manufacturing. Nucleoside modified mRNAs encode the same protein as the protein encoded by the non-nucleoside modified mRNA variant. Certain modified nucleosides are considered to enhance the stability of the mRNA and / or increase the efficiency of its translation which in turn enhances the production of the desired protein in the cell comprising the mRNA. Certain nucleoside modifications are considered to alter the immunogenicity profile of vaccines which comprise such nucleoside modified mRNA, and can be particularly useful to reduce the innate immune response often elicited by non-nucleoside modified mRNA variants. This may be achieved by decreasing the recognition of the mRNA as foreign by pattern recognition receptors which would normally result in the activation of innate immunity. Commonly employed such nucleoside modifications include for example 5- methoxyuridine, which is a modified nucleoside triphosphate (NTP) for incorporation into mRNA using T7 RNA polymerase. Incorporation of 5-methoxyuridine can reduce the immunogenicity of the resulting mRNA. 5-methylcytidine (m5C) is also one example of such nucleoside modifications that involves the addition of a methyl group to cytidine. Additional non-limiting examples are N6-methyladenosine (m6A), which involves the addition of a methyl group to adenosine, 5-methyluridine (m5U), which involves the addition of a methyl group to uridine, 2-thiouridine (s2U), which contains a sulfur group and is known to increase mRNA stability, 2'-O-methylguanosine (m2,2G), which involves the addition of a methyl group to the 2'-oxygen of guanosine, 2'-O-methylcytidine (m2,2C), which involves the addition of a methyl group to the 2'-oxygen of cytidine, 2'-O-methyluridine (m2,2U), which involves the addition of a methyl group to uridine at the 2'-oxygen, and 5-methoxycytidine (m5oC), which contains a methoxy group. Pseudouridine (abbreviated by the Greek letter psi, UJ) is an isomer of the nucleoside uridine in which the uracil is attached via a carboncarbon instead of a nitrogen-carbon glycosidic bond. Pseudouridine, which is the most abundant RNA modification in cellular RNA, can regulate RNA expression post- transcriptionally and plays a variety of roles in the cell including translation, localization and stabilization of RNA. Without being bound by theory, the present inventors envision that modified nucleosides which have one or more of the above discussed beneficial effects on mRNA stability, translation efficiency and / or immunogenicity profile are advantageous in the context of the present disclosure. As used herein, the term "nucleoside modified" in relation to a nucleic acid sequence is meant to be understood that said nucleic acid sequence comprises at least one nucleoside modification as discussed above. In one non-limiting example, at least one uridine in an RNA sequence is replaced by pseudouridine. In another non-limiting example, all uridines in an RNA sequence are replaced by pseudouridines. Thus, in on embodiment, said nucleic acid as defined in a) and / or b) is a nucleoside modified mRNA comprising one or more modified nucleoside(s), such as a nucleoside modified mRNA comprising one or more pseudouridine residue(s). In one embodiment, said one or more modified nucleoside(s) is / are selected from a group consisting of 5-methoxyuridine, 5- methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), 2-thiouridine (s2U), 2'-O-methylguanosine (m2,2G), 2'-O-methylcytidine (m2,2C), 2'-O-methyluridine (m2,2U), 5-methoxycytidine (m5oC), 1-methylpseudouridine (mlUJ), l-methyl-3-(3-amino-5- carboxypropyl)pseudouridine (mlacp3UJ), 2'-0-methylpseudouridine (UJm), 5- methyldihydrouridine (m5D) and (3-methylpseudouridine (m3UJ). In one embodiment, the nucleic acid as defined in a) and / or b) comprises a poly-A tail. In one embodiment, the nucleic acid as defined in a) and / or b) comprises a 5' untranslated region, such as wherein said 5' untranslated region enhances translation. In one embodiment, the nucleic acid as defined in a) and / or b) comprises an mRNA cap. Said mRNA cap may be an m7GpppG cap, a 3'-0-methyl-m7GpppG cap or an anti-reverse cap analog. In one embodiment, the nucleic acid as defined in a) and / or b) comprises a cap-independent translational enhancer. As discussed above as well as demonstrated in the appended Examples, the immunogenic composition as disclosed herein may comprise one or more the antigens in the form of polypeptides. Thus, in one embodiment, said first component is selected from the group consisting of a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; such as wherein said first component is a polypeptide corresponding to the PnrA protein of 5. pneumoniae. In one embodiment, said second component is selected from the group consisting of a polypeptide corresponding to the AliA protein of 5. pneumoniae and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, such as wherein said second component is a polypeptide corresponding to the AliA protein of 5. pneumoniae.

[0041] In one embodiment, said first component is selected from the group consisting of a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and said second component is selected from the group consisting of a polypeptide corresponding to the AliA protein of 5. pneumoniae and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae. In one embodiment, said first component is a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and said second component is a polypeptide corresponding to the AliA protein of 5. pneumoniae. In one particular embodiment of the immunogenic composition as disclosed herein said first component and said second component are in the form of a fusion polypeptide.

[0042] It is also envisioned that the compositions may comprise one component in the form of a polypeptide and another component in the form of a polynucleotide. In one embodiment, said first component is a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and said second component is a nucleic acid encoding the AliA protein of 5. pneumoniae. In another embodiment, said first component is a nucleic acid encoding the PnrA protein of 5. pneumoniae; and said second component is polypeptide corresponding to the AliA protein of 5. pneumoniae. For the sake of clarity, as used herein, the term "corresponding to" in relation to a polypeptide is to be understood as the polypeptide is or makes up the protein referred to. For example, a polypeptide corresponding to the PnrA protein of 5. pneumoniae is to be understood as the polypeptide is the PnrA protein of 5. pneumoniae and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae is to be understood as the polypeptide is an immunogenic fragment of the PnrA protein of 5. pneumoniae.

[0043] The skilled person will understand that various modifications and / or additions can be made to an immunogenic polypeptide as defined herein, in order to tailor the polypeptide to a specific application without departing from the scope of the present disclosure. For example, an immunogenic polypeptide as defined herein may comprise additional amino acid residues for the purpose of improving production, purification and / or stabilization in vivo or in vitro of the polypeptide. Thus, an immunogenic polypeptide may comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue may individually or collectively be added in order to, for example, improve production, purification, solubility and / or stabilization in vivo or in vitro. Thus, in one embodiment, there is provided an immunogenic composition as disclosed herein, wherein said first and / or second component further comprises additional amino acid residues at the N- and / or C-terminus thereof. Such an immunogenic component should be understood as a polypeptide having one or more additional amino acid residues at the very first and / or the very last position in the polypeptide chain, i.e. at the N- and / or C-terminus. For the sake of clarity, the presence of an additional amino acid residue at the N- and / or C- terminus of said immunogenic polypeptide does not preclude the presence of additional N- or C-terminal extensions, for example in the form a "tag" for purification as described above. For example, in the case where first and / or second component is selected from immunogenic fragments as defined herein, said additional amino acid residue at the N- terminus may be a methionine. Thus, in one embodiment, said immunogenic fragment comprises a methionine residue at the N-terminus. In one embodiment, said additional amino acid residue(s) at the N- terminus and / or C-terminus or at the N-terminal and / or C- terminal improve production, purification and / or stabilization in vivo or in vitro of said immunogenic polypeptide. In one embodiment, said additional amino acid residue(s) improve for purification of said immunogenic polypeptide. Such additional amino acid residues may also provide a "tag" for purification, for example a His-tag (such as for example a His6-tag (SEQ ID NO:134) or a His8-tag (SEQ ID NO:149) or a "myc" (c-myc)-tag (SEQ ID NO:150) or a "FLAG" tag (SEQ ID NO:151) for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of the His-tag. For example PnrA further comprising SpyTag and a His6 tag corresponds to SEQ ID NO:135 and Ali A further comprising SpyTag and a His6 tag corresponds to SEQ ID NO:136 as defined herein. For the sake of clarity, the presence of additional amino acid residues at the N- and / or C- terminus of said immunogenic polypeptide or fragments as defined herein, does not preclude the presence of additional N- or C-terminal extensions, for example in the form of a "tag" for purification as described above. As shown in the appended Examples, the presence of a tag in the first and / or second component does not influence the immunogenic properties thereof.

[0044] As used herein, the term "coupling system" refers to any system comprising at least a first and a second molecular part or "moiety" which have affinity for each other and between which a bond of some sort or association between said molecular parts or moieties may be formed. Said association may be via non-covalent interaction, such as for example hydrogen bonds. The coupling system may for example consist of two moieties between which a covalent bond may form spontaneously or with the assistance of a moiety with catalytic activity, such as an enzyme, or where chemical crosslinking means are used to link different molecules together. The coupling system may for example consists of or comprises two moieties between which form a high affinity complex, which complex is based on non- covalent interaction. Non-limiting examples of such coupling systems include biotinstreptavidin and biotin-avidin. The skilled person is familiar variants of avidins, such as NeutrAvidin, CatpAvidin, and also knows that thus anti-biotin antibodies may be utilized. Thus, it is also possible that the coupling system may be based on antibody-antigen interaction, wherein the first moiety is an antibody, and a second moiety is an antigen, which the antibody is capable of binding to. Non-limiting examples include FLAG-tag and anti-FLAG antibody system. The skilled person is familiar with suitable coupling system may be based on antibody-antigen interaction. In some aspects of the present disclosure, a coupling system specifically refers to a coupling moiety and a complementary moiety that have the functionality to spontaneously form an isopeptide bond or that are able to form an isopeptide bond in the presence of a moiety with catalytic activity, such as an enzyme (e.g. a ligase, such as SpyLigase). In other words, at least one coupling moiety and at least one complementary moiety are derived from proteins which are capable of spontaneously forming one or more isopeptide bonds. It is further possible to link additional molecules to each of the two moieties, creating a complex wherein it is the two moieties with affinity for each other which constitute the ligate of the coupling system.

[0045] In the present disclosure, one of the two moieties of the coupling system is referred "coupling moiety" and the other moiety of the coupling system is referred to as "complementary moiety", wherein the coupling moiety is comprised in the first and / or second component and the complementary moiety may optionally link an additional molecule, such as a vesicle, to said first and / or second component. It should be noted that equally well, the two moieties of the coupling system may be referred inversely; such that one of the two moieties of the coupling system is referred "coupling moiety" and the other moiety of the coupling system is referred to as "complementary moiety", wherein the complementary moiety is comprised in the first and / or second component and the coupling moiety may optionally link an additional molecule, such as a vesicle, to the first and / or second component. As used herein, the term "complementary coupling moiety" and "complementary moiety" are used interchangeably in the context of the coupling system as discussed above. An isopeptide bond is a very robust covalent bond, providing a strong connection between the parts of the coupling system, and anything else linked to the respective moieties.

[0046] As mentioned, the term "coupling moiety" is used herein to describe one of the two moieties constituting the coupling system. More specifically, a coupling moiety is a polymer of amino acids having the distinct functionality of binding the other moiety of the coupling system, i.e. the "complementary moiety". In the context of the invention, the coupling moiety is comprised in said first and / or second component. Typically, the coupling moiety comprises one residue involved in the isopeptide bond while the complementary moiety comprises the other residue involved in the isopeptide bond. For example, when the coupling moiety comprises a reactive lysine residue, the complementary moiety comprises a reactive asparagine, aspartic acid, glutamine or glutamic acid residue, or when the coupling moiety comprises a reactive asparagine, aspartic acid, glutamine or glutamic acid residue, the complementary moiety comprises a reactive lysine residue or a reactive alpha-amino terminus. Thus, the coupling moiety may comprise a reactive asparagine residue while the complementary moiety may comprise a reactive lysine residue, or the coupling moiety may comprise a reactive lysine residue while the complementary moiety may comprise a reactive asparagine residue. Hence, by utilizing a coupling system by designing first and / or second component as defined herein comprising a coupling moiety with affinity for a complementary moiety, wherein said complementary moiety is in turn displayed on the surface of a vesicle, optionally via means of an autotransporter, as exemplified in the appended Examples.

[0047] Thus, in one embodiment there is provided an immunogenic composition as disclosed herein, wherein said first component further comprises at least one coupling moiety of a coupling system, wherein the coupling system allows for formation of at least one isopeptide bond between said at least one coupling moiety of said coupling system and at least one complementary moiety of said coupling system. In one embodiment, said first component is a nucleic acid as defined in a) and further comprises a nucleic acid sequence encoding said at least one coupling moiety of a coupling system. In one embodiment, said first component is a polypeptide as defined in a) and further comprises an amino acid sequence corresponding to said at least one coupling moiety of a coupling system.

[0048] In one embodiment there is provided an immunogenic composition as disclosed herein, wherein said second component further comprises at least one coupling moiety of a coupling system, wherein the coupling system allows for formation of at least one isopeptide bond between said at least one coupling moiety of said coupling system and at least one complementary moiety of said coupling system. In one embodiment, said second component is a nucleic acid as defined in b) and further comprises a nucleic acid sequence encoding said at least one coupling moiety of a coupling system. In one embodiment, said second component is a polypeptide as defined in b) and further comprises an amino acid sequence. Thus, in one embodiment, there is provided an immunogenic composition as disclosed herein, wherein a) said first component is at least one component selected from the group consisting of a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and wherein said first component further comprises a polypeptide corresponding to at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a polypeptide corresponding to the Al i A protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, and wherein said second component further comprises a polypeptide corresponding to at least one coupling moiety of a coupling system.

[0049] In another embodiment, wherein said first and / or second component are in the form of a fusion polypeptide, said fusion polypeptide comprises an amino acid sequence corresponding to at least one coupling moiety of a coupling system, wherein the coupling system allows for formation of at least one isopeptide bond between said at least one coupling moiety of said coupling system and at least one complementary moiety of said coupling system.

[0050] As exemplified in Example 3 it is possible that said first component and second component comprised in the immunogenic composition comprise coupling moieties of different coupling systems. In this manner, targeted coupling of said first component and said second component, each to different target via means of their respectively complementary coupling moieties, can be achieved. Thus, in one embodiment there is provided an immunogenic composition as disclosed herein, wherein said first component and second component comprise coupling moieties of different coupling systems. It is also possible that the said first component and second component comprise coupling moieties of the same coupling system. For example, said first component and second component comprise different coupling moieties of the same coupling systems, such that said first component comprises the coupling moiety and the second component comprises the complementary coupling moiety of the same system. In this manner, target coupling may also be achieved. In yet another embodiment, said first component and second component comprise the same coupling moieties of the same coupling system. In this way random coupling to the same target via means of the complementary coupling moieties can be achieved. As is appreciated by the skilled person, random coupling can be steered by adjusting the stoichiometry of the first and second components.

[0051] In one embodiment, said at least one coupling moiety of a coupling system is located C- or N-terminally of said polypeptide as defined in a), b) and / or said fusion polypeptide, such as is located N-terminally of said polypeptide defined in a), b) and / or said fusion polypeptide component.

[0052] In one embodiment, said at least one coupling moiety of a coupling system is located at the C- or N-terminus of said polypeptide as defined in a), b) and / or said fusion polypeptide, such as is located at the N-terminus of said polypeptide defined in a), b) and / or said fusion polypeptide component. In one embodiment, said at least one coupling moiety of a coupling system is located at the C- or N-terminus of said polypeptide defined in a), such as is located at the N-terminus in said polypeptide defined in a). In one embodiment, said at least one coupling moiety of a coupling system is located at the C- or N-terminally of said polypeptide defined in b), such as is located at the N-terminus in said polypeptide defined in b). In one embodiment, said at least one coupling moiety of a coupling system is located at the C- or N- terminally of said fusion polypeptide component, such as is located at the N-terminus in said fusion polypeptide component.

[0053] In one embodiment, said at least one coupling moiety of a coupling system is located internally in said polypeptide defined in a), b) and / or said fusion polypeptide, such as located internally in said polypeptide defined in a) and / or b).

[0054] As used herein, it is to be understood that when it is written that said at least one coupling moiety is "located C- or N-terminally" said moiety is located near said C- or N-terminus, such as within 30 amino acid residues (aa) of said C- or N-terminus, such as within 20 aa of said C- or N-terminus, such as within 10 aa of said C- or N-terminus, such as within 5 aa of said C- or N-terminus, such as within 4 aa of said C- or N-terminus, such as within 3 aa of said C- or N- terminus, such as within 1 aa of said C- or N-terminus. The skilled person appreciates that when it is written that said at least one coupling moiety is "located at the C- or N-terminus", is to be interpreted as located directly adjacent to the C- or N-terminus. A person of skill in the art will understand that by using the language "located C- or N-terminally" or "located at the C- or N-terminus" one does not imply a strict interpretation of the location of said moiety in relation to other moieties or structures comprised in a construct. In other words, the skilled person will understand that the expressions "located C- or N-terminally" or to express the location of said at least one coupling moiety of a coupling system in relation to said at least one polypeptide as defined in a), b) and / or said fusion polypeptide, may imply that said at least one coupling moiety of a coupling system may be directly at the C- or N- terminus of said at least one polypeptide as defined in a), b) and / or said fusion polypeptide, or may be indirectly at the C- or N-terminus of said at least one polypeptide as defined in a), b) and / or said fusion polypeptide such as separated by one or several amino acid residues. For example, said coupling moiety may be separated or flanked by a linker or spacer sequence from the N- and / C -terminus of said at least one polypeptide as defined in a), b) and / or said fusion polypeptide. An illustrative non-limiting example of such arrangement is [polypeptide]-[spacer or li nker]-[coupling moiety] or [coupling moiety]-[spacer or linker]- [polypeptide] or [spacer or li nker]-[coupling moiety]-[spacer or li nker]-[polypeptide] . In another embodiment, said at least one coupling moiety of a coupling system is located internally in said at least one polypeptide as defined in a), b) and / or said fusion polypeptide. By way of illustration, for example, said at least one target polypeptide moiety may be located between two domains of the said polypeptide or between tandem repeats of said polypeptide. It is also envisioned that in the fusion polypeptide as defined hereon, said at least one coupling moiety may be located between the polypeptides as defined in a) and b) fused.

[0055] With reference to the above explanation of how the terms "located C- or N-terminally" or "located at the C- or N-terminus" are used herein, the skilled person will also understand that by using the language "located internally in said at least one polypeptide", it is to be further understood that said at least one coupling moiety of a coupling system may be located within the sequence of said at least one polypeptide. For the sake of clarity, the coupling system comprises at least one coupling moiety and a complementary moiety. The designation coupling moiety and complementary moiety as used herein are to be understood to be merely for the purpose of indicating the interrelation between said moieties, such that the coupling moiety is compatible with its complementary moiety for the formation of an isopeptide bond between said moieties. For example, if a specific coupling system comprises the moieties A and B, then the specific moiety, herein referred to A may be equally well designated a coupling moiety as a complementary moiety and will be compatible with B, which is its complementary moiety or coupling moiety, respectively (depending on the designation chosen for A). In the present context, the designation coupling moiety or complementary moiety may merely be due which of the coupling moiety and complementary moiety is first mentioned, for example in the context of the method of the first aspect as disclosed herein. For example, in the context of reciting specific coupling systems, such as the system C-D, if C is denoted coupling moiety, then D is denoted the complementary moiety and inversely if D is denoted coupling moiety, then C is the denoted complementary moiety. As illustrated by the specific example of SpyTag- SpyCatcher (SEQ ID NO:10-SEQ ID NO:11), herein SpyTag is denoted coupling moiety, then SpyCatcher is the complementary moiety and inversely if SpyCatcher is denoted coupling moiety, then SpyTag is the complementary moiety.

[0056] In one embodiment, said coupling system is derived from pilus proteins from Gram positive bacteria, such as derived from pilus proteins from Gram positive bacteria selected from the group consisting of pilus proteins from Lactiplantibacillus, pilus proteins from Bacillus, pilus proteins from Ruminococcus, and pilus proteins from Streptococcus, such as preferably derived from pilus proteins from Streptococcus (also referred herein to as Gram positive Streptococcal pilus proteins). In one embodiment, said coupling system is derived from a one or more Gram positive Streptococcal pilus proteins, such as from a Gram positive Streptococcal pilus protein, selected from the group consisting of the major pilin protein Spy0128 of Streptococcus pyogenes , the fibronectin binding protein FbaB of Streptococcus pyogenes, the fibronectin-binding protein from Streptococcus dysgalactiae, the pilus-subunit RrgA from 5. pneumoniae, and the pilus subunit RrgC from 5. pneumoniae. A coupling system may comprise two moieties (in other words units) in the form of a coupling moiety - complementary moiety pair, also referred to as the coupling moiety and complementary moiety of the coupling system. Coupling systems may comprise more than two moieties, for example as in the form of a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first (the coupling moiety) and second (the complementary moiety) moieties are capable of being isopeptide bonded by catalytic means of the third moiety. This is discussed in more detail below.

[0057] Coupling moiety-complementary moiety pairs, also referred to as Catcher / Tag ligation pairs in the literature, can be derived from any protein that is capable of spontaneously forming isopeptide bonds, such as for instance described in Prbschel et al PLoS One. 2017.12(6), Tan et al PLoS One. 2016. 11(10) and WO 2011 / 098772. Particular suitable coupling moiety- complementary moiety pairs or so called Catcher / Tag ligation pairs are the peptide tag and corresponding peptide catcher pairs described in WO 2011 / 098772, the Spy0128 Catcher / Tag pair described in Zakeri & Howarth 2010 J Am Chem Soc 132(13), the FbaB Spy Catcher / Tag pair described in Zakeri et al Proc Natl Acad Sci USA. 2012. 109(12), and the Sdy Catcher / Tag pair derived from Fibronectin binding protein (UniProtKb Q53971) described in Tan et al. (2016) supra. Preferably the coupling moiety-complementary moiety pair (in other words Catcher / Tag ligationpair) is a SpyCatcher / SpyTag pair as described in Zackeri et al. (2012) supra, a SdyCatcher / SdyTag pair described in Tan et al. (2016) supra, or a SnoopCatcher / SnoopTag pair as described in Veggiani et al. Proc Natl Acad Sci USA 2016, 113(5):1202-7, more preferably the SpyCatcher / SpyTag pair (Zackeri et al. (2012) supra) or the SnoopCatcher / SnoopTag pair (Veggiani et al. (2016) supra).

[0058] In one embodiment, there is provided an immunogenic composition as described herein, wherein the coupling moiety and complementary moiety of the coupling system are selected from the group consisting of SnoopTag2 (SEQ ID NO:7), DogTag2 (SEQ ID NO:8), SnoopLigase2 (SEQ ID NO:9), SpyTag (SEQ ID NQ:10), SpyCatcher (SEQ ID NO:11), SnoopTag (SEQ ID NO:12), SnoopCatcher (SEQ ID NO:13), SpyTag002 (SEQ ID NO:14), SpyCatcher002 (SEQ ID NO:15), SpyTag003 (SEQ ID NO:16), SpyCatcher003 (SEQ ID NO:17), SdyTag (SEQ ID NO:18), SdyCatcher (SEQ ID NO:19), SilkTag (SEQ ID NQ:20), SilkCatcher (SEQ ID NO:21), DogTag (SEQ ID NO:22), DogCatcher (SEQ ID NO:23), SnoopTagJr (SEQ ID NO:24), SnoopLigase (SEQ ID NO:25), Jo (SEQ ID NO:26), In (SEQ ID NO:27), NGTag (SEQ ID NO:28), NGCatcher (SEQ ID NO:29), SpyCatcher-N (SEQ ID NQ:30), SpyCatcher-21 (SEQ ID NO:31), SpyStapler (SEQ ID NO:32), SpyLigase (SEQ ID NO:33), MoonCake (SEQ ID NO:34), Katl (SEQ ID NO:35), QueenCatcher (SEQ ID NO:36), PsCsCatcher (SEQ ID NO:37), Ktag (SEQ ID NO:38), BDTag (SEQ ID NO:39), RumTrunkTagD9N (SEQ ID NQ:40), RumTrunkTag (SEQ ID NO:41), RumTag (SEQ ID NO:42), Rum2Tag (SEQ ID NO:43), Rum3Tag (SEQ ID NO:44), Rum4Tag (SEQ ID NO:45), Rum5Tag (SEQ ID NO:46), Rum6Tag (SEQ ID NO:47), Rum7Tag (SEQ ID NO:48), BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NQ:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), Bac5Tag (SEQ ID NO:53), Clib9 (SEQ ID NO:54), PhoTag (SEQ ID NO:55), PsCsTag (SEQ ID NO:56), and SpyCatcher_orig (SEQ ID NO:57), and variants thereof exhibiting at least 70% identity thereto, wherein said moieties are selected such that said moieties have the capacity of forming an isopeptide bond between each other, and wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70% identity thereto.

[0059] In one embodiment said variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one embodiment, said a variant of a coupling moiety or of a complementary moiety exhibits at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to said coupling moiety or said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto. In one embodiment, said a variant of a coupling moiety or of a complementary moiety exhibits at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to said coupling moiety or said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one embodiment, said coupling system is a coupling moiety-complementary moiety pair.

[0060] The terms "SpyTag-SpyCatcher coupling system", "SpyTag" and "SpyCatcher" describe the first part (coupling moiety) and second part (complementary moiety), respectively, of a particular coupling system which may be used in the current disclosure. The coupling system is derived from a CnaB domain present in the Streptococcus pyogenes fibronectin-binding protein FbaB. Within the hydrophobic core of this domain, a triad of amino acids (lysine, aspartate and a catalytic glutamate) spontaneously form an isopeptide bond (Hagan et al. 2010 Angew Chem Int Ed Engl Nov 2;49(45):8421-5). The isolated CnaB domain was converted into a protein coupling system by splitting it into a peptide, the so called "SpyTag", sometimes abbreviated "SpT", and the remaining protein partner called the "SpyCatcher", sometimes abbreviated "SpC" (Zakeri et al., Proc Natl Acad Sci USA (2012) 109(12):E690-7). Similarly, "SpyTag002" or "SpyTag2" may be abbreviated "SpT2" and its partner "SpyCatcher002" or "SpyCatcher2" may be abbreviated "SpC2" and these terms are used interchangeably. Similar shorthand and logic applies to other Tags and Catchers such as: SpyCatcher003, SpyTag003, SnoopCatcher002. The two peptides possess the ability to spontaneously form an isopeptide bond between each other. In the context of the invention, the SpyTag (coupling moiety) and the target polypeptide moiety comprised in a POI may form a recombinant polypeptide wherein the SpyTag acts as the coupling moiety and is accessible to the complementary moiety. The SpyCatcher acts as the complementary moiety and may form a robust bond to the SpyTag.

[0061] The term "SnoopTag-SnoopCatcher coupling system", "SnoopTag" and "SnoopCatcher" refer to the first part (coupling moiety) and second part complementary moiety) respectively of another coupling system, which has been derived from the D4 Ig-like domain of the adhesin RrgA from 5. pneumoniae (Veggiani et al. Proc Natl Acad Sci USA (2016) 113(5):1202-7). To create this system, the D4 Ig-like domain was cleaved to create a coupling moiety called "SnoopTag", sometimes abbreviated "SnT", and a remaining complementary moiety "SnoopCatcher", sometimes abbreviated "SnC". In the context of the invention, the SnoopTag (coupling moiety) and the target polypeptide moiety comprised in a POI may form a recombinant polypeptide, wherein the SnoopTag is accessible to the complementary moiety.

[0062] The terms "KTag" and "SpyLigase" refer to a peptide tag and an enzyme, respectively. After the successful adaptation of the CnaB domain into the SpyTag / SpyCatcher protein coupling system, the SpyCatcher was further split up into the "KTag", sometimes abbreviated "KT", and "SpyLigase" (Fierer et al., Proc Natl Acad Sci USA (2014) 111(13):E1176-81). Ktag acts as a coupling moiety which may form a covalent bond with a complementary moiety in the presence of SpyLigase, which is needed to catalyze the bond formation. It has also been discovered that, in the presence of SpyLigase, both Ktag and SpyTag may alternate between acting as a coupling moiety and acting as a complementary moiety. However, SpyLigase remains a polypeptide separate from the coupling system or may remain associated with the complex in non-covalent fashion upon bond formation. This type of coupling system, wherein there are more than two moieties (such as a coupling moiety and a complementary moiety) are referred to as a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first (the coupling moiety) and second (the complementary moiety) moieties are capable of being isopeptide bonded by catalytic means of the third moiety (such as SpyLigase; SEQ ID NO:33). Other tripartite coupling systems exist, wherein the same logic is applied. Examples of such tripartite coupling systems are KTag-SpyTag (SEQ ID NO:38-SEQ ID NO:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a ligase. Examples of such ligases are as mentioned above SpyLigase (SEQ ID NO:33), but also SpyStapler (SEQ ID NO:32), SnoopLigase (SEQ ID NO:25), and SnoopLigase2 (SEQ ID NO:9).

[0063] In one embodiment, said coupling system is selected from the group consisting of SpyTag- SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag-SpyCatcher_orig (SEQ ID NQ:10-SEQ ID NO:57), SpyTag-SpyCatcher002 (SEQ ID NQ:10-SEQ ID NO:15), SpyTag-SpyCatcher003 (SEQ ID NQ:10-SEQ ID NO:16), SpyTag-SpyCatcher-N (SEQ ID NQ:10-SEQ ID NQ:30), SpyTag- SpyCatcher-21 (SEQ ID NQ:10-SEQ ID NO:31), SpyTag-SdyCatcher (SEQ ID NQ:10-SEQ ID NO:19), SpyTag002-SpyCatcher002 (SEQ ID NO:14-SEQ ID NO:15), SpyTag002-SpyCatcher (SEQ ID NO:14-SEQ ID NO:11), SpyTag002-SpyCatcher003 (SEQ ID NO:14-SEQ ID NO:17), SpyTag002-SdyCatcher (SEQ ID NO:14-SEQ ID NO:19), SpyTag003-SpyCatcher003 (SEQ ID NO:16-SEQ ID NO:17), SpyTag003-SpyCatcher (SEQ ID NO:16-SEQ ID NO:11), SpyTag003- SpyCatcher002 (SEQ ID NO:16-SEQ ID NO:15), SpyTag003-SdyCatcher (SEQ ID NO:16-SEQ ID NO:19), SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13), Sil kTag-Sil kCatcher (SEQ ID NQ:20-SEQ ID NO:21), DogTag-DogCatcher (SEQ ID NO:22-SEQ ID NO:23), DogTag2- DogCatcher (SEQ ID NO:8-SEQ ID NO:23), Jo-In (SEQ ID NO:26-SEQ ID NO:27), NGTag- NGCatcher (SEQ ID NO:28-SEQ ID NO:29), SdyTag-SdyCatcher (SEQ ID NO:18-SEQ ID NO:19), SdyTag-SpyCatcher (SEQ ID NO:18-SEQ ID NO:11), SdyTag-SpyCatcher002 (SEQ ID NO:18-SEQ ID NO:15), SdyTag-SpyCatcher003 (SEQ ID NO:18-SEQ ID NO:17), KTag-SpyTag (SEQ ID NO:38-SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SnoopTag2- SnoopCatcher (SEQ ID NO:7-SEQ ID NO:13), DogTag2-SnoopCatcher (SEQ ID NO:8-SEQ ID NO:13), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), MoonCake-RumTrunkTagD9N (SEQ ID NO:34-SEQ ID NO:40), MoonCake-RumTag (SEQ ID NO:34-SEQ ID NO:42), MoonCake-SpyTag (SEQ ID NO:34-SEQ ID NQ:10), MoonCake-SdyTag (SEQ ID NO:34-SEQ ID NO:18), Katl-SpyTag (SEQ ID NO:35-SEQ ID NQ:10), Katl-SdyTag (SEQ ID NO:35-SEQ ID NO:18), Katl- RumTrunkTagD9N (SEQ ID NO:35-SEQ ID NQ:40), Katl-RumTag (SEQ ID NO:35-SEQ ID NO:42), and variants thereof exhibiting at least 70% identity thereto, wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively, and exhibits at least 70% identity thereto, such as a variant selected from the group consisting of SEQ ID NQ:10-19, 22-25, 34 and 38-40 and variants exhibits at least 70% identity thereto. In one embodiment, said at least 70% identity is as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0064] A coupling system may also be a tripartite system and such tripartite system may be used in the context of the present invention as discussed above. Thus, in one embodiment, said coupling system is a tripartite system comprising a first moiety, a second moiety, and a third moiety, wherein said first and second moieties are capable of being isopeptide bonded by means of the third moiety. In another embodiment, said tripartite system comprises a first and second moiety selected from the group consisting of Ktag-SpyTag (SEQ ID NO:38-SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SpyTag-BDTag (SEQ ID NQ:10- SEQ ID NO:39), DogTag2-SnoopTagJr (SEQ ID NO:8-SEQ ID NO:24), and wherein the tripartite system further comprises a third moiety which is a catalytic moiety. In one embodiment, said catalytic moiety is a ligase. Suitable ligases which can act as a third moiety that catalyzes isopeptide bonding between the first and the second moiety of the tripartite coupling system are ligases such as SpyLigase (SEQ ID NO:33), but also SpyStapler (SEQ ID NO:32), SnoopLigase (SEQ ID NO:25), and SnoopLigase2 (SEQ ID NO:9).

[0065] In one embodiment, said coupling system is selected from the group consisting of SpyTag- SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag-SpyCatcher_orig (SEQ ID NQ:10-SEQ ID NO:57), SpyTag002-SpyCatcher2 (SEQ ID NO:14-SEQ ID NO:15) and SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13) and variants thereof exhibiting at least 70% identity thereto, wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and SpyTag002-SpyCatcher2 (SEQ ID NO:14-SEQ ID NO:15) and variants thereof exhibiting at least 70% identity thereto, wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively. In one embodiment, said at least 70% identity is as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%. In one embodiment, said coupling system is selected from the group consisting of SpyTag- SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11) and variants thereof exhibiting at least 70% identity thereto, wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as wherein said coupling system is SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11). In one embodiment, said coupling system is selected from the group consisting of SpyTag002-SpyCatcher2 (SEQ ID NO:14-SEQ ID NO:15) and variants thereof exhibiting at least 70% identity thereto, wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming an isopeptide bond to its complementary moiety or to its coupling moiety, respectively; such as wherein said coupling system is SpyTag-SpyCatcher2 (SEQ ID NQ:10- SEQ ID NO:15). In one particular embodiment, said coupling moiety is SpyTag (SEQ ID NQ:10) and said complementary moiety is SpyCatcher (SEQ ID NO:11). In one embodiment, said coupling moiety is SpyCatcher (SEQ ID NO:11) and said complementary moiety is SpyTag (SEQ ID NQ:10). In one particular embodiment, said coupling moiety is SpyTag (SEQ ID NQ:10) and said complementary moiety is SpyCatcher2 (SEQ ID NO:15). In one embodiment , said coupling moiety is SpyCatcher2 (SEQ ID NO:15) and said complementary moiety is SpyTag (SEQ ID NQ:10). In one embodiment , said coupling moiety is SnoopTag (SEQ ID NO:12) and said complementary moiety is SnoopCatcher (SEQ ID NO:13). In one embodiment, said coupling moiety is SnoopCatcher (SEQ ID NO:13) and said complementary moiety is SnoopTag (SEQ ID NO:12).

[0066] In particular embodiments of the immunogenic composition as disclosed herein, said first component comprises or consists of said polypeptide as defined in a) further comprising the coupling moiety selected from the group consisting of SpyTag having the amino acid sequence SEQ ID NQ:10, SpyCatcher2 having the amino acid sequence SEQ ID NO:15 and SpyCatcher having the amino acid sequence SEQ ID NO:11, SnoopCatcher having the amino acid sequence SEQ ID NO:13 and SnoopTag having the amino acid sequence SEQ ID NO:12; such as wherein said first component comprises or consists of said polypeptide as defined in a) further comprising the coupling moiety selected from the group consisting of SpyTag having the amino acid sequence SEQ ID NQ:10, SpyCatcher2 having the amino acid sequence SEQ ID NO:15 and SpyCatcher having the amino acid sequence SEQ ID NO:11; such as wherein said first component comprises or consists of said polypeptide as defined in a) further comprising the coupling moiety SpyTag having the amino acid sequence SEQ ID NO:10 or the coupling moiety SpyCatcher having the amino acid sequence SEQ ID NO:15. In one embodiment, said first component comprises or consists of said polypeptide corresponding to the PnrA protein having the amino acid sequence SEQ ID NO:1 further comprising the coupling moiety SpyTag having the amino acid sequence SEQ ID NQ:10 or the coupling moiety SpyCatcher? having the amino acid sequence SEQ ID NO:15.

[0067] Said polypeptide as defined in a) and said coupling moiety may be directly or indirectly fused, such as indirectly fused via a linker sequence or a spacer sequence.

[0068] In one particular embodiment there is provided an immunogenic composition as defined herein, wherein said first component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:58 , SEQ ID NO:135, and any amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:58 or SEQ ID NO:135; such as wherein said first component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:58 and any amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:58.

[0069] In particular embodiments of the immunogenic composition as disclosed herein, said second component comprises or consists of said polypeptide as defined in b) further comprising the coupling moiety selected from the group consisting of SpyTag having the amino acid sequence SEQ ID NQ:10, SpyCatcher? having the amino acid sequence SEQ ID NO:15 and SpyCatcher having the amino acid sequence SEQ ID NO:11, SnoopCatcher having the amino acid sequence SEQ ID NO:13 and SnoopTag having the amino acid sequence SEQ ID NO:12; such as wherein said first component comprises or consists of said polypeptide as defined in b) further comprising the coupling moiety selected from the group consisting of SpyTag having the amino acid sequence SEQ ID NQ:10, SnoopCatcher having the amino acid sequence SEQ ID NO:13 and SpyCatcher having the amino acid sequence SEQ ID NO:11; such as wherein said first component comprises or consists of said polypeptide as defined in a) further comprising the coupling moiety SpyTag having the amino acid sequence SEQ ID NQ:10 or the coupling moiety SnoopCatcher having the amino acid sequence SEQ ID NO:13; such as wherein said first component comprises or consists of said polypeptide corresponding to the AliA protein having the amino acid sequence SEQ ID NO:2 or SEQ ID NO:152 further comprising the coupling moiety SpyTag having the amino acid sequence SEQ ID NQ:10 or the coupling moiety SnoopCatcher having the amino acid sequence SEQ ID NO:13; such as wherein said first component comprises or consists of said polypeptide corresponding to the AliA protein having the amino acid sequence SEQ ID NO:2 further comprising the coupling moiety SpyTag having the amino acid sequence SEQ ID NQ:10 or the coupling moiety SnoopCatcher having the amino acid sequence SEQ ID NO:13.

[0070] In one embodiment, said polypeptide as defined in b) and said coupling moiety are directly or indirectly fused, such as indirectly fused via a linker sequence or a spacer sequence.

[0071] In one particular embodiment there is provided an immunogenic composition as defined herein, wherein said second component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:59, SEQ ID NO:136; and any amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:59 or SEQ ID NO:136; 7 such as wherein said first component comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:59 and any amino acid sequence having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to SEQ ID NO:59.

[0072] As discussed above, the first component, second component and / or fusion polypeptide as defined herein further comprising a coupling moiety may via the means of the coupling system be coupled by the formation of an isopeptide bond to a desired target, for example a desired target may be a vesicle. In this context, the vesicle displays the complementary moiety of the coupling system. By bringing the polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide as defined in contact with said vesicle under conditions which allow for the formation of an isopeptide bond , a vesicle displaying said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or said fusion polypeptide may be obtained. In this way, said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide as defined herein may be displayed on at least one vesicle by means of the coupling system.

[0073] Thus, in one embodiment there is provided an immunogenic composition as defined herein, wherein the composition further comprises at least one vesicle, such as at least two vesicles. For example, said at least two vesicles may be different in from each other in at least one property. It is to be understood that the display of any one of said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide on said at least one vesicle is dependent on the formation of an isopeptide bond between the respective moieties of the coupling system present on said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide and said at least one vesicle. Said at least one complementary moiety of the coupling system which said at least one vesicle comprises is accessible for binding to the coupling moiety of said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide, thus it is understood to be present on the outer surface of said at least one vesicle. It may be directly or indirectly coupled to said at least one vesicle, for example via means of a membrane bound protein or via a protein anchored in the membrane (such as a protein comprising a transmembrane domain). Thus, in one embodiment, there is provided an immunogenic composition as define herein, which composition further comprises at least one vesicle, such as at least two vesicles, wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component; and wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said second component. In one particular embodiment, said first and second components are coupled to said at least one vesicle. It will be understood that said first and second components may be coupled to the same vesicle or may be coupled to different vesicles. If the vesicle displays complementary moieties which can form isopeptide bonds with coupling moiety comprised in said first component and with the coupling moiety comprised in said second component, the (same) vesicle may display both the first and second component. This may occur if the coupling moiety comprised in said first component and coupling moiety comprised in said second component are different, in which case the vesicle displays both their respective complementary coupling moieties. This may also occur randomly if the coupling moiety comprised in said first component and coupling moiety comprised in said second component are same. Thus, in one embodiment, said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component and at least one complementary moiety to the coupling moiety comprised in said second component. In one embodiment there is provided an immunogenic composition as disclosed herein, wherein said first component and second component comprise coupling moieties of different coupling systems or wherein said first component and second component comprise coupling moieties of the same coupling system. In one particular embodiment, said first component and second component comprise the same coupling moieties of the same coupling system. As discussed and exemplified extensively above, the coupling system may be a coupling system derived from pilus proteins from Gram positive bacteria. In one particular embodiment of the first aspect as disclosed herein, there is provided an immunogenic composition capable of eliciting an immune response against 5. pneumoniae in a subject when administered to said subject, said immunogenic composition comprising a first component and a second component, wherein a) said first component is at least one component selected from the group consisting of a nucleic acid encoding a PnrA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the PnrA protein of 5. pneumoniae; a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a nucleic acid encoding an AliA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae; a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of S. pneumoniae, and wherein said second component further comprises at least one coupling moiety of a coupling system; wherein said PnrA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:1 and said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2 or SEQ ID NO:152; wherein the coupling system comprises at least one coupling moiety and at least one complimentary moiety and allows for the formation of at least one isopeptide bond between said at least one coupling moiety and at least one complementary moiety, and wherein the coupling system is derived from Gram positive bacterial pilus proteins which are capable of spontaneously forming one or more isopeptide bonds; and wherein the composition further comprises at least one vesicle; and wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component; and wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said second component; and wherein said first and second components are coupled to a vesicle / vesicles.

[0074] Thus, said first and second components may be coupled to the same vesicle. Alternatively, said first and second components may be coupled to different vesicles. It will be appreciated that the immunogenic composition as disclosed herein may comprise a mixture of vesicles which have only the first component coupled thereto and vesicles which have only the second component coupled thereto; or vesicles which have both the first component and the second component coupled thereto; or any mixture thereof.

[0075] As discussed above, it will be understood that said least one coupling moiety of a coupling system comprised in the first and / or second component may be a polypeptide or peptide. Thus, in one embodiment said first component further comprises a polypeptide or peptide corresponding to at least one coupling moiety of a coupling system. In one embodiment said second component further comprises a polypeptide or peptide corresponding at least one coupling moiety of a coupling system.

[0076] In the present case, wherein said polypeptide or fragment thereof as defined in a), said polypeptide or fragment thereof as defined in b) and / or fusion polypeptide, is an antigen moiety, it may be particularly useful to display said target polypeptide moiety on outer membrane vesicles (OMVs) as the OMVs may be used to deliver the antigen to the subject and at the same time make use of the immune system stimulating properties of said vesicles. Outer membrane vesicles (OMVs) are ubiquitously released from the outer membrane (OM) of Gram-negative bacteria. OMVs comprise intrinsic adjuvant activity provided by the presence of various pathogen recognition receptor ligands, such as lipopolysaccharide and / or immunogenic surface proteins. This provides an attractive combination with the nonliving, particulate nature of the OMVs. OMVs have been described to protect animals and humans against various pathogens. Thus, OMVs are considered suitable for use in vaccines as well as in targeted drug delivery.

[0077] Of note, outer membrane vesicles (OMVs) and protein bodies (PBs) are both utilized as scaffolds in vaccine design and production, but differ significantly in their structure, function, and immunogenic properties. As described earlier, OMVs are spherical lipid bilayer nanostructures naturally secreted by Gram-negative bacteria. They comprise various bacterial components such as proteins, lipopolysaccharides (LPS), and periplasmic constituents, which contribute to their inherent immunogenicity. OMVs can be engineered to express specific antigens, making them versatile platforms for vaccine development. In contrast, protein bodies are dense, protein-rich inclusions found in bacterial cells. They are formed by the aggregation of recombinant proteins and can be used to produce vaccines by expressing antigens within these bodies. However, protein bodies lack the intrinsic immunogenic properties of OMVs and often require additional adjuvants to enhance their immunogenicity.

[0078] OMVs are generally considered more preferable as vaccine scaffolds due to several advantages. Firstly, OMVs are inherently immunogenic, which means they can elicit strong innate and adaptive immune responses without the need for additional adjuvants. This is partly due to the presence of LPS and other bacterial components that act as pathogen- associated molecular patterns (PAMPs), stimulating the immune system. Secondly, OMVs can be produced in a scalable and cost-effective manner, with high yields and purity. They also maintain their structural integrity during storage and transport, which simplifies vaccine distribution. In contrast, protein bodies often require complex purification processes and may face stability issues. Additionally, OMVs can be engineered to carry multiple antigens, providing broad protection against various pathogens. These attributes make OMVs a more versatile and efficient scaffold for vaccine development compared to protein bodies.

[0079] OMVs have been successfully used in approved vaccines and are compatible with Good Manufacturing Practice (GMP) standards, making them suitable for large-scale production and regulatory approval. They can be administered mucosally, which is advantageous for inducing both systemic (IgG) and mucosal (IgA) immune responses. This dual response is crucial for protecting against pathogens that enter through mucosal surfaces. In contrast, protein bodies are typically administered via injection and primarily induce systemic IgG responses.

[0080] Characterization and sterilization of OMVs are feasible, ensuring high purity and safety of the final vaccine product. Protein bodies, however, pose challenges in characterization due to impurities and particle heterogeneity, and their large size complicates sterilization by conventional methods. The proper folding of antigens within OMVs ensures a high-quality immune response, whereas protein bodies may contain misfolded antigens, potentially leading to lower efficacy.

[0081] OMVs require soluble antigens for effective vaccine production, whereas protein bodies do not have this requirement, allowing for faster screening of multiple candidates. Despite this advantage, the difficulty in purifying protein bodies and the presence of impurities trapped within them can compromise the quality of the vaccine.

[0082] OMVs offer several advantages over protein bodies as vaccine scaffolds, including inherent immunogenicity, compatibility with GMP manufacturing, and the ability to induce both systemic and mucosal immune responses. These attributes make OMVs a more versatile and efficient platform for vaccine development.

[0083] Thus, in one embodiment of the immunogenic composition as disclosed herein, said at least one vesicle is a self-adjuvating vesicle. As described herein, a "self-adjuvating vesicle" is a vesicle for which there is no need for a further separate adjuvant in order to elicit an immune response, such as a therapeutically effective immune response, to an antigen in the context of a composition, such as a vaccine. It is to be understood that the adjuvant function is provided by the self-adjuvating vesicle itself, for example via means of lipopolysaccharide and immunogenic surface proteins present thereon. In one embodiment, said at least one vesicle is an Outer Membrane Vesicle (OMV). In one embodiment, said at least one vesicle is derived from a bacterium. As used herein, the term "derived from" is to be interpreted to encompass "originated from", "obtained from", or "isolated from". In one embodiment, said at least one vesicle is derived from a Gram-negative bacterium. It is also possible that the immunogenic composition comprises at least two, such as at least two different vesicles, such as at least two different OMVs, which are derived from a Gram-negative bacterium or from different Gram-negative bacteria. In another embodiment, said at least one vesicle is, such as at least two vesicles are, selected from the group consisting of vesicles derived from Escherichia coli, Neisseria meningitidis, Neisseria gonorrhea and Salmonella spp, such as selected from the group consisting of vesicles derived from Escherichia coli and Salmonella spp, such as wherein said at least one vesicle is derived from Salmonella spp. In one embodiment, said at least one vesicle is, such as at least two vesicles are, selected from the group consisting of vesicles derived from Escherichia coli and Salmonella spp. In a further embodiment, said at least one vesicle is a vesicle derived from Escherichia coli. In one embodiment, said at least two vesicles are derived from Escherichia coli. In another further embodiment, said at least one vesicle is a vesicle derived from Salmonella spp. In one embodiment, said at least two vesicles are derived from Salmonella spp. In one embodiment, said at least one vesicle is derived from a subspecies of 5. enterica subsp. Enterica, such as from Salmonella enterica subsp. Enterica serovar Typhimurium (5. Typhimurium). In one embodiment, said at least two vesicles are derived from a subspecies of 5. enterica subsp. Enterica, such as from Salmonella enterica subsp. Enterica serovar Typhimurium (5. Typhimurium). It is to be understood that in the present context, embodiments of immunogenic composition recited to comprise at least one vesicle are equally applicable to the immunogenic composition comprising at least two vesicles, for example two different vesicles. The skilled person appreciates that vesicles may differ for example due to their origin and / or due to the components displayed thereon. In one embodiment there is provided an immunogenic composition as defined herein, wherein said at least one vesicle displays on its outer surface at least one complementary moiety of the coupling system as defined herein. In one embodiment, said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component. In one embodiment, said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said second component. In one embodiment, said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component and at least one complementary moiety to the coupling moiety comprised in said second component. In one embodiment, said complementary moieties may be the same or may be different. In one embodiment, said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said fusion polypeptide.

[0084] In Gram-negative bacteria, secretion and surface display of heterologous proteins are difficult to achieve due to the presence of a complex, multi-layered cell envelope consisting of an inner membrane, an outer membrane, and a gel like substance between the membranes called the periplasm. Several secretion systems have evolved to overcome this barrier and deliver proteins in the extracellular environment. Of these the Autotransporter (AT) pathway (also known as the type V secretion system), is suitable for piggy-back transport of cargo proteins, because it combines simplicity with a high transport capacity. Autotransporter proteins are large proteins that are secreted by Gram-negative bacteria, such as E. coli, and are synthesized as large precursor proteins that contain three domains: an N-terminal signal peptide that targets the protein to the Sec translocon and initiates transfer across the inner membrane; a passenger domain, which comprises the "cargo" protein that is to be secreted; and a C-terminal beta-domain comprising a beta-barrel structure that integrates into the outer membrane and plays an important role in translocation of the passenger domain across the outer membrane into extracellular space. After translocation, the passenger domain is cleaved from the translocator domain and is released into the extracellular environment. Cleavage may be the action of an (external) protease or a protease motif situated between the translocator domain and the passenger domain. Alternatively, cleavage takes place through an intramolecular autocatalytic event at a specific site between the translocator domain and the passenger domain. Autotransporter proteins may be modified by mutations which prevent said cleavage, such that the passenger domain is not released to the extracellular environment and instead remains as part of the autotransporter protein anchored into the membrane (in other words is displayed on the membrane for example the membrane of an OMV). The skilled person appreciates that OMVs with Ats may be derived from host cells producing the AT or, alternatively, Ats can be brought into the membrane of an OMV or lipid vesicle by reconstitution. It is possible to replace the passenger domain, or a part thereof, with a desired polypeptide such that the autotransporter thereby displays a desired polypeptide on the membrane it is anchored into, for example the membrane of an OMV. Alternatively, it is possible to fuse desired polypeptide to said autotransporter such that said autotransporter thereby displays a desired polypeptide on the membrane it is anchored into, for example the membrane of an OMV.

[0085] Suitable autotransporter proteins that can be used to obtain the bacterial cells and OMVs of the present invention are proteins that belong to the pfam autotransporter family ('Autotransporter' PF03797). Non-limiting examples of suitable AT classes include: the classical aTs (Va), where the translocator that forms a 12-stranded p-barrel in the outer membrane, and a mostly p-helical passenger, are part of one polypeptide (e.g. Hbp, Tsh, EspC, IgA protease, Pet, EspP, EstA, Ag43, AIDA-I, IcsA, VacA); trimeric aTs (Vc), which require three polypeptides to constitute a full 12-stranded p-barrel translocator to secrete the passengers which includes a coiled-coil stalk and p-helical head regions (YadA, BadA, and Hia).; patatin-like aTs (Vd), with similar domain architecture to Va but where the translocator is a 16-stranded p-barrel that contains a POTRA domain (e.g. PIpD); inverse aTs (Ve), which comprise an inverted domain organization with an N-terminal signal sequence followed by the translocator, then the linker and a C-terminal passenger Intimin, Invasin); and Hop-family aTs (Vf) possessing an interrupted p-barrel translocator where the passenger is inserted in the loop joining the 1st and second p-strands, and therefore resembling a prolonged loop protruding from the 8-stranded p-barrel (see Clarke et al. Front Immunol. 2022 Jul 1;13:921272). Particularly useful autotransporter proteins in the present context are classical autotransporters, such as serine protease autotransporters of the Enterobacteriacea ('SPATE') such as described in Yihfen et al., Trends in Microbiol. (2008) 16(8): 370-9. The skilled person appreciates that it would be possible to utilize N-terminally truncated autotransporters, such that at least part of the passenger domain is truncated, such as the entire passenger domain is deleted. To such a truncated autotransporter the complementary moiety can be fused. It is also possible that the entire passenger domain can be replaced by a heterologous moiety and still accomplish display. The skilled person appreciate that the complementary moiety may be fused to such a heterologous moiety. It may be preferred to keep at least the linker domain of said autotransporter intact in order to realize display. Thus, in one embodiment, there is provided an immunogenic composition as defined herein, wherein said at least one vesicle displays on its outer surface an autotransporter (AT) fusion protein, wherein said AT fusion protein comprises an AT protein and said at least one complementary moiety of the coupling system. The coupling system may be as defined herein. Thus, the first and / or second component comprising a coupling moiety or the fusion protein as defined herein may thus be coupled to the AT protein via an isopeptide bond to complementary moiety in the AT fusion protein.

[0086] In one embodiment, said autotransporter protein is a serine protease autotransporter of the Enterobacteriaceae (SPATE), such as a SPATE protein from Escherichia coli. SPATE proteins contain a highly conserved channel-forming C-terminal domain, which facilitates secretion of the passenger domain to the cell surface and autoproteolytic cleavage, which releases the passenger from the bacterial cell. The passenger folds into a characteristic beta stem structure with an N-terminal globular domain that performs serine proteolytic activity and on the cell surface, the passenger of a SPATE is cleaved from the translocator and released into the extracellular environment. Cleavage occurs in a conserved site located between two consecutive asparagine residues in the linker region joining the passenger and the translocator domains.

[0087] In one embodiment as disclosed herein, said SPATE is selected from the group consisting of hemoglobin-binding protease (Hbp) from E. coli, EspP from E. coli, Pet from E. coli, EspC from E. coli, Tsh from E. coli, Hap from Haemophilus influenzae, Hap from Neisseria gonorrhoeae, Hap from Neisseria meningitidis, IgA protease from Haemophilus influenzae, IgA protease from Neisseria gonorrhoeae, IgA protease from Neisseria meningitidis and SepA from Shigella flexneri, any N-terminally truncated variant thereof retaining ability to translocate the complementary moiety to the vesicle outer surface and polypeptide exhibiting at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, thereto; such as the group consisting of hemoglobin-binding protease (Hbp) from E. coli, EspP from E. coli, Pet from E. coli, EspC from E. coli, Tsh from E. coli, Hap from Haemophilus influenzae, Hap from Neisseria gonorrhoeae, Hap from Neisseria meningitidis, IgA protease from Haemophilus influenzae, IgA protease from Neisseria gonorrhoeae, IgA protease from Neisseria meningitidis and SepA from Shigella flexneri, any N-terminally truncated variant thereof retaining ability to translocate the complementary moiety to the vesicle outer surface.

[0088] In one embodiment, said SPATE protein is selected from the group consisting of hemoglobinbinding protease (Hbp) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60, extracellular serine protease (EspC) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65 and temperature-sensitive hemagglutinin (Tsh) comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66, any N- terminally truncated variant of SEQ ID NQ:60 retaining ability to translocate the complementary moiety to the vesicle outer surface, any N-terminally truncated variant of SEQ ID NO:65 retaining ability to translocate the complementary moiety to the vesicle outer surface, any N-terminally truncated variant of SEQ ID NO:66 retaining ability to translocate the complementary moiety to the vesicle outer surface, and any proteins exhibiting at least such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% , identity to any one of said SEQ ID NQ:60, SEQ ID NO:65,SEQ ID NO:66 and said variants thereof; such as wherein the SPATE protein is selected from the group consisting of Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60, EspC comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65 and Tsh comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66. In one embodiment, said SPATE protein is EspC comprising or consisting of the amino acid sequence as defined in SEQ ID NO:65. In another embodiment, said SPATE protein is Tsh comprising or consisting of the amino acid sequence as defined in SEQ ID NO:66. In a further embodiment, said SPATE protein is selected from the group consisting of Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60 and any proteins exhibiting at least such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto; such as wherein the SPATE protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:60.

[0089] In one embodiment, said autotransporter protein comprises at least one mutation that prevents cleavage at the autocatalytic cleavage site. In another embodiment, said autotransporter protein comprises a mutation that disrupts the autocatalytic cleavage site such that cleavage at the autocatalytic cleavage site is prevented. It is known in the art that said autocatalytic cleavage site of SPATE proteins is part of an invariant motif ("linker domain") in SPATES that attains an alpha helical conformation (said linker domain comprising the amino acid sequence "EVNNLNKRMGDLRD", SEQ ID NO:195), as described by Kostakioti, M. and Stathopoulos, C. Infect. Immun. (2006) 74(9): 4961-4969. In the SPATE protein Hbp said autocatalytic cleavage site is specifically between the two Asparagine residues of the aforementioned motif, namely Asnl048 and Asnl049. In Hbp, Asnl048 and Aspll45 form an unusual catalytic dyad that mediates the autocatalytic cleavage event through the cyclization of the asparagine. A person skilled in the art will understand that mutations, in particular non-conservative, in the linker domain motif or outside this motif that affect, directly or indirectly, the formation of the catalytic dyad between Asnl048 and Aspll45, may also prevent cleavage. For example, mutations affecting the alpha-helical conformation of the linker domain or the positioning of the cleavage site residues in the beta-domain pore may prevent cleavage. In one embodiment, said autotransporter protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60 and further comprises at least one mutation in a position selected from the group consisting of N1048, N1049, K1052, R1053, G1055, L1057, R1069, D1145, K1149, E1179, and E1197, which at least one mutation results in prevention of cleavage at the autocatalytic cleavage site, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NQ:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G, N1048A, N1048D, N1048S, N1049S, K1052A, R1053A, G1055R, L1057R, and D1145X wherein X is any amino acid other than Glu (E), such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NO:60 and further comprises at least one mutation in a position selected from the group consisting of N1048G and N1049S, such as wherein said autotransporter protein is Hbp comprising the amino acid sequence as defined in SEQ ID NO:60 and further comprises mutations in positions N1048G and N1049S. In one preferred embodiment, said autotransporter protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NQ:60 and further comprises mutations in positions N1048G and N1049S. This mutated Hbp corresponds to SEQ ID NO:61 as defined herein. In a further embodiment, said Hbp comprises or consists of the amino acid sequence as defined in SEQ ID NO:61 and any proteins exhibiting at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto, provided said proteins comprise an autocatalytic cleavage site which has been mutated such that cleavage is prevented.

[0090] As it is known to the person skilled in the art, an alternative way to removing of the autocatalytic function by introducing at least one mutation that prevents cleavage at the autocatalytic cleavage site, is the partial or complete deletion of the autocatalytic site. Thus, in one embodiment, the SPATE protein comprises a deletion mutation of the autocatalytic cleavage site, such that the autocatalytic cleavage site is partially or completely deleted. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:62-64 and any proteins exhibiting at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity to any one of said sequences SEQ ID NO:62-64.

[0091] In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:62. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:63. In one embodiment, said Hbp comprising a deletion mutation of the autocatalytic cleavage site comprises or consists of an amino acid sequence according to SEQ ID NO:64.

[0092] As explained above, the present inventors envision that said at least one complementary binding moiety of said coupling system may be incorporated into the AT protein by one or several of N-terminal fusion to said AT protein; internal incorporation of the said at least one complementary binding moiety into the AT protein; and the replacement of part of the AT protein by said at least one complementary binding moiety of said coupling system. For example, complementary binding moiety of said coupling system may be incorporated at the location of one of the side domains of the passenger domain of the AT protein.

[0093] In one embodiment, said AT fusion protein comprises at least one complementary binding moiety of said coupling system located N-terminally in said AT protein, such as located at the N-terminus of the said AT protein. In one embodiment, said AT fusion protein comprises at least one complementary binding moiety of said coupling system located internally in said AT protein, such as internally in the portion of said AT protein displayed on said vesicle. It is possible to insert or completely or partially replace one or more of the side domains of the AT protein, such as of the SPATE protein, with said at least one complementary binding moiety of said coupling system. Thus, in one embodiment, there is provided a method as disclosed herein, wherein said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein.

[0094] For the sake of clarity, the skilled person is aware of that the above discussion in relation to the N-terminal location of said at least one complementary binding moiety of said coupling system, such as located N-terminally or located at the N-terminus of the AT protein, is to be interpreted as relating to the location in the AT protein as displayed on the outer membrane (in other words, relating to located N-terminally of or at the N-terminus of the passenger domain, since the signal peptide is not part of the passenger domain). The skilled person is aware that all AT proteins are synthesized with an N-terminal signal peptide that is cleaved off when passing the inner membrane. In the present context, the displayed AT fusion protein does not comprise the signal peptide as it has been cleaved of during the processing thereof.

[0095] In particular, the Hbp comprises at least five side domains located at amino acid positions 53-308 (domain dl), 533-608 (domain d2), 657-697 (domain d3), 735-766 (domain d4) and 898-992 (domain d5) of said Hbp as defined in SEQ ID NO 126:, thus comprising the signal peptide corresponding to amino acid residues 1-52. As displayed on the outer membrane, the five side domains are located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683-714 (domain d4) and 846-940 (domain d5) of Hbp (SEQ ID NO:60) or HbpD (SEQ ID NO:61) (as described in WQ2019081685). The AT fusion protein used in the present method may comprise a complementary moiety of the coupling system at the location of one of the side domains of said passenger domain.

[0096] The Hbp comprising at least one moiety of a coupling system can be obtained by insertion into at least one side domain or by replacing the whole or part of at least one side domain in the passenger domain with a moiety of the coupling system. Thus, in one embodiment, said at least one complementary binding moiety of said coupling system is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein, such as replaces the whole of at least one of the side domains of the AT protein. Each of the side domains dl, d2, d3, d4 or d5 of the Hbp as defined herein, such as wild-type Hbp (SEQ ID NQ:60) or HbpD (SEQ ID NO:61), or other loops projecting from the beta-stem domain may be used for inserting one of the moieties of the coupling system. In one embodiment, said AT fusion protein is selected from the group consisting of AT fusion proteins comprising or consisting of an amino acid sequence selected from SEQ ID NQ:60, SEQ ID NO:61 and any proteins exhibiting at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto and wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683-714 (domain d4) and 846-940 (domain d5), such as wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), amino acid positions 481-556 (domain d2) and amino acid positions 683-714 (domain d4), such as wherein said side domain is located at amino acid positions 1-256 (domain dl). In one embodiment, said AT fusion protein is selected from the group consisting of AT fusion proteins comprising of an amino acid sequence selected from SEQ ID NO:67 and any proteins exhibiting at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, identity thereto.

[0097] Thus, in one embodiment, said at least one complementary binding moiety of said coupling system replaces a part of at least one of the side domains of the AT protein. In another embodiment, said AT comprises at least first and a second complementary binding moiety of a coupling system as defined herein, wherein the first at least one complementary binding moiety is different from the second at least one complementary binding moiety. In this way, a bivalent immunogenic compositions can be achieved, for example as described in appended Example 3, comprising bivalent vesicles, such as bivalent OMVs.

[0098] In one embodiment, said first at least one complementary binding moiety is inserted into or replaces part of or the whole of at least one of the side domains of the AT protein and said second at least one complementary binding moiety is inserted into or replaces part of or the whole of at least one of the side other domains of the AT protein; such as wherein said first at least one complementary binding moiety replaces part of or the whole of at least one of the side domains of the AT protein and said second at least one complementary binding moiety replaces part of or the whole of at least one other of the side domains of the AT protein. In one embodiment, said at least one AT fusion protein comprises of Hbp as defined herein, such as SEQ ID NO:61, and wherein said at least side domain is selected from side domains located at amino acid positions 1-256 (domain dl), 481-556 (domain d2), 605-645 (domain d3), 683-714 (domain d4) and 846-940 (domain d5), such as wherein said side domain is located at amino acid positions 1-256 (domain dl).

[0099] In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 1-255 or 2-255 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain dl. In other words, said at least one complementary binding moiety replaces essentially whole domain dl. In one embodiment, said at least one AT fusion protein comprises of Hbp as defined herein, such as SEQ ID NO:67. In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 482-555 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain d2. In other words, said at least one complementary binding moiety replaces essentially whole domain d2. In one embodiment, said at least one complementary binding moiety of said coupling system replaces amino acid residues 708-712 of SEQ ID NO:61. In other words, said at least one complementary binding moiety replaces part of domain d4.

[0100] In one embodiment, said AT fusion protein comprises two complementary moieties, which may be the same or different. In one embodiment, said AT fusion protein comprises two complementary moieties inserted into or replacing the whole of or part of domain 1 and domain 2 as defined herein. In one embodiment, said AT fusion protein comprises two complementary moieties inserted into or replacing the whole of or part of domain 1 and domain 4 as defined herein.

[0101] The term "linker " or "linker polypeptide" as used herein denotes peptide linkers of natural and / or synthetic origin. Such linkers consist of a linear amino acid chain wherein the 20 naturally occurring amino acids are the monomeric building blocks. The chain has a length of from 1 to 50 amino acids, in one embodiment between 1 and 28 amino acids, in one embodiment between 3 and 25 amino acids. The linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides, such as polypeptides with a hinge-function. The linker has the function to ensure that the individual entities of a fusion polypeptide can perform their biological activity by allowing the entities to fold correctly and to be presented properly. In one embodiment the linker polypeptide is a "synthetic linker polypeptide" that is designated to be rich in glycine, threonine, glutamine, and / or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids, such as GSS, GGGGS (SEQ ID NO:109), QQQQG (SEQ ID NO:110), GSGSS (SEQ ID NO:111), GSGSG (SEQ ID NO:112), or SSSSG (SEQ ID NO:113). This small repetitive unit may be repeated for two to five times to form a multimeric unit. At the amino- and / or carboxyterminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added.

[0102] Other synthetic peptidic linkers are composed of a single amino acid, which is repeated between 10 to 20 times and may comprise at the amino- and / or carboxy-terminal end up to six additional arbitrary, naturally occurring amino acids, such as e.g. serine in the linker GS15G (SEQ ID NO:114). All linker polypeptides can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the linker polypeptides are themselves polypeptides, the entities of the fusion polypeptide are connected to the linker via a peptide bond that is formed between two amino acids. In one embodiment the polypeptide linker has an acid sequence selected from the group comprising GSGSSGSASG (SEQ ID NO:115), GEGTGGSGSG (SEQ ID NO:116), GSGSSGSGTS (SEQ ID NO:117), GSSGSGSGSG (SEQ ID NO:118), (G3S)3(SEQ ID NO:119), (G3S)4(SEQ ID NQ:120), (G3S)5(SEQ ID NO:121), (G3S)6(SEQ ID NO:122), (G4S)3(SEQ ID NO:123), (G4S)4(SEQ ID NO:124), (G4S)5(SEQ ID NO:125), (G5S)2(SEQ ID NO:126), (G5S)3(SEQ ID NO:127), and (G5S)4(SEQ ID NO:128).

[0103] As used herein, the term "spacer" refers to shorter amino acid stretch, comprising one or several amino acid residues, such as 1, 2 or 3 amino acid residues and can be inserted between different parts in fusion polypeptides.

[0104] Suitable linkers are known in the art and are for example described in the review article entitled "Fusion protein linkers: Property, design and functionality." by Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369. In one particular embodiment, said one complementary moiety is flanked by linker or spacers. Without being bound, it is considered that linkers aid in providing optimal exposure of the complementary moiety improving accessibility for binding of the coupling moiety thereto. In one particular embodiment, said one complementary moiety is flanked by linkers or spacers having a length of 3 to 10 amino acid residues. In one embodiment, said linkers or spacers have a length of 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. In one embodiment, said linkers or spacers have a length of 5 amino acid residues. In one embodiment, said linkers or spacers have a length of 10 amino acid residues. In one embodiment, said linkers or spacers are selected from the group consisting of GSS, GGGGS (SEQ ID NO:109), QQQQG (SEQ ID NO:110), GSGSS (SEQ ID NO:111), GSGSG (SEQ ID NO:112), SSSSG (SEQ ID NO:113), GS15G (SEQ ID NO:114), GSGSSGSASG (SEQ ID NO:115), GEGTGGSGSG (SEQ ID NO:116), GSGSSGSGTS (SEQ ID NO:117), GSSGSGSGSG (SEQ ID NO:118), (G3S)3(SEQ ID NO:119), (G3S)4(SEQ ID NQ:120), (G3S)5(SEQ ID NO:121), (G3S)6(SEQ ID NO:122), (G4S)3(SEQ ID NO:123), (G4S)4(SEQ ID NO:124), (G4S)5(SEQ ID NO:125), (G5S)2(SEQ ID NO:126), (G5S)3(SEQ ID NO:127), and (G5S)4(SEQ ID NO:128). In particular, in one embodiment, said linkers or spacers are selected from the group consisting of GSGSS (SEQ ID NO:111), GSGSG (SEQ ID NO:112), GSS, GSGSSGSASG (SEQ ID NO:115), GEGTGGSGSG (SEQ ID NO:116), GSGSSGSGTS (SEQ ID NO:117) and GSSGSGSGSG (SEQ ID NO:118).

[0105] In one particular embodiment, said at least one AT fusion protein is Hbp comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein amino acid positions 1-255 or 2-255 are replaced by said at least one complementary binding moiety of said coupling system and wherein said at least one complementary binding moiety is optionally flanked with one or two linkers or spacers.

[0106] In one embodiment, said AT fusion protein is selected from the group consisting of AT fusion proteins comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:68, 70-76 and 101-108, such as the group consisting of SEQ ID NO:68 and 70, or the group consisting of SEQ ID NO:71 and 72, or the group consisting of SEQ ID NO:73 and 74. In one embodiment, said AT fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:68, 71 and 72. In one embodiment, said AT fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:70, 71 and 72.

[0107] For example, an AT protein comprising SEQ ID NO:67 is a Hbp protein wherein positions corresponding to positions in 1-255, such as in 2-255, of SEQ ID NO:61 have been deleted. In one embodiment, said AT protein comprises or consists of amino acid SEQ ID NO:67. In one embodiment, said AT fusion protein comprises or consists of amino acid SEQ ID NO:68. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 683-714 (domain d4) are replaced, such as partially or completely replaced, by at least one said at least one complementary binding moiety of said coupling system. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein said at least one complementary binding moiety of said coupling system has been inserted into domain d4 as defined herein. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence has been inserted into domain d4 as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NO:75 or SEQ ID NQ:106 or SEQ ID NQ:108.

[0108] In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 481-556 (domain d2) are replaced, such as partially or completely replaced, by at least one said at least one complementary binding moiety of said coupling system. In one embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein said at least one complementary binding moiety of said coupling system has been inserted into domain d2 as defined herein. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into domain d2 as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NQ:101 or SEQ ID NQ:107. In one embodiment, said AT fusion protein comprises a plurality of complementary moieties of said coupling system, such as at least two, such at least three, such as at least four, such as at least five, complementary moieties of said coupling system. Said plurality of complementary moieties may be inserted into or replace, partially or completely, any one of or all of side domains dl, d2, d3, d4 or d5 of Hbp as discussed above. In particular embodiments, wherein said at least two, such as at least three, such as at least four, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the different amino acid sequences.

[0109] In one embodiment, said AT fusion protein comprises a plurality of complementary moieties of said coupling system, such as at least two, such at least three, such as at least five, complementary moieties of said coupling system. It will be understood that the plurality of complementary moieties may be comprised of several copies of the same moiety or of copies of different moieties. In one particular embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-256 (domain dl) and positions 683-714 (domain d4) are partially or completely replaced by at least one complementary binding moiety of said coupling system. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into said domains as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NO:76 or SEQ ID NQ:102 or SEQ ID NQ:105. In one particular embodiment, said AT fusion protein is HbpD comprising or consisting of the amino acid sequence as defined in SEQ ID NO:61, wherein positions 1-256 (domain dl) and positions 481-556 (domain d2) are partially or completely replaced by at least one complementary binding moiety of said coupling system. In one embodiment, said at least one complementary binding moiety of said coupling system and at least one linker sequence, such as two flanking linker sequences, have been inserted into said domains as defined herein. In one embodiment, said AT fusion protein comprises of consists of the amino acid sequence SEQ ID NQ:103 or SEQ ID NQ:104.

[0110] In one particular embodiment, said at least two, such at least three, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the same amino acid sequence. In another embodiment, at least two, such at least three, such as at least five, such as all, of said plurality of complementary moieties of said coupling system comprise the different amino acid sequences.

[0111] In one embodiment, there is provided the immunogenic composition as disclosed herein, wherein said first component, said second component and / or said fusion polypeptide are / is coupled via an isopeptide bond to said vesicle via means of the complementary moiety of a coupling system on said vesicle, such as an isopeptide bond between the coupling moiety of the coupling system on said first component, said second component and / or said fusion polypeptide and the complementary moiety on said vesicle.

[0112] In one embodiment, there is provided an immunogenic composition as described herein, wherein said first component is a polypeptide as defined in a) comprising at least one coupling moiety of a coupling system as defined herein and is coupled to a vesicle as defined herein via means of an isopeptide bond between said coupling moiety and said complementary moiety on said vesicle.

[0113] In one embodiment, there is provided an immunogenic composition as described herein, wherein said second component is a polypeptide as defined in b) comprising at least one coupling moiety of a coupling system as defined herein and is coupled to a vesicle as defined herein, via means of an isopeptide bond between said coupling moiety and said complementary moiety on said vesicle.

[0114] In one particular embodiment, there is provided an immunogenic composition as defined herein, wherein said first component is a polypeptide as defined in a) comprising at least one coupling moiety of a coupling system as defined herein and said second component is a polypeptide as defined in b) comprising at least one coupling moiety of a coupling system as defined herein and wherein said first and second components are coupled to a vesicle via means of an isopeptide bond between their respective coupling moieties and complementary moieties on said vesicle. In one embodiment, said first component and said second component are coupled to the same vesicle. In one embodiment, said first component and said second component are coupled to different vesicles. Said vesicles may be vesicles as defined herein, such as OMVs. In yet another embodiment, there is provided an immunogenic composition as defined herein comprising the fusion polypeptide, which fusion polypeptide comprises a first component which is a polypeptide as defined in a) and a second component which is a polypeptide as defined in b), which further comprises at least one coupling moiety of a coupling system and is coupled to a vesicle as defined herein via means of an isopeptide bond between said at least one coupling moiety and the complementary moiety on said vesicle. Said vesicle may be a vesicle as defined herein, such as an OMV.

[0115] Utilizing a coupling system during polypeptide or protein synthesis to fuse various moieties and parts together to form a final product, such as an immunogenic composition comprising OMVs displaying antigens, can be advantageous when compared to, for example, designing a polypeptide or protein which is synthesized as one contiguous sequence, or as "one unit". This way, utilizing the coupling system has advantages of not only in the synthesis step, but furthermore offers flexibility in terms of which antigens are displayed and how these are displayed on the vesicle, for example in monovalent or bivalent fashion. A coupling system, comprising at least a first and a second molecular part or "moiety" which have affinity for each other and between which a bond may be formed allows for great flexibility when synthesizing the product. It can allow the user to keep synthesized parts of a final complex separate until it is desired that they are combined and bonded to each other. For example, a antigen (herein represented by the first and second components as defined herein) comprising a first molecular part or "moiety" of the coupling system, can be synthesized separately from a vesicle comprising the second molecular part or "moiety" of the coupling system, to be later combined and allowed to interact and bond via the respective coupling moieties and form the final complex or product. Particularly, this enables mass-production of certain parts, for example the vesicles can be prepared in advance and stored until needed and then brought into contact with first and second components comprising corresponding moieties to those displayed on the vesicles of the coupling system. Alternatively, the product may be synthesized and coupled in real-time. In one embodiment of the present disclosure, the present inventors utilize such a coupling system to obtain an immunogenic composition as disclosed herein. In a second related aspect of the present disclosure, there is provided outer membrane vesicles (OMVs) which display one or both of the antigens as disclosed herein according to first component as defined in a) and component as defined in b). Thus, there are provided OMVs, which display one component each and such OMVs can be pooled to prepared a bivalent compositions of monovalent OMVs. Alternately, the OMVs may display both the first component as defined in a) and component as defined in b) and in which case the OMVs themselves a bivalent and also provide for a bivalent composition. Examples of both types as disclosed in the appended Example 3.

[0116] Thus, in one embodiment of this aspect, there is provided an outer membrane vesicle which displays at least one first component according to a) as described herein on its outer surface. In another embodiment of this aspect, there is provided an outer membrane vesicle which displays at least one second component according to b) as described herein on its outer surface. In yet another embodiment, there is provided an outer membrane vesicle which displays at least one first component according to a) and at least one second component according to b) as described herein on its outer surface.

[0117] In this context, the first component is polypeptide as defined in a) and the second component is a polypeptide as defined in b). Thus, said first component may be a polypeptide corresponding to the PnrA protein of 5. pneumoniae or a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae, wherein said PnrA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:1. Said second component may be a polypeptide corresponding to the AliA protein of 5. pneumoniae or a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, wherein said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2 or SEQ ID NO:152. The outer membrane vesicles according to this second aspect provide thus for an outer membrane vesicle displaying antigens and which themselves can be comprised in an immunogenic composition or vaccine composition which can elicit an immune response against 5. pneumoniae in a subject when administered to said subject and thus be useful in treatment and / or prevention of pneumococcal infections. As described herein, it is possible to couple the at least one polypeptide as defined in a) and / or the at least one polypeptide as defined in b) to the OMV via means of a coupling system as described herein. The skilled person will appreciate that the details of said coupling system discussed in the context of the first aspect as disclosed herein are equally relevant for this second aspect and are not repeated here merely for the sake of brevity. Likewise, the features of the OMV as such are extensively discussed herein in the context of the first aspect and the skilled person appreciates that that disclosure is equally relevant for this second aspect and is not repeated herein merely for the sake of brevity.

[0118] Thus, in one embodiment there is provided an OMV as disclosed herein, wherein said OMV vesicle is as defined herein and wherein said at least one polypeptide as defined in a) is coupled to the outer surface of said vesicle via an isopeptide bond formed between a coupling moiety comprised in said at least one polypeptide as defined in a) and a complementary moiety on said vesicle. In one embodiment there is provided an OMV as disclosed herein, wherein said OMV vesicle is as defined herein and wherein said at least one polypeptide as defined in b) is coupled to the outer surface of said vesicle via an isopeptide bond formed between a coupling moiety comprised in said at least one polypeptide as defined in b) and a complementary moiety on said vesicle.

[0119] In this context, the OMV may be as discussed for the first aspect. Additionally, the coupling moiety and complementary moiety may be part of the coupling system as discussed in the context of the first aspect. As also explained in the context of the first aspect, the coupling moiety or complementary moiety of the coupling system which is present on the vesicle, may form part of an autotransporter (AT) fusion protein, which AT fusion protein comprises an autotransporter protein and the complementary moiety.

[0120] In one embodiment, there is provided an outer membrane vesicle as disclosed herein, wherein said at least one vesicle displays on its outer surface an autotransporter (AT) fusion protein and wherein said at least one polypeptide as defined in a) and / or at least one polypeptide as defined in b) is / are coupled to the AT fusion proteins by means of a coupling system as defined herein. In other words, the outer membrane vesicle may display on its outer surface an autotransporter (AT) fusion protein and wherein said at least a polypeptide as defined in a) and / or at least a polypeptide as defined in b) are coupled to the AT fusion protein via an isopeptide bond formed between a coupling moiety comprised in said at least a polypeptide as defined in a) and / or b) and a complementary moiety in said AT fusion protein. It will be appreciated that the disclosure of the AT fusion protein in the context of the first aspect, is also relevant for the outer membrane vesicle of the second aspect and is not repeated here for brevity alone.

[0121] In one particular embodiment, said at least one polypeptide as defined in a) and at least one polypeptide as defined in b) are bound to the same AT fusion protein as defined herein.

[0122] In another embodiment, the outer membrane vesicle displays on its outer surface at least one fusion polypeptide comprising at least one polypeptide as defined in a) and at least one polypeptide as defined in b). In one embodiment, said at least one fusion polypeptide is coupled to the outer surface of the outer membrane vesicle via an isopeptide bond formed between a coupling moiety of comprised in said a fusion polypeptide and a complementary moiety on said vesicle, preferably wherein said coupling moiety and complementary moiety are part of the coupling system as defined herein.

[0123] The present inventors found that the immunogenic composition as disclosed in the first aspect of the disclosure is surprisingly advantageous for a vaccine composition against an 5. pneumoniae infection. Especially, the inventors have found that a vaccine composition comprising the above described immunogenic composition elicits a superior therapeutic effect in comparison to known 5. pneumoniae vaccines in the prior art. As shown in the appended Examples 4 and 5 at least, the immunogenic composition of the present disclosure elicits both cellular and antibody responses, including serum IgG and IgA in the nose, as well as reduces colonization in the nose. In addition, the present inventors expect based on the data presented in appended Example 9 that the immunogenic composition as disclosed herein will be efficient in protecting against infections by a large variety of serotypes of 5. pneumoniae, including serotypes for which no vaccines exist at present. The skilled person is aware of the fact that in order to elicit an immune response in a subject, an agent with adjuvant properties may be provided to said subject together with immunogenic compositions as disclosed herein. Thus, in one embodiment, said immunogenic composition as described herein further comprises an agent with adjuvant effect. In particular, the agent with an adjuvant effect may be present in an immune-effective amount. It will be appreciated that the immunogenic compositions as disclosed herein, may be useful as a medicament, as discussed below in relation to the first aspect of the disclosure.

[0124] In a related third aspect of the present disclosure, there is provided a vaccine composition comprising the immunogenic composition as defined herein or the outer membrane vesicle as defined herein and a pharmaceutically acceptable carrier or excipient. The term "vaccine composition" as used herein relates to a composition comprising the immunogenic composition of the present disclosure which can be used to prevent or treat a pathological condition associated with 5. pneumoniae in a subject. The vaccine composition may be in a liquid form, but may also be in lyophilized form, freeze-dried form, powder form or other suitable forms, which may be reconstituted before administration if suitable. The vaccine composition may or may not include one or more additional components that enhance the immunological activity of the active component or such as buffers, reducing agents, stabilizing agents, chelating agents, bulking agents, osmotic balancing agents (tonicity agents); surfactants, polyols, anti-oxidants; lyoprotectants; anti-foaming agents; preservatives; and colorants, detergents, sodium salts, and / or antimicrobials etc. The vaccine composition may additionally comprise further components typical to pharmaceutical compositions. The vaccine of the present invention is, preferably, for human and / or veterinary use. The vaccine composition may be sterile and / or pyrogen-free. The vaccine composition may be isotonic or hypotonic with respect to humans.

[0125] In one particular embodiment, said vaccine compositions comprises an agent with adjuvating effect. In one embodiment, said vaccine composition further comprises an immune-effective amount of an agent with adjuvant effect. As used herein, the term "immune-effective" refers a sufficient amount of an adjuvant to increase the vaccine's immunogenicity to a level high enough to effectively vaccinate a typical patient. As discussed above, an immunogenic composition as disclosed herein and / or a vaccine composition as disclosed herein may comprise an agent with adjuvant effect in an amount that is immunoeffective. Suitably, said adjuvant stimulates systemic or mucosal immunity. The skilled person is aware of suitable adjuvants. Non-limiting examples of suitable adjuvant in the context of the present disclosure include polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, immunostimulating sequences (ISS), an oil in water emulsion, cation lipids containing a quaternary ammonium salt, cytokines, aluminum hydroxide or aluminum phosphate, saponin or nanoparticles or any combinations or mixtures thereof. As explained in the context of outer membrane vesicles (OMVs), OMVs have adjuvant properties and can thus have an adjuvant effect (such as self-adjuvating effect) in some embodiment of the composition as disclosed herein.

[0126] As discussed above, the term "adjuvant" as used herein refers to a substance that enhances, augments or potentiates the host's immune response (antibody and / or cell-mediated) to an antigen or fragment thereof. Exemplary adjuvants for use in accordance with the present disclosure further include inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, the TLR9 agonist CpG oligodeoxynucleotide, the TLR4 agonist monophosphoryl lipid (MPL), the TLR4 agonist glucopyranosyl lipid (GLA), the water in oil emulsions Montanide ISA 51 and 720, mineral oils, such as paraffin oil, virosomes, bacterial products, such as killed bacteria Bordetella pertussis, Mycobacterium bovis, toxoids, nonbacterial organics, such as squalene, thimerosal, detergents (Quil A), cytokines, such as IL-1, IL-2, IL-10 and IL-12, and complex compositions such as Freund's complete adjuvant, and Freund's incomplete adjuvant. Generally, the adjuvant used in accordance with the present invention preferably potentiates the immune response to the multimeric complex of the invention and / or modulates it towards the desired immune responses.

[0127] The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the immunogenic composition according to the present disclosure. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art. In another embodiment, composition does not comprise any additional agent with adjuvant effect. For example, this may be the case when the vaccine composition comprises an immunogenic composition as defined herein which comprises vesicles, which are self- adjuvating vesicles, such as outer membrane vesicles. This may also be the case when the vaccine composition comprises at least one outer membrane vesicle displaying at least one polypeptide as defined in a) and at least one polypeptide as defined in b) as defined herein.

[0128] The inventors envision that there are several manners suitable for the delivery of the vaccine composition as disclosed herein and the vaccine composition may be formulated according to the chosen alternative. Thus, in one embodiment there is provided the vaccine composition as disclosed herein, wherein vaccine composition is formulated for parenteral administration, mucosal administration or topical administration; such as formulated for parenteral administration or mucosal administration; or wherein vaccine composition is formulated for oral administration, buccal administration, gastrointestinal administration, intrapulmonary administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intravaginal administration, intra rectal administration or topical administration; such as intramuscular administration, intrapulmonary administration or intranasal administration; such as intranasal administration. In one particular embodiment, said vaccine composition is formulated for intranasal administration. It may be beneficial to administer the vaccine composition to the nasal mucosa and thereby achieve the reduction to nasal load of bacteria as demonstrated in the appended examples. In another embodiment, said vaccine composition is formulated for intramuscular administration.

[0129] The ability to induce a humoral and / or cell mediated immune response in vivo can be determined using a mouse or rat model of 5. pneumoniae infection, which mirrors the disease in humans and represents a system to examine pathogenesis and therapeutic efficacy of candidate vaccines. As shown in the Examples disclosed herein, the administration of the immunogenic composition as defined herein or of the vaccine composition as defined herein leads to 5. pneumoniae specific antibody response of serum IgG and mucosal IgA.

[0130] Thus, in one embodiment, the immunogenic composition or the vaccine composition as disclosed herein is capable of eliciting a 5. pneumoniae specific antibody response, such as a serum IgG antibody response and / or a mucosal IgA response, in a subject upon administration thereof. In one embodiment, said 5. pneumoniae specific antibody response is a serum IgG antibody response.

[0131] In one embodiment, said serum IgG response is an AliA specific serum IgG response. In one embodiment, the elicited level of a nti-Ali A IgG antibodies in the serum of said subject is at least 10 times, such as at least 25 times, such as at least 50 times, such as at least 75 times, such as at least 100 times, such as at least 250 times, such as at least 500 times, such as at least 750 times higher upon administration thereof compared to the level in said subject before administration.

[0132] In one embodiment, said serum IgG response is an PnrA specific serum IgG response. In one embodiment, the elicited level of anti-PnrA IgG antibodies in the serum of said subject is at least 2 times, such as at least 4 times, such as at least 5 times, such as at least 8 times, such as approximately 10 times higher upon administration thereof compared to the level in said subject before administration. Alternatively, the elicited levels of anti-PnrA IgG antibodies may be at least 2 times, such as at least 4 times, such as at least 5 times, such as at least 8 times, such as approximately 10 times higher upon administration thereof compared to the level in a control sample.

[0133] In one embodiment, said serum IgG response is measured after at least 43, such as at least 70, such as at least 98, such as at least 126 days after administration of said composition to said subject. In one embodiment, the serum IgG titer is measured on after one, two or three or more administrations of said composition, for example after two or more administrations. As explained in the appended Examples, it is surprising and beneficial that an IgG response is detectable a very long time after administration of the composition as disclosed herein.

[0134] In one embodiment as disclosed herein, said 5. pneumoniae specific antibody response is a mucosal IgA response, such as a nasal IgA response. In one embodiment, said the elicited level of anti-AliA IgA antibodies, such as nasal anti-AliA IgA antibodies, is at least 2 times higher, such as 4 times higher, such as 5 times higher, such as 8 times higher, such as approximately 10 times higher in a subject after administration of said composition, compared to a control subject or a control sample. In one embodiment, said control subject is a subject who has not been administered said composition. In one embodiment, the nasal anti-AliA IgA antibodies are measured in said subject at least 15 weeks post administration, such as wherein increased levels of said nasal anti-AliA IgA antibodies can be detected in a subject at least 15 weeks post administration compared to the level in said subject before administration. In one embodiment said IgA antibodies are measured on after one, two or three or more administrations of said composition, such as after two or more administrations.

[0135] As indicated above, in one embodiment, the immunogenic composition as disclosed herein elicits a 5. pneumoniae specific antibody response in the subject who has been administered the immunogenic composition. 5. pneumoniae specific antibody response may be a 5. pneumoniae specific serum IgG response and / or IgA response. In one embodiment, said serum IgG response is measured in comparison to serum IgG levels detectable in a control sample, for example a control serum sample. In one embodiment, said serum IgA response is measured in comparison to serum IgG levels detectable in a control sample, for example a control mucosal sample. The term "control serum sample" and "control mucosal sample", as used herein, refer to a serum sample or mucosal sample which may be obtained from a subject who has not been administered any immunogenic composition and / or vaccine composition against 5. pneumoniae, nor has been challenged or infected with 5. pneumoniae. Such subject may thus be a naive control. Said control serum sample or mucosal sample may also be obtained from a subject who has been vaccinated against 5. pneumoniae, using a vaccine that does not comprise the first component and / or the second component according to the present disclosure. It is appreciated by those skilled in the art, that these subjects are considered naive with respect to the immunogenic composition according to the present disclosure. In one embodiment, said control serum or mucosal sample is obtained from a naive control. In certain embodiments, said control serum or mucosal sample is obtained from a subject prior to the administration of the immunogenic composition as disclosed herein to the subject. In the case of a serum sample, such control sample is also known as a "pre-bleed sample", which may be used as an internal control for evaluating the effect of administering an immunogenic composition to a subject, and is well known in the art.

[0136] As shown appended Examples 1 and 2, the immunogenic composition as disclosed herein, or a vaccine composition comprising said immunogenic composition, is able to significantly reduce the bacterial load of 5. pneumoniae in the nose of a subject thereof post-challenge. Importantly, said effect is also observed long time after the administration of the compositions, which demonstrates the longevity of the immune response elicited.

[0137] Thus, in one embodiment there is provided the immunogenic composition or the vaccine composition as disclosed herein, which immunogenic composition or vaccine composition upon administration is capable of reducing the bacterial load of 5. pneumoniae in the nose of a subject. In one embodiment there is provided the immunogenic composition or the vaccine composition as disclosed herein, which immunogenic composition or vaccine composition upon administration is capable of reducing the bacterial load of 5. pneumoniae in the nose of a subject compared to the bacterial load of 5. pneumoniae in the nose of unvaccinated control subjects or compared to the bacterial load of 5. pneumoniae in the nose of the same subject prior to administration. For example, in one embodiment there is provided the immunogenic composition or the vaccine composition as disclosed herein, which immunogenic composition or vaccine composition upon administration is capable of reducing the bacterial load of 5. pneumoniae in the nose of a subject post challenge compared to the bacterial load of 5. pneumoniae in the nose of unvaccinated control subjects post challenge as shown in the appended Examples. In one embodiment, the reduction of the bacterial load of 5. pneumoniae in the nose is measured 3 days post challenge. In other words, an effect which is considered to be clinically relevant is attained at least 3 days post challenge, but may be attained earlier. Thus, in a clinical setting, a preexisting colonization is expected to be reduced by the administration of said composition and / or new colonization is expected to be reduced and / or prevented. It is expected that an effect will be observed in a clinical setting which is in the same range as observed in the appended Examples. Thus in one embodiment, the reduction of the bacterial load of 5. pneumoniae in the nose is at least a 0.75 log reduction, such as at least a 1.0 log reduction, such as at least 1.25 log reduction, such as at least 1.5 log reduction, such as at least 2.0 log reduction, in the number of colony forming units (CFU) in the nose compared to compared to the bacterial load of 5. pneumoniae in the nose of unvaccinated control subjects. Importantly, this effect detectable at least 3 weeks after the administration of the composition and also 15 weeks after the administration thereof as shown in Example 4. Thus, in one embodiment, the reduction of the bacterial load of 5. pneumoniae in the nose is detectable at least 3 weeks after the administration of the composition, for example after a first, second or third administration of the composition. In one embodiment, there is provided the immunogenic composition or the vaccine composition as disclosed herein, wherein the reduction of the bacterial load of 5. pneumoniae in the nose is detectable at least 15 weeks after the administration of the composition, for example after a first, second or third administration of the composition.

[0138] As discussed above, the present disclosure relates to an immunogenic composition and related vaccine composition as disclosed herein, which the present inventors expect to be useful for the treatment and / or prevention of infections by many serotypes of 5. pneumoniae, including serotypes for which no vaccine product exists today. The present inventors expect that the compositions of the present disclosure will be useful for the treatment and / or prevention of infections by more serotypes of 5. pneumoniae than are covered by the prior art vaccines individually as well as collectively. Thus, in one embodiment there is provided an immunogenic composition or vaccine composition as disclosed herein, which is capable of eliciting antibodies which are capable of binding to, in other words have affinity for, PnrA and / or antibodies which are capable of binding to, in other words have affinity for, Ali A; from at least 24 serotypes, such as at least 25, such as at least 26, such as at least 27 serotype, such as at least 28, such as at least 29, such as at least 30, such as at least 31, such as at least 32, such as at least 33, such as at least 34, such as at least 35, such as at least 36, such as at least 37, such as at least 38, such as at least 39, such as at least 40, such as at least 41, such as at least 42, such as at least 43 serotypes or more serotypes of 5. pneumoniae. In one embodiment, said immunogenic composition or vaccine composition is capable of eliciting antibodies which are capable of binding to PnrA and antibodies which are capable of binding to Ali A.

[0139] In one embodiment, said immunogenic composition or vaccine composition is capable of eliciting antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA, such as capable of eliciting antibodies which are capable of binding to PnrA and antibodies which are capable of binding to AliA; from at least one serotype of 5. pneumoniae selected from the group consisting of serotype 12A / F, 35B, 9N, 7F, 15A, 19F, 14, 5, 19A, 22A / F, 9V, 6C, 23F, 33A / F, 7C, 10, 2, 1, 8, 15B / C, 11A / D, 6B, 23A, 23B, 4, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F; -such as selected from group consisting of serotype 35B, 9N, 7F, 15A, 19F, 14, 5, 22A / F, 6C, 23F, 33A / F, 7C, 10, 2, 1, 8, 15B / C, 11A / D, 6B, 23A, 23B, 4, 18A, 24B, 25A, 34, 35F, 38, 40 and 42, such as selected from group consisting of serotype 35B, 9N, 7F, 15A, 19F, 14, 5, 22A / F, 6C, 23F, 33A / F, 7C, 10, 2, 1, 8, 15B / C, 11A / D, 6B, 23A, 23B, 4, 24B, 25A, 34, 35F, 38, 40 and 42, such as selected from group consisting of serotype 35B, 9N, 7F, 15A, 19F, 14, 5, 22A / F, 6C, 23F, 33A / F, 7C, 10, 2, 1, 8, 15B / C, 11A / D, 6B, 23A, 4, 24B, 25A, 34, 35F, 38, 40 and 42.. As explained in the context of the eighth aspect, the present inventors expect that the immunogenic composition or vaccine composition as defined herein is capable of eliciting antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA also of other serotypes in addition to those exemplified in Example 9.

[0140] As discussed above, said immunogenic composition or vaccine composition is capable of eliciting antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA from serotype(s) of 5. pneumoniae for which no vaccine drug product exists per today.

[0141] Thus in one embodiment, said immunogenic composition or vaccine composition is capable of eliciting antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA, such as capable of eliciting antibodies which are capable of binding to PnrA and antibodies which are capable of binding to AliA; from at least one serotype of 5. pneumoniae selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F, such as selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 24B, 25A, 34, 35F, 38, 40 and 42, such as selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 24B, 25A, 34, 35F, 38, 40 and 42.

[0142] In one particular embodiment, said immunogenic composition or vaccine composition is capable of eliciting antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA, such as capable of eliciting antibodies which are capable of binding to PnrA and antibodies which are capable of binding to AliA; from at least one serotype of 5. pneumoniae selected from the group consisting of 35B, 15A, 23A, 23B, 16F and 34, such as the group consisting of 35B, 15A, 23A, 23B and 34, such as the group consisting of 35B, 15A, 23A and 34.

[0143] As discussed above, and also shown in appended Example 4, the immunogenic composition and / or vaccine composition as disclosed herein is also able to elicit a 5. pneumoniae specific cellular response upon administration to a subject. Thus, in one embodiment, there is provided an immunogenic composition or a vaccine composition as disclosed herein, wherein immunogenic composition or the vaccine composition induces T cell based immunity comprising an AliA specific T cell response upon restimulation of said subject with AliA or whole 5. pneumoniae. In one embodiment, said AliA-specific T cell response comprises inducing an increase in IFN-y and / or IL17A secretion by AliA-specific T cells.

[0144] In one embodiment, there is provided an immunogenic composition or a vaccine composition as disclosed herein, wherein the immunogenic composition or the vaccine composition induces T-cell based immunity comprising a PnrA specific T cell response upon restimulation of said subject with PnrA or whole 5. pneumoniae. In one embodiment, said PnrA-specific T cell response comprises inducing an increase in the number of IFN-y and / or IL17A secreting PnrA-specific T cells.

[0145] It will be appreciated that said increase may be assessed in an ex vivo experimental model, for example measured in a sample obtained by biopsy from the subject, such as wherein said subject is a human subject. The inventors envision that the degree of said increase is a clinically significant increase in comparison to any relevant control which has not been administered the immunogenic composition as disclosed herein. A sample obtained from a control is termed the "control sample". In a fourth aspect the present disclosure also relates to a method for the production of an antiserum, said method comprising administering an immunogenic composition or the vaccine composition as described herein to mammalian host to produce antibodies in said host and recovering antiserum containing the antibodies produced in said animal host. Within the scope of the present disclosure is also an antiserum obtainable by said method. Thus, in a fifth aspect there is provided an antiserum obtainable by the method of aspect four. Said antiserum comprises antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to Ali A, such as capable of eliciting antibodies which are capable of binding to PnrA and antibodies which are capable of binding to Ali A From at least 24 serotypes, such as at least 25, such as at least 26, such as at least 27 serotype, such as at least 28, such as at least 29, such as at least 30, such as at least 31, such as at least 32, such as at least 33, such as at least 34, such as at least 35, such as at least 36, such as at least 37, such as at least 38, such as at least 39, such as at least 40, such as at least 41, such as at least 42, such as at least 43 or more serotypes of 5. pneumoniae.

[0146] Said antiserum comprises antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA from at least one serotype of 5. pneumoniae selected from the group consisting of serotype 12A / F, 35B, 9N, 7F, 15A, 19F, 14, 5, 19A, 22A / F, 9V, 6C, 23F, 33A / F, 7C, 10, 2, 1, 8, 15B / C, 35B, 3, 11A / D, 6B, 23A, 23B, 4, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F, or subsets thereof as disclosed in the context of the third aspect as disclosed herein.

[0147] In addition, said antiserum comprises antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA; from at least one serotype of 5. pneumoniae selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F; or subsets thereof as disclosed in the context of the third aspect as disclosed herein.

[0148] The skilled person appreciates that antibodies may for example be isolated from the antiserum and an antibody preparation can be obtained and used in the treatment of 5. pneumoniae infection by passive immunization. Thus, in a related sixth aspect there is provided an antibody preparation comprising antibodies which are capable of binding to PnrA and / or antibodies which are capable of binding to AliA, such as antibodies capable of binding to PnrA and antibodies which are capable of binding to AliA, for use in the treatment of a 5. pneumoniae infection, such as for use in the preventive treatment of a 5. pneumoniae infection. In one embodiment, said antibody preparation comprises antibodies capable of binding to PnrA and antibodies which are capable of binding to AliA from at least 24 serotypes, such as at least 25 serotypes, such as at least 26, such as at least 27 serotype, such as at least 28, such as at least 29, such as at least 30, such as at least 31, such as at least 32, such as at least 33, such as at least 34, such as at least 35, such as at least 36 serotypes, such as at least 37 serotypes of 5. pneumoniae.

[0149] In a related seventh aspect there is provided a method for prophylactic treatment of a 5. pneumoniae infection in a mammalian subject, comprising passive immunization by administering to said mammalian subject in need thereof an antibody preparation as defined herein.

[0150] As demonstrated in the appended Examples, the present inventors have found that the immunogenic composition and / or the vaccine composition as disclosed herein is advantageous as a medicament. Thus, in an eight aspect of the present disclosure, there is provided the immunogenic composition and / or the vaccine composition according to the present disclosure including the embodiments discussed above in relation to the first, second and third aspect of the disclosure for use as a medicament.

[0151] Moreover, as demonstrated in the appended Examples, the present inventors have found that the immunogenic composition and / or the vaccine composition as disclosed herein is particularly advantageous in the therapeutic treatment and / or prophylactic treatment of a 5. pneumoniae in a subject in need thereof. Accordingly, in a ninth aspect of the present disclosure, there is provided the immunogenic composition and / or the vaccine composition according to the present disclosure for use in the treatment of a 5. pneumoniae infection. Said use comprises administration of the immunogenic composition or the vaccine composition to a subject, such as a subject in need thereof. The subject in need thereof, may be a subject susceptible to a 5. pneumoniae infection or a subject already infected with 5. pneumoniae. Thus, said use may be in the treatment of a 5. pneumoniae infection, such as for use in the preventive treatment of a 5. pneumoniae infection. In one embodiment said use is in therapeutic treatment of a 5. pneumoniae infection. In one embodiment said use is in preventive (also referred to as prophylactic) treatment a 5. pneumoniae infection. Based on Example 9, the present inventors expect that the immunogenic composition and / or the vaccine composition according to the present disclosure may be useful for the protection against 5. pneumoniae infection caused by various serotypes. Non-limiting examples of such serotypes include the ones recited in Table 1. Importantly, the present inventors also expect that the immunogenic composition according to the present disclosure is able to elicit a protective immune response against a large variety of pneumococcal serotypes not explicitly mentioned in Example 9. Non-limiting examples of such serotypes are 13, 20, 31, 39 10A, 10B, 10F, 11A, 11D, 11E, 12A 12F, 15B, 15C, 17F, 18C, 20B, 22A, 22F, 24A, 24F, 28F, 33A, 33B, 33C, 33F, 35A, 35C, 6A, 6D, 6E, 7B, 7F, 9L.

[0152] In one particular embodiment, said 5. pneumoniae infection is selected from an infection by at least one 5. pneumoniae serotype selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F; such as selected from group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 19B, 24B, 25A, 28A, 29, 34, 35F, 36, 38, 40 and 42-, such as selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 24B, 25A, 34, 35F, 38 40 and 42. In one specific embodiment, said 5. pneumoniae infection is selected from an infection by at least one 5. pneumoniae serotype selected from the group consisting of serotype 35B, 15A, 23A, 23B, 16F and 34, such as selected from the group consisting of serotype 35B, 15A, 23A, 16F and 34, or selected from the group consisting of 35B, 15A, 23A, 23B and 34. In some embodiments, said treatment is preventive treatment of infection by additional serotypes, in addition to prevalent serotypes for which prior art vaccines exist per today. In one embodiment, said use may be in the treatment is preventive treatment of infection by 5. pneumoniae serotype 19A, 19F, 14 and 1 and of at least one serotype selected from the group consisting of group consisting of serotype 35B, 15A, 6C, 7C, 9A, 18A, 19B, 21, 24B, 25A, 28A, 29, 35F, 36, 37, 38, 40, 42 and 47F; such as selected from group consisting of serotype 6C, 7C, 9A, 18A, 19B, 21, 24B, 25A, 28A, 29, 35F, 36, 37, 38, 40, 42 and 47F; such as selected from group consisting of serotype 6C, 7C, 9A, 18A, 19B, 24B, 25A, 28A, 29, 35F, 36, 38, 40 and 42, such as selected from the group consisting of serotype 6C, 7C, 18A, 24B, 25A, 35F, 38, 40 and 42. In one embodiment, said use may be in the preventive treatment of infection by 5. pneumoniae serotype 8, 10A, 11 a, 12F, 15B, 22F and 33F and of at least one serotype selected from the group consisting of group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F, such as selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 24B, 25A, 34, 35F, 38, 40 and 42, such as selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 24B, 25A, 34, 35F, 38, 40 and 42. It is known in the art that the prevalence of serotypes varies between different geographical regions as well as populations. For example, in Europe, serotypes 8, 10A, 11A, 12F, 15B, 22F and 33F are common. Thus, in one particular embodiment, said use is in the preventive treatment of infection by 5. pneumoniae serotype 8, 10A, 11A, 12F, 15B, 22F and 33F and of at least one serotype selected from the group consisting of serotype 35B, 15A, 23A, 23B, 16F and 34, such as selected from the group consisting of 35B, 15A, 23A, 23B and 34. In one embodiment, said treatment is preventive treatment of infection by 5. pneumoniae serotype 19A, 19F, 14 and 1 and of at least one serotype selected from the group consisting of serotype 35B, 15A, 23A, 23B, 16F and 34. In pediatric populations, the serotypes 6A, 6B, 9V, 14, 19A, 19F and 23F have been reported to prevail. Thus, in one particular embodiment, said treatment is preventive treatment of infection by 5. pneumoniae serotype 6B, 9V, 14, 19A, 19F and 23F and at least one serotype selected from the group consisting of group consisting of serotype 35B, 15A, 23A, 23B, 16F and 34.

[0153] In one embodiment, said treatment is preventive treatment of infection by at least 24 serotypes, such as at least 25, such as at least 26, such as at least 27 serotype, such as at least 28, such as at least 29, such as at least 30, such as at least 31, such as at least 32, such as at least 33, such as at least 34, such as at least 35, such as at least 36, such as at least 37, such as at least 38, such as at least 39, such as at least 40, such as at least 41, such as at least 42, such as at least 43 or more serotypes of 5. pneumoniae. The exact dose of the immunogenic composition and / or vaccine composition which is administered to the subject may depend on the purpose of the treatment (e.g. treatment of acute disease vs. prophylactic vaccination), route of administration, age, body weight, general health, gender, diet, time of administration, drug interaction and the severity of the condition, and will be ascertainable with routine experimentation by those skilled in the art. In particular, said administration is administration of a therapeutically effective amount of said immunogenic composition or vaccine composition. In one embodiment, said effective amount of said immunogenic composition or said vaccine composition is an amount which elicits an immune response resulting in therapeutic treatment and / or prophylactic treatment of 5. pneumoniae, for example resulting in ameliorating and / or eliminating symptoms associated with said 5. pneumoniae infection or preventing symptoms associated with said 5. pneumoniae infection. It is envisioned that the above mentioned therapeutically effective dose is in the range of about 10 ng to about 1000 pg, or in the range of about 50 pg to about 1000 pg, total amount of the polypeptide as defined in a) and of the polypeptide as defined in b) each per administration. In one embodiment, said use comprises administration of the immunogenic composition or the vaccine composition to said subject in a dose corresponding to a total amount of at least approximately 10 ng, such as at least 50 pg, and at most approximately 1000 pg of the polypeptide corresponding to the PnrA protein and a total amount of at least approximately 10 ng, such as at least 50 pg, and at most approximately 1000 pg of the polypeptide corresponding to the Al iA protein.

[0154] In one embodiment, said use comprises administration of the immunogenic composition or the vaccine composition to said subject in a dose corresponding to a total amount of at least approximately 50 ng and at most approximately 1000 pg of the polypeptide corresponding to the PnrA protein and a total amount of at least approximately 100 ng and at most approximately 1000 pg of the polypeptide corresponding to the Al iA protein. In another embodiment, said use comprises administration of the immunogenic composition or the vaccine composition to said subject in a dose corresponding to a total amount of at least approximately 180 ng and at most approximately 1000 pg of the polypeptide corresponding to the PnrA protein and a total amount of at least approximately 300 ng and at most approximately 1000 pg of the polypeptide corresponding to the Al iA protein. The immunogenic composition and / or the vaccine composition of the present disclosure may be administered to the subject one or more times, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Accordingly, in one embodiment, said use comprises multiple administration of the immunogenic composition or the vaccine composition to said subject.

[0155] The vaccine composition of the disclosure may be used in a prime-boost regimen. In the prime-boost regimen, a prime / boost vaccine is used which is composed of two or more types of vaccines including a vaccine used in primary immunization (prime or priming) and a vaccine used in booster immunization (boost or boosting). The vaccine used in primary immunization and the vaccine used in booster immunization may differ from each other or may be the same. Primary immunization and boosting immunization may be performed sequentially, this is, however, not mandatory. In one embodiment, said multiple administration regimen comprises a prime administration and at least one boost administration, such as wherein said multiple administration regimen is a two-dose primeboost regimen comprising said prime administration and one boost administration, such as wherein said multiple administration regimen is a three-dose prime-boost regimen comprising said prime administration and two boost administrations or such as wherein said multiple administration regimen is a dose prime-boost regimen comprising said prime administration and at least three boost administrations.

[0156] In one embodiment, the time interval between said prime vaccination and a first boost administration is less than about 18 months, such as less than 12 months, such as less than 10 months, such as less than 8 months, such as less than 6 months, such as less than 3 months, such as less than 2 months; or such as a time interval between about 2 months and about 12 months, such as between about 4 months and about 10 months, between about 6 months and about 8 months. The term "1 month" is to be interpreted as approximately a calendar month, thus such as 25-35 days, for example 28-31 days. In one embodiment, the time interval between the first boost administration and the second boost administration, is between about 1 month and about 10 years, such as between about 1 month and about 5 years, such as between about 2 months and about 5 years, such as between about 3 months and about 5 years, such as between about 6 months and about 4 years, such as between about 12 months and about 3 years, such as between about 12 months and about 18 months; or wherein the time interval between the first boost administration and the second boost administration, is between about 1 month and about 16 months, such as between about 1 months and about 12 months, such as between about 1 months and about 8 months, such as between about 2 months and about 12 months, such as between about 2 months and about 6 months, such as between about 2 months and about 3 months. In a particular embodiment, said prime-boost regimen comprises, within a year from the administration of the prime dose, the administration of the first boost dose which is followed by subsequent administration repeated on an annual, biannual or triannual basis of boost doses to said subject.

[0157] In one embodiment, the time interval between administration subsequent to said second boost administration is at least as long as said time interval between the first boost administration and the second boost administration or longer. In one embodiment, a prime dose and optionally a first boost dose are administered year 1, a second boost dose is administered year 2, and subsequent doses are administered annually 1, 2 or 3 years after administration of the second boost dose. For example, said boost doses can be administered year 2, 3, 4, 5 etc.; or year 2, 4, 6, 8 etc.; or year 3, 6, 9, 12 etc.; or year 2, 3, 5, 8 etc.; or according to any other regimen. In one embodiment, the time interval between said prime vaccination and said first boost vaccination is less than about 10 weeks, such as less than about 9 weeks, such as less than about 8 weeks, such as a time interval between about 2 weeks and about 8 weeks. In one embodiment the time interval between said prime vaccination and said first boost vaccination is about 14 to about 42 days, such as is about 28 days.

[0158] As discussed in the context of the first and third aspect as disclosed herein, several different routes of administration are contemplated. Thus, in one embodiment, said use comprises parenteral administration, mucosal administration or topical administration; such as parenteral administration or mucosal administration. In one embodiment, said uses comprises oral administration, buccal administration, gastrointestinal administration, intrapulmonary administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intravaginal administration, intra rectal administration or topical administration; such as intramuscular administration, intrapulmonary administration or intranasal administration; such as intranasal administration. In one particular embodiment, said use comprises intranasal administration. The present inventors envision that in addition to preventing future infections, the immunogenic composition or the vaccine composition as disclosed herein, may be useful in the treatment of ongoing infections. In this context, without being bound by theory, it is envisioned that administration is to a subject that already suffers from pneumococcal infection and results in lowering of pneumococcal load.

[0159] Thus, in one embodiment, said treatment is curative treatment administered to a patient suffering from a 5. pneumoniae infection, such as curative treatment of an infection by a serotype selected from the group of serotypes consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and serotype 47F, or from a subset thereof as defined above.

[0160] In a related tenth aspect there is provided a method of treatment and / or prevention of a 5. pneumoniae infection, comprising administration therapeutically effective dose of an immunogenic composition as defined herein or of a vaccine composition as defined herein to as subject in need thereof. The skilled person appreciated that the embodiments and disclosures in relation to the ninth aspects of the present disclosure are equally relevant for this tenth aspect and are not repeated here merely for the sake of brevity.

[0161] It is to be understood that embodiments discussed above in relation to the ninth and tenth aspects of the disclosure relating to the immunogenic composition as disclosed herein or the vaccine composition as disclosed herein for use in the therapeutic treatment and / or prophylactic treatment of an 5. pneumoniae infection and to the method of treatment and / or prevention of an 5. pneumoniae infection are equally relevant for eleventh aspect of the disclosure. For the sake of brevity, these will not be repeated herein. Thus, in the eleventh aspect of the present disclosure, there is provided a use of the immunogenic composition as disclosed herein or the vaccine composition as disclosed herein in the manufacture of a medicament for the therapeutic treatment and / or prophylactic treatment of a 5. pneumoniae infection in a subject susceptible thereto.

[0162] In a related aspect of the present disclosure, there is also provided a method of displaying a first and / or and second component on an outer membrane vesicle, comprising the steps of -providing an immunogenic composition as defined herein, wherein said first component is a polypeptide corresponding to a PnrA protein of 5. pneumoniae or an immunogenic fragment thereof as defined in a) and said second component is a polypeptide corresponding to an AliA protein of 5. pneumoniae or an immunogenic fragment thereof as defined in b), and wherein each of said first and second component comprises at least one coupling moiety of a coupling system;

[0163] -providing at least one outer membrane vesicle as defined herein comprising at least one complementary moiety of said coupling system; and

[0164] -contacting said immunogenic composition with said at least one outer membrane vesicle under condition permissive of formation of an isopeptide bond between said at least one coupling moiety and said at least one complementary moiety and thereby obtaining at least one outer membrane vesicle displaying at least one of said first and / or and second component, preferably wherein said coupling moiety and complementary moiety are part of the coupling system as defined herein.

[0165] In a thirteenth aspect there is also provided a nose spray comprising the immunogenic composition as defined herein or the vaccine composition as defined herein. The composition of said nose spray may comprise suitable excipients and / or carriers as defined for the vaccine composition as disclosed herein. The nose spray may comprise a container which holds the composition and an administering means, such as a pumping or spraying means, and container may be adapted for single use or repeated use, such as two uses for example corresponding one administration in each nostril.

[0166] Encompassed by the present disclosure are also kits of parts as detailed in the fourteenth, fifteenth and sixteenth aspects as disclosed herein. Thus, the fourteenth aspect there is provided a kit of parts, comprising: - at least one outer membrane vesicle displaying on its outer surface at least one polypeptide as defined in a);

[0167] - at least one outer membrane vesicle displaying on its outer surface at least one polypeptide as defined in b); and instructions for use.

[0168] Thus, the kit comprises two different outer membrane vesicles, which may be present in two separate compositions in the kit. Alternatively, as provided by the fifteenth aspect as disclosed herein, the kit may comprise:

[0169] - an immunogenic composition comprising a polypeptide as defined in a) and a polypeptide as defined in b), each further comprising to a coupling moiety of a coupling system;

[0170] - at least one outer membrane vesicle which displays on the surface at least one complementary coupling moiety of the coupling system; and

[0171] - instructions for use. It is to be understood that the polypeptide as defined in a) and the polypeptide as defined in b) may comprise the same or different coupling moieties of the same or of different coupling systems as defined herein. Consequently, the at least one outer membrane vesicle may display one or several types of complementary coupling moieties, which together with said coupling moieties may be coupled via means of isopeptide bonds. In this way, the immunogenic composition may be provided separately from the outer membrane vesicles (or compositions comprising said outer membraned vesicles). These compositions may be combined to obtain outer membrane vesicles which display the polypeptides as defined in a) and b) (in other words, which display the antigens PnrA or immunogenic fragments thereof and Ali A or immunogenic fragments thereof).

[0172] It is also envisioned that instead of the immunogenic composition, the kit may comprise two compositions comprising the bespoke antigens, namely the first composition comprising a polypeptide as defined in a) further comprising to a coupling moiety of a coupling system; the second compositions comprising a polypeptide as defined in b) further comprising to a coupling moiety of a coupling system; and at least one outer membrane vesicle which displays on the surface at least one complementary coupling moiety of the coupling system; and instructions for use. It is possible that the outer membrane vesicle is tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in a); to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in b); or to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in a) and b). Thus, the at least one outer membrane vesicle of the kit, may be provided in a first composition comprising at least one outer membrane vesicle tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in a) and a second composition comprising at least one outer membrane vesicle tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in b); alternatively in one composition comprising at least one outer membrane vesicle tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in a) and at least one outer membrane vesicle tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in b); or alternatively in one composition comprising at least one outer membrane vesicle tailored to comprise the complementary coupling moiety which is capable of forming an isopeptide bond with the coupling moiety of polypeptide as defined in a) and b).

[0173] Provided in the sixteenth aspect of the present disclosure is a kit of parts, comprising

[0174] - a first component as defined in a) which component is a nucleic acid further comprising a nucleic acid sequence encoding least one coupling moiety of said coupling system, wherein said coupling system is as defined herein;

[0175] - a second component as defined in b) which component is a nucleic acid further comprising a nucleic acid sequence further comprising a nucleic acid sequence encoding least one coupling moiety of said coupling system, wherein said coupling system is as defined herein;

[0176] - at least one outer membrane vesicle as defined herein; and

[0177] - instructions for use.

[0178] In a related aspect there is provided a kit of parts, comprising - an immunogenic composition comprising a first and second component as defined herein wherein said first and second component are polypeptides comprising coupling moieties of a coupling system as defined herein;

[0179] - at least one outer membrane vesicle as defined herein which displays on the surface at least one complementary coupling moiety of the coupling system as defined herein; and

[0180] - instructions for use.

[0181] It will be appreciated that two separate compositions (an immunogenic composition and a second compositions) may be provided in said kit, such that the immunogenic composition comprises as first component a polypeptide corresponding to the PnrA protein of 5. pneumoniae or a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae and as a second component a polypeptide corresponding to the AliA protein of 5. pneumoniae or a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, wherein each of said first and second component comprises coupling moieties of a coupling system as defined herein, which may the same or different; and the second composition comprises at least one outer membrane vesicle which displays on the surface at least one complementary coupling moiety of the coupling system; and instructions for use. The second composition may thus comprise least one OMV which displays a complementary coupling moiety to the coupling moiety of the first component and a complementary coupling moiety to the coupling moiety of the second component, which may be the different complementary coupling moieties. Alternatively, the second compositions may comprise least one OMV displaying complementary coupling moieties of the coupling moiety of the first and second component, which may be the same complementary coupling moieties.

[0182] In another aspect of the present disclosure, there is provided a polynucleotide encoding a polypeptide in fusion with at least one coupling moiety of a coupling system as defined herein, wherein said polypeptide is selected from the group consisting of: i) a polypeptide corresponding to the PnrA protein of 5. pneumoniae; ii) a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; iii) a polypeptide corresponding to the AliA protein of 5. pneumoniae; iv) a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, and v) a fusion polypeptide comprising at least one polypeptide as defined in i) or ii) in fusion with at least one of polypeptide as defined in iii) or iv) wherein said PnrA protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and said AliA protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:2 or to SEQ ID NO:152.

[0183] Also encompassed is an expression vector comprising said polynucleotide as well as a host cell comprising said expression vector.

[0184] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an expression cassette" includes one or more of the expression cassettes disclosed herein and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0185] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[0186] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes also the concrete number, e.g., about 20 includes 20.

[0187] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well- known in the art. Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0188] The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art.

[0189] While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims.

[0190] List of references Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402

[0191] Bogaert et al., Lancet. 2004 Jun 5; 363 (9424):1871-2.

[0192] Chen et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369

[0193] Dua et al. Int J Pharm. 1997 147(2): 233-242.

[0194] Hashemzadeh-Bonehi et al. Mol Microbiol. (1998) 30(3):676-8

[0195] Kuipers et al., Infect Immun. 2017 Sep 20; 85(10).

[0196] Nishibe et al., 2002. jibi inkoka tembo 45((Supplement 1)): 46-49.

[0197] Ngocho et al., J Infect. 2020 Aug ; 81 (2) :213-220.

[0198] Presnyak et al., Cell, 160(6):llll-24 (2015)

[0199] Prbschel et al., PLoS One. 2017.12(6),

[0200] Tan et al., PLoS One. 2016. 11(10).

[0201] Van beek et al., Virulence. 2020 Dec;ll(l):1310-1328.

[0202] Veggiani et al., Proc Natl Acad Sci USA 2016, 113(5):1202-7.

[0203] Voss et al., Front Immunol. 2018 Oct 18; 9:2405.

[0204] Yihfen et al., Trends in Microbiol. (2008) 16(8): 370-9.

[0205] Zakeri & Howarth 2010 J Am Chem Soc 132(13).

[0206] Zakeri et al., Proc Natl Acad Sci USA. 2012. 109(12).

[0207] Brief Description of the Figures

[0208] Figure 1: Coomassie stained SDS-PAGE gels (Figure 1A and Figure IB) demonstrating successful expression of HbpD-SpC in an OMV preparation (in Figure 1A, lane 1: ladder and lane 2: OMV preparation; in Figure IB, lanes 1, 8, and 13: ladder and lanes 2 and 9: OMV preparation). Successful expression of Spy-tagged antigens and conjugation of said Spy- tagged antigens to HbpD-SpC displayed on OMVs as described in Example 1 is shown in Figure 1A lanes 3-7 and in Figure IB lanes 3-7, 10-12 and 14. Adducts comprising the respective Spy-tagged antigen coupled to HbpD-SpC (HbpD-SpC-SpT-antigen fusions) displayed on OMVs are indicated with black arrowheads in respective lanes, and nonconjugated HbpD-SpC displayed on OMVs is indicated with white arrowheads in respective lanes. Possible degradation products are indicated with open arrowheads and asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane. Molecular mass (kDa) weight markers are indicated left of the first molecular mass ladders in each of Figure 1A and IB. Scatter plots depicting the formation of 5. pneumoniae colony forming units (CFU) in nasal tissue as described in Example 1 (Figure 1C and Figure ID). The Y-axis depicts measured Logio CFU in nasal tissue, and the X-axis indicates various cohorts comprising either a non-vaccinated control group or groups vaccinated with solutions comprising OMV only, or groups vaccinated with immunogenic compositions comprising OMV conjugated to one of the respective tested candidate antigens. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Figure 2: Coomassie stained SDS-PAGE gel (Figure 2A) demonstrating successful expression of HbpD-SpC in an OMV preparation (in Figure 2A, lane 1: ladder and lane 2: OMV preparation). Successful expression of Spy-tagged antigens and conjugation of said Spy- tagged antigens to HbpD-SpC displayed on OMVs as described in Example 2 is shown in Figure 2A lanes 3 and 5-7. Adducts comprising the respective Spy-tagged antigen (H6-SpT- AliA, H6-SpT-PnrA, H6-SpT-DiiA or H6-SpT-SP1690) coupled to HbpD-SpC (HbpD-SpC-SpT- antigen fusions) displayed on OMVs are indicated with open arrowheads in respective lanes, and non-conjugated HbpD-SpC displayed on OMVs is indicated with a solid black circle in lane 2 (•). Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane. Molecular mass (kDa) weight markers are indicated left of the molecular mass ladders. Coomassie stained SDS-PAGE gel (Figure 2B) demonstrating the result of combining monovalent antigen formulations to produce bivalent (lanes 5-13 and 15-23) or tetravalent (lanes 24-26) immunogenic compositions. Non-conjugated HbpD-SpC displayed on OMVs is indicated with a solid black circle (•) (lanes 2-4). Bands representing HbpD-SpC conjugates to H6-SpT-AliA (A), H6-SpT-PnrA (P), H6-SpT-DiiA (D) and H6-SpT-SP1690 (5) are indicated with open arrowheads and their corresponding capital letter initial in respective lanes. Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane.

[0209] Molecular mass (kDa) weight markers are indicated on top of the molecular mass ladders, in the mass ladder lanes (lane 1 and 14). Scatter plot (Figure 2C) depicting the load of 5. pneumoniae in NALT (nasal tissue) measured as colony forming units (CFU) as described in Example 2. The Y-axis depicts measured Logio CFU in NALT (nasal tissue), and the X-axis indicates various cohorts comprising either an unvaccinated control group or groups vaccinated with monovalent, bivalent or tetravalent immunogenic compositions comprising monovalent OMVs conjugated to one of the respective tested candidate antigens. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0210] Figure 3: Schematic overview (Figure 3A) of a bivalent immunogenic composition comprising monovalent vesicles (antigens PnrA and AliA displayed on separate vesicles) and a bivalent immunogenic composition comprising a bivalent vesicle (antigens PnrA and AliA displayed on same vesicle). Coomassie stained SDS-PAGE gels (Figure 3B and Figure 3C) demonstrating successful expression of HbpD-SpC in an OMV preparation (in Figure 3B, lane 1: ladder and lane 2: OMV preparation) or successful expression of HbpD-SnT(dl)-SpT(d4) in an OMV preparation (in Figure 3C, lane 1: ladder and lane 2: OMV preparation). Successful expression of Spy-tagged antigens and conjugation of said Spy-tagged antigens to HbpD-SpC or HbpD- SnT(dl)-SpT(d4) displayed on OMVs as described in Example 3 is shown in Figure 3B lanes 3- 4 and Figure 3C lane 3. Adducts comprising the respective Spy-tagged or SpyTag2-tagged antigen(s) coupled to HbpD-SpC or HbpD-SnT(dl)-SpT(d4) displayed on OMVs are indicated with open arrowheads or with letters (H6-SnC-AliA = a, H6-SpC2-PnrA = p, H6-SnC-AliA and H6-SpC2-PnrA = a+p) in respective lanes. Non-conjugated HbpD-SpC or HbpD-SnT(dl)- SpT(d4) displayed on OMVs is indicated with a solid black circle in lane 2 (•) of Figure 3B and Figure 3C respectively. Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane. Molecular mass (kDa) weight markers are indicated left of the molecular mass ladders. Scatter plot (Figure 3D) depicting 5. pneumoniae load in NALT (nasal tissue) measured as colony forming units (CFU) as described in Example 3. The Y-axis depicts measured Logio CFU in NALT (nasal tissue), and the X-axis indicates various cohorts comprising either an unvaccinated control group or groups vaccinated with bivalent immunogenic composition comprising monovalent vesicles or a bivalent immunogenic composition comprising bivalent vesicles. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. (*, p < 0.05; **, p < 0.01; *** , p < 0.001).

[0211] Scatter plot (Figure 3E) depicting a nti-Ali A and / or anti-PnrA IgG mediated response in serum in cohorts vaccinated with either a bivalent immunogenic composition comprising monovalent vesicles or an immunogenic composition comprising bivalent vesicles as described in Example 3. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Scatter plot (Figure 3F) depicting a nti-Ali A or anti- PnrA IgA responses in nasal homogenates in cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles as described in Example 3. Horizontal bars represent mean per group, and symbols represent samples from individual mice.

[0212] Figure 4: Schematic overview (Figure 4A) of a bivalent immunogenic composition comprising a bivalent vesicle (antigens PnrA and AliA displayed on same vesicle). Coomassie stained SDS gel (Figure 4B) confirming the protein profile of the produced bivalent immunogenic composition comprising bivalent OMVs carrying SpT-AliA and SpT-PnrA, as well as OMVs not coupled to antigen (control in lane 2). Adducts comprising the respective Spy-tagged antigens coupled to HbpD-SpC displayed on OMVs are indicated with open arrowheads and with letters (H6-SnC-AliA = A, H6-SpC2-PnrA = P). Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane. Molecular mass ladder in lane 1. Scatter plots (Figure 4C) depicting the formation of 5. pneumoniae colony forming units (CFU) in NALT (nasal tissue) as described in Example 4. The Y-axis depicts measured Logio CFU in NALT (nasal tissue), and the X-axis indicates various cohorts comprising either an unvaccinated control group or groups vaccinated with bivalent immunogenic composition comprising bivalent OMVs after 3 weeks and 15 weeks. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Graph (Figure 4D) depicting a nti-Ali A and anti-PnrA IgG mediated response in cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles at day 43, 70, 96 and 126 as described in Example 4. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Scatter plot (Figure 4E) depicting a nti-Ali A IgA mediated humoral responses in cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles after 3 and 15 weeks as described in Example 4. Horizontal bars represent mean per group, and symbols represent samples from individual mice. Scatter plots (Figure 4F) depicting cellular response (IL-17A and IFN-y) in spleen from cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles after challenge at 15 weeks as described in Example 4. Mock (negative control), PMA / lnonomycin (P / l; positive control); Heat killed pneumococci (Sp) with multiplicity of infection (MOI) of 1 (Spl) or 10 (SplO); purified AliA antigen at 1 pg / mL (AliAl) or 10 pg / mL (AliAlO); or purified PnrA antigen at 1 pg / mL (PnrAl) or 10 pg / mL (PnrAlO). Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice.

[0213] Figure 5: Coomassie stained SDS-PAGE gel (Figure 5A) demonstrating successful expression of HbpD-SpC in an OMV preparation (in Figure 5A, lane 1: ladder and lane 2: OMV preparation). Successful expression of Spy-tagged antigens, with (lane 3) or without a His6- tag (lane 4), and conjugation of said Spy-tagged antigens to HbpD-SpC displayed on OMVs as described in Example 5. Adducts comprising the respective Spy-tagged antigens (H6-SpT- AliA, H6-SpT-PnrA, SpT-AliA or SpT-PnrA) coupled to HbpD-SpC displayed on bivalent OMVs or non-conjugated HbpD-SpC displayed on OMVs are indicated with open arrowheads in respective lanes. Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane. Molecular mass (kDa) weight marker are indicated left of the molecular mass ladder. Scatter plot (Figure 5B) depicting the formation of 5. pneumoniae colony forming units (CFU) in NALT (nasal tissue) as described in Example 5. The Y-axis depicts measured Logio CFU in NALT (nasal tissue), and the X-axis indicates various cohorts comprising either an unvaccinated control group or groups vaccinated with bivalent immunogenic composition comprising bivalent OMVs wherein: the Spy-tagged antigens comprised a His6-tag and a 3 dose vaccine regime was utilized ("OMV-AliA-PnrA (His6): 3X imm."), the Spy-tagged antigens did not comprise a His6-tag and a 3 dose vaccine regime was utilized ("OMV-AliA-PnrA: 3X imm."), or the Spy-tagged antigens did not comprise a His6-tag and a 2 dose vaccine regime was utilized ("OMV-AliA-PnrA: 2X imm."). All groups were challenged after 3 weeks. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Scatter plot (Figure 5C) depicting the formation of 5. pneumoniae colony forming units (CFU) in NALT (nasal tissue) as described in Example 5. The Y-axis depicts measured Logio CFU in NALT (nasal tissue), and the X-axis indicates various cohorts comprising either an unvaccinated control group, a group vaccinated with bivalent immunogenic composition comprising bivalent OMVs wherein the Spy-tagged antigens did not comprise a His6-tag and a 3 dose vaccine regime was utilized ("OMV-AliA-PnrA"), or a group vaccinated with Prevnar ("PCV"). All groups were challenged after 15 weeks. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Scatter plot (Figure 5D) depicting a nti-Ali A IgG response in serum of unvaccinated control ("control") or cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles wherein the Spy-tagged antigens comprised a His6- tag and a 3 dose vaccine regime was utilized ("OMV-AliA-PnrA (His6): 3X imm."), the Spy- tagged antigens did not comprise a His6-tag and a 3 dose vaccine regime was utilized ("OMV-AliA-PnrA: 3X imm."), or the Spy-tagged antigens did not comprise a His6-tag and a 2 dose vaccine regime was utilized ("OMV-AliA-PnrA: 2X imm."), as described in Example 5. Al i A-specific IgG responses were analyzed on post-immune serum, taken after the third vaccination (day 44). Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Scatter plot (Figure 5E) depicting cellular response (IL-17A and IFN-y) in spleen from cohorts vaccinated either two times or three times with a bivalent immunogenic composition comprising bivalent vesicles or unvaccinated control as described in Example 5. Challenge after 3 weeks. Mock (negative control), PMA / lnonomycin (PMA / lono; positive control); Heat killed pneumococci (Sp); purified AliA antigen (AliA); or purified PnrA antigen (PnrA). Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Scatter plot (Figure 5F) depicting a nti-Ali A IgG response in unvaccinated control ("control") or cohort vaccinated with a bivalent immunogenic composition comprising bivalent vesicles ("OMV-AliA-PnrA"), as described in Example 5. AliA-specific IgG was analyzed on post-immune serum, taken at day 39, day 67, day 95 and day 136. Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Scatter plot (Figure 5G) depicting cellular response (IL-17A and IFN-y) in spleen from cohorts vaccinated three times with a bivalent immunogenic composition comprising bivalent vesicles or unvaccinated control, as described in Example 5. Challenge after 15 weeks. Mock (negative control), PMA / lnonomycin (PMA / lono; positive control); Heat killed pneumococci (Sp); purified AliA antigen (AliA); or purified PnrA antigen (PnrA). Horizontal bars represent geometric mean per group, and symbols represent samples from individual mice. Figure 6: Line plot (Figure 6A) depicting the survival curve of mice involved in the Murine lethal pneumonia co-infection experiment as described in Example 6. Boxplots (Figure 6B) depicting bacterial load (CFUs / organ or CFUs / ml) in nasal tissue, blood and lungs of mice immunized with PCV13 intramuscularly ("IM:PCV13", a bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA intranasally (IN:OMVs), a protein body formulation intradermally (I D :PBs) or unvaccinated control as described in Example 6. Boxplot (Figure 6C) depicting IgG antibody deposition on the bacterial surface in serum of mice immunized with a bivalent immunogenic composition comprising bivalent vesicles or unvaccinated control, as described in Example 6. MFI: Mean fluorescence intensity.

[0214] Figure 7: Scatter plots (Figure 7A and Figure 7B) depicting a nti-Ali A IgG and IgA response in unvaccinated control ("control") or cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles in low (3 pg) medium (6 pg) or high (12 pg) doses for IgG, and low (3 pg) or high (12 pg) doses for IgA, as described in Example 7. Horizontal bars in panels A and B represent geometric mean per group, and symbols represent samples from individual mice. Detection limits are indicated with a dashed line. Line plots (Figure 7C) depicting a nti-Ali A IgG response at 0, 14, 28 and 42 days in unvaccinated control ("control") or cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles in low (3 pg) medium (6 pg) or high (12 pg) doses, as described in Example 7. Horizontal bars in panels A and B represent geometric mean per group, and symbols represent samples from individual mice. Detection limits are indicated with a dashed line. Table at bottom indicates number of mice in each cohort displaying a nti-Ali A response (IgG mediated response in serum) after the first, second and third immunization, as described in Example 7.

[0215] Figure 8: Line plots (Figure 8A) depicting a nti-Ali A IgG mediated response in rats at 14, 28 and 42 days in unvaccinated control ("control") or cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles in low (20 pg) or high (100 pg) doses, as described in Example 8. Titers are displayed as the geometric mean with standard deviation of an experiment with 4 animals, and symbols represent samples from individual rats. Detection limits are indicated with a dashed line. Table at bottom indicates number of rats in each cohort displaying a nti-Ali A and anti-PnrA response (IgG response in serum) after the first, second and third immunization, as described in Example 8. Scatter plots (Figure 8B) depicting a nti-Ali A or anti-PnrA IgA response in unvaccinated control ("vehicle") or cohorts vaccinated with a bivalent immunogenic composition comprising bivalent vesicles in low (20 pg) or high (100 pg) doses, as described in Example 8. Horizontal bars in the panels represent geometric mean per group, and symbols represent samples from individual rats. Detection limits are indicated with a dashed line.

[0216] Figure 9: Diagram (Figure 9A) detailing Streptococcus pneumoniae serotypes covered by current known polysaccharide based vaccines. Diagram (Figure 9B) detailing a panel of 51 Streptococcus pneumoniae clinical isolates (representing 44 different serotypes) applied to mouse sera (Example 7) and rat sera (Example 8) immunized with a bivalent immunogenic composition comprising bivalent vesicles. References a-e at bottom of figure indicate origin of the cohorts. Western blots (Figure 9C) detailing the a nti-Ali A and anti-PnrA antibody responses in mouse sera (sera from Example 7) and rat sera (sera from Example 8) to a panel of 51 Streptococcus pneumoniae clinical isolates (representing 44 different serotypes).

[0217] Figure 10: Bar graphs (Figure 10) detailing amino acid sequence% identity of the two antigens present in the bivalent immunogenic composition, AliA and PnrA, as well as two other antigens not present in the bivalent immunogenic composition, Dii A and crNanA. X- axis: number of isolates (in 1000s); Y-axis:% identity. Y-axis has been truncated to only show values of 50% identity and above.

[0218] Examples

[0219] The following examples disclose the development of an immunogenic composition. The composition according to the present disclosure is shown herein to elicit antibody responses and cellular responses in vaccinated subjects, as well as reduce the bacterial load of pneumococci in the nose and improve survival compared to unvaccinated control subjects.

[0220] In addition, the data presented supports that the immunogenic composition according to the present disclosure is capable of eliciting protective immune response against a large number of serotypes of 5. pneumococcus. Thus, the present inventors consider said composition suitable to be utilized as a pneumococcal vaccine. Provided below is a list of selected general materials and methods applied in the following examples, more detailed lists of materials and methods can be found in each respective example text:

[0221] General materials and methods

[0222] Bacterial strains and growth conditions: For mouse infection experiments, serotype 4 Pneumococcal Bacteremia Collection Nijmegen (PBCN) isolate 0231 cells (PBCN0231) were cultured in Todd-Hewitt broth (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with yeast extract to OD620 0.25 and stored in glycerol at -80 °C.

[0223] Streptococcus pneumoniae strains used for Western blotting analysis (Example 9) were from the lab collection of Radboud University Nijmegen and grown in the same medium. E. coli strain TOP1OF' (Life Technologies) was used as the host strain for cloning procedures and E. coli strain BL21( DE3) (Thermo Scientific) was used for antigen production. Outer membrane vesicles (OMVs) were produced using strain Salmonella Typhimurium SL3261 AtoIRA AmsbB (Kuipers et al, Infect Immun. 2017 Sep 20; 85(10)).

[0224] Expression Plasmids: The autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:69), comprising an autotransporter protein in the form of HbpD (a double substitution mutant, N1048G and N1049S, also with domain 1 partially deleted, Adi) (SEQ ID NO:84) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was expressed from the medium-copy pEH3 vector backbone (described in Hashemzadeh-Bonehi et al. Mol Microbiol. (1998) 30(3) :676-8) that drives expression under control of a isopropyl P-D-l-thiogalactopyranoside-(IPTG)-inducible LacUV5 promoter. A person skilled in the art will understand that the autotransporter fusion protein "HbpD-SpC" is initially expressed with a signal peptide (SEQ ID NO:69), but is presented on the surface of OMVs without a signal peptide (SEQ ID NO:68), see section entitled "Autotransporter fusion protein production" for more information.

[0225] All antigen fusion proteins, SpT-PnrA (SEQ ID NO:58) , SpT-AliA (SEQ ID NO:59), SpT-ala2 (SEQ ID NO:139), SpT-DiiA (SEQ ID NO:137), SpT-SP1690 (SEQ ID NO:138), SpT-crNanA (SEQ ID NQ:140), H6-SpT-PnrA (SEQ ID NO:135), H6-SpT-AliA (SEQ ID NO:136), H6-SpT-DiiA (SEQ ID NO:137), H6-SpT-SP1690 (SEQ ID NO:138), SnC-ala2-SpT-H6 (SEQ ID NO:139) H6-SpT-crNanA (SEQ ID NQ:140), H6-SnC-AliA (SEQ ID NO:155), H6-SpC2-PnrA (SEQ ID NO:156), H6-SpT2-AliA (SEQ ID NO:157), H6-SpT2-SpuA (SEQ ID NO:158), H6-SpT2-TprX (SEQ ID NO:159), H6-SpT2- MetQ (SEQ ID NQ:160), H6-SpT2-LivJ (SEQ ID NO:161), H6-SpT2-PcsB (SEQ ID NO:162), H6- SpT2-AdcAII (SEQ ID NO:163), H6-SpT2-PrtA(N') (SEQ ID NO:164), and H6-SpT2-PsaA (SEQ ID NO:165 were expressed from a pET28 expression vector derivative under control of a T7 promoter. Fusion proteins TrM-AliA-TrM (SEQ ID NO:166), TrM-PnrA-TrM (SEQ ID NO:167), TrM-MetQ-TrM (SEQ ID NO:168) and TrM-AliA-TrM (SEQ ID NO:169) destined to be produced as protein bodies were expressed from a pASK-IBA3 backbone under tet-promoter control (IBA GmbH, Gottingen, Germany).

[0226] Outer Membrane Vesicle (OMV) production: OMVs displaying the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68) were produced using Salmonella Typhimurium strain SL3261 AtoIRA AmsbB and transformed with plasmid pEH3-HbpD-SpC. Bacterial cells were grown at 30 °C overnight in TYMC-medium (10 g / L Tryptone, 5 g / L Yeast Extract, 2mM MgCl2, 2mM CaCl2, 0.2% glucose, Km25, Cm30, and 30 pg / mL of chloramphenicol) (Kuipers et al, Infect Immun. 2017 Sep 20; 85(10)) or an animal component-free and fermentation- optimized version of that. Where appropriate, the medium further comprised IPTG (f.c. 100 pM) to drive expression of HbpD-SpC. To harvest OMVs, cultures of "HbpD-SpC" (SEQ ID NO:68) expressing Salmonella were initially subjected to low-speed centrifugation to remove most cells. The supernatant was then filtered on a cellulose-acetate membrane with a pore size of 0.45 pm to remove residual bacteria. OMVs were isolated from the filtered medium by ultracentrifugation (Beckmann Rotor 45Ti, 45.000 rpm, 4 °C, 1 hour) and the OMV pellet was resuspended in PBS / 15% glycerol stored at < -70 °C until further use.

[0227] Alternatively, the 0.45 pm-filtered supernatant was further concentrated and diafiltrated (PBS) by tangential flow filtration (TFF) with a molecular weight cutoff (MWCO) of 500 kDa to obtain an OMV suspension, which was stored at <-70°C until further use.

[0228] Autotransporter fusion protein production: As described in the section entitled "Expression Plasmids", the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:69), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:84) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was expressed from vector pEH3. Following expression, the autotransporter fusion protein "HbpD-SpC" (SEQ ID NO:68), comprising an autotransporter protein in the form of HbpD (SEQ ID NO:67) and a complementary moiety of the coupling system in the form of SpyCatcher ("SpC", SEQ ID NO:11), was displayed on the surface of OMVs. However, before being displayed on the surface of OMVs, the fusion protein "HbpD-SpC" was first expressed from the plasmid "pEH3-HbpD-SpC". The expressed but pre-display version of "HbpD-SpC" comprised a cleavable signal peptide (SEQ ID NO:69). When the autotransporter fusion protein was displayed on the surface of OMVs, the signal peptide was cleaved, resulting in an autotransporter fusion protein sequence without the signal peptide displayed on the OMV membrane (SEQ ID NO:68). Herein, whenever reference is made to OMVs displaying autotransporter fusion proteins, it should be assumed that the autotransporter fusion protein sequence does not comprise a signal peptide sequence. Whenever reference is made to "expressed" autotransporter fusion proteins which have yet to be displayed on the surface of OMVs, it should be assumed that the autotransporter fusion protein sequence further comprises a signal peptide sequence.

[0229] Antigen production: Recombinant pneumococcal antigens, AliA (SEQ ID NO:2), PnrA (SEQ ID NO:1), Dii A (SEQ ID NQ:130) SP1690 (SEQ ID NO:131), ala2 (SEQ ID NO:132), crNanA (SEQ ID NO:133), SpuA (SEQ ID NO:141), TprX (SEQ ID NO:142), MetQ (SEQ ID NO:143), LivJ (SEQ ID NO:144), PcsB (SEQ ID NO:145), AdcAII (SEQ ID NO:146), PrtA(N') (SEQ ID NO:147) and PsaA (SEQ ID NO:148) were designed as polypeptides comprising said antigen sequence together with a coupling moiety of a coupling system, such as SpyTag (SpT, SEQ ID NQ:10; or SpT2, SEQ ID NO:14), SnoopCatcher (SnC, SEQ ID NO:13) or SpyCatcher002 (SpC2, SEQ ID NO:15). Optionally, the polypeptides further comprised a Hise-tag (SEQ ID NO:134). Fusion polypeptides were isolated from Escherichia coli BL21(DE3) cells transformed with an appropriate pET28-based expression plasmid. Each polypeptide comprising a respective antigen was isolated individually.

[0230] Workflow for Hise-taqqed antigens: Escherichia coli: BL21(DE3) cells were grown at 37 °C in LB-medium (10 g / L Tryptone, 5 g / L Yeast Extract and 10 g / L NaCI) further comprising 0.2% glucose and 50 pg / mL kanamycin. Cells were induced for protein expression for 2.5 hours by addition of IPTG (final concentration, f.c., 0.5 mM) when cells reached an QD660 value of ~0.6. Harvested cells were suspended in a phosphate binding buffer (50 mM NaPO4, 300 mM NaCI, pH 7.4) comprising PMSF (f.c. 1-10 mM) and lysed through high-pressure cell disruption using a OneShot cell disruptor. Following low-speed and high-speed centrifugation steps to remove debris and membrane material, the resulting cleared lysate was subjected to Hise-tag based affinity purification to isolate the antigens. The cleared lysate was incubated with TALON superflow beads (Cytiva Life Sciences) in the presence of 5 mM Imidazole and the bound Hise-tagged antigens were subsequently eluted from the beads using 200 mM Imidazole. Eluates were dialyzed against PBS to remove imidazole (sample:dialysis buffer volume ratio = 1:500-1,000). Dialyzed eluates were supplemented with glycerol (f.c. 10% v / v) and stored frozen until further use.

[0231] Workflow for antigens without Hise-taqs Escherichia coli: BL21(DE3) cells were grown at 37 °C in LB-medium (10 g / L Tryptone, 5 g / L Yeast Extract and 10 g / L NaCI) further comprising 0.2% glucose and 50 pg / mL kanamycin. Cells were induced for protein expression for 2.5 hours by addition of IPTG (f.c. 0.5 mM) when cells reached an OD660 value of ~0.6.

[0232] Harvested cells were resuspended in 20mM Tris. Cl pH 8.0 and lysed using high pressure. Lysate was clarified using low-speed centrifugation and microfiltration and the resulting cleared lysate were purified using anion-exchange (AEX) chromatography. Bound antigens were subsequently eluted from the AEX beads using 20mM Tris. Cl pH 8.0 comprising an appropriate excess concentration of sodium chloride. Eluates were stored at 2-8 °C until further use.

[0233] Conjugation: To obtain conjugation of polypeptides comprising antigens to vesicles (OMVs), OMVs displaying the autotransporter fusion protein "HbpD-SpC" (HbpD-SpC, SEQ ID NO:68) (prepared as described in the above sections entitled “Outer Membrane Vesicle (OMV) production" and “Autotransporter fusion protein production") were first thawed. Said thawed OMVs displaying "HbpD-SpC" (HbpD-SpC, SEQ ID NO:68) were then incubated with a molar excess of purified Spy-tagged antigen (prepared as described in the section entitled “Antigen production” , with or without a Hise-tag). When producing bivalent vesicles, such as bivalent OMVs displaying Spy-tagged AliA and PnrA simultaneously, both Spy-tagged antigens were added to the OMVs simultaneously. Conjugation mixtures were incubated at 2-8 °C for 16-24h. In the case of OMVs displaying HbpD-SnT(dl)-SpT(d4) (SEQ ID NO:76), a molecular excess of purified H6-SnC-AliA (SEQ ID NO:155) was added and the mixture was incubated at room temperature for 2h. A same amount of H6-SpC2-PnrA (SEQ ID NO:156) was then added and incubation was continued for another 2h. Subsequently, an excess amount of H6-SpC2-PnrA was again added and the mixture was incubated for 24h at 4°C. Following incubations, PBS was added to a final volume of 25 mL and the vesicle-polypeptide mixture was filtered through a 0.45 pm filter. The filtrate was centrifuged (70Ti rotor, 45,000 rpm [208,000 x g], 75 min, 4 °C), and sedimented vesicle-polypeptide conjugates were resuspended in 0.5 mL of PBS+15% glycerol. To allow salt washing, the vesicle-polypeptide suspension was mixed with 5.5 mL of PBS supplemented with 410 mM of NaCI (total 550 mM NaCI) and centrifuged (MLA-80 rotor, 65.000 rpm [293.000 xg], 60 min, 4 °C). Vesicle- polypeptide pellet was resuspended in PBS + 15% glycerol to a concentration of 2 OD units / pl and stored at <-70°C until use in vaccination studies of Examples 1-3. OMVs used in Example 5 were suspended in an alternative final formulation buffer comprising 10 mM Na- PO4, 2% sucrose, pH 7.4 as specified in the Example description.

[0234] Alternatively, to wash and remove non-bound antigen from bivalent OMVs, such as bivalent OMVs displaying AliA and PnrA simultaneously (OMV-AliA-PnrA), conjugation mixtures were subjected to diafiltration by TFF (500 MWCO). These OMVs were subjected to further purification using size-exclusion chromatography and finally suspended in 0.75X PBS, 15% v / v glycerol. The resulting conjugated bivalent vesicles were stored at <-70°C until further use in vaccination studies described in Example 4 and Examples 6-8.

[0235] A control sample of thawed vesicles displaying the autotransporter fusion protein "HbpD- SpC" (HbpD-SpC, SEQ ID NO:68), prepared as described in the above sections entitled “Outer Membrane Vesicle (OMV) production" and “Autotransporter fusion protein production”, was incubated under identical conditions but without the addition of a polypeptide comprising an antigen sequence.

[0236] The vesicle-fusion polypeptide formulations were judged to be suitable for further use upon SDS-PAGE / Coomassie staining analysis.

[0237] Where OMV vaccine doses are stated in OD units, 1 OD unit equals to the amount of OMVs that is derived from one OD600 or OD660 unit of SL3261 AtoIRA AmsbB (pEH3-HbpD-SpC) cells. OMV vaccine doses stated in pg refer to the total protein content of the OMV formulation as determined by DC Protein Assay (Biorad) according to manufacturer's recommendations.

[0238] ELISA: Detection of antibody responses by enzyme-linked immunosorbent assay analysis (ELISA). Maxisorp high binding affinity plates (Nunc) were generally coated with 5 pg / mL purified Ali A or PnrA fusions in carbonate coating buffer (0.1 M carbonate / bicarbonate pH 9.6) or PBS at 4°C overnight. In the case of Example 7, panel C the same procedure applied, except that plates were coated with 2.5 pg / mL purified antigen fusions. The next day, wells were blocked with 1% BSA (Sigma) and subsequently incubated for 1-2 h at 37°C with serum, nasal homogenates or nasal lavage samples from individual animals diluted in PBS 1% BSA.

[0239] Thereafter, for mouse samples, the wells were incubated with primary anti-mouse IgG- alkaline phosphatase (Sigma) or primary anti-mouse IgA-alkaline phosphatase (Southern Biotech) in PBS 1% BSA for 1 h at 37°C. Samples were developed using 1 mg / mL p- nitrophenylposphate in substrate buffer (1 M diethanolamine, 0.5 mM MgCI2 pH 9.8) (Calbiochem, VWR) and the optical density was measured at 405 nm 20 minutes after substrate addition. For rat samples, wells were incubated with anti-rat IgG-HRP (Bio-Rad) or Goat anti-Rat IgA, HRP (PAI-84707; ThermoFisher) for 30 min at room temperature.

[0240] The ELISA was developed using with 0.1 mg TMB / mL in phosphate citrate buffer and the reaction was stopped after 15 min using 2M H2SO4, whereafter the optical density at 450 nm and 690 nm (for reference) was measured. For both mouse and rat samples, all wells were washed with PBS containing 0.05% Tween-20 (Merck) between and after the incubations steps. For IgG, serum antibody levels compared to pre-immune serum were measured. For nasal IgA, antibody levels compared to the non-vaccinated control group were measured.

[0241] SDS-PAGE and Western Blotting: SDS-PAGE was performed using standard 10% SDS-PAGE gels. Cell or protein samples were solubilized in SDS-PAGE sample buffer (50 mM TrisHCI pH 6.8, 2% w / v SDS, 10% glycerol, 0.01% w / v bromophenol blue, 50 mM DTT) and boiled for at least 10 min. Western blotting was performed as described (Towbin et al. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4.) using specified antisera / antibodies and LumiLight Western Blotting Substrate (Roche). Prior to the antisera / antibody incubations, proper protein transfer was confirmed by reversible protein staining on blotting membranes using Ponceau- S solution (Serva, Germany). Images were captured using an AI600 imager (Amersham). Animal studies: For experiments involving mice, female C57BI / 6 J mice of 7 weeks old were obtained from Charles River in France (Example 2) or Germany (Example 1 and 3-7). Upon arrival, mice were randomized over individually ventilated cages. Mice were allocated to control and experimental groups randomly and the investigators were not blinded. Sterile water and food were provided ad libitum. Prior to experimentation, the mice had at least 5 days to acclimate. For rat studies, female and male Wistar-Han rats of 8 weeks old were obtained from Charles River in Germany. Animals were left to acclimatize for at least 5 days before start of the study.

[0242] Mice were intranasally immunized three times with 5 pL (2.5 pL per nostril) vaccine formulation comprising OMVs in either PBS / 15% glycerol or 0.75X PBS / 15% glycerol while under inhalation anesthesia (isoflurane), with a 2-week interval between each immunization. Doses of OMVs administered were as specified in the respective Example descriptions. Mice receiving PCV were intramuscularly immunized three times with 30-50 pL of Prevnar (l / 15th- l / 10thof human dose) with two-week intervals. At specified time points, blood was collected from the tail vein for antibody analysis by ELISA. Whereas mice of Example 7 were only used to study induction of antibody responses upon vaccine administration, mice of Examples 1-5 and Example 6 were subjected to challenge with 5. pneumoniae to study induction of protection against pneumococcal colonization or invasive pneumococcal disease, respectively.

[0243] For colonization experiments (Examples 1-5), 3 weeks or 15 weeks after the last vaccination, as indicated, mice were intranasally challenged with 1 x 106CFU 5. pneumoniae PBCN0231 in 5 pL PBS under inhalation anesthesia. Three days post-challenge, mice were euthanized, and nasal tissue was harvested and homogenized using an IKA T10 basic blender. To allow bacterial enumeration (log CFU / animal), samples were serially diluted on blood agar plates containing gentamicin (Mediaproducts, Groningen, the Netherlands) and incubated at 37°C overnight. Lower limit of detection: 22 CFU / animal. Where indicated, nasal tissue homogenates or nasal lavage samples were also used for detection of IgA levels by ELISA. For the pneumococcal disease experiment (Example 6), 18 days after the third immunization, mice were infected with 104-5PFU of influenza virus strain A / Udorn / 307 / 72 (H3N2) in 10 pL PBS without anesthesia. Three days post-influenza infection, mice were infected with 3 x 105CFU of 5. pneumoniae PBCN0231 in 15 pL PBS under inhalation anesthesia (isoflurane). Over the course of 160 hours, mice were scored based on clinical symptoms indicating the development of invasive disease (bodyweight loss, hunched back, ruffled coat, reduced mobility) and were removed from the experiment just before they reached their humane endpoint. Blood, homogenized nasal tissue and homogenized lung tissue of sacrificed mice were plated to determine the bacterial load as described above. Lower limit of detection: nasal tissue 22 CFU / animal, lung tissue 44 CFU / animal and blood 22 CFU / animal.

[0244] Rats (Example 8) were intranasally immunized four times with 22.2 pL (11.1 pL per nostril) of vaccine formulation containing OMVs in 0.75X PBS / 15% glycerol at specified doses, while under inhalation anesthesia (isoflurane), with a 2-week interval between each immunization. At specified time points, blood was collected from the tail vein for IgG analysis by ELISA. Moreover, nasal lavage was performed on a panel of rats at day 45 to measure local levels of IgA by ELISA.

[0245] Statistics and plots: Unless stated otherwise, statistical analyses were performed using GraphPad Prism version 5.03 (GraphPad Software) on loglO transformed data. Details on the statistical parameters used can be found in the Example descriptions. P-value < 0.05 was considered significant and p-values are expressed as follows: * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001. In scatter plots displaying levels of bacterial CFUs, antibodies or cytokines, dashed lines indicate detection limit, symbols represent individual mice and horizontal bars indicate the geometric mean of each group.

[0246] Example 1

[0247] Efficacy of immunogenic compositions comprising pneumococcal antigens without an external adjuvant

[0248] In a previously published study (Van beek et al., Virulence. 2020 Dec;ll(l):1310-1328) a panel of well-conserved pneumococcal proteins was tested (Ali A (SEQ ID NO:2), SpuA (SEQ ID NO:141), TprX (SEQ ID NO:142), MetQ (SEQ ID NO:143), LivJ (SEQ ID NO:144), PcsB (SEQ ID NO:145), AdcAII (SEQ ID NO:146), PrtA(N') (SEQ ID NO:147), PsaA (SEQ ID NO:148) in the context of their suitability as vaccine antigens, inducing protection against nasal colonization of S. pneumoniae (see Fig. IB). The antigens were comprised in the following respective polypeptides (H6-SpT2-AliA (SEQ ID NO:157), H6-SpT2-SpuA (SEQ ID NO:158), H6-SpT2-TprX (SEQ ID NO:159), H6-SpT2-MetQ (SEQ ID NQ:160), H6-SpT2-LivJ (SEQ ID NO:161), H6-SpT2- PcsB (SEQ ID NO:162), H6-SpT2-AdcAII (SEQ ID NO:163), H6-SpT2-PrtA(N') (SEQ ID NO:164), H6-SpT2-PsaA (SEQ ID NO:165) carrying a translationally fused Spytag002 (SpT2, SEQ ID NO:14) and a His6 purification tag (SEQ ID NO:134). The results showed that all of the antigens tested in the panel induced protection against nasal colonization compared to the non-vaccinated control, with the composition comprising the antigen AliA being the most potent. This Example demonstrates the suitability of a further panel of well-conserved pneumococcal proteins PnrA (SEQ ID NO:1), ala2 (SEQ ID NO:132), DiiA (SEQ ID NQ:130), SP1690 (SEQ ID NO:131) and crNanA (SEQ ID NO:133) respectively in the context of an immunogenic composition without an additional) external adjuvant.

[0249] In the development of an immunogenic composition comprising at least the antigen AliA, the selection of further components of said immunogenic composition, for example, further antigens, is not an obvious choice. The above-mentioned previously published data, as well as complementary data from Example 1, show that many equally suitable candidates exist and that it is not obvious which candidate(s) should be further included in an immunogenic composition.

[0250] Material and Methods

[0251] Pneumococcal antigens PnrA (SEQ ID NO:1), ala2 (SEQ ID NO:132), DiiA (SEQ ID NQ:130), SP1690 (SEQ ID NO:131), and crNanA (SEQ ID NO:133) comprised in the following respective polypeptides H6-SpT-PnrA (SEQ ID NO:135), SnC-ala2-SpT-H6 (SEQ ID NO:139), H6-SpT-DiiA (SEQ ID NO:137), H6-SpT-SP1690 (SEQ ID NO:138), and H6-SpT-crNanA (SEQ ID NQ:140) were expressed in translational fusion with a SpyTag (SEQ ID NQ:10) and a His6 purification tag (SEQ ID NO:134). The polypeptides were purified and coupled to vesicles displaying HbpD- SpC (SEQ ID NO:68) as described in the relevant sub-headings of the "General Materials and Methods" section. To analyze the protein profiles of the produced respective monovalent immunogenic compositions, equivalent volumes of vaccine stocks containing equivalent amounts of OMVs, based on OD units, were run on a SDS-PAGE gel followed by Coomassie staining. SDS-PAGE / Coomassie staining was carried out as described in the "General Materials and Methods" section.

[0252] Murine model for pneumococcal colonization: The resulting immunogenic compositions were tested for protective efficacy following intranasal administration in a murine nasal pneumococcal challenge model. Mice received three doses of 8 OD660 units of an immunogenic composition comprising an OMV conjugated to one of the tested candidate antigens in PBS / 15% glycerol formulation buffer. Three weeks after the last vaccination, the animals were intranasally challenged with 1 x 106CFU of 5. pneumoniae PBCN0231. Three days after challenge, nasal tissue was harvested and the bacterial load was determined. More detailed information can be found under relevant sub-headings of the "General Materials and Methods" section.

[0253] Results

[0254] SDS-PAGE / Coomassie staining was carried out to analyze the protein profiles of the produced respective monovalent immunogenic compositions and demonstrated successful expression of HbpD-SpC in the OMV preparation (Figure 1A and IB). The antigens PnrA, ala2, Dii A, SP1690, and crNanA were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight (see lanes 3-7 in Figure 1A). Similarly, the antigens AliA, SpuA, TprX, MetQ, LivJ, PcsB, AdcAII, PrtA(N'), and PsaA were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight (see lanes 3-7, 10-12 and 14 in Figure IB). Conjugation was efficient as the vast majority of HbpD-SpC (128 kDa) migrated as higher molecular weight species upon addition of the respective SpyTagged antigen. The amount of OMV material per volume of monovalent immunogenic compositions respectively (OMVs displaying HbpD- SpC-H6-SpT-PnrA, HbpD-SpC-H6-SpT-ala2, HbpD-SpC-H6-SpT-DiiA, HbpD-SpC-H6-SpT- SP1690, or HbpD-SpC-H6-SpT-crNanA, respectively), was highly similar as judged by similar intensities of the major outer membrane proteins intrinsically present in the OMV membrane (see Figure 1A and IB). Judged by identical means, the amount of OMV material per volume of the respective monovalent immunogenic compositions comprising OMVs displaying HbpD-SpC-H6-SpT2-AliA, HbpD-SpC-H6-SpT2-SpuA, HbpD-SpC-H6-SpT2-TprX, HbpD-SpC-H6-SpT2-MetQ, HbpD-SpC-H6-SpT2-LivJ, HbpD-SpC-H6-SpT2-PcsB, HbpD-SpC-H6- SpT2-AdcAII, HbpD-SpC-H6-SpT2-PrtA(N'), and HbpD-SpC-H6-SpT2-PsaA was highly similar. Thus, monovalent immunogenic compositions comprising monovalent vesicles were successfully obtained.

[0255] Murine model for pneumococcal colonization: Efficacy was demonstrated by a significant reduction in 5. pneumoniae colony forming units (CFU) in the nasal cavity upon vaccination with the immunogenic composition comprising an OMV conjugated to one of the tested candidate antigens compared to a non-vaccinated control group (see Figure 1C).

[0256] As shown in Figure 1C, suitability of the well-conserved pneumococcal proteins PnrA, DiiA, SP1690 and crNanA in the context of an immunogenic composition without an additional external adjuvant were demonstrated by inducing a 1.5 - 2 logw reduction of nasal colonization by 5. pneumoniae. Antigenic fragment ala2, representing the N-terminal alphahelical domain of pneumococcal protein PspA, appeared even more efficacious, yielding a ~ 3 logw reduction in CFU. However, despite its immunogenicity, ala2 is the most variable region of PspA and not well conserved among circulating pneumococcal strains (Kuipers et al, Infect Immun. 2017 Sep 20;85(10):e00281-17). Hence, it is not suitable for production of a broadly protective pneumococcal vaccine. Figure ID, demonstrates that well-conserved pneumococcal proteins SpuA (SEQ ID NO:141), TprX (SEQ ID NO:142), MetQ (SEQ ID NO:143), LivJ (SEQ ID NO:144), PcsB (SEQ ID NO:145), AdcAII (SEQ ID NO:146), PrtA(N') (SEQ ID NO:147), PsaA (SEQ ID NO:148) are also suitable as vaccine candidates since they also result in reduction of nasal colonization by 5. pneumoniae in the same range. Overall, non-vaccinated animals showed approximately lxlO5CFU of 5. pneumoniae in the nasal tissue whereas animals vaccinated with one of the candidate pneumococcal proteins (excluding ala2) showed approximately lxlO3CFU in the nasal tissue.

[0257] Thus, the studies showed that there were several similarly suitable candidate antigens in the context of an immunogenic composition without an additional external adjuvant.

[0258] It was concluded that in particular antigens DiiA (SEQ ID NQ:130), PnrA (SEQ ID NO:1) and SP1690 (SEQ ID NO:131) may present as suitable partners to Al iA (SEQ ID NO:2) for the development of a multivalent immunogenic composition, capable of addressing a broad band of serotypes, as described in following examples and were investigated further.

[0259] Example 2

[0260] Efficacy of immunogenic compositions comprising multiple antigens without an external adjuvant

[0261] This Example demonstrates the successful deployment and efficacy of immunogenic compositions comprising at least two antigens, i.e. multivalent or bivalent immunogenic compositions / vaccine formulations. In particular, bivalent immunogenic compositions comprising combinations of the pneumococcal antigens Ali A, PnrA, DiiA and SP1690 were tested to identify the most optimal antigen pairing for the further development of a pneumococcal vaccine candidate.

[0262] Materials and Methods

[0263] Four pneumococcal antigens, PnrA (SEQ ID NO:1), AliA (SEQ ID NO:2), DiiA (SEQ ID NO:130), and SP1690 (SEQ ID NO:131), comprised in the following respective polypeptides H6-SpT- PnrA (SEQ ID NO:135), H6-SpT2-AliA (SEQ ID NO:157), H6-SpT-DiiA (SEQ ID NO:137), and H6- SpT-SP1690 (SEQ ID NO:138), were expressed. Each polypeptide comprised a coupling moiety of a coupling system in the form of SpyTag (SEQ ID NQ:10) and a His6-tag (SEQ ID NO:134) to allow purification. Said polypeptides were in turn conjugated to vesicles (OMVs) displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68), resulting in monovalent antigen formulations for each respective pneumococcal antigen. Said autotransporter fusion protein HbpD-SpC in turn comprised an autotransporter protein in the form of HbpD (SEQ ID NO:67) coupled to a complementary moiety of a coupling system in the form of SpyCatcher (SpC; SEQ ID NO:11). Expression, purification and coupling procedures were performed as described in the relevant sub-headings of the "General Materials and Methods" section.

[0264] Bivalency or tetravalency was reached by combining monovalent antigen formulations comprising any of the antigens in a 1:1 or 1:1:1:1 ratio (per OD unit of OMVs), respectively. To analyze the protein profiles of the produced respective bivalent or tetravalent immunogenic compositions, equivalent amounts of OMV material, based on OD units, were run on an SDS-PAGE gel followed by Coomassie staining. SDS-PAGE / Coomassie staining was carried out as described in the "General Materials and Methods".

[0265] Preparation of composition of administration: All formulations (monovalent, bivalent and tetravalent immunogenic compositions) comprised the same total amount of OMVs (per OD units of OMVs).

[0266] Murine model for pneumococcal colonization: The multivalent and bivalent antigen formulations were tested for efficacy in a mouse model for pneumococcal colonization. Female C57BI / 6 J mice were intranasally immunized three times (day 0, day 14 and day 28) with an 8 OD units dose of OMVs in PBS / 15% glycerol. Mice in the unvaccinated control group were not immunized. Three weeks after the final immunization, mice were challenged intranasally with the 5. pneumoniae serotype 4 PBCN0231 strain. Three days after challenge, nasal tissue was harvested and the bacterial load was determined. More detailed information concerning animal models can be found under the relevant sub-heading of the "General Materials and Methods" section.

[0267] Results

[0268] SDS-PAGE / Coomassie staining was carried out to analyze the protein profiles of the produced respective monovalent immunogenic compositions and demonstrated successful expression of HbpD-SpC in the OMV preparation (see lane 2 in Figure 2A). The antigens Ali A, PnrA, Dii A and SP1690 were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight. Conjugation was efficient as the vast majority of HbpD-SpC (128 kDa) migrated as higher molecular weight species upon addition of the respective SpyTagged antigen (see lanes 3 and 5-7 in Figure 2A). The amount of OMV material per volume of the respective monovalent immunogenic compositions (OMV-AliA, OMV-PnrA, OMV-DiiA and OMV-SP1690), was highly similar as judged by similar intensities of the major outer membrane proteins intrinsically present in the OMV membrane (see bands indicated with an * in lanes 2-3 and 5-7 in Figure 2A). Thus, monovalent immunogenic compositions comprising monovalent vesicles were successfully obtained. To analyze the protein profile of the subsequently produced bivalent OMV vaccine formulations (immunogenic compositions) (OMV-AliA + OMV-PnrA, OMV-AliA + OMV-DiiA, OMV AliA + OMV-SP1690, OMV-PnrA + OMV-DiiA, OMV-PnrA + OMV-SP1690, OMV-DiiA + OMV-SP1690) and tetravalent vaccine formulation (immunogenic composition) (OMV-AliA + OMV-PnrA + OMV-DiiA + OMV-SP1690), equal volumes of the respective vaccine formulations used in the mouse experiment at days 0, 14 and 28 were subjected to SDS- PAGE and Coomassie staining (Figure 2B). Per vaccine formulation, samples used at the various timepoints were loaded in random order. Each respective formulation contained highly similar amounts of OMV material per pl as judged by the similar intensity of Coomassie stained bands representing the major outer membrane proteins intrinsically present in the OMV membrane (marked with an * in lanes 2-13 and 15-26 in Figure 2B). Moreover, presence of two or four antigens in the respective vaccine formulations, in similar amounts was confirmed. This followed from the presence of Coomassie stained bands of similar intensity representing adducts of HbpD-SpC with the respective antigens. No antigen adducts were observed for control, which was a formulation comprising OMV-HbpD-SpC and no antigen (-) (Figure 2B, lanes 2-4).

[0269] Murine model for pneumococcal colonization: Recovery of live pneumococci from nasal tissue revealed that mice immunized with the multivalent immunogenic composition were significantly more protected against colonization compared to the unvaccinated control (see Figure 2C). Specifically, the bivalent immunogenic compositions comprising antigens AliA and PnrA and bivalent immunogenic composition comprising antigens AliA and Dii A were the most efficacious, yielding a ~2.5 logw CFU reduction in the nasal cavity compared to the CFU in the nasal cavity of non-vaccinated animals. However, the bivalent immunogenic composition comprising AliA and PnrA demonstrated the least spread in protection level among the individual mice. These results were surprising considering that monovalent immunogenic compositions investigated in Example 1, indicated that antigens ala2 and Dii A would be preferred candidates for the development of a bivalent immunogenic composition together with the antigen AliA. Interestingly, the immunogenic composition comprising antigens AliA, PnrA, Di iA and SP1690 (i.e. four antigens instead of two), provided for a lower logw CFU reduction in the nasal cavity when compared to bivalent counterparts. Thereof, the present inventors concluded that an immunogenic composition is not necessarily improved by simply adding more antigens to the composition. These data highlight the potential of a bivalent immunogenic composition comprising the Al iA and PnrA antigens for development of a pneumococcal vaccine.

[0270] Example 3

[0271] Superior efficacy of bivalent immunogenic composition comprising bivalent vesicles compared to monovalent vesicles

[0272] This Example demonstrates the superior efficacy of a bivalent immunogenic composition comprising bivalent vesicles displaying both antigens PnrA and AliA compared to a bivalent immunogenic composition comprising monovalent vesicles displaying either PnrA or AliA.

[0273] Material and Methods

[0274] Two bivalent immunogenic compositions were prepared comprising the antigens AliA (SEQ ID NO:2) and PnrA (SEQ ID NO:1). Said immunogenic compositions were either: (1) a mixture of monovalent vesicles (OMVs) displaying polypeptides comprising either AliA or PnrA, or (2) bivalent vesicles (OMVs) displaying polypeptides comprising both antigens (AliA and PnrA) simultaneously coupled to the same autotransporter fusion protein (HbpD) (see schematic illustration in Figure 3A). Efficacy was tested in a murine model for pneumococcal colonization.

[0275] Production of (l) a bivalent immunogenic composition comprising monovalent vesicles: A bivalent immunogenic composition comprising monovalent outer membrane vesicles (OMVs) was produced by mixing two separately prepared monovalent immunogenic compositions. In one first composition, polypeptides (H6-SpT2-AliA; SEQ ID NO:157) comprising the antigen AliA (SEQ ID NO:2), a SpyTag002 (SpT) (SEQ ID NO:14) and a His6-tag (SEQ ID NO:134) were conjugated to OMVs displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68), resulting in a first monovalent immunogenic composition. In one second composition, polypeptides (H6-SpT-PnrA; SEQ ID NO:135) comprising the antigen PnrA (SEQ ID NO:1), a SpyTag (SpT) (SEQ ID NQ:10) and a His6-tag (SEQ ID NO:134) were conjugated to OMVs displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68), resulting in a second monovalent immunogenic composition.

[0276] The bivalent immunogenic composition comprising monovalent vesicles (OMVs) displaying polypeptides comprising either Al iA (H6-SpT2-AliA; SEQ ID NO:157) or PnrA (H6-SpT-PnrA, SEQ ID NO:135) was produced by mixing the above-described first and second monovalent immunogenic compositions in a 1:1 ratio (per total protein content).

[0277] To analyze the protein profiles of the produced immunogenic compositions, equivalent amounts of OMV material (2 pg, based on of total protein content) were run on an SDS- PAGE gel followed by Coomassie staining. SDS-PAGE / Coomassie staining was carried out as described in the "General Materials and Methods" (see Figure 3B).

[0278] Production of (2) a bivalent immunogenic composition comprising bivalent vesicles: A bivalent immunogenic composition comprising bivalent vesicles, in other words vesicles displaying both AliA and PnrA simultaneously, was produced. One first polypeptide (H6-SnC- AliA; SEQ ID NO:155) comprising AliA (SEQ ID NO:2), a His6 purification tag (SEQ ID NO:134) and a coupling moiety, SnoopCatcher (SEQ ID NO:13)), and one second polypeptide (H6- SpC2-PnrA; SEQ ID NO:156) comprising PnrA (SEQ ID NO:1), a His6 purification tag (SEQ ID NO:134) and a coupling moiety SpyCatcher002 (SpC2) SEQ ID NO:15), were conjugated to OMVs displaying an autotransporter fusion protein comprising HbpD and two separate types of complementary moieties of a coupling system, namely SnoopTag (SnT (SEQ ID NO:12)) and SpyTag002 (SpT2 (SEQ ID NO:14) ((HbpD-SnT(dl)-SpT2(d4); (SEQ ID NO:76)) (see Figure 3A).

[0279] SDS-PAGE / Coomassie staining was carried out as described in the "General Materials and Methods" section to confirm the protein profiles of the produced conjugated vesicles (OMVs) (see Figure 3C).

[0280] Murine model for pneumococcal colonization: The immunogenic compositions were tested for immunogenicity and efficacy in a mouse model for pneumococcal colonization. Female C57BI / 6 J mice were intranasally immunized three times (day 0, 14 and 28) with a 12 pg dose of conjugated vesicles (OMVs) suspended in PBS / 15% glycerol. Mice in the unvaccinated control group received PBS intranasally. Three weeks after the final immunization, mice were challenged intranasally with the 5. pneumoniae strain PBCN0231 (serotype 4). Three days after challenge (day 52), nasal tissue was harvested and the bacterial load was determined (see Figure 3D). More detailed information concerning animal models can be found under the relevant sub-heading of the "General Materials and Methods" section. IgG Humoral response: To evaluate the induction of a humoral response, serum taken 16 days after the third vaccination (day 44) was used to measure the antigen-specific IgG levels by ELISA (Figure 3E). ELISA was performed as detailed in the section entitled "ELISA" under "General Materials and Methods".

[0281] IgA Humoral response: To test for induction of local IgA, nasal tissue homogenates of mice vaccinated with the bivalent immunogenic composition comprising bivalent vesicles displaying both Ali A and PnrA simultaneously and challenged 3 weeks after vaccination, were analyzed by ELISA (see Figure 3F). ELISA was performed as detailed in the section entitled "ELISA" under "General Materials and Methods".

[0282] Results

[0283] (1) bivalent immunogenic composition comprising monovalent vesicles: SDS- PAGE / Coomassie staining was carried out to analyze the protein profiles of the produced respective monovalent immunogenic compositions and demonstrated successful expression of HbpD-SpC in the OMV preparation. The antigens AliA and PnrA were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight. Conjugation was efficient as the vast majority of HbpD-SpC (128 kDa) migrated as higher molecular weight species upon addition of the respective SpyTagged antigen (see Figure 3B). The amount of OMV material per pg of sample loaded (total protein c)ntent) in the monovalent immunogenic compositions, OMV-AliA and OMV-PnrA, respectively, was highly similar as judged by the similar intensity of Coomassie stained bands representing the major outer membrane proteins intrinsically present in the OMV membrane. Non-conjugated HbpD-SpC is indicated (•). Bands representing HbpD-SpC conjugates to H6-SpT-AliA and H6-SpT-PnrA are indicated (<). Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane.

[0284] The bivalent immunogenic composition comprising monovalent vesicles (OMVs) displaying polypeptides comprising either AliA (H6-SpT2-AliA; SEQ ID NO:157) or PnrA (H6-SpT-PnrA, SEQ ID NO:135) was subsequently obtained by mixing the above-described first and second monovalent immunogenic compositions in a 1:1 ratio (per total protein content).

[0285] Thus, a bivalent immunogenic composition comprising monovalent vesicles was successfully obtained.

[0286] (2) bivalent immunogenic composition comprising bivalent vesicles: SDS-PAGE / Coomassie staining was carried out to confirm the protein profile of the produced bivalent immunogenic composition and demonstrated successful expression of HbpD-SnT(dl)- SpT2(d4) (SEQ ID NO:76) in the vesicle (OMV) preparation when no antigens were added (see control (ctrl.) in Figure 3C). Upon addition of H6-SpC-AliA and H6-SpC2-PnrA, efficient conjugation to HbpD-SnT(dl)-SpT2(d4) (120 kDa) occurred as demonstrated by the emergence of higher molecular weight species on SDS-PAGE. Moreover, adducts comprising HbpD-SnT(dl)-SpT2(d4) and both H6-SpC-AliA and H6-SpC2-PnrA simultaneously (a+p; Figure 3C) were successfully formed. A significant portion of adducts only containing H6-SpC-AliA (p; Figure 3C) was also observed. Although the branched nature of conjugates carrying two antigens at different positions of HbpD made interpretation difficult, the identity of the antigen conjugates could be deduced from the apparent molecular weight on SDS-PAGE. Moreover, the presence of AliA in HbpD-SnT(dl)-SpT2(d4)- H6-SpC-AliA-H6-SpC2-PnrA conjugates could be established by western blotting (data not shown).

[0287] Thus, a bivalent immunogenic composition comprising bivalent valent vesicles was successfully obtained.

[0288] Murine model for pneumococcal colonization: Recovery of live pneumococci from nasal tissue revealed that mice immunized with the bivalent immunogenic composition comprising monovalent vesicles displaying each antigen on separate vesicles, as well as mice immunized with the bivalent immunogenic composition comprising bivalent vesicles displaying the antigens simultaneously on the same vesicle, were significantly protected against colonization compared to the unvaccinated control (see Figure 3D). In both cases a reduction of ~1.5 logw CFU was observed compared to the unvaccinated control.

[0289] Thus, the data show that both compositions resulted in efficient reduction of nasal colonization by 5. pneumoniae in vaccinated subjects, indicating protective effects of the vaccine. IgG Humoral response: Vaccination with the bivalent immunogenic composition comprising monovalent vesicles displaying each antigen on separate vesicles yielded high AliA-directed IgG titers (~ 1 x 105ng / ml) and low but significant levels of PnrA directed antibodies (~ 1 x 103ng / ml). Remarkably, the bivalent immunogenic composition comprising bivalent vesicles displaying the antigens simultaneously on the same vesicle yielded higher levels of anti-AliA (>105ng / ml) and induced a prominent, 15-fold increased level for PnrA-directed antibodies (>104ng / ml). In particular the difference in anti-PnrA titer between the two formulations is striking (see Figure 3E).

[0290] In conclusion, Example 3 shows the efficacy of both bivalent immunogenic compositions, both the composition comprising bivalent vesicles displaying both antigens PnrA and AliA and the bivalent immunogenic composition comprising monovalent vesicles displaying either PnrA or AliA, in providing a reduction of nasal colonization by 5. pneumoniae in vaccinated subjects compared to non-vaccinated subjects. Example 3 further demonstrates that both immunogenic compositions induced an IgG-mediated humoral response in vaccinate subjects (see Figure 3E). However, it also demonstrates that the bivalent immunogenic composition comprising bivalent vesicles displaying both antigens PnrA and AliA induced a superior anti-PnrA antibody response in vaccinated subjects compared to the anti-PnrA antibody response in subjects vaccinated with the bivalent immunogenic composition comprising vesicles displaying the antigens on separate vesicles.

[0291] IgA Humoral response: Induction of local IgA antibody responses has been suggested to be important for prevention against mucosal pathogens. Clearly detectable levels of antigenspecific (anti-AliA and anti-PnrA) IgA was observed (see Figure 3F), demonstrating that the bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously also induced a local humoral (IgA) immune response.

[0292] Hence, immunogenic compositions comprising vesicles (OMVs) carrying AliA and PnrA antigens simultaneously or on separate vesicles are both suitable candidates for pneumococcal vaccine development. Example 4 Immunogenicity and protective efficacy of bivalent immunogenic composition comprising bivalent vesicles over time

[0293] This Example demonstrates the immunogenicity and protective efficacy of a bivalent immunogenic composition comprising bivalent vesicles displaying the antigens Al iA and PnrA simultaneously over time.

[0294] Materials and Methods

[0295] To produce the bivalent vesicles in Example 4, polypeptides comprising the antigen Ali A and a coupling moiety of a coupling system, SpyTag (SpT) (SpT-Ali A; SEQ ID NO:59) and polypeptides comprising the antigen PnrA and a coupling moiety of a coupling system, SpyTag (SpT) (SpT-PnrA; SEQ ID NO:58), were incubated with vesicles (OMVs) displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68) as described in the appropriate sections under "General Materials and Methods". Whereas polypeptides comprising antigens used in Example 1-3 further comprised a His6-tag to allow isolation by affinity chromatography, the polypeptides of Example 4 did not further comprise an affinity tag and were isolated using anion exchange chromatography, as detailed in the sections "Antigen Production" and "Workflow for antigens without Hise-tags" under "General Materials and Methods".

[0296] SDS-PAGE / Coomassie staining was carried out as detailed in the section "SDS-PAGE and Western Blotting" under "General Materials and Methods" to confirm the protein profile of the produced bivalent immunogenic composition and demonstrate successful expression of HbpD-SpC (SEQ ID NO:68) in the vesicle (OMV) preparation. An amount of 4 g (per total protein content) of OMVs carrying SpT-AliA and SpT-PnrA, as well as OMVs not coupled to antigen, were loaded (Fig. 4B).

[0297] Murine model for pneumococcal colonization: Protective efficacy and underlying immunogenicity was investigated using the murine pneumococcal challenge model as described in Examples 1-3, analyzing reduction of nasal pneumococcal load induced by the immunogenic composition (12 pg doses of conjugated OMVs in 0.75X PBS / 15% glycerol) in the nasal tissue 3 weeks after vaccination. However, as effective vaccines are expected to protect against infection over a long period of time, longevity of protective responses was also investigated by further analyzing immunogenic composition-induced reduction of nasal pneumococcal load in the nasal tissue at a time interval longer than 3 weeks, namely an extended 15-week interval between vaccination and pneumococcal challenge (Fig. 4C). These two time intervals were also utilized when investigating further protective responses, such as IgG and IgA humoral responses and systemic cellular responses, as described below. IgG Humoral response: To evaluate the underlying IgG humoral immune responses, blood taken before vaccination (day -4), and at several timepoints after third vaccination (day 43, day 70, day 98 and day 126), was used to measure the antigen-specific IgG levels in serum by ELISA (see Figure 4D).

[0298] IgA Humoral response: To test for induction of local IgA, nasal tissue homogenates of mice vaccinated with the bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously and challenged either 3 weeks or 15 weeks after vaccination, were analyzed by ELISA (see Figure 4E).

[0299] Cellular immune response: Induction of cellular immune responses was investigated in mice vaccinated with the bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously and challenged with pneumococci 15 weeks after vaccination (see Figure 4F). Three days post-challenge, mice were euthanized, and spleens were harvested. Isolated spleens were directly transferred to culture medium (RPMI 1640 Glutamax + 3% FCS + 1% Pen / Strep) and kept on ice until further processing for in vitro stimulation. Single cell suspensions of spleens were prepared in culture medium using a Falcon® 70 pm cell strainer (Fisher Scientific, Schwerte, Germany) followed by incubation with red blood cell lysis buffer (Invitrogen, Karlsruhe, Germany) for 5 min. Splenocytes (5 x 105in final volume of 200 pL) were stimulated for 72 h at 37 °C and 5% CO2. Supernatant was collected and analyzed by luminex for presence of IL-17A, and IFN-y. Stimuli were the following: Mock (negative control), PMA / lnonomycin (P / l; positive control); Heat killed pneumococci (Sp) with multiplicity of infection (MOI) of 1 or 10; purified AliA antigen at 1 pg / mL or 10 pg / mL; or purified PnrA antigen at 1 pg / mL or 10 pg / mL. To be able to detect responses induced by the vaccine rather than the pneumococci used for challenge, samples from mice immunized with OMV-AliA-PnrA were compared with samples from mice immunized with non-conjugated OMV-HbpD-SpC (OMV).

[0300] Results

[0301] Polypeptides (SpT-AliA; SEQ ID NO:59) comprising the antigen AliA and the coupling moiety SpyTag (SpT) and polypeptides (SpT-PnrA; SEQ ID NO:58) comprising the antigen PnrA and the coupling moiety SpyTag (SpT), were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight (HbpD-SpC- SpT-AliA: 199 kDa, HbpD-SpC-SpT-PnrA: 162 kDa). Conjugation was efficient as the vast majority of HbpD-SpC (126 kDa), as present in control OMVs (-), migrated as higher molecular weight species upon addition of the respective SpyTagged antigens. The resulting bivalent immunogenic composition comprised bivalent vesicles and a similar content of SpT- AliA and SpT-PnrA as judged by the similar intensities of the Coomassie stained bands representing HbpD-SpC-SpT-AliA and HbpD-SpC-SpT-PnrA adducts, respectively (see Fig. 4B). Thus, a bivalent immunogenic composition comprising bivalent vesicles was successfully obtained. Adducts HbpD-SpC-SpT-AliA (<A) and HbpD-SpC-SpT-PnrA (<P) are indicated. Asterisks indicate major outer membrane proteins intrinsically present in the OMV membrane.

[0302] Murine model for pneumococcal colonization: Mice immunized with a bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA showed considerably reduced numbers (>2.5 logw CFU) of live pneumococci in nasal tissue, compared to nonvaccinated animals, when challenged 3 weeks after vaccination (see Figure 4C). These results mirrored the results observed in Example 3, where mice immunized with another bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA also showed considerably reduced numbers (>2.5 logw CFU) of live pneumococci in nasal tissue. Importantly, mice challenged 15 weeks after vaccination according to Example 4 still showed a significant reduction of pneumococcal load, indicating that the bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously induced durable immune responses that protect against pneumococcal colonization over a prolonged period of time (see Figure 4C). Thus, the data shows that the bivalent immunogenic composition resulted in efficient protection against and / or reduction of nasal colonization by 5. pneumoniae in vaccinated subjects over a prolonged period of time.

[0303] IgG Humoral response: Approximately two weeks after the third vaccination (day 43) high levels of AliA-directed IgG antibodies (~ 1 x 105ng / ml) were observed. Significant levels of PnrA directed IgG antibodies (~ 1 x 103ng / ml) were also detected. Thus, potent induction of antigen-specific antibodies by OMV-AliA-PnrA was demonstrated (see Figure 4D). Importantly, similar levels of both antibodies were measured in later serum samples, including samples taken 14 weeks after vaccination (day 126). From this data, the inventors were able to conclude that bivalent immunogenic compositions comprising bivalent vesicles displaying both AliA and PnrA simultaneously induced a persistent humoral (IgG) response. IgA Humoral response: Induction of local IgA antibody responses has been suggested to be important for prevention against mucosal pathogens. Clearly detectable levels of antigenspecific IgA were observed. Importantly, said levels only slightly declined over time from ~200 ng / ml (3 weeks) to ~ 80 ng / ml (15 weeks), demonstrating that the bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously also induced an enduring local humoral (IgA) immune response.

[0304] Cellular immune response: Moderate but apparent elevation of IFN-gamma cytokine levels was observed in splenocytes isolated from vaccinated animals upon stimulation with the antigens or the heat-killed bacteria, demonstrating antigen-specific responses (Fig. 4F). Particularly strong antigen-specific IL-17A responses were observed upon stimulation with whole pneumococci (Sp) or AliA, demonstrating clear induction of systemic cellular responses by the OMV-AliA-PnrA vaccine.

[0305] In conclusion, Example 4 demonstrates that a bivalent immunogenic composition comprising bivalent vesicles displaying both AliA and PnrA simultaneously provided broad protection against 5. pneumoniae infection, even after an extended period of time (15 weeks). All assays, humoral and cellular, demonstrated the efficacy of the referred to immunogenic composition which was able to activate many protective responses in the vaccinated subjects. Such protective responses were efficient protection against and / or reduction of nasal colonization by 5. pneumoniae, induced and persistent humoral (IgG and IgA) responses, and strong antigen-specific IL-17A responses. For an immunogenic composition to be considered a viable candidate for further vaccine development, it must prove that it can provide or induce such a span of protective responses in a subject, and furthermore, it must also prove that the provided or induced protective responses are retained over an extended period of time.

[0306] Example 5

[0307] Formulation effects on the longevity of protective responses induced by a bivalent immunogenic composition

[0308] This Example investigated whether or not an alternative formulation would affect the longevity of protective responses induced by a bivalent immunogenic composition comprising bivalent vesicles displaying the antigens AliA and PnrA simultaneously. It has been suggested in the art that low osmolality buffers favor intranasal adsorption (Nishibe et al. 2002. jibi inkoka tembo 45((Supplement 1)): 46-49; Dua et al. IntJ Pharm. 1997147(2): 233-242). In this example, the protective effective efficacy of a bivalent immunogenic composition comprising bivalent vesicles displaying antigens AliA and PnrA simultaneously, formulated in a slightly hypotonic solution made of 2% Sucrose and 10 mM sodium phosphate buffer (pH 7.4), with a target osmolality of 90 mOsm / kg, was investigated. Moreover, protective efficacy of OMVs carrying His6-tagged and non-His6-tagged antigens were compared side, as well as vaccine regimes comprising two versus three dose administrations.

[0309] Materials and Methods

[0310] A bivalent immunogenic composition comprising bivalent vesicles with an even distribution of AliA and PnrA at the vesicle surface was produced as follows: Polypeptides (SpT-Ali A; SEQ ID NO:59) comprising the antigen AliA and a coupling moiety of a coupling system, SpyTag (SpT) and polypeptides (SpT-PnrA; SEQ ID NO:58) comprising the antigen PnrA and a coupling moiety of a coupling system, SpyTag (SpT), were simultaneously incubated with vesicles (OMVs) displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68) for 24h at 4 °C with continuous agitation. To achieve efficient conjugation, an 8-fold molecular excess of SpT-AliA over HbpD-SpC was used and a 3-fold molecular excess of SpT-PnrA over HbpD-SpC.

[0311] A bivalent immunogenic composition comprising bivalent vesicles with an even distribution of His6-tagged antigens AliA and PnrA at the vesicle surface was produced as follows: Polypeptide (H6-SpT-AliA; SEQ ID NO:136) comprising the antigen AliA, a His6-tag and a coupling moiety of a coupling system, SpyTag (SpT), and polypeptide (H6-SpT-PnrA; SEQ ID NO:135) comprising the antigen PnrA, a His6-tag and a coupling moiety of a coupling system, SpyTag (SpT), were simultaneously incubated with vesicles (OMVs) displaying an autotransporter fusion protein HbpD-SpC (SEQ ID NO:68) for 24h at 4 °C with continuous agitation. To achieve efficient conjugation, an 4-fold molecular excess of H6-SpT-AliA over HbpD-SpC was used and a 3-fold molecular excess of H6-SpT-PnrA over HbpD-SpC. This resulted in a bivalent immunogenic composition comprising bivalent vesicles with an even distribution of AliA and PnrA at the vesicle surface (essentially as described in Example 4). The text below provides for details related to the alternative formulation.

[0312] Incubated OMVs displaying polypeptides comprising the two antigens were salt-washed using 20 mM TrisCi pH 8.0 (~550 mM NaCI) and collected by ultracentrifugation (MLA-80 rotor, 65.000 rpm [293.000 x g], 60 min, 4 °C). Subsequently, said OMVs were resuspended in phosphate-sucrose buffer (10 mM Na-PO4, 2% sucrose, pH 7.4). To remove potential aggregates, the formulation was filtered through a 0.45 pm filter (Sarstedt, Flitropur S 0.45, REF 83.1826, LOT 00525103). OMVs displaying polypeptides comprising the two antigens were collected again by ultracentrifugation (MLA-80 rotor, 65.000 rpm [293.000 x g], 60 min, 4 °C) and resuspended in phosphate-sucrose buffer (10 mM Na-PO4, 2% sucrose, pH 7.4). SDS-PAGE / Coomassie staining was carried out as detailed in the section "SDS-PAGE and Western Blotting" under "General Materials and Methods" to confirm the protein profile of the produced bivalent immunogenic compositions. An amount of 2 pg (per total protein content) of OMVs not carrying antigens, OMVs carrying His6-tagged SpT-AliA and SpT-PnrA, or OMVs carrying non-His6-tagged SpT-AliA and SpT-PnrA were loaded (Fig. 5A).

[0313] Murine model for pneumococcal colonization: Female C57BI / 6 J mice were intranasally immunized three times under inhalation anesthesia (isoflurane) with 5 pL OMVs (2.5 pL per nostril) with a 2 week interval (day 0, day 14 and day 28). Where indicated (2X imm; see Fig. 5B), mice were intranasally immunized two times (day 14, day 28). Each dose comprised 12 pg of OMVs in 10 mM Na-PO4, 2% sucrose, pH 7.4. Mice in the unvaccinated control group were immunized intranasally with 10 mM Na-PO4, 2% sucrose, pH 7.4. Mice in the PCV group were intramuscularly immunized three times with 30 pl of Prevnar (l / 15thof human dose) with 2 week intervals (day 0, day 14 and day 28). 3 weeks (day 49; see Fig. 5B) or 15 weeks after the third vaccination (day 133; see Fig. 5C), mice were intranasally challenged with 1 x 106CFU Streptococcus pneumoniae PBCN0231 (Serotype 4) in 5 pL PBS under inhalation anesthesia (isoflurane). Three days post-infection, mice were euthanized, and nasal tissue was harvested, homogenized and used for plating on blood agar plates (containing gentamycin) to determine the bacterial load. Lower limit of detection: 22 CFU / animal.

[0314] IgG Humoral response: For mice challenged 3 weeks after vaccination, induction of AliA- specific IgG responses was analyzed on post-immune serum, taken after the third vaccination (day 44) by ELISA (see Figure 5D). For mice challenged 15 weeks after vaccination, AliA-specific IgG was analyzed on post-immune serum, taken at day 39, day 67, day 95 and day 136 by ELISA (see Figure 5F). Control samples concern sera from mice vaccinated with OMV-HbpD-SpC not decorated with AliA and PnrA antigens (see Figure 5D and Figure 5F).

[0315] Cellular immune response: Induction of cellular immune responses was investigated in unvaccinated and vaccinated mice challenged with pneumococci either 3 weeks (see Figure 5E) or 15 weeks (see Figure 5G) after vaccination. Three days post-challenge, mice were euthanized, and spleens were harvested. Isolated spleens were directly transferred to culture medium (RPMI 1640 Glutamax + 3% FCS + 1% Pen / Strep) and kept on ice until further processing for in vitro stimulation. Single cell suspensions of spleens were prepared in culture medium using a Falcon® 70 pm cell strainer (Fisher Scientific, Schwerte, Germany) followed by incubation with red blood cell lysis buffer (Invitrogen, Karlsruhe, Germany) for 5 min. Splenocytes (5 x 105in final volume of 200 pL) were stimulated for 72 h at 37 °C and 5% CO2. Supernatant was collected and analyzed by luminex for presence of IL-17A, and IFN-y. Stimuli were the following: Mock (negative control), PMA / lnonomycin (PMA / lono; positive control); Heat killed pneumococci (Sp); purified AliA antigen; or purified PnrA antigen.

[0316] Samples from unvaccinated mice were analyzed as controls.

[0317] Results

[0318] SDS-PAGE / Coomassie staining was carried out to confirm the protein profile of the produced bivalent immunogenic compositions with and without His6-tagged antigens. Successful expression of HbpD-SpC (SEQ ID NO:68) could be was demonstrated in control OMVs not incubated with antigen (Figure 5A). Moreover, polypeptides comprising the antigen AliA and the coupling moiety SpyTag (SpT) (SpT-AliA; SEQ ID NO:59) and polypeptides comprising the antigen PnrA and the coupling moiety SpyTag (SpT) (SpT-PnrA; SEQ ID NO:58), were successfully conjugated to HbpD-SpC as demonstrated by the emergence of adducts of the expected molecular weight. Conjugation was efficient as the vast majority of HbpD-SpC (126 kDa) migrated as higher molecular weight species upon addition of the respective SpyTagged antigens. Using the same strategy, successful conjugation of polypeptides comprising a His6- tagged and SpyTagged version of AliA (H6-SpT-AliA; SEQ ID NO:136) or PnrA (H6-SpT-PnrA; SEQ ID NO:135) could be demonstrated (Figure 5A). Each resulting bivalent immunogenic composition - either carrying His6-tagged antigens or not - comprised bivalent vesicles and a similar content of AliA and PnrA as judged by the similar intensities of the coomassie stained bands representing HbpD-SpC-SpT-AliA(- / + H6) and HbpD-SpC-SpT-PnrA(- / + H6) adducts, respectively (see Figure 5A). In addition, each respective formulation comprised highly similar amounts of OMV material (per g of total protein content) as judged by the similar intensity of Coomassie stained bands representing the major outer membrane proteins intrinsically present in the OMV membrane. Thus, as the resulting OMVs were suspended in 10 mM Na-PO4, 2% sucrose, pH 7.4, bivalent immunogenic compositions comprising bivalent vesicles in an alternative formulation was successfully obtained.

[0319] Moreover, bivalent immunogenic compositions comprising bivalent vesicles carrying either His6-tagged antigens or non-His6-tagged antigens were successfully obtained.

[0320] Murine model for pneumococcal colonization: Mice immunized with the bivalent immunogenic composition comprising bivalent vesicles displaying AliA and PnrA simultaneously showed statistically significantly reduced numbers (~1 logw CFU) of live pneumococci in nasal tissue compared to non-vaccinated animals, in animals challenged 3 weeks after vaccination, indicative of protective responses against pneumococcal colonization (see Figure 5B). In addition, a similar reduction of pneumococcal colonization in nasal tissue was observed irrespective if...

Claims

CLAIMS1. An immunogenic composition capable of eliciting an immune response, such as a protective immune response, against 5. pneumoniae in a subject when administered to said subject, said immunogenic composition comprising a first component and a second component, wherein a) said first component is at least one component selected from the group consisting of a nucleic acid encoding a PnrA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the PnrA protein of 5. pneumoniae; a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system or a nucleic acid encoding at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a nucleic acid encoding an AliA protein of 5. pneumoniae; a nucleic acid encoding an immunogenic fragment of the AliA protein of 5. pneumoniae; a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of S. pneumoniae, and wherein said second component further comprises at least one coupling moiety of a coupling system or a nucleic acid encoding at least one coupling moiety of a coupling system; wherein said PnrA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:1 and said AliA protein comprises or consists of an amino acid sequence having at least 80% identity to SEQ ID NO:2 or SEQ ID NO:152; wherein the coupling system comprises at least one coupling moiety and at least one complimentary moiety and allows for the formation of at least one isopeptide bond between said at least one coupling moiety and at least one complementary moiety, and wherein thecoupling system is derived from pilus proteins from Gram positive bacteria, which proteins are capable of spontaneously forming one or more isopeptide bonds.

2. The immunogenic composition according to claim 1, wherein a) said first component is at least one component selected from the group consisting of a polypeptide corresponding to the PnrA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the PnrA protein of 5. pneumoniae; and wherein said first component further comprises at least one coupling moiety of a coupling system; and b) said second component is at least one component selected from the group consisting of a polypeptide corresponding to the AliA protein of 5. pneumoniae; and a polypeptide corresponding to an immunogenic fragment of the AliA protein of 5. pneumoniae, and wherein said second component further comprises at least one coupling moiety of a coupling system.

3. The immunogenic composition according to claim 1 or 2, wherein said first component is said PnrA protein comprising or consisting of SEQ ID NO:1 and said second component is said AliA protein comprising or consisting of SEQ ID NO:2.

4. The immunogenic composition according to any one of claims 1-3, further comprising at least one vesicle, such as at least two vesicles, wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component; and wherein at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said second component.

5. The immunogenic composition according to any one of clams 1-4, wherein said first and second components are coupled to different vesicles.

6. The immunogenic composition according to any one of claims 1-3, further comprising at least one vesicle, wherein said at least one vesicle displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component and at least one complementary moiety to the coupling moiety comprised in said second component.

7. The immunogenic composition according to any one of clams 1-4 and 6, wherein said first and second components are coupled to the same at least one vesicle.

8. The immunogenic composition according to any one of claims 1-7, wherein said first component and second component comprise coupling moieties of different coupling systems or wherein said first component and second component comprise coupling moieties of the same coupling system.

9. The immunogenic composition according to any one of claims 1-8, wherein said first component and second component comprise the same coupling moieties of the same coupling system.

10. The immunogenic composition according to any one of claims 1-9, wherein the coupling system is derived from pilus proteins from Gram positive bacteria selected from the group consisting of pilus proteins from Lactiplantibacillus, pilus proteins from Bacillus, pilus proteins from Ruminococcus, and pilus proteins from Streptococcus.

11. The immunogenic composition according to any one of claims 1-10, wherein the coupling system is derived from pilus proteins from Streptococcus.

12. The immunogenic composition according to any one of claims 10-11, wherein said pilus proteins from Streptococcus are selected from the group consisting of the major pilin protein Spy0128 of Streptococcus pyogenes, the fibronectin binding protein FbaB of Streptococcus pyogenes, the fibronectin-binding protein from Streptococcus dysgalactiae, the pilus-subunit RrgA from 5. pneumoniae, and the pilus subunit RrgC from 5. pneumoniae.

13. The immunogenic composition according to any one of claims 1-12, wherein the coupling moiety and complementary moiety of the coupling system are selected from the group consisting of SnoopTag2 (SEQ ID NO:7), DogTag2 (SEQ ID NO:8), SnoopLigase2 (SEQ ID NO:9), SpyTag (SEQ ID NO:10), SpyCatcher (SEQ ID NO:11), SnoopTag (SEQ ID NO:12), SnoopCatcher (SEQ ID NO:13), SpyTag002 (SEQ ID NO:14), SpyCatcher002 (SEQ ID NO:15), SpyTag003 (SEQ ID NO:16), SpyCatcher003 (SEQ ID NO:17), SdyTag (SEQ ID NO:18), SdyCatcher (SEQ ID NO:19), SilkTag (SEQ ID NQ:20), SilkCatcher (SEQ ID NO:21), DogTag (SEQ ID NO:22), DogCatcher (SEQ ID NO:23), SnoopTagJr (SEQ ID NO:24), SnoopLigase (SEQ ID NO:25), Jo (SEQ ID NO:26), In (SEQ ID NO:27), NGTag (SEQ ID NO:28), NGCatcher (SEQ ID NO:29), SpyCatcher-N (SEQ ID NQ:30), SpyCatcher-21 (SEQ ID NO:31), SpyStapler (SEQ ID NO:32), SpyLigase (SEQ ID NO:33), MoonCake (SEQ ID NO:34), Katl (SEQ ID NO:35), QueenCatcher (SEQ ID NO:36), PsCsCatcher (SEQ ID NO:37), Ktag (SEQ ID NO:38), BDTag (SEQ ID NO:39), RumTrunkTagD9N (SEQ ID NQ:40), RumTrunkTag (SEQ ID NO:41), RumTag (SEQ ID NO:42), Rum2Tag (SEQ ID NO:43), Rum3Tag (SEQ ID NO:44), Rum4Tag (SEQ ID NO:45), Rum5Tag (SEQ ID NO:46), Rum6Tag (SEQ ID NO:47), Rum7Tag (SEQ ID NO:48), BacTag (SEQ ID NO:49), Bac2Tag (SEQ ID NQ:50), Bac3Tag (SEQ ID NO:51), Bac4Tag (SEQ ID NO:52), Bac5Tag (SEQ ID NO:53), Clib9 (SEQ ID NO:54), PhoTag (SEQ ID NO:55), PsCsTag (SEQ ID NO:56), and SpyCatcher_orig (SEQ ID NO:57), and variants thereof exhibiting at least 70% identity thereto, wherein said moieties are selected such that said moieties have the capacity of forming an isopeptide bond between each other, and wherein a variant of a coupling moiety exhibiting at least 70% identity to said coupling moiety or a variant of a complementary moiety exhibiting at least 70% identity to said complementary moiety is a functional variant which retains the capacity of forming anisopeptide bond to its complementary moiety or to its coupling moiety, respectively, or to a moiety exhibiting at least 70% identity thereto.

14. The immunogenic composition according to any one of claims 1-13, wherein coupling system is a coupling moiety-complementary moiety pair.

15. The immunogenic composition according to any one of claims 1-14, wherein the coupling system is selected from the group consisting of SpyTag-SpyCatcher (SEQ ID NQ:10-SEQ ID NO:11), SpyTag-SpyCatcher_orig (SEQ ID NO:10-SEQ ID NO:57), SpyTag-SpyCatcher003 (SEQ ID NQ:10-SEQ ID NO:16), SpyTag-SpyCatcher-N (SEQ ID NQ:10-SEQ ID NQ:30), SpyTag- SpyCatcher-21 (SEQ ID NQ:10-SEQ ID NO:31), SpyTag-SdyCatcher (SEQ ID NQ:10-SEQ ID NO:19), SpyTag002-SpyCatcher002 (SEQ ID NO:14-SEQ ID NO:15), SpyTag002-SpyCatcher (SEQ ID NO:14-SEQ ID NO:11), SpyTag002-SpyCatcher003 (SEQ ID NO:14-SEQ ID NO:17), SpyTag002-SdyCatcher (SEQ ID NO:14-SEQ ID NO:19), SpyTag003-SpyCatcher003 (SEQ ID NO:16-SEQ ID NO:17), SpyTag003-SpyCatcher (SEQ ID NO:16-SEQ ID NO:11), SpyTag003- SpyCatcher002 (SEQ ID NO:16-SEQ ID NO:15), SpyTag003-SdyCatcher (SEQ ID NO:16-SEQ ID NO:19), SnoopTag-SnoopCatcher (SEQ ID NO:12-SEQ ID NO:13), Sil kTag-Sil kCatcher (SEQ ID NQ:20-SEQ ID NO:21), DogTag-DogCatcher (SEQ ID NO:22-SEQ ID NO:23), DogTag2- DogCatcher (SEQ ID NO:8-SEQ ID NO:23), Jo-In (SEQ ID NO:26-SEQ ID NO:27), NGTag- NGCatcher (SEQ ID NO:28-SEQ ID NO:29), SdyTag-SdyCatcher (SEQ ID NO:18-SEQ ID NO:19), SdyTag-SpyCatcher (SEQ ID NO:18-SEQ ID NO:11), SdyTag-SpyCatcher002 (SEQ ID NO:18-SEQ ID NO:15), SdyTag-SpyCatcher003 (SEQ ID NO:18-SEQ ID NO:17), KTag-SpyTag (SEQ ID NO:38-SEQ ID NQ:10), DogTag-SnoopTagJr (SEQ ID NO:22-SEQ ID NO:24), SnoopTag2- SnoopCatcher (SEQ ID NO:7-SEQ ID NO:13), DogTag2-SnoopCatcher (SEQ ID NO:8-SEQ ID NO:13), SpyTag-BDTag (SEQ ID NQ:10-SEQ ID NO:39), MoonCake-RumTrunkTagD9N (SEQ ID NO:34-SEQ ID NQ:40), MoonCake-RumTag (SEQ ID NO:34-SEQ ID NO:42), MoonCake-SpyTag (SEQ ID NO:34-SEQ ID NQ:10), MoonCake-SdyTag (SEQ ID NO:34-SEQ ID NO:18), Katl-SpyTag (SEQ ID NO:35-SEQ ID NQ:10), Katl-SdyTag (SEQ ID NO:35-SEQ ID NO:18), Katl- RumTrunkTagD9N (SEQ ID NO:35-SEQ ID NQ:40), Katl-RumTag (SEQ ID NO:35-SEQ ID NO:42).

16. The immunogenic composition according to any one of claims 1-15, wherein said coupling moiety is SpyTag (SEQ ID NO:10) and said complementary moiety is SpyCatcher (SEQ ID NO:11) or wherein said coupling moiety is SpyCatcher (SEQ ID NO:11) and said complementary moiety is SpyTag (SEQ ID NQ:10).

17. The immunogenic composition according to any one of claims 1-16, wherein the said at least one vesicle is at least one Outer Membrane Vesicle (OMV) derived from a gramnegative bacterium, such as wherein said at least two vesicles are at least two Outer Membrane Vesicles (OMVs) derived from a gram-negative bacterium.

18. The immunogenic composition according to claim 17, wherein said gram negative bacterium is selected from the group consisting of Escherichia coli, Neisseria meningitidis, Neisseria gonorrhea and Salmonella spp.

19. The immunogenic composition according to any one of claims 17-18, wherein said gram negative bacterium is Salmonella Typhimurium.

20. The immunogenic composition according to any one of claims 17-19, wherein said least one OMV displays on its outer surface at least one complementary moiety of the coupling system as defined in any one of claims 10-16, such as wherein said least one OMV displays on its outer surface at least one complementary moiety to the coupling moiety comprised in said first component and at least one complementary moiety to the coupling moiety comprised in said second component.

21. The immunogenic composition according to any one of claims 1-20, wherein said least one vesicle displays on its outer surface an autotransporter (AT) fusion protein and wherein said AT fusion protein comprises an AT protein and said at least one complementary moiety of the coupling system, such as of the coupling system as defined in any one of claims 10-16.

22. The immunogenic composition according to claim 21, wherein the autotransporter protein is a serine protease autotransporter of the Enterobacteriaceae (SPATE), such as a SPATE protein from Escherichia coli.

23. The immunogenic composition according to claim 21 or 22, wherein the autotransporter protein is hemoglobin-binding protease (Hbp) from Escherichia coli.

24. The immunogenic composition according to claim 23, wherein said Hbp comprises the amino acid sequence as defined in SEQ ID NO:67.

25. Outer membrane vesicle (OMV) which displays on its outer surface at least one first component which is a polypeptide as defined in claim 1 a) and at least one second component which is a polypeptide as defined in claim 1 b).

26. Vaccine composition comprising the immunogenic composition as defined in any one of claims 1 to 24 or OMV as defined in claim 25 and a pharmaceutically acceptable carrier or excipient.

27. Immunogenic composition according to any one of claims 1-24, OMV according to claim 25, or the vaccine composition according to claim 26, for use as a medicament.

28. Immunogenic composition according to any one of claims 1-24, OMV according to claim 25, or the vaccine composition according to claim 26, for use in the treatment of a 5. pneumoniae infection, such as for use in the preventive treatment of a 5. pneumoniae infection.

29. Immunogenic composition for use according to claim 28, wherein said 5. pneumoniae infection is selected from an infection by at least one 5. pneumoniae serotype selected fromthe group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 9A, 16F, 18A, 19B, 21, 24B, 25A, 28A, 29, 34, 35F, 36, 37, 38, 40, 42 and 47F.

30. Immunogenic composition for use according to any one of claims 28-29, wherein said 5. pneumoniae infection is selected from an infection by at least one 5. pneumoniae serotype selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 23B, 24B, 25A, 34, 35F, 38, 40 and 42.

31. Immunogenic composition for use according to any one of claims 28-30, wherein said 5. pneumoniae infection is selected from an infection by at least one 5. pneumoniae serotype selected from the group consisting of serotype 35B, 15A, 6C, 7C, 23A, 24B, 25A, 34, 35F, 38, 40 and 42.

32. Kit of parts, comprising- at least one outer membrane vesicle displaying on its outer surface at least one polypeptide as defined in claim 1 a) or claim 2 a);- at least one outer membrane vesicle displaying on its outer surface at least one polypeptide as defined in claim 1 b) or claims 2 b); and instructions for use.

33. Kit of parts, comprising- an immunogenic composition comprising a first and second component as defined in any one of claims 2-3 wherein said first and second component comprise coupling moieties of a coupling system as defined in any one of claims 10-16;- at least one outer membrane vesicle as defined in any one of claims 17-24 which displays on the surface at least one complementary coupling moiety of the coupling system as defined in any one of claims 10-16; and- instructions for use.

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