Immunogenic fusion proteins

Abstract

The present invention relates to an immunogenic fusion protein comprising or consisting of an amino acid sequence comprising: i. a first amino acid sequence portion consisting of 170-178 amino acids, preferably 174-175 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7; ii. a second amino acid sequence portion consisting of 165-174 amino acids, preferably 169-170 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 14; and optionally, iii. a linker amino acid sequence portion consisting of 1-20 amino acids and separating the first amino acid sequence portion from the second amino acid sequence portion. The immunogenic fusion protein preferably consists of 335-372 amino acids, preferably 343-353 amino acids, more preferably 343-347 amino acids. The present invention further relates to a nucleic acid molecule, a vector, a host cell, a vaccine, and a method of vaccinating against or treating a group B Streptococcus infection encoding the immunogenic fusion protein. It is proposed that Figure 3 be published together with the abstract.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the fields of microbiology and vaccine technology and to the development of immunogenic fusion proteins capable of conferring immunity against group B Streptococcus infections. More specifically, the present invention relates to immunogenic fusion proteins that confer immunity against invasive strains of group B Streptococcus. The present invention further relates to nucleic acid molecules encoding the immunogenic fusion proteins, vectors, host cells, vaccines, and methods of vaccinating against or treating group B Streptococcus infections. [Background technology]

[0002] 2. Background of the Invention Group B Streptococcus (Streptococcus agalactiae) (GBS) is the leading cause of invasive bacterial infections, including meningitis, in the neonatal period. Today, in the United States alone, approximately 5,000 cases of invasive disease are caused by this bacterium annually. The overall mortality rate from this infection is approximately 10%, and many of the children who survive have permanent neurological sequelae. In light of this, there have been significant efforts to find ways to prevent and treat it, and to understand the mechanism of infection caused by GBS.

[0003] GBS can also cause mastitis in cattle, a bovine disease of significant economic importance, so the development of a vaccine against GBS infection is also of interest in veterinary medicine.

[0004] Approximately 20% of all women are vaginal carriers of GBS, and vertical transmission from the maternal genital tract is probably the most common source of neonatal disease caused by this bacterium. However, only about 1% of children colonized with GBS at birth develop serious infections. Therefore, other factors besides exposure to the bacterium at birth must contribute to the development of neonatal disease.

[0005] Group B Streptococcus strains are divided into nine serotypes (Ia, Ib, and II-VIII) based on the structure of the polysaccharide capsule. The four "classical" serotypes Ia, Ib, II, and III occur in approximately equal proportions among strains within normal flora, but type III is the most clinically important serotype, especially since it is responsible for most cases of meningitis. Since the capsule is a known virulence factor, it has been studied in some detail, especially in type III strains. Efforts are being made to develop a vaccine, in which the type III polysaccharide capsule is an essential component.

[0006] European Patent No. 0 866 133 discloses a vaccine capable of protecting a recipient against infections caused by group B Streptococcus. The invention relates to the combined use of polysaccharides with fragments of epsilon protein. The invention further discloses epidemiological data suggesting that type-specific capsules play an important role in immunity against group B Streptococcus infections (see page 7, lines 2-3). Furthermore, there are several different combinations between different proteins and polysaccharides described in the application, but all claims include polysaccharides indicating the importance of that particular component. However, the use of polysaccharide capsules as vaccines can be problematic due to cross-reactivity with human tissues (Pritchard et al., Infect Immun, 1992, vol. 60:1598). It would therefore be very useful to develop a vaccine based on proteins rather than polysaccharides.

[0007] Gravekamp et al., Infection and Immunity, December 1997, pp. 5216-5221, discloses an evaluation of the immunogenicity and protection of the repeat number and the N-terminal portion alone of the alpha (α)C protein. It was found that immunogenicity decreased with increasing repeat number (see FIG. 2B). However, it was also found in protection assays that antibodies against the N-terminal region were primarily responsible for protection compared to antibodies against the N-terminal region (see p. 5219, left column, line 6 from the bottom, and p. 5220, right column, lines 26-29).

[0008] WO 9410317 describes the use of alpha protein (GBS surface protein) in the development of a conjugate vaccine. A drawback of this protein is that it is not normally expressed by the type III strain, which is responsible for many severe GBS infections. Therefore, protective immunity against this strain is not induced by the alpha protein vaccine.

[0009] WO 9421685 describes the use of Rib protein (GBS surface protein) in the development of a vaccine. This protein induces immunity when administered with alum. However, Rib protein has the drawback of not eliciting protective immunity against any of the GBS strains.

[0010] Lindahl et al., Nonimmunodominant Regions Are Effective As Building Blocks In A Streptococcal Fusion Protein Vaccine, Cell Host & Microbe, Vol. 2, pp. 427-434, December 2007, discloses fusion proteins comprising the N-terminal regions of group B streptococcal surface proteins, Rib and AlpC.

[0011] Similarly, WO2008127179 describes a fusion protein comprising at least one first N-terminal region fragment of a Group B Streptococcus surface protein, or an analog, homolog, derivative, or immunologically related amino acid sequence, or fragment thereof, fused to at least one second N-terminal region fragment of a Group B Streptococcus surface protein, or an analog, homolog, derivative, or immunologically related amino acid sequence, or fragment thereof, wherein the first and second at least one N-terminal region fragment of the Group B Streptococcus surface protein are derived from different Group B Streptococcus lineages, and the fusion protein has the ability to induce protective immunity against Group B Streptococcus. Summary of the Invention [Problem to be solved by the invention]

[0012] Despite progress in moving toward a suitable vaccine for the prevention of GBS disease, there remains a need for additional methods and vaccines for preventing and treating GBS infections. Thus, there remains a need to explore vaccine strategies capable of inducing protective immunity against a broad range of GBS strains.

[0013] It is therefore a primary object of the present invention to provide immunogenic fusion proteins that can be used in vaccines capable of eliciting protective immunity against GBS infection.

[0014] It is a further object of the present invention to provide a vaccine that induces protective immunity against many clinically important GBS strains.

[0015] Another object of the present invention is to provide a vaccine comprising one or several immunogenic fusion proteins that induce protective immunity against GBS infection. A single or several proteins have several advantages over vaccines composed of numerous proteins, such as production costs and safety.

[0016] It is a further object of the present invention to provide immunogenic fusion proteins capable of providing improved protection against GBS.

[0017] The means by which each of the above objectives, as well as others, will become apparent from the description of the invention which follows. [Means for solving the problem]

[0018] Summary of the Invention The present invention is based on further studies by the inventors on the fusion proteins disclosed in WO2008127179. In particular, immunogenic fusion proteins comprising shorter amino acid sequences of the N-terminal regions of Rib and AlpC have been found to be suitable. Such immunogenic fusion proteins may be easier to produce and use for vaccination. Moreover, as shown in Examples 4 and 5, such shorter fusion proteins provide improved protection against GBS, in particular by providing improved protection against adhesion to and / or invasion of endothelial cells.

[0019] Additionally, a corresponding codon-optimized nucleic acid molecule has been devised that encodes an immunogenic fusion protein, which may be provided within a vector. A host cell may then express the immunogenic fusion protein or contain the vector. Each of the immunogenic fusion proteins, nucleic acid molecules, vectors, and host cells may be used in a vaccine. The immunogenic fusion proteins, nucleic acid molecules, vectors, host cells, and / or vaccines may further be used in a method of vaccinating against or treating a group B Streptococcus infection.

[0020] Thus, a first aspect of the present invention provides a method for producing a composition comprising the steps of: i. a first amino acid sequence portion consisting of 170 to 178 amino acids, preferably 174 to 175 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7; ii. a second amino acid sequence portion consisting of 165 to 174 amino acids, preferably 169 to 170 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 14; and optionally iii. a linker amino acid sequence portion consisting of 1 to 20 amino acids and separating the first amino acid sequence portion from the second amino acid portion; An immunogenic fusion protein comprising or consisting of an amino acid sequence consisting of: The present invention relates to an immunogenic fusion protein consisting preferably of 335 to 372 amino acids, preferably of 343 to 353 amino acids, more preferably of 343 to 347 amino acids.

[0021] As pointed out above, immunogenic fusion proteins may be easier to produce and use for vaccination. Immunogenic fusion proteins have the advantage that they are immunogenic without the use of adjuvants, however, they can also be used with adjuvants to increase immunogenicity, such as alum or aluminum hydroxide (Al(OH)3).

[0022] Another advantage of the immunogenic fusion proteins is that they can be combined with further immunogenic fusion proteins comprising the N-terminal regions of Alp1 and Alp2. As seen in the examples, the combination of two such immunogenic fusion proteins leads to a synergistic effect on the number of antibodies obtained (antibody titer) when used together in immunization.

[0023] The invention is described in more detail below. [Brief description of the drawings]

[0024] [Figure 1]FIG. 1 shows the fold increase in IgG obtained 29 days after immunization using a single administration of an immunogenic fusion protein according to the first aspect of the invention, either alone or in combination with a further immunogenic fusion protein comprised in a vaccine according to the fifth aspect of the invention, with / without adjuvant, at different dosages. [Diagram 2] FIG. 1 shows the fold increase in IgG obtained on day 57 after immunization using the first and second doses of an immunogenic fusion protein according to the first aspect of the invention either alone or in combination with a further immunogenic fusion protein comprised in a vaccine according to the fifth aspect of the invention, with / without adjuvant, at different dosages. [Diagram 3] Figure 1 shows mouse pup survival following neonatal bacterial infection with group B Streptococcus strain BM110 (8x103 cfu). Pups were born from female mice immunized with either a) immunogenic fusion protein of SEQ ID NO:21 + adjuvant (aluminum hydroxide (Al(OH)3)), b) or control (adjuvant (aluminum hydroxide) only). [Figure 4A] FIG. 2 shows the results obtained in a human epithelial cell invasion assay using clinical isolates and antisera obtained from immunization with the immunogenic fusion protein of SEQ ID NO:21. [Figure 4B] FIG. 2 shows the results obtained in a human epithelial cell invasion assay using clinical isolates and antisera obtained from immunization with the immunogenic fusion protein of SEQ ID NO:21. [Figure 4C] FIG. 2 shows the results obtained in a human epithelial cell invasion assay using clinical isolates and antisera obtained from immunization with the immunogenic fusion protein of SEQ ID NO:21. [Figure 4D] FIG. 2 shows the results obtained in a human epithelial cell invasion assay using clinical isolates and antisera obtained from immunization with the immunogenic fusion protein of SEQ ID NO:21. [Figure 4E]FIG. 2 shows the results obtained in a human epithelial cell invasion assay using clinical isolates and antisera obtained from immunization with the immunogenic fusion protein of SEQ ID NO:21. [Figure 5A] FIG. 1 shows a drawing of a fusion protein based on WO2008127179, including a leader sequence based on SEQ ID NO: 43 and a shorter amino acid sequence of the fusion protein based on SEQ ID NO: 21. [Figure 5B] FIG. 1 shows a drawing of a fusion protein based on WO2008127179, including a leader sequence based on SEQ ID NO: 43 and a shorter amino acid sequence of the fusion protein based on SEQ ID NO: 21. [Figure 5C] FIG. 1 shows a drawing of a fusion protein based on WO2008127179, including a leader sequence based on SEQ ID NO: 43 and a shorter amino acid sequence of the fusion protein based on SEQ ID NO: 21. [Figure 6] FIG. 2 shows the interaction between the shorter fusion protein of SEQ ID NO: 21 and α1β1 integrin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention As used herein, unless otherwise specified, "a" or "an" means "one or more." Throughout this specification, any and all references are specifically incorporated by reference into this patent application.

[0026] As described above, the first aspect of the present invention is i. a first amino acid sequence portion consisting of 170 to 178 amino acids, preferably 174 to 175 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7; ii. a second amino acid sequence portion consisting of 165 to 174 amino acids, preferably 169 to 170 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 14; and optionally iii. a linker amino acid sequence portion consisting of 1 to 20 amino acids and separating the first amino acid sequence portion from the second amino acid portion; An immunogenic fusion protein comprising or consisting of an amino acid sequence consisting of: The present invention relates to an immunogenic fusion protein consisting preferably of 335 to 372 amino acids, preferably of 343 to 353 amino acids, more preferably of 343 to 347 amino acids.

[0027] The term "immunogenic" is intended to mean having the ability to induce an immune response. The immunogenic fusion protein of the present invention is immunogenic and is characterized by its ability to induce a protective immune response against GBS containing at least a surface protein (the N-terminal region of which is included in the immunogenic fusion protein). Inducing protective immunity against group B streptococci is understood to include inducing protective immunity against group B streptococci carrying at least one of the surface proteins Rib and AlpC. "Capable of" means that the immunogenic fusion protein induces protective immunity in an individual having an immune system when administered in a suitable dose to said individual. The immunogenic fusion protein is preferably capable of inducing protective immunity against group B streptococci.

[0028] The term "protective immunity" in the context of the present invention refers to the ability of serum antibodies and / or cytotoxic T cell responses elicited during immunization to protect (partially or fully) against disease caused by an infectious agent, such as Group B Streptococcus. That is, a vertebrate immunized with a vaccine of the present invention experiences limited growth and spread of Group B Streptococcus. To determine whether protective immunity is elicited by a fusion protein or vaccine, techniques well known to those skilled in the art can be used. For example, to determine whether immunization with a fusion protein or vaccine of the present invention elicits protective immunity against Group B Streptococcus infection, Group B Streptococcus can be challenged in an immunized test animal, and the growth and spread of Group B Streptococcus is measured. For example, to determine whether protective immunity is elicited, a method based on the method described in the Examples below can be used.

[0029] For the purposes of the present invention, the term "fusion protein" refers to an assembly of two or more protein regions or fragments thereof. As described above, an immunogenic fusion protein comprises (or preferably consists of) an amino acid sequence consisting of at least a first and a second amino acid sequence portion, which are associated with the N-terminal regions of the group B Streptococcus surface proteins Rib and AlpC, respectively.

[0030] Combining amino acid sequence portions to obtain a fusion protein can be achieved by fusing, i.e. linking (covalently coupling, binding or linking or conjugating) the amino acid sequence portions together at one or both ends, or by linking together separate nucleic acid molecules encoding each amino acid sequence portion such that when the linked nucleic acid molecules are translated, a single continuous expression product is produced.

[0031] In the fusion protein, the first and second amino acid sequence portions may be separated by a linker amino acid sequence portion.

[0032] It is further envisaged in the context of the present invention that a fusion protein can be obtained by cross-linking the amino acid sequence parts or by entrapment thereof on a macromolecular structure.

[0033] For the purposes of the present invention, the term "protein" refers to a molecular chain of amino acids. A protein does not have a specific length and can be modified in vivo or in vitro, if necessary, for example by glycosylation, amidation, carboxylation, or phosphorylation. In particular, peptides, oligopeptides, and polypeptides are included in the definition.

[0034] The term "N-terminal region" in the context of the present invention refers to the N-terminal region (N) of a protein.

[0035] Group B Streptococcus strains, also referred to herein as GBS, are known and can be isolated from the blood of infected humans. GBS is the most common cause of neonatal sepsis in the United States and is responsible for approximately 5,000 cases annually.

[0036] The designation "Group B Streptococcus" comes from the fact that streptococci are divided into immunological groups based on the presence of specific carbohydrate antigens on their cell surfaces. Currently, groups A through O are recognized.

[0037] The Rib protein of group B Streptococcus, also referred to herein as Rib and Rib protein, is a surface protein known in the art and described, for example, in WO 9421685. The designation "Rib" stands for protease resistance, immunity, and group B. The Rib protein was first isolated as a characteristic 95 kDa protein from group B Streptococcus strains of serotype III. The Rib protein is expressed by almost all group B Streptococcus strains that fall into the clinically important serotype III, which is responsible for most cases of meningitis, and by several lineages of other serotypes, such as II. In addition, Rib is expressed by all lineages of the hypervirulent clone of type III. Methods have been devised for purifying the Rib protein, and antibodies against this protein have been demonstrated to protect against lethal infections with strains expressing the Rib protein (for further details, e.g., DNA and protein sequences, see WO 9421685).

[0038] SEQ ID NO:7 shows the shorter N-terminal region of Rib: MAEVISGSAVTLNTNMTKNVQNGRAYIDLYDVKNGKIDPLQLITLNSPDLKAQYVIRQGGNYFTQPSELTTVGAASINYTVLKTDGSPHTKPDGQVDIINVSLTIYNSSALRDKIDEVKKKAEDPKWDEGSRDKVLISLDDIKTDIDNNPKTQSDIANKITEVTNLEKILVPRIP Nucleic acid DNA molecules encoding the amino acid sequence of SEQ ID NO:7 are represented by SEQ ID NOs:1, 3 and 5, where SEQ ID NO:3 is codon-optimized for expression in E. coli and SEQ ID NO:5 is codon-optimized for expression in Cricetulus griseus (Chinese hamster ovary) cells.

[0039] Corresponding nucleic acid RNA molecules encoding the amino acid sequence of SEQ ID NO:7 are represented by SEQ ID NOs:2, 4 and 6, where SEQ ID NO:4 is codon-optimized for expression in E. coli and SEQ ID NO:6 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0040] The difference between the shorter N-terminal region of Rib, i.e. SEQ ID NO:7, and the sequence presented for the N-terminus of Rib in WO2008127179 corresponds to a 5 amino acid long leading sequence GPLGS, SEQ ID NO:44.

[0041] The AlpC protein of group B Streptococcus, also known as the alpha protein, is a group B Streptococcus surface protein known in the art. WO 9410317 describes a conjugate vaccine composition comprising the alpha protein. The native group B Streptococcus AlpC precursor protein as described in WO 9410317 has a molecular weight of 108 kDa. Cleavage of the putative signal sequence of 41 amino acids results in a mature protein of 104 kDa. (Note: however, the signal sequence was subsequently found to be 56 amino acid residues in length: Stalhammar-Carlemalm et al., J Exp Med 177:1593; 1993). The 20 kDa N-terminal region of the AlpC antigen shows no homology to previously described protein sequences and is followed by a series of nine tandemly repeated units that account for 74% of the mature protein. Each repeat unit (represented herein as "R") is identical and consists of 82 amino acids with a molecular mass of approximately 8500 daltons, encoded by 246 nucleotides. The C-terminal region of the AlpC antigen contains a cell wall anchor domain motif present in several Gram-positive surface proteins.

[0042] SEQ ID NO: 14 shows the shorter N-terminal region of AlpC: STIPGSAATLNTSITKNIQNGNAYIDLYDVKLGKIDPLQLIVLEQGFTAKYVFRQGTKYYGDVSQLQSTGRASLTYNIFGEDGLPHVKTDGQIDIVSVALTIYDSTTLRDKIEEVRTNANDPKWTEESRTEVLTGLDTIKTDIDNNPKTQTDIDSKIVEVNELEKLLVLS The difference between the shorter N-terminal region of AlpC, i.e. SEQ ID NO: 14, and the sequence presented for the N-terminus of AlpC in WO2008127179 corresponds to the 5 amino acid long leader sequence GPLGS, SEQ ID NO: 44.

[0043] Nucleic acid DNA molecules encoding the amino acid sequence of SEQ ID NO:14 are represented by SEQ ID NOs:8, 10 and 12, where SEQ ID NO:10 is codon-optimized for expression in E. coli and SEQ ID NO:12 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0044] Corresponding nucleic acid RNA molecules encoding the amino acid sequence of SEQ ID NO:14 are represented by SEQ ID NOs:9, 11 and 13, where SEQ ID NO:11 is codon-optimized for expression in E. coli and SEQ ID NO:13 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0045] The term "sequence identity" refers to a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. If the lengths of the two sequences to be compared are not equal, the two sequences must be aligned to obtain the best possible fit. Sequence identity can be calculated, for example, by BLAST programs, such as BLASTP or BLASTN programs (Pearson WR and DJ Lipman (1988) PNAS USA, vol. 85:2444-2448) (www.ncbl.nlm.nlh.gov / BLAST).

[0046] Preferably, the immunogenic fusion protein consists of an amino acid sequence: a first amino acid sequence portion consisting of 170 to 178 amino acids, preferably 174 to 175 amino acids, having at least 90% sequence identity with the full-length amino acid sequence shown in SEQ ID NO:7.

[0047] The second amino acid sequence portion consisting of 165 to 174 amino acids, preferably 169 to 170 amino acids, has at least 90% sequence identity with the full-length amino acid sequence shown in SEQ ID NO:14.

[0048] The first amino acid sequence portion should consist of a maximum of 178 amino acids, for example 170-178 amino acids, preferably 174-175 amino acids. As shown, SEQ ID NO: 7 contains 175 amino acids. The first M in the sequence is optional, thus resulting in 174 amino acids in this case.

[0049] The first amino acid sequence portion has at least 90%, such as at least 95%, such as at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0050] The second amino acid sequence portion should consist of a maximum of 174 amino acids, for example 165-174 amino acids, preferably 169-170 amino acids. As shown, SEQ ID NO: 14 contains 170 amino acids. The first A in the sequence may be optional, thus resulting in 169 amino acids in this case.

[0051] The second amino acid sequence portion has at least 90%, such as at least 95%, such as at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:14.

[0052] The linker amino acid sequence portion is optional. In other words, if included, the linker amino acid sequence portion may consist of 0 to 20 amino acids. The amino acids in the linker amino acid sequence portion may be selected randomly, i.e. to obtain a random coil formation. If present, the linker amino acid sequence portion separates the first amino acid sequence portion from the second amino acid portion. In other words, the linker amino acid sequence portion is provided between the first amino acid sequence portion and the second amino acid portion.

[0053] One of the first and second amino acid sequence portions may be provided with a leading / following amino acid sequence portion located at an end furthest from the other of the first and second amino acid sequence portions, which may comprise one or more amino acids.

[0054] The immunogenic fusion protein preferably consists of 335 to 372 amino acids, preferably 343 to 353 amino acids, and more preferably 343 to 347 amino acids.

[0055] In a preferred embodiment of the immunogenic fusion protein according to the first aspect of the invention, - the first amino acid sequence portion comprises, or preferably consists of, amino acids 1 to 175 or 2 to 175 of the sequence represented by SEQ ID NO: 7, and the second amino acid sequence portion comprises, or preferably consists of, amino acids 1 to 170 or 2 to 170 of the sequence shown in SEQ ID NO:14;

[0056] SEQ ID NO: 7 contains 175 amino acids. Amino acids 2 to 175 correspond to the sequence of amino acids in SEQ ID NO: 7, except for the initial methionine (M).

[0057] In a preferred embodiment of the immunogenic fusion protein according to the first aspect of the present invention, the amino acid sequence comprises a linker amino acid sequence portion, and the linker amino acid sequence portion consists of 1 to 10 amino acids, preferably 2 to 5 amino acids, more preferably 2 amino acids, and most preferably the amino acid sequence EF.

[0058] When the linker amino acid sequence portion consists of the amino acids EF (glutamic acid-phenylalanine), the second amino acid sequence portion preferably comprises, or preferably consists of, amino acids 2 to 170 of the sequence shown in SEQ ID NO:14.

[0059] Preferably, the immunogenic fusion protein has at least 90% sequence identity with the entire amino acid sequence, preferably as set forth in SEQ ID NO:21.

[0060] In a preferred embodiment, the immunogenic fusion protein consists of the sequence of amino acids set forth in SEQ ID NO:21, or the immunogenic fusion protein consists of amino acids 2 to 347 of SEQ ID NO:21.

[0061] As shown in connection with the figures, such immunogenic fusion proteins are shorter than the immunogenic fusion proteins disclosed in WO2008127179, yet still have good efficacy and are suitable for use in vaccines against GBS.

[0062] Furthermore, as shown in Examples 4 and 5, such shorter amino acid sequences, by removing the leader sequence (SEQ ID NO: 43), are more effective and improved in providing protection against GBS, particularly by providing more effective and / or improved protection against adhesion to and / or invasion of endothelial cells.

[0063] SEQ ID NO:21 is the amino acid sequence: MAEVISGSAVTLNTNMTKNVQNGRAYIDLYDVKNGKIDPLQLITLNSPDLKAQYVIRQGGNYFTQPSELTTVGAASINYTVLKTDGSPHTKPDGQVDIINVSLTIYNSSALRDKIDEVKKKAEDPKWDEGSRDKVLISLDDIKTDIDNNPKTQSDIANKITEVTNLEKILVPR IPEFSTIPGSAATLNTSITKNIQNGNAYIDLYDVKLGKIDPLQLIVLEQGFTAKYVFRQGTKYYGDVSQLQSTGRASLTYNIFGEDGLPHVKTDGQIDIVSVALTIYDSTTLRDKIEEVRTNANDPKWTEESRTEVLTGLDTIKTDIDNNPKTQTDIDSKIVEVNELEKLLVLS Corresponds to.

[0064] The difference between the shorter fusion protein based on SEQ ID NO:21 and the sequence presented for the fusion protein in WO2008127179 is the 32 amino acid long leading sequence: GPLGSASVLIGISFLGGFTQGQFNISTDTVFA-SEQ ID NO:43, a sequence not found in SEQ ID NO:21.

[0065] In further embodiments of the immunogenic fusion protein according to the first aspect of the invention, the immunogenic fusion protein is modified by glycosylation, amidation, carboxylation, or phosphorylation, or by conjugation to a capsular polysaccharide or RSV antigen as further described with respect to the third aspect of the invention below.

[0066] This is advantageous because such fusion proteins may have enhanced immunogenicity. Numerous techniques are available and well known to those skilled in the art that may be used without undue experimentation to substantially increase the immunogenicity of a fusion protein. For example, the fusion protein may be modified by coupling with dinitrophenol groups or arsanilic acid, or by denaturation with heat and / or SDS. Alternatively, the fusion protein may be coupled to an immunogenic carrier. For a review of some general considerations in coupling strategies, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988). Useful immunogenic carriers are well known in the art. Examples of such carriers are keyhole limpet hemocyanin (KLH); albumins, such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated charcoal; or bentonite.

[0067] In a further embodiment, the immunogenic fusion protein is conjugated to a capsular polysaccharide, preferably a bacterial polysaccharide, more preferably a group B Streptococcus polysaccharide.

[0068] By polysaccharide is meant any linear or branched polymer of monosaccharide residues usually linked by glycosidic bonds, and thus includes oligosaccharides. Preferably, polysaccharides contain 2 to 50 monosaccharide units, more preferably 6 to 30 monosaccharide units.

[0069] The polysaccharide component may be based on or derived from the polysaccharide component of the polysaccharide capsule obtained from a number of Gram-positive and Gram-negative bacterial pathogens, such as H. influenzae, N. meningitidis, and S. pneumoniae. Other bacterial sources of polysaccharide components that may be conjugated to the fusion proteins of the invention include Staphylococcus aureus, Klebsiella, Pseudomonas, Salmonella typhi, Pseudomonas aeruginosa, and Shigella dysenteriae. Suitable polysaccharide components for use in accordance with this aspect of the invention include Hib oligosaccharides, lipopolysaccharides from Pseudomonas aeruginosa, lipopolysaccharides from Salmonella, and O-specific polysaccharides from Shigella dysenteriae.

[0070] Other polysaccharide components suitable for use in accordance with the present invention will be known to those skilled in the art.

[0071] Fragments of bacterial capsular polysaccharides may be generated by any suitable method, such as by acid hydrolysis or ultrasonic irradiation, etc. Other methods of preparing polysaccharide components are well known to those skilled in the art.

[0072] The polysaccharide is preferably a capsular polysaccharide from group B Streptococcus or an equivalent thereof. The polysaccharide should be coupled to the fusion protein, preferably by a covalent bond. A particularly preferred method for coupling polysaccharides to proteins is reductive amination. Other methods include activation of the polysaccharide with cyanogen bromide followed by reaction with adipic acid dihydrazide (spacer) and conjugation to the carboxylate groups of the carrier protein using a soluble carbodiimide; functionalization of the carrier protein with adipic acid dihydrazide followed by coupling with cyanogen bromide-activated polysaccharides; chemical modification of both the carrier protein and the polysaccharide followed by coupling thereof.

[0073] The polysaccharide molecule may be coupled to the fusion protein by a spacer molecule, such as adipic acid, which can be used to facilitate coupling of the fusion protein to the polysaccharide. After performing the coupling reaction, the conjugate can be purified by diafiltration or other known methods to remove unreacted protein or polysaccharide components.

[0074] If the polysaccharide is derived from a bacterial pathogen other than GBS, the conjugate may elicit immunity against more than one pathogen, e.g., multiple types of bacteria, a potentially important application of immunogenic fusion proteins.

[0075] A second aspect of the invention relates to a nucleic acid molecule comprising a polynucleotide encoding an immunogenic fusion protein according to the first aspect of the invention.

[0076] Such nucleic acid molecules can be used in producing immunogenic fusion proteins, for example directly in the cell culture from which the immunogenic fusion protein is isolated and obtained, or indirectly within a subject in which the immunogenic fusion protein is expressed, for example for immunization.

[0077] The nucleic acid molecule can be, for example, a DNA molecule or an RNA molecule. The RNA molecule can be an mRNA molecule.

[0078] The nucleic acid molecule may comprise, or preferably consist of, a first polynucleotide having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to one of SEQ ID NOs: 1-6 fused, directly or via a linker polynucleotide sequence, to a second polynucleotide having at least 90%, for example at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to one of SEQ ID NOs: 8-13.

[0079] The linker polynucleotide sequence is optional, but is included where the immunogenic fusion protein encoded by the nucleic acid molecule has a linker provided between the first and second amino acid sequence portions, where the first polynucleotide is directly fused to the second polynucleotide, there is no linker polynucleotide sequence that would be present in the nucleic acid molecule.

[0080] Each of the above-cited SEQ ID NOs:1-6 and 8-13 may be optionally truncated by removing the first three bases which code for an initial methionine (M) in the first amino acid sequence portion and an initial alanine (A) in the second amino acid sequence portion of the immunogenic fusion protein encoded by the nucleic acid molecule.

[0081] The nucleic acid molecule may optionally include one or more stop codons. The nucleic acid molecule may optionally include one or more start codons.

[0082] The nucleic acid molecule may be codon optimized for expression in a particular organism, such as E. coli or Cricetulus griseus (Chinese hamster ovary) cells.

[0083] In a preferred embodiment of the nucleic acid molecule according to the second aspect of the invention, the nucleic acid molecule has at least 90% sequence identity with a sequence of nucleotides represented by one of SEQ ID NOs: 15 to 20. Preferably, the nucleic acid molecule has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity with a sequence of nucleotides represented by one of SEQ ID NOs: 15 to 20. Each of SEQ ID NOs: 15 to 20 cited in these preferred embodiments may be optionally truncated by removing the first three bases that code for an initial methionine (M) in the first amino acid sequence portion of the immunogenic fusion protein encoded by the nucleic acid molecule. Furthermore, each of SEQ ID NOs: 15 to 20 cited in these preferred embodiments may be optionally truncated by removing the last 1 to 6 bases of the two stop codons. This may be used, for example, when the nucleic acid molecule is used as a DNA or RNA vaccine - transcription and / or translation stops at the ends of the nucleic acid molecule.

[0084] The nucleic acid molecule may preferably consist of 1044 (excluding the initial M) to 1047 (including the initial M) bases.

[0085] A third aspect of the invention relates to a vector comprising a nucleic acid molecule according to the second aspect of the invention.

[0086] A wide variety of expression host / vector combinations may be employed in expressing the nucleic acid molecules. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Useful expression vectors for bacterial hosts include bacterial plasmids such as those derived from E. coli, including pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof, broader host plasmids such as RP4, phage DNA, including numerous derivatives of phage lambda, such as lambda GT10 and lambda GT11, NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells include the 2.mu. plasmid and its derivatives. Useful vectors for insect cells include pVL941.

[0087] In addition, any of a wide variety of expression control sequences may be used to express the nucleic acid molecules in these vectors. Useful expression control sequences include those associated with the structural genes of the expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter regions of phage lambda, the control region of the fd coat protein, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters, e.g., Pho5, the yeast alpha mating system promoter, and other constitutive and inducible promoter sequences known to control the expression of such genes in prokaryotic or eukaryotic cells, or viruses thereof, and various combinations thereof.

[0088] A fourth aspect of the invention relates to a host cell which expresses an immunogenic fusion protein according to the first aspect of the invention or which comprises a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0089] The host cell may be a gram-negative bacterial cell, a gram-positive bacterial cell, a yeast cell, an insect cell, an animal cell, an African green monkey cell, a human cell, or a plant cell. The host cell may be selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, Aspergillus, Lactobacillus, Shigella, Salmonella, Listeria, Streptococcus, Staphylococcus, and fungi. However, E. coli is preferred.

[0090] Additionally, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, African green monkey cells such as COS1, COS7, BSC1, BSC40, and BMT10, human cells, and plant cells in tissue culture may be used. Preferred host organisms include bacteria such as E. coli and B. subtilis, and mammalian cells in tissue culture.

[0091] When the host cell is used to produce an immunogenic fusion protein, or to produce the first and second amino acid sequence portions thereof, or to produce a linker amino acid sequence portion, the produced amino acid sequence portion can be isolated from the microbial culture or cell culture and purified using any of a variety of conventional methods, including liquid chromatography (e.g., normal phase or reverse phase, etc.) using HPLC, FPLC, etc.; affinity chromatography (e.g., with inorganic ligands or monoclonal antibodies, etc.); ion exchange chromatography, size exclusion chromatography; immobilized metal chelate chromatography; gel electrophoresis; tangential flow filtration, etc. One skilled in the art can select the most suitable isolation and purification techniques without departing from the scope of the invention.

[0092] In addition, the immunogenic fusion protein, its first and second amino acid sequence portions, or the linker amino acid sequence portion may be produced by any of a number of chemical techniques, for example, they may be prepared using solid phase synthesis techniques or may be prepared by synthesis in solution.

[0093] A fifth aspect of the invention relates to a vaccine comprising one or more of an immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention.

[0094] The amount of each of the immunogenic fusions, nucleic acid molecules, vectors, and / or host cells is preferably a pharma- ceutically effective amount, or an amount capable of inducing protective immunity against group B Streptococcus, or an amount sufficient to induce an immune response. Further, the amount can be, for example, an amount sufficient to reduce, and most preferably prevent, a clinically significant defect in activity and response in a vaccinated subject. Alternatively, the amount is an amount sufficient to cause an improvement in a clinically significant condition in the host or subject.

[0095] As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dosage may contain a predetermined amount of active composition calculated in association with the required diluent; i.e., carrier or additive, to produce the desired therapeutic effect. Furthermore, the dosage administered will vary depending on the active ingredient(s) used, the age, weight, etc. of the individual to be treated. The vaccine preferably has the ability to induce protective immunity against group B Streptococcus.

[0096] As indicated in the figure, the immunogenic fusion protein elicits an immune response when administered to a subject. Such an immune response can also be obtained indirectly by administering a nucleic acid molecule, either as a DNA nucleic acid molecule that is subsequently transcribed and translated to express the immunogenic fusion protein, or as an RNA or mRNA nucleic acid molecule that is translated into and expresses the immunogenic fusion protein. The nucleic acid molecule can be provided within a vector to enhance control of transcription and / or translation, which in turn can be provided within a host cell to further control and / or increase expression of the immunogenic fusion protein.

[0097] The vaccine may further comprise a pharma- ceutically acceptable vehicle and, optionally, an adjuvant. The term "pharma- ceutically acceptable vehicle" is intended to mean any suitable acceptable excipient, adjuvant, carrier, diluent commonly used in pharmaceutical preparations.

[0098] The vaccine may include a vaccine composition. Vaccines may further contain other pharmacologically acceptable components, such as salts, buffers, immunologically active components, adjuvants (AlOH), wetting agents, emulsifying and suspending agents, or sweetening, flavoring, perfuming agents, or other substances desirable to improve the effectiveness of the composition, etc. A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient individual.

[0099] Adjuvants are substances that can be used to specifically enhance a particular immune response. Usually, the adjuvant and the composition are mixed before being presented to the immune system, or are provided separately, but in the same site of the animal or human being to be immunized. Adjuvants can be roughly divided into several groups based on their composition. Such groups include oil adjuvants (e.g., Freund's complete and incomplete adjuvants), inorganic salts such as AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), AlOH, silica, kaolin, and carbon), polynucleotides (e.g., poly IC and poly AU acids), and certain natural substances (e.g., wax D from Mycobacterium tuberculosis), as well as substances found in Corynebacterium parvum or Bordetella pertussis, and members of the Brucella genus.

[0100] Alhydrogel (e.g. aluminium hydroxide, Al(OH)3) is a particularly effective adjuvant for the immunogenic fusion proteins according to the first aspect of the invention and is therefore particularly useful in vaccines according to the fifth aspect of the invention.

[0101] Methods for preparing and formulating vaccines and vaccine compositions are well known to those skilled in the art. The choice of ingredients varies, for example, depending on the route of administration of the composition. For example, compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally include liposome solutions containing liquid dosage forms. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water.

[0102] The vaccines may be administered to humans or animals, including mammals and birds, such as rodents (mice, rats, guinea pigs, or rabbits); birds (turkeys, hens, or chickens); other livestock (cattle, horses, pigs, or piglets); pets (dogs, cats, and other pets); and humans. Although many animals may be treated with the vaccines of the present invention, the preferred individuals to be treated are humans or commercially useful animals and livestock, such as fish, e.g., tilapia and camels.

[0103] The vaccine can be administered to an individual according to methods known in the art. Such methods include, for example, parenteral, for example, injection into or through the skin, application through all routes: for example, intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal, or subcutaneous. Such methods may also be applied by topical administration as drops, sprays, gels, or ointments on the mucosal epithelium of the eye, nose, mouth, anus, or vagina, or on the epidermis of the external skin located in any part of the body. Another possible application route is by application of sprays, aerosols, or powders through inhalation via the respiratory tract. In this last case, the particle size used determines how deep the particles penetrate into the respiratory tract. Alternatively, application may be via the digestive route, for example, by combining with food, feed, or drinking water as a powder, liquid, or tablet, or by administering directly into the mouth as a liquid, gel, tablet, or capsule, or into the anus as a suppository.

[0104] Many different techniques exist for timing immunizations. It is possible to administer the vaccine or components a-d more than once to increase the level and diversity in expression of the immunoglobulin repertoire expressed by the immunized animal. Generally, when multiple immunizations are performed, the immunizations may be performed at intervals of 1 week to 3 months, such as at intervals of 2 weeks to 2 months, such as at intervals of 1-2 months, or at intervals of 4 weeks.

[0105] Furthermore, one or possibly more booster injections of the vaccine or components a-d may be administered. Preferably, the booster injections may be administered about 1 to 6 months after the initial injection(s).

[0106] A vaccine according to the fifth aspect of the invention may preferably comprise one or more additional immunoactive components, which may be an antigen, an immune enhancer and / or a vaccine, any of which may include an adjuvant.

[0107] Thus, in a preferred embodiment of the vaccine according to the fifth aspect of the invention, the vaccine comprises a) a further immunogenic fusion protein comprising or consisting of an amino acid sequence having at least 90%, such as 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42; b) a further nucleic acid molecule encoding a further immunogenic fusion protein, which preferably has at least 90% sequence identity with a nucleotide sequence represented by one of SEQ ID NOs: 36 to 41; c) a further vector comprising a further nucleic acid molecule; or d) a further host cell expressing a further immunogenic fusion protein or containing a further vector The present invention further includes one or more of the following:

[0108] As discussed further below, the further immunogenic fusion proteins are derived from the N-terminal regions of GBS surface proteins Alp1 and Alp2 / 3, as also disclosed in WO 2017 / 068112. As shown in the figures and discussed below, synergistic effects are observed when using a vaccine comprising both the immunogenic fusion protein and the further immunogenic fusion protein.

[0109] Moreover, by including one or more of the additional components a-d, the vaccine may provide broader protection against all group B Streptococcus lineages.Similar to what is described above for immunogenic fusion proteins, further immunogenic fusion proteins may be expressed indirectly by further nucleic acid molecules, further vectors, and further host cells.

[0110] The amount of each of a-d should preferably be a pharma- ceutically effective amount, or an amount capable of inducing protective immunity against group B Streptococcus, or an amount sufficient to induce an immune response, or at least an amount sufficient to increase the general immune response to the vaccine.

[0111] As described above, further immunogenic fusion proteins have at least 90%, such as 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence depicted in SEQ ID NO:42, which has the amino acid sequence: MAEVISGSAATLNSALVKNVSGGKAYIDIYDVKNGKIDPLNLIVLTPSNYSANYYIKQGGRIFTSVNQLQTPGTATITYNILDENGNPYTKSDGQIDIVSLVTTVYDTTELRNNINKVIENANDPKWSDDSRKDVLSKIEVIKNDIDNNPKTQSDIDNKIVEVNELEKLLVLP EFSTIPGSAATLNTSITKNIQNGNAYIDLYDVKNGLIDPQNLIVLNPSSYSANYYIKQGAKYYSNPSEITTTGSATITFNILDETGNPHKKADGQIDIVSVNLTIYDSTALRNRIDEVINNANDPKWSDGSRDEVLTGLEKIKKDIDNNPKTQIDIDNKINEVNEIEKLLVVSL Represents.

[0112] Nucleic acid DNA molecules encoding the amino acid sequence of SEQ ID NO:42, i.e., the nucleic acid DNA molecules encompassed in (b), are represented by SEQ ID NOs:36, 38, and 40, where SEQ ID NO:38 is codon-optimized for expression in E. coli, and SEQ ID NO:40 is codon-optimized for expression in Cricetulus griseus (Chinese hamster ovary) cells.

[0113] Corresponding nucleic acid RNA molecules encoding the amino acid sequence of SEQ ID NO:42 are represented by SEQ ID NOs:37, 39, and 41, where SEQ ID NO:39 is codon-optimized for expression in E. coli and SEQ ID NO:41 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0114] As mentioned above, further immunogenic fusion proteins are derived from the N-terminal regions of the GBS surface proteins Alp1 and Alp2 / 3.

[0115] The Alp1 protein is also known as the epsilon protein and is a group B streptococcal alpha protein-like protein.

[0116] The amino acid sequence of the N-terminal region of Alp1 is SEQ ID NO:28: MAEVISGSAATLNSALVKNVSGGKAYIDIYDVKNGKIDPLNLIVLTPSNYSANYYIKQGGRIFTSVNQLQTPGTATITYNILDENGNPYTKSDGQIDIVSLVTTVYDTTELRNNINKVIENANDPKWSDDSRKDVLSKIEVIKNDIDNNPKTQSDIDNKIVEVNELEKLLVLP Presented by.

[0117] Nucleic acid DNA molecules encoding the amino acid sequence of SEQ ID NO:28 are represented by SEQ ID NOs:22, 24, and 26, where SEQ ID NO:24 is codon-optimized for expression in E. coli and SEQ ID NO:26 is codon-optimized for expression in Cricetulus griseus (Chinese hamster ovary) cells.

[0118] Corresponding nucleic acid RNA molecules encoding the amino acid sequence of SEQ ID NO:28 are represented by SEQ ID NOs:23, 25, and 27, where SEQ ID NO:25 is codon-optimized for expression in E. coli and SEQ ID NO:27 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0119] The Alp2 protein is another alpha protein-like protein. Like other family members, the Alp2 protein has an N-terminal domain and several repeated domains toward the C-terminus. The N-terminal regions of Alp2 and Alp3 are identical. Alp3 is another alpha protein-like protein, also known as R28. Alp3 is very similar to the R28 protein, which is also found in S.pyogenes.

[0120] The amino acid sequence of the N-terminal region of Alp2 is SEQ ID NO:35: STIPGSAATLNTSITKNIQNGNAYIDLYDVKNGLIDPQNLIVLNPSSYSANYYIKQGAKYYSNPSEITTTGSATITFNILDETGNPHKKADGQIDIVSVNLTIYDSTALRNRIDEVINNANDPKWSDGSRDEVLTGLEKIKKDIDNNPKTQIDIDNKINEVNEIEKLLVVSL Presented by.

[0121] Nucleic acid DNA molecules encoding the amino acid sequence of SEQ ID NO:35 are represented by SEQ ID NOs:29, 31, and 33, where SEQ ID NO:31 is codon-optimized for expression in E. coli and SEQ ID NO:33 is codon-optimized for expression in Cricetulus griseus (Chinese hamster ovary) cells.

[0122] Corresponding nucleic acid RNA molecules encoding the amino acid sequence of SEQ ID NO:35 are represented by SEQ ID NOs:30, 32, and 34, where SEQ ID NO:32 is codon-optimized for expression in E. coli and SEQ ID NO:34 is codon-optimized for expression in Cricetulus griseus (Chinese Hamster Ovary) cells.

[0123] SEQ ID NO: 42 therefore corresponds to SEQ ID NO: 28 (N-terminus of Alp1) fused to SEQ ID NO: 35 (N-terminus of Alp2 / 3) via a linker amino acid sequence part consisting of the amino acids EF.

[0124] The second immunogenic fusion protein is i. a further first amino acid sequence portion having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 28; ii. a second additional amino acid sequence portion having at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO: 35, and optionally iii. a further linker amino acid sequence portion consisting of 1 to 20 amino acids and separating the further first amino acid sequence portion from the further second amino acid portion; It may comprise or consist of an amino acid sequence consisting of:

[0125] In each of SEQ ID NOs: 28 and 42, the initial M may be optional. Thus, in a preferred embodiment of the vaccine according to the fifth aspect of the invention, the vaccine comprises a) an immunogenic fusion protein according to the first aspect of the present invention, which comprises an amino acid sequence represented by SEQ ID NO:21, or an immunogenic fusion protein comprising amino acids 2 to 347 of SEQ ID NO:21; b) a further immunogenic fusion protein comprising, and preferably consisting of, the sequence of amino acids set forth in SEQ ID NO: 42, or comprising, and preferably consisting of, amino acids 2 to 347 of SEQ ID NO: 42; Includes.

[0126] In such embodiments, preferably no other proteins or amino acid sequences different from the defined immunogenic fusion protein and the defined further immunogenic fusion protein are present in the vaccine.

[0127] In a preferred embodiment of the vaccine according to the fifth aspect of the invention, the vaccine further comprises aluminium hydroxide as an adjuvant, in such an embodiment the vaccine is preferably a pharmaceutical composition and further comprises a pharma- ceutically acceptable excipient, diluent or carrier.

[0128] A sixth aspect of the invention relates to an immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a vaccine according to the fifth aspect of the invention for use in i) vaccination against or ii) treatment of a group B Streptococcus infection.

[0129] The immunogenic fusion proteins, nucleic acid molecules, vectors, host cells, or vaccines may be used to effect such vaccination or treatment by administering the immunogenic fusion proteins, nucleic acid molecules, vectors, host cells, or vaccines to a subject in need thereof.

[0130] A corresponding seventh aspect of the invention relates to a method of i) vaccinating against or ii) treating a group B Streptococcus infection by administering to a subject in need thereof an immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a vaccine according to the fifth aspect of the invention.

[0131] A corresponding eighth aspect of the invention relates to the use of an immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention for the manufacture of a pharmaceutical composition or medicament, such as a vaccine, for i) vaccination against group B Streptococcus infection or ii) treatment of group B Streptococcus infection.

[0132] An immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a vaccine according to the fifth aspect of the invention may therefore be used in a method of preventing or treating an infection caused by group B Streptococcus.

[0133] Maternal immunoprophylaxis with a vaccine has long been proposed as a potential route to protection against group B streptococcal infections in both mothers and young children.

[0134] Thus, in preferred embodiments of the sixth, seventh and eighth aspects of the invention, an immunogenic fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a vaccine according to the fifth aspect of the invention is administered to a female human subject.

[0135] Administration thus has the potential to confer immunity to GBS infection on the unborn children of human females. Additionally, administration can also be performed on males when they are elderly.

[0136] According to this embodiment, an immunogenic fusion protein, nucleic acid molecule, vector, host cell, or vaccine is administered to a non-pregnant or pregnant woman under conditions and in an amount sufficient to induce the production of antibodies (via passive transfer of antibodies across the placenta) that serve to protect both the woman and her fetus or newborn.

[0137] The term "preventing or treating" in its various grammatical forms in the context of the present invention refers to preventing, curing, reversing, attenuating, mitigating, ameliorating, inhibiting, minimizing, suppressing, or halting (1) the deleterious effects of a disorder associated with Group B Streptococcus infection, (2) the progression of the disorder, or (3) the disorder pathogen (Group B Streptococcus). Additionally, the term "preventing or treating" is intended to include building up an individual's total or partial immunity against Group B Streptococcus infection.

[0138] A ninth aspect of the invention relates to a method for preventing or treating an infection caused by Group B Streptococcus, comprising administering to an individual in need thereof an effective amount of antibodies elicited from exposing a second individual to an immunogenic fusion protein, nucleic acid molecule, vector, host cell or vaccine according to the first to fifth aspects of the invention.

[0139] According to this embodiment, resistance or immunity to group B Streptococcus is conferred to an individual by passive immunization, i.e., the induced antisera are collected and provided directly to a recipient suspected of having an infection caused by group B Streptococcus. It is envisioned that such antisera may be administered to pregnant women (at or before birth) under conditions of sufficient time and amount that the antisera serves to protect either the fetus or newborn (through passive transfer of antibodies across the placenta).

[0140] The vaccine or antisera of the invention may therefore be provided either before the onset of infection (to prevent or attenuate an anticipated infection) or after an actual infection has begun.

[0141] A tenth aspect of the invention provides a method for producing an immunogenic fusion protein, comprising the steps of: - providing a host cell according to the fourth aspect of the invention; - multiplying the host cells; - purifying the immunogenic fusion protein according to the first aspect of the invention; - obtaining an immunogenic fusion protein; The present invention relates to a method including the steps of:

[0142] Alternatively, immunogenic fusion proteins can be produced in cell-free expression systems, which contain all the necessary elements for expression from a recombinant nucleic acid of interest, operably linked to a promoter that functions in that particular system. EXAMPLES

[0143] Example 1 - Immunization of healthy women Healthy adult females were immunized with an immunogenic fusion protein consisting of SEQ ID NO:21, alone or together with a further immunogenic fusion protein based on SEQ ID NO:42, with or without adjuvant (aluminum hydroxide).

[0144] FIG. 1 shows the fold increase in IgG obtained on day 29 after immunization with a single dose of an immunogenic fusion protein according to the first aspect of the invention, either alone or in combination with a further immunogenic fusion protein comprised in a vaccine according to the fifth aspect of the invention, with / without adjuvant (aluminium hydroxide), at different dosages.

[0145] In FIG. 1, the first seven results were obtained by administering an immunogenic fusion protein consisting of SEQ ID NO:21 alone or together with adjuvant (aluminum hydroxide) (0.5 mg).

[0146] As can be seen from Figure 1, the use of aluminum hydroxide is beneficial as it boosts the IgG increase fold at the 10 μg dose to a value comparable to that of the 50 μg dose. Similarly, the 50 μg dose in combination with aluminum hydroxide achieves an increase fold comparable to that of the 250 μg dose.

[0147] Figure 1 further shows that when a vaccine containing aluminum hydroxide and both an immunogenic fusion protein based on SEQ ID NO: 21 and an additional immunogenic fusion protein based on SEQ ID NO: 42 is used, a dose of 25 μg of each of the immunogenic fusion proteins achieves a similar or higher fold increase compared to a dosage of 250 μg of the immunogenic fusion protein based on SEQ ID NO: 21. The highest fold increase was achieved on day 29 using aluminum hydroxide and 50 μg of each immunogenic fusion protein. The number of subjects (n) for each experiment is presented in the figure.

[0148] FIG. 2 shows the fold IgG increase obtained on day 57 after immunization with the first and second doses of an immunogenic fusion protein according to the first aspect of the invention, either alone or in combination with a further immunogenic fusion protein comprised in a vaccine according to the fifth aspect of the invention, with / without adjuvant, at different dosages.

[0149] In FIG. 2, the first seven experiments are obtained by administering an immunogenic fusion protein consisting of SEQ ID NO: 21 alone or together with 0.5 mg of adjuvant (aluminum hydroxide).

[0150] As can be seen from Figure 2, the administration of an immunogenic fusion protein consisting of SEQ ID NO: 21 together with aluminum hydroxide in two doses of 50 μg and 100 μg results in an approximately 100-fold increase in immunogenicity. However, when using a vaccine containing aluminum hydroxide and both an immunogenic fusion protein based on SEQ ID NO: 21 and a further immunogenic fusion protein based on SEQ ID NO: 42, a higher increase in immunogenicity is observed, even though the doses of the respective immunogenic fusion proteins are only 25 μg or 50 μg.

[0151] The number of subjects (n) for each experiment is presented within the figure. Example 2 - Immunization of female mice to protect their young Figure 3 shows the group B streptococcus strain BM110 (8 × 10 3Figure 3 shows the survival rate of mouse pups after neonatal bacterial infection with 10000 cfu of GBS. Pups were born to female mice immunized with either a) the immunogenic fusion protein of SEQ ID NO: 21 in combination with adjuvant (aluminum hydroxide (Al(OH)3)), b) or control (adjuvant (aluminum hydroxide) only). As can be seen in Figure 3, mouse pups born to mothers immunized with the immunogenic fusion protein based on SEQ ID NO: 21 (dashed line) had a significantly higher survival rate than mouse pups born to mothers receiving the control (solid line) throughout the experiment (0-48 hours post-infection). Thus, the immunogenic fusion protein of SEQ ID NO: 21, when administered to female mice, significantly protected mouse pups born to such female mice from GBS infection and death. The immunogenic fusion proteins provided similar protection against GBS infection and death as reported for mice in Figure 2, Panels C and E, by Lindahl et al., Nonimmunodominant Regions Are Effective As Building Blocks In A Streptococcal Fusion Protein Vaccine, Cell Host & Microbe, Vol. 2, pp. 427-434, December 2007.

[0152] Example 3 - Epithelial cell invasion assay Figures 4A-4E show the results obtained in a human epithelial cell invasion test using antisera obtained from immunization with a clinical isolate and an immunogenic fusion of SEQ ID NO: 21. Each bar shows the relative epithelial cell invasion rate (%) for the control (no serum), the pre-immunization stage at day 0 (D0) and the post-immunization stage at day 57 (D57). A good effect on the inhibition of endothelial cell invasion is observed. Thus, the immunogenic fusion protein achieved an inhibitory effect on epithelial cell invasion similar to that reported in panel B of Figure 3 by Lindahl et al., Nonimmunodominant Regions Are Effective As Building Blocks In A Streptococcal Fusion Protein Vaccine, Cell Host & Microbe, Vol. 2, pp. 427-434, December 2007.

[0153] Thus, Figures 1 and 2 show that it is advantageous to administer the immunogenic fusion protein together with an additional fusion protein, and also to use an adjuvant, such as aluminum hydroxide, although the immunogenic fusion protein induced an immune response, such as an IgG fold increase, even at low dosages (10 μg) and without the inclusion of an adjuvant.

[0154] Furthermore, Figures 3 and 4A-4E show that the immunogenic fusion proteins achieve similar effects as those observed for the immunogenic fusion proteins disclosed in Lindahl et al., Nonimmunodominant Regions Are Effective As Building Blocks In A Streptococcal Fusion Protein Vaccine, Cell Host & Microbe, Vol. 2, pp. 427-434, December 2007, and WO2008127179.

[0155] The human dosage of each of the immunogenic fusion protein and the further immunogenic fusion protein (if administered) may be in the range of 10 μg to 250 μg, such as 10 μg to 100 μg, for example 15 μg to 75 μg, preferably 25 μg to 75 μg, for example 25 μg to 50 μg.

[0156] The preferred dosage for humans of each of the immunogenic fusion protein and the further immunogenic fusion protein (if administered) in the presence of an adjuvant, such as aluminum hydroxide, is therefore within the range of 10 to 250 μg, preferably 10 to 150 μg, preferably 25 to 100 μg, or 25 to 75 μg.

[0157] When the immunogenic fusion protein is administered together with a further immunogenic fusion protein and in the presence of an adjuvant, such as aluminum hydroxide, the dosage of each fusion protein is preferably in the range of 15 μg to 75 μg, preferably 25 μg to 75 μg, for example 25 μg to 50 μg.

[0158] In the absence of an adjuvant, such as aluminum hydroxide, the dosage of each of the immunogenic fusion protein and further immunogenic fusion protein (if administered) to a human is 10 to 1000 μg, preferably 50 to 500 μg, or preferably 100 to 250 μg.

[0159] When the immunogenic fusion protein and further immunogenic fusion proteins (if administered) are administered indirectly as by administration of the corresponding nucleic acid molecule, vector, or host cell, the dosage of the corresponding nucleic acid molecule, vector, or host cell is preferably selected to obtain an immune response similar to the immune response that would be obtained if the immunogenic fusion protein and further immunogenic fusion proteins (if administered) were administered directly to a subject.

[0160] Preferably, the dosage of each of the immunogenic fusion protein and the further immunogenic fusion protein (if administered) is repeated. Thus, the immunogenic fusion protein and the further immunogenic fusion protein (if administered) are administered in a first dose and repeated as a second dose some time after the first dose. The first and second doses may be spaced apart by 1 week to 3 months, such as 2 weeks to 2 months, for example 1 to 2 months, or 4 weeks.

[0161] Example 4 - Fusion proteins based on SEQ ID NO: 21 provide reduced shielding of the conserved region at the N-terminus of Rib As mentioned at the beginning, the present invention is based on further studies on the fusion proteins disclosed in WO2008127179. In this study, an immunogenic fusion protein sequence (SEQ ID NO: 21) was identified, which is shorter than the immunogenic fusion proteins disclosed in WO2008127179 but still has good efficacy and is suitable for use in a vaccine against GBS, as shown in the figures discussed above.

[0162] This example provides further insight into the differences between the shorter immunogenic fusion proteins and those of WO2008127179.

[0163] In particular, the difference between the sequences is the 32 amino acid long leading sequence: GPLGSASVLIGISFLGGFTQGQFNISTDTVFA-SEQ ID NO:43, which is not found in SEQ ID NO:21.

[0164] Computer simulations were performed on sequences for immunogenic fusion proteins of WO2008127179, including a lead sequence based on SEQ ID NO: 43. The simulations were based on molecular dynamics, where each atom in the system is explicitly modeled. The forces that neighboring atoms exert on each atom are also calculated and used to update the relative positions of each atom at short time intervals (typically femtoseconds). The simulations generate molecular orbitals, and all conformations are written out at defined time intervals.

[0165] In particular, a high-quality model of the full-length fusion protein of WO2008127179 was prepared using Alphafold2, version v2.1.0, Jumper et al., Nature, vol. 596, pp. 583-589 (2021). The C-terminal domain, i.e., the N-terminus of AlpC, is located far from the leader sequence and was therefore removed, leaving a 32 amino acid leader sequence (SEQ ID NO: 43) and an N-terminal rib domain containing a total of 204 amino acids. Gromacs 2021.5 was used to prepare the system as follows; the Alphafold2 model was solvated in a cubic unit cell using water (gmx solvate) and ions were added to make the system neutral (gmx genion). Energy was minimized by keeping the heavy atoms confined and subsequent equilibration steps (pressure and temperature (300K)). After verifying that the system was under steady state, we proceeded to the main simulation as follows. The molecular motion of the global system was simulated for 100 ns with a delta time of 2 fs for a total of 50 million steps, and the system conformation was saved every 5000 steps, resulting in 10000 model trajectories.

[0166] MDanalysis v2.0.0 was used to analyze the global trajectories and verify that the rib domain oscillates around the ground state, showing that the molecule is stable under the current conditions. The analysis was repeated three times (Sim1-3).

[0167] Partial results showed that the lead sequence (amino acids 1-32) found a stable conformation after about 60 ns after undergoing extensive conformational changes. Gromacs Mindist (gmx Mindist) was used to calculate the interactions between the lead sequence and the rib domain. To find tight and stable interactions between the lead sequence and the rib domain, the last 30 ns (3000 models) of the simulation were considered and the interactions that occurred at least 30% of the time (interactions in more than 900 models) were calculated based on an average of three simulations. The interaction at this point was defined as the minimum distance (0.3 nm or less) between any atoms of the amino acids. The results are presented in Table 1 below and in Figure 5A, which represents the Rib N-terminus in a surface depiction (with the lead sequence represented in a ball and stick depiction). The surface depiction of the Rib N-terminus is shaded from white, corresponding to high frequency contacts (high percentage), to black, corresponding to no or rare contacts.

[0168] Figure 5B shows an alternative depiction of the Rib N-terminus (shown as a cartoon depiction) and the leader sequence (shown as a ball-and-stick depiction). The cartoon depiction of the Rib N-terminus is shaded from white, corresponding to frequent contacts (high percentage), to black, corresponding to no or rare contacts.

[0169] Additionally, the amino acid conservation of the rib domains was calculated as follows; 144 complete Streptococcus agalactiae genomes were downloaded from PatricBRC, and 95 rib domains were found using blastp 2.9.0+. The 95 sequences were aligned using Muscle v3.8.1551, and the normalized information content per amino acid was computed using logomaker 0.8. The conservation scores are reported in the Cons. column of Table 1 below and are shown in Figure 5C, which represents the Rib N-terminus in a surface depiction (with the lead sequence represented in ball and stick representation). The surface depiction of the Rib N-terminus is shaded from white, corresponding to a high conservation score, to black, corresponding to no or low conservation scores.

[0170] [Table 1-1]

[0171] [Table 1-2]

[0172] Table 1 shows the stable interactions between the leader sequence (amino acids 1-32) and the rib domain (amino acids 33-204). Each pair of amino acids between the leader sequence and the rib domain that interacts for at least 30% of the time in the last 30 ns of the simulation is listed. The highest percentage for each leader sequence amino acid is shown in bold. The conservation of the amino acids is estimated and shown in the Cons. line.

[0173] As can be seen in Table 1, four pairs of structural elements govern the interaction; the first is a stretch of the leader sequence (29-32) closest to the domain that fits into one of the major beta-sheets of the Rib domain (129-133). The second element is leader sequence amino acids 23-26 that interact with a loop region (126-127) of the Rib domain that connects the two beta-sheets. The third interaction is leader sequence amino acids 12-16 that interact with several beta-sheets (84, 113, 127). Finally, the first part of the leader sequence (1-5) interacts with a loop (90-93) of the Rib domain.

[0174] These interacting pairs associate with the N-terminal portion of Rib, which contains a high proportion of conserved amino acids, suggesting that these regions are important for the intended purpose of the Rib proteins (facilitating infection of cells by GBS bacteria).

[0175] Thus, from the results in Table 1 it is clear that the 32 amino acid leader sequence of the RibN-AlpCN fusion protein of WO2008127179 fits into and interacts with these critical portions of the Rib portion of the fusion protein at least partially. Thus, the leader sequence interacts with and reduces access to these portions temporarily, i.e., at least 30% of the time. Thus, when used to elicit an immune response, the presence of the leader sequence reduces the access of the immune system to these critical portions of the Rib protein.

[0176] In contrast, shorter immunogenic fusion proteins based on SEQ ID NO:21 do not contain a leader sequence and therefore are unlikely to have any interactions that reduce access to these important parts of the immune system, resulting in a better immune response.

[0177] In particular, the interactions between the leader sequence and the Rib domain occur at different amino acids in the leader sequence. Thus, truncating the immunogenic fusion proteins of WO2008127179 results in a reduction in interactions in each case and allows the immune system to have better access to all parts of the RibN domain of the immunogenic fusion protein. Thus, even minor truncations, such as removing fewer amino acids of the leader sequence, still allow the immune system to have better access to the RibN domain of the immunogenic fusion protein.

[0178] As shown in Figures 5A-C, the leader sequence interacts with one endcap at the N-terminus of Rib, and the significance of this observation is investigated in Example 5 below.

[0179] Example 5 - A conserved region at the N-terminus of Rib, masked by the leader sequence, is involved in adhesion to and invasion of epithelial cells when Rib proteins interact with α1β1 integrin We investigated the significance of the conserved region at the N-terminus of Rib found in Example 4. Keeping in mind that Rib is a GBS surface protein and that antibodies to RibN provide protection against GBS infection, including invasion of epithelial cells, we hypothesized that the conserved region could be involved in promoting adhesion to and invasion of epithelial cells.

[0180] The alpha-fold structure for α1β1 integrin was obtained from the sequence cited in Bolduc et al., Microbiology (2007), vol. 153, pp. 4039-4049, and an interaction simulation was performed between the N-terminus of Rib contained in the fusion moiety based on SEQ ID NO: 21 and α1β1 integrin. FIG. 6 shows the results, with α1β1 integrin shown as a black surface drawing and the N-terminus of Rib shown as a light grey surface drawing. In FIG. 6A, the N-terminus of Rib is shown in a similar orientation to FIG. 5A-FIG. 5C. Thus, although not shown in FIG. 6, a leader sequence is provided at the right end cap of the N-terminus of Rib. By comparing with FIG. 5A-FIG. 5C, it is further revealed that the conserved region identified in Example 4 is involved in binding with α1β1 integrin. The results further revealed that the N-terminus binds to domain 2 (von Willebrand factor, type A) and domain 3 of α1β1 integrin.

[0181] Thus, Examples 4 and 5 show that the leader sequence interacts with and shields a conserved region at the N-terminus of Rib that is involved in binding to α1β1 integrin and thus in adhesion to and invasion of epithelial cells.

[0182] Thus, when immunizing with the N-terminal region of Rib provided with the leader sequence, these conserved regions will be less exposed due to the shielding effect of the leader sequence, which will result in a less efficient immune system response and / or antibody response against the conserved regions.

[0183] In contrast, when immunization is performed using the N-terminus of Rib in which the leader sequence (SEQ ID NO: 43) has been removed or at least truncated, using such an N-terminus alone or as part of a fusion protein of the first aspect of the invention or SEQ ID NO: 21, these conserved regions become more accessible to the immune system. Thus, it becomes easier for the immunized host to form antibodies against the conserved regions. More efficient and complete neutralization of the conserved regions increases the protection against epithelial cell adhesion and invasion via α1β1 integrin.

[0184] In summary, the use of a fusion protein based on the first aspect of the invention, in which the leading sequence of the Rib component (SEQ ID NO: 43) has been removed or at least shortened, as exemplified by SEQ ID NO: 21, can improve protection, particularly against adhesion to and / or invasion of endothelial cells, thereby improving protection against GBS.

Claims

1. i. a first amino acid sequence portion consisting of 170-178 amino acids, preferably 174-175 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 7; ii. a second amino acid sequence portion consisting of 165-174 amino acids, preferably 169-170 amino acids, and having at least 90% sequence identity with the amino acid sequence represented by SEQ ID NO: 14; and optionally iii. a linker amino acid sequence portion consisting of 1 to 20 amino acids and separating the first amino acid sequence portion from the second amino acid portion; An immunogenic fusion protein comprising or consisting of an amino acid sequence consisting of: An immunogenic fusion protein, preferably consisting of 335-372 amino acids, preferably 343-353 amino acids, more preferably 343-347 amino acids.

2. The first amino acid sequence portion comprises, or preferably consists of, amino acids 1 to 175 or 2 to 175 of the sequence represented by SEQ ID NO:7; and 2. The immunogenic fusion protein of claim 1, wherein the second amino acid sequence portion comprises, or preferably consists of, amino acids 1 to 170, or 2 to 170 of the sequence represented by SEQ ID NO:

14.

3. 3. The immunogenic fusion protein according to claim 1 or 2, wherein the amino acid sequence comprises a linker amino acid sequence portion, and wherein the linker amino acid sequence portion consists of 1 to 10 amino acids, preferably 2 to 5 amino acids, more preferably 2 amino acids, and most preferably the amino acid sequence EF.

4. The immunogenic fusion protein of any one of claims 1 to 3, having at least 90% sequence identity to SEQ ID NO:

21.

5. 5. The immunogenic fusion protein according to any one of claims 1 to 4, which consists of the amino acid sequence represented by SEQ ID NO:21, or consists of amino acids 2 to 347 of SEQ ID NO:

21.

6. A nucleic acid molecule comprising a polynucleotide encoding an immunogenic fusion protein described in any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, having at least 90% sequence identity with a nucleotide sequence represented by one of SEQ ID NOs: 15 to 20.

8. A vector comprising the nucleic acid molecule according to any one of claims 6 to 7.

9. A host cell expressing an immunogenic fusion protein according to any one of claims 1 to 5, or comprising a nucleic acid molecule according to any one of claims 6 to 7, or a vector according to claim 8.

10. A vaccine comprising one or more of the immunogenic fusion protein of any one of claims 1 to 5, the nucleic acid molecule of any one of claims 6 to 7, the vector of claim 8, or the host cell of claim 9.

11. a) a further immunogenic fusion protein comprising or consisting of an amino acid sequence having at least 90%, such as 95, 96, 97, 98, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42; b) a further nucleic acid molecule encoding said further immunogenic fusion protein, said further nucleic acid molecule preferably having at least 90% sequence identity with a nucleotide sequence represented by one of SEQ ID NOs: 36 to 41; c) a further vector comprising said further nucleic acid molecule, or d) a further host cell expressing said further immunogenic fusion protein or containing said further vector.

11. The vaccine of claim 10, further comprising one or more of:

12. a) an immunogenic fusion protein according to claim 5; b) a further immunogenic fusion protein comprising, preferably consisting of, the sequence of amino acids set forth in SEQ ID NO: 42, or comprising, or preferably consisting of, amino acids 2 to 347 of SEQ ID NO: 42; The vaccine of claim 11 , comprising:

13. The vaccine according to any one of claims 10 to 12, further comprising aluminium hydroxide as an adjuvant.

14. An immunogenic fusion protein according to any one of claims 1 to 5, a nucleic acid molecule according to any one of claims 6 to 7, a vector according to claim 8, a host cell according to claim 9 or a vaccine according to any one of claims 10 to 13 for use in i) vaccination against group B Streptococcus infection or ii) treatment of group B Streptococcus infection.

15. 15. The immunogenic fusion protein of any of claims 1 to 5, the nucleic acid molecule of any of claims 6 to 7, the vector of claim 8, the host cell of claim 9, or the vaccine of any of claims 10 to 13 for use according to claim 14, wherein the immunogenic fusion protein of any of claims 1 to 5, the nucleic acid molecule of any of claims 6 to 7, the vector of claim 8, the host cell of claim 9, or the vaccine of any of claims 10 to 13 is administered to a female human subject.

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