Highly stable antigen-presenting fusion proteins and uses thereof as vaccine components for the therapy and / or prophylaxis of pathogenic bacteria

Abstract

The present invention relates to antigens, more particularly protein antigens of enterococcal pathogens derived from the protein AdcA that are particularly useful as vaccine components and constructs for therapy and / or prophylaxis of bacterial infections. The present invention further relates to antibodies directed against the antigens of the invention and medical uses thereof.

Description

[0001] Highly stable antigen-presenting fusion proteins and uses thereof as vaccine components for the therapy and / or prophylaxis of pathogenic bacteria

[0002] The present invention relates to antigens, more particularly protein antigens of enterococcal pathogens derived from the protein AdcA that are particularly useful as vaccine components and constructs for therapy and / or prophylaxis of bacterial infections. The present invention further relates to antibodies directed against the antigens of the invention and medical uses thereof.

[0003] Background of the invention

[0004] Enterococci are Gram-positive, facultative anaerobic microorganisms colonizing a broad range of hosts, from invertebrates to mammals, including humans. They are part of commensal microbiota in the gastrointestinal (GI) tract, where they help to stabilize physiological pH, stimulate the immune system, and participate in reducing the number of potential ecological niches for pathogens (Krawczyk et al., 2021; Lebreton et al., 2014; Wan et al., 2016).

[0005] When the delicate host bacteria equilibrium is disturbed, Enterococcus sp. can become dangerous opportunistic pathogens, especially through the acquisition of antibiotic resistance. Indeed, enterococci constitute one of the major etiological agents of nosocomial infections (Weiner et al., 2016; Zhou et al., 2020), as they easily infect patients with recent surgery, organ transplantation, diabetes, malignancy, and renal insufficiency (Chiiambi et al., 2020; Kodali et al., 2015). E.faecium infections have higher rates of antibiotic resistance and mortality (Jabbari Shiadeh et al., 2019), with resistance to aminoglycoside and beta-lactams (Ch’ng et al., 2019), sulfonamides and glycopeptides, including vancomycin (Ben Braiek and Smaoui, 2019).

[0006] Consistently, the World Health Organization (WHO) has declared vancomycin-resistant E. faecium (VREfm) a threat to humankind for which rapid actions are needed (Tacconelli et al., 2018). With the rise of antibiotic resistance, new treatments were developed and introduced (quinupristin-dalfopristin, linezolid, daptomycin, tigecycline), but these further accelerated the selection of multidrug or even pan-resistant enterococci (“Global burden of bacterial antimicrobial resistance in 2019,” 2022). Prevention of these infections through vaccines can diminish the usage of antibiotics, minimize high-cost treatments and reduce lengths of hospitalizations (Vekemans et al., 2021).

[0007] So far, several vaccine antigen candidates against E. faecium have been investigated (Kalfopoulou and Huebner, 2020; Kazemian et al., 2019; Nallapareddy et al., 2011; Romero- Saavedra et al., 2014; Romero-Saavedra et al., 2015; Theilacker et al., 2012), albeit there is hitherto no available vaccine (World Health Organization, 2022).

[0008] Over the past 20 years, there has been an explosion in the prevalence of antibiotic resi stant bacterial infections, both in the hospital and in the general community. Notably, the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baiimannii, Pseudomonas aeruginosa, and Enterobacter species) are responsible for a substantial percentage of nosocomial infecti ons and present serious therapeutic challenges for physicians. These multi-drug resistant infections increase morbidity and mortality and require broad-spectrum antimicrobial coverage, or the increased usage of antibiotic agents.

[0009] The d-block transitional metal ion Zinc (Zn), essential to all forms of life, plays a structural and catalytic role in about 5% of bacterial proteins, where it participates in multiple biological processes such as carbon metabolism and regulation of DNA transcription (Andreini et al., 2006). Therefore, pathogenic bacteria need to hunt for Zn through Zn-binding proteins (Capdevila et al., 2016) to be able to survive in the inhospitable environment and counteract the host-derived limitation of the ion (Xia et al., 2021).

[0010] Consistently, mutations in Zn-regulating genes impair virulence and increase susceptibility to the host defense machinery in several bacterial species (Bayle et al., 2011; Burcham et al., 2020; Gao et al., 2018; Grim et al., 2017; Makthal et al., 2020). In E. faecium, Zn homeostasis is regulated by the surface-exposed lipoprotein AdcA, a member of substrate-binding proteins (SBPs) of ATP -binding cassette (ABC) transporters which is upregulated during infection (Romero-Saavedra et al., 2015). Similarly, the core genome of E.faecalis encodes a conserved AdcACB system and an orphan substrate-binding lipoprotein AdcAII (Lam et al., 2022). Inactivation of genes encoding these proteins results in severe growth and survival defects and a decreased resistance to ampicillin, bacitracin, and daptomycin (Lam et al., 2022). Analogous zinc uptake mechanisms have been observed in S. aureus. Indeed, S. aureus possesses two ABC transporters for Zn acquisition, CntABCDF and AdcABC; while the CntABCDF system uses the chelating molecule staphylopine for ion uptake, the AdcABC transporter uses the same mechanism of direct binding to Zn2+, as observed in Enterococci (Grim et al., 2017). Simultaneous deletion of AdcABC and CntABCDF systems resulted in a growth impairment under Zn-restricted conditions and attenuated virulence (Grim KP, et al. The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity. mBio. 2017 Oct 31 ;8(5):e01281 - 17. doi: 10.1128 / mBio.01281-17. PMID: 29089427; PMCID: PMC5666155.).

[0011] Besides contributing to zinc homeostasis, AdcA of S. aureus was shown to bind human plasminogen and the human negative complement regulator factor H (FH), and thus may support S. aureus invasion and colonization (Salazar et al., 2022). Also in Streptococcus pneumoniae and S. pyogenes, the ABC transporter proteins AdcAI and AdcAII are both required for full pathogenic potential and are differently regulated as a response to metal abundance (Luo et al., 2021; Ong et al., 2018).

[0012] Yousief SW, et al. (in: Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model. Microbes Infect. 2020 Apr;22(3): 127-136. doi: 10.1016 / j.micinf.2019.09.002. Epub 2019 Oct 1. PMID: 31585177) disclose the basic membrane protein (BMP) family ABC transporter substrate-binding protein, as a potential vaccine candidate against E. faecium. Recombinant BMP of E. faecium was expressed in Escherichia coli and purified by metal affinity chromatography. Swiss albino mice were immunized with the recombinant BMP combined with Bacillus Calmette-Guerin (BCG) and / or alum as adjuvants. Mice immunized with BMP combined with alternating BCG and alum developed BMP-specific IgG and were protected against E. faecium challenge as evidenced from organ bioburden and histopathological examination. Furthermore, serum from immunized mice showed enhanced opsonophagocytic activity and protected mice against E. faecium. challenge by passive immunization. Bioinformatic analysis revealed appreciable degrees of homology between E. faecium BMP and proteins from other pathogens which suggests BMP could be a useful vaccine against multiple pathogens.

[0013] Romero- Saavedra F, et al. (in: Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections. PLoS One. 2015 Aug 31;10(8):e0136625. doi: 10.1371 / journal. pone.0136625. PMID: 26322633; PMCID: PMC4556446) disclose the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm) that were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E.faecium E155 as well as clinical strains E. faecium El 162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens.

[0014] WO 2010 / 127784A1 discloses a medicament for the treatment or the prevention of a bacterial infection is disclosed which contains a polypeptide having a contiguous sequence of at least six amino acids of SEQ ID NO: 1 as disclosed. The polypeptide can be used for the preparation of a vaccine against an Enterococcus infection.

[0015] As mentioned above, Enterococci - as part of the ESKAPE set of bacteri a - are among the m ost important pathogens associated with infections in hospitalized patients. Especially the presence of multiple antibiotic resistance determinants in most clinically relevant isolates urges the development of alternative treatment and prevention strategies to combat these sometimes untreatable infections.

[0016] The opsonophagocytic assay has been used to simulate the immune response in vitro and to identify enterococcal virulence factors. However, some antigens have been identified so farthat may offer the potential of inducing a protective immune response, and therefore would be promising vaccine targets.

[0017] It is therefore an object of the present invention to provide a new promising vaccine target antigen for an active or passive immunotherapy of bacteria, and in particular ESKAPE pathogens. It is another object of the present invention, to provide novel and effective vaccines based on said target antigen and medical uses thereof. According to a first aspect of the present invention, the above object is solved by providing an antibacterial antigen comprising a continuous stretch of at least 6 amino acids of the amino acid sequence GSEEEDHDHGEEDHHHE (SEQ ID NO: 2) or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to said continuous stretch of SEQ ID NO: 2, wherein the length of the antigen is between about 300 and 6 amino acids, preferably between about 50 and 6 amino acids, and most preferred about 17 amino acids. Preferably, the antibacterial antigen produces opsonic antibodies, as described herein.

[0018] According to a second aspect of the present invention, the above object is solved by providing an antigenic protein construct, comprising at least one antibacterial antigen sequence according to the present invention integrated into the DI domain of a bacterial arginine binding protein (ArgBP), without a destabilization of the protein structure, preferably wherein the at least one antibacterial antigen sequence is integrated into the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima. Preferred is the antigenic protein construct according to the present invention, wherein the construct shows no substantial sign of degradation when stored for more than 30 days at 37°C.

[0019] According to a third aspect of the present invention, the above object is solved by providing an antibody or antigen binding fragment thereof binding specifically to a contiguous sequence of at least six amino acids of SEQ ID NO: 2, the antibacterial antigen according to the present invention and / or the antigenic protein construct according to the present invention. Preferably, the antibody or antigen binding fragment thereof is opsonic.

[0020] According to a fourth aspect of the present invention, the above object is solved by providing a nucleic acid encoding the antibacterial antigen according to the present invention, the antigenic protein construct according to the present invention or the antibody or antigen binding fragment thereof according to the present invention, wherein preferably the nucleic acid is selected from DNA, RNA, stabilized RNA and combinations thereof.

[0021] According to a fifth aspect of the present invention, the above object is solved by providing a pharmaceutical composition comprising the antibacterial antigen according to the present invention, the antigenic protein construct according to the present invention or the antibody or antigen binding fragment thereof according to the present invention or the nucleic acid according to the present invention, and a pharmaceutically acceptable carrier, adjuvant and / or diluent, wherein said composition preferably is a vaccine.

[0022] According to a sixth aspect of the present invention, the above object is solved by the antibacterial antigen according to the present invention, the antibody or antigen binding fragment thereof according to the present invention, the nucleic acid according to the present invention or the pharmaceutical composition according to the present invention for use in the prophylaxis or treatment of diseases, in particular for the prophylactic or therapeutic treatment of a disease or condition caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. fciecium. E. fctecalis. S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.

[0023] According to a seventh aspect of the present invention, the above object is solved by providing a method for treating or preventing a disease or condition in a subject that is caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof, comprising administering to the subject an effective amount of the antibacterial antigen according to the present invention, the antibody or antigen binding fragment thereof according to the present invention, the nucleic acid according to the present invention or the pharmaceutical composition according to the present invention.

[0024] As mentioned above, the present invention relates to an antibacterial peptide / protein antigen comprising a continuous stretch of at least 6 amino acids of the amino acid sequence GSEEEDHDHGEEDHHHE (SEQ ID NO: 2) or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to said continuous stretch of SEQ ID NO: 2. The overall length of the antibacterial peptide / protein antigen sequence is between about 300 and 6 amino acids, preferably between about 50 and 6 amino acids, and most preferred about 17 amino acids. The protein Adc A of E. faecium had proven to elicit specific, opsonic, and protective antibodies, with extensive cross-reactivity and serotype-independent coverage among the homologous strain E. faecium El 55 and the clinical isolates E. faecium El 162, E.faecalis 12030, type 2 and type 5 (Romero-Saavedra et al., 2015, 2014). In view of the observed antigenicity of AdcA lipoprotein, its specific location, and its crucial role in many bacterial species, the inventors decided to use AdcA an attractive candidate for vaccine development.

[0025] AdcA as i solated from the Enterococcus faecium has the following amino acid sequence (SEQ ID NO: 1) according to the NCBI Accession number WP_130587550.

[0026] MKKISVGLIGVAALGLLGACSSTNDAKVSNDKDGKLEIVTTFYPMYDFTKNIVGDEA NVDLMVPAGSEPHDYEPSAI<DMAI<AHDADVFVYHNENMESWVPI<AI<ESWI<I<AGP NVVEGTKDMILLPGSEEEDHDHGEEDHHHELDPHTWVSPKMAIKEVSNIKDQLVKLY PKKAKVFETNAEKYLTKLKRLDADYTTSLKEAKQKSFVTQHAAFGYLALDYGLIQVP IAGLSPEEEPSSGRLAELKEYVKKNKINYIYFEKNANDKIAKTLANEAGIKLEVLNPLE SLTKEQMDNGEDYVSVMEDNLKALEKTTMVAGKEVVPEKEAKDEKTVANGYFKDV DVKDRELSDYTGEWQSVYPLLKDGILDEVFDYKAKLNKDMTAAEYKDYYTTGYKT DIDTINIKDNTIDFVVKGEHHQYTYKYVGYKILNYEKGNRGVRFNFETDDAGAGRYK YVQFSDHGIAPSKAEHFHIFFGGESQEKLYNELHNWPTFYPASLSKHEIAQEMLAH

[0027] Homologous AdcA sequences could be identified, for example, in E. faecalis (Sequence ID: WP_010710535.1), E. caecorum (Sequence ID: WP_047340993.1), E. avium (Sequence ID: WP_227132389.1), Enterococcus casseliflavus (Sequence ID: WP_154694472.1), Enterococcus hirae (Sequence ID: WP 161637762.1), and Enterococcus sp. 10A9 DIV0425 (Sequence ID: WP_086283622.1). Therefore, AdcA seems to be present in several pathogenic bacteria, such as, for example, Gram-positive bacteria, such as E. faecalis and E. faecium strains, and thus provides the basis as a promising vaccine target for active or passive immunotherapy of, for example, enterococci, as disclosed herein.

[0028] Here, the inventors found that AdcA can also elicit opsonic antibodies against MRS A and other gram-positive pathogens of clinical relevance. Furthermore, the inventors used different bioinformatic analyses that ultimately identified a main epitope responsible for the cross- reactive immunological activity of AdcA, designated herein as the EH-motif. The amino acid sequence comprising the epitope was identified as GSEEEDHDHGEEDHHHE (SEQ ID NO: 2) or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to SEQ ID NO: 2. This sequence was then used to further study and develop antibacterial approaches.

[0029] Preferred is the antibacterial antigen according to the present invention, wherein said antigen sequence is fused to the amino acid sequence of a suitable fibrillising peptide (see, for example Collier JH, Messersmith PB. Enzymatic modification of self-assembled peptide structures with tissue transglutaminase. Bioconjug Chem. 2003 Jul-Aug;14(4):748-55. doi:

[0030] 10.1021 / bc034017t. PMID: 12862427; Smith A. Fibril formation by short synthetic peptides. Subcell Biochem. 2012;65:29-51. doi: 10.1007 / 978-94-007-5416-4_2. PMID: 23224998; Nasstrom T, et al. Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts. Int J Mol Sci. 2021 Aug 28;22(17):9334. doi: 10.3390 / ijms22179334. PMID: 34502242; PMCID: PMC8431055), in particular to the amino acid sequence QQKFQFQFEQQ (SEQ ID NO: 4). Fibrillising activity may be observed using Dynamic Light scattering (DLS), as shown herein.

[0031] Further preferably, a linker peptide was added between the antigen and fibrillising peptide sequence, in order to maintain the optimal immunogenicity of the antigen sequence. Furthermore, acetylation and amidation of the N- and C-terminal ends, respectively, may be added in order to keep the neutrality of terminal ends. Therefore, the structure of one preferred construct was as follows (MW 3821.73):

[0032] Ac-GSEEEDHDHGEEDHHHE-SGSG-QQKFQFQFEQQ-NH2 (SEQ ID NO: 3)

[0033] In addition to the above, the inventors sought to further improve the immunogenicity and suitability of the EH-antigen and ultimately engineered a hyper-stable and multi-presenting version of the EH-motif, Sc(EH)3 (SEQ ID NO: 5), which embeds three EH-motifs in its sequence. The results obtained clearly characterized the Sc(EH)3 antigen construct as one promising strategy for the development of a vaccine active against different Gram-positive ESKAPE pathogens, as disclosed herein.

[0034] MGSEEEDHDHGEEDHHHEAIDEIKSRGYLLVGLSADFPPFEFVDEGSEEEDHDHGEED HHHENGNIVGFDVDLAKEIARRLGVELKIVDMTFDGLIPSLLTKKIDVIISGMTITEERK KVVAFSDPYFDAGGGSEEEDHDHGEEDHHHEGGSGEQYGIAVRKEDTDLLEFINSVL RELKKLEHHHHHH (SEQ ID NO: 5) Another preferred aspect of the present invention thus relates to an antigenic protein construct, comprising at least one antibacterial antigen sequence according to the present invention integrated into the DI domain of a bacterial arginine binding protein (ArgBP), without that a substantial destabilization of the protein structure takes place. Destabilization may be analyzed using methods known in the state of the art and the literature, such as the resulting change in folding free energy (AAG) as calculated. The construct may comprise one, two or three copies of the antibacterial antigen sequence according to the present invention integrated. The sequences may be from one bacterial AdcA protein or from a AdcA protein of different homologs from different bacterial strains.

[0035] Preferred is an antigenic protein construct according to the present invention, wherein the at least one antibacterial antigen sequence is integrated into the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima (see, for example, Jaworek MW , et al. On the extraordinary pressure stability of the Thermotoga maritima arginine binding protein and its folded fragments - a high-pressure FTIR spectroscopy study. Phys Chem Chem Phys. 2020 May 28;22(20): 11244-11248. doi: 10.1039 / d0cp01618g. Epub 2020 May 13. PMID: 32400824). More preferred is an antigenic protein construct according to the present invention, wherein the continuous stretch of the amino acid sequence GSEEEDHDHGEEDHHHE (SEQ ID NO: 2) as defined herein or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to SEQ ID NO: 2 is integrated in the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima between amino acids E46 and N47, G97 and G98, and at the N-terminal end of the protein.

[0036] Further preferred is the antigenic protein construct according to the present invention which is engineered as a hyper-stable and multi-presenting version of the EH-motif. The construct according to the present invention showed no substantial sign of degradation when stored for at least 30 days at 37°C.

[0037] The inventors in the context of the present invention proved that antibodies raised against an epitope in the antigen sequence according to the present invention, as particularly exemplified using the Sc(EH)3 construct (SEQ ID NO: 5), possess cross-reactive properties that mediate the opsonic killing of a wide spectrum of Gram-positive pathogens, including VRE E. faecium and MRS A, and confer protection in a mouse infection model. This highlights the epitope in the SC(EH)3 as a promising antigen for the development of a vaccine active against different Grampositive ESKAPE pathogens.

[0038] Another aspect of the present invention therefore relates to an antibody or antigen binding fragment thereof binding specifically to a contiguous sequence of at least six amino acids of SEQ ID NO: 2, the antibacterial antigen according to the present invention as described herein and / or the antigenic protein construct according to the present invention as described herein. The antibody may be a polyclonal mix of antibodies or a monoclonal antibody and / or an antibody or antigen binding fragment thereof that is opsonic and exhibits opsophagocytotic activity. Antigen binding fragments may be selected from an scFV fragment and a Fab-fragment of the antibody. Antibodies may be raised according to methods known in the art, see also below in the examples.

[0039] Since appropriate animal models for an effective analysis of the immunogenicity and protective efficacy towards bacterial virulence factors are missing, the opsonophagocytic assay is used as a surrogate to simulate the mammalian in vivo immune response in vitro, and to identify and confirm enterococcal protein antigens, in this context AdcA, that can be developed into, for example, effective antibacterial vaccines (Markus Hufnagel, Steffi Koch, Andrea Kropec, Johannes Huebner: Opsonophagocytic assay as a potentially useful tool for assessing safety of enterococcal preparations. International Journal of Food Microbiology 88 (2003) 263- 267). OPA and OPIA are widely used to present op sonophagocytic-dep endent bacteria-killing with the usage of chosen agent and the specificity of this action. Full protocols of both assays were previously described elsewhere (Kropec A, Sava IG, Vonend C, Sakinc T, Grohmann E, Huebner J. Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections. Microbiology 2011; 157:3429-34). See also Figure 3 and the examples, below.

[0040] Another aspect of the present invention then relates to a nucleic acid encoding the antibacterial antigen according to the present invention, the antigenic protein construct according to the present invention or the antibody or antigen binding fragment thereof according to the present invention, wherein preferably the nucleic acid is selected from DNA, RNA, stabilized RNA and combinations thereof. The nucleic acids according to the present invention may be used to produce the peptides or proteins according to the present invention, for example as part of suitable expression constructs (see for example below). The nucleic acid may also be used as nucleic acid vaccines, for example a stabilized RNA vaccines (see, for example, Gote V, et al. A Comprehensive Review of mRNA Vaccines. Int J Mol Sci. 2023 Jan 31;24(3):2700. doi: 10.3390 / ijms24032700. PMID: 36769023; PMCID: PMC9917162).

[0041] Another aspect of the invention then relates to a method to produce the peptides or proteins according to the present invention, comprising synthesizing the peptides or proteins or expressing the peptides or proteins in a suitable cellular system, and subsequent purification of the peptides or proteins as is known in the state of the art (see also below).

[0042] Another aspect of the invention then relates to a pharmaceutical composition comprising the antibacterial antigen according to the present invention, the antigenic protein construct according to the present invention or the antibody or antigen binding fragment thereof according to the present invention or the nucleic acid according to the present invention, and a pharmaceutically acceptable carrier, adjuvant and / or diluent, wherein said composition preferably is a vaccine. Vaccines of the invention may be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, or buccal or oral. Preferably, the pharmaceutical composition according to the present invention is a vaccine, wherein said vaccine is formulated for administration via intramuscular, subcutaneous, or inhalation routes. Suitable adjuvants include oils i.e. Freund’s complete or incomplete adjuvant; salts i.e. A1K(SO4)2, AlNa(S04)2, A1NH4(SO4)2, silica, kaolin, carbon polynucleotides, i.e. poly IC and poly AU. Preferred adjuvants include QuilA and Alhydrogel.

[0043] Preferably, said pharmaceutical composition is effective for the prevention of a bacterial, and particularly an enterococcal and / or staphylococcal infection.

[0044] Another aspect of the present invention then relates to a pharmaceutical composition, wherein the peptide or protein is covalently bound or conjugated to an immunocarrier, such as capsular polysaccharides.

[0045] Another aspect of the present invention then relates to a method for producing a pharmaceutical composition according to the present invention, preferably a vaccine, comprising admixing at least one of the antibacterial antigen according to the present invention, the antigenic protein construct according to the present invention or the antibody or antigen binding fragment thereof according to the present invention or the nucleic acid according to the present invention with a pharmaceutically acceptable carrier diluent or adjuvant as described above.

[0046] The present invention provides new prophylactic and therapeutic approaches in order to treat bacterial infections. It is therefore a further aspect of the present invention to particularly provide a prophylaxis or treatment of such bacteria.

[0047] Another aspect of the present invention thus relates to the antibacterial antigen according to the present invention, the nucleic acid according to the present invention or the pharmaceutical composition according to the present invention for use in the prophylaxis or treatment of diseases, in particular for the prophylactic or therapeutic treatment of a disease or condition caused by a bacterium, such as, for example, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Staphylococcus aureus (e.g., MRSA) and coagulase-negative staphylococci, as well as strains of Clostridia, Listeria or S. pyogenes, and in particular bacterial infections that are caused by strains showing resistance against multiple antibiotics, such as multiple resistant Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and coagulase- negative staphylococci, as well as strains of Clostridia or Listeria. Respective multiple resistant strains are also described in the literature. Preferably the bacterial infection is by a bacterium selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.

[0048] Another aspect of the present invention thus relates to the antibody or antigen binding fragment thereof according to the present invention, in particular the antibody or antigen binding fragment thereof that specifically bind to the antigenic peptides or proteins according to the present invention, the nucleic acid according to the present invention or the pharmaceutical composition according to the present invention for use in the prophylaxis or treatment of diseases, in particular for the prophylactic or therapeutic treatment of a disease or condition caused by a bacterium as described above. Preferred is an antibody or antigenic fragment thereof that is selected from a monoclonal antibody, an scFV fragment and a Fab-fragment. The antibodies can further be human or humanized antibodies or fragments. Methods to produce antibodies are known to the person of skill and described in the respecti ve li terature, above, and below. Another aspect of the present invention then relates to a method for treating or preventing a disease or condition in a subject that is caused by a bacterium as described above, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. fciecium. E. fctecalis. S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof, comprising administering to the subject an effective amount of the antibacterial antigen according to the present invention, the antibody or antigen binding fragment thereof according to the present invention, the nucleic acid according to the present invention or the pharmaceutical composition according to the present invention.

[0049] In a particularly preferred embodiment, the pharmaceutical composition as administered is a vaccine and is administered to those individuals at risk of bacterial, and in particular enterococcal infection, such as infants, elderly and immunocompromised individuals.

[0050] Vaccine compositions are preferably administered in a unit dosage form of about 0.001 to 100 Pg / kg (Peptide / body weight) and more preferably 0.01 to 10 Pg / kg and most preferably 0.1 to 1 Pg / kg 1 to 3 times with an interval of about 1 to 6 week interval s between immunizations.

[0051] Preferred is a method for the treatment and / or prophylaxis as described herein, wherein said method comprises administering intramuscularly, subcutaneously, or via inhalation to said vertebrate a therapeutically effective amount of a pharmaceutical composition, and preferably a vaccine, according to the present invention.

[0052] Further preferred is a method for the treatment and / or prophylaxis as described herein, wherein said method further comprises the administration of an active agent selected from additional antibiotics and / or preparations improving the activity of the immune system of the individual.

[0053] A further aspect of the present invention relates to diagnostic assay for detecting a bacterial infection in a subject, comprising identifying the antibacterial antigen according to the present invention and / or antibodies against the antibacterial antigen according to the present invention in a sample derived from a subject to be diagnosed, wherein the presence of the antibacterial antigen according to the present invention and / or antibodies against the antibacterial antigen according to the present invention, optionally when compared to a control sample, detects a bacterial infection in the subject. The subject may be a human patient, and the assay may be used to monitor the infection, wherein several samples are analyzed over the course of a bacterial infection.

[0054] Bacterial infections to be detected are infections caused by Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus (e.g., MRSA) and coagulase-negative staphylococci, as well as strains of Clostridia, Listeria or S. pyogenes, and in particular bacterial infections that are caused by strains showing resistance against multiple antibiotics, such as multiple resistant Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus and coagulase- negative staphylococci, as well as strains of Clostridia or Listeria. Respective multiple resistant strains are also described in the literature. Preferably the bacterial infection is by a bacterium selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.

[0055] Preferred is a diagnostic assay according to the present invention, wherein identifying the antibacterial antigen according to the present invention comprises the use of an antibody or antigen binding fragment thereof as described herein, and preferably said antibody or fragment thereof is imm obilized on a solid matrix. Both or either of the antibody or fragment thereof or the peptide (antigen) or a homolog (antigen) thereof can be labeled (e.g. with a fluorescent label, an enzymatic label, a mass label, or the like) for the diagnosis.

[0056] The ESKAPE pathogens are a high-priority group of concern defined by the World Health Organisation. They are responsible for the majority of nosocomial infections worldwide, and are capable to evade the biocidal action of most antibiotics commonly used in the clinical settings. Therefore, alternative non-antibiotic treatments against these pathogens are urgently needed. Vancomycin-resistant E. faecium (VREfm), together with methicillin-resistant Staphylococcus aureus (MRSA) are the two Gram-positive pathogens belonging to this group. The inventors previously showed that AdcA, a protein in the Zinc regulating system of E. faecium, can elicit specific, opsonic, and protective antibodies against E. faecium and E. faecalis. Here, the inventors found that AdcA can also elicit opsonic antibodies against MRSA and other gram-positive pathogens of clinical relevance. Later the inventors used different bioinformatic analysis to identify the main epitope responsible for the cross-reactive immunological activity of AdcA, from here after called the EH-motif. Also, the inventors engineered a hyper-stable and multi-presenting version of the EH-motif, Sc(EH)s (SEQ ID NO: 5), which embeds three EH-motifs in its sequence. Finally, the inventors proved that antibodies raised against the Sc(EH)s possess cross-reactive properties that mediate the opsonic killing of a wide spectrum of Gram-positive pathogens, including VRE E. faecium and MRS A, and confer protection in a mouse infection model. Highlighting the Sc(EH)s as a promising antigen for the development of a vaccine active against different Gram-positive ESKAPE pathogens.

[0057] MRS A and VREfm are an increasing problem and serious threat in hospitals and long term care units among patients of all ages, and urgent actions are required to prevent an acceleration of this problem (Centers for Disease Control and Prevention (U.S.), 2019; Tacconelli et al., 2018). Although quite a few candidate vaccine antigens have been proposed against E. faecium (Kalfopoulou and Huebner, 2020; Kazemian et al., 2019; Nallapareddy et al., 2011; Romero- Saavedra et al., 2014; Theilacker et al., 2012), and diverse and complex designs against S. aureus are investigated in clinical trials (Clegg et al., 2021; Jahantigh et al., 2022), no vaccine is still available (Bacterial vaccines in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO, n.d.), particularly since preventing S. aureus infection has proven to be challenging, leading to the high rate of failures in the clinical trial stage (Miller et al., 2020).

[0058] Hence, in response to this urgent and unmet need for non-antibiotic approaches to stop VRE and MRSA, the inventors searched for vaccine antigens with a potential use against multiple pathogens, with the idea to design an antigen that would act against a wide panel of important gram-positive pathogens. Indeed, pan-vaccinomic strategies are currently an important challenge for the development of a universal vaccine candidate against WHO priority pathogens (Ismail et al., 2021).

[0059] With this aim, the inventors targeted the lipoprotein AdcA from E. faecium, starting from the preliminary observation that the antibodies raised against this protein mediate opsono- phagocytosis in different S. aureus strains. Consistently, using a structural vaccinology approach, the inventors identified a region of AdcA, the EH-motif, that is predicted to be highly immunogenic and is highly conserved among a wide spectrum of pathogens. Therefore, the inventors produced a new molecule with multiple presentation of the EH-motif, Sc(EH)3 (SEQ ID NO: 5), that presents a rigid core and flexible EH-motifs located in two loops and on the N- terminus of the protein, as evidenced by MD simulations. Flexibility of antigens is a greatly neglected feature that may improve immunological response. Indeed, multiple potential conformations of antigens seem beneficial as they increase variability of antigenic form during recognition by B-cells, which can broaden the diversity of antibodies in the immune response (Fedechkin et al., 2020).

[0060] Importantly, the inventors then showed in in vitro and in vivo assays that Sc(EH)3 is immunogenic in rabbits, and produces antibodies that successfully induce opsonic dependent killing against E. faecium 11236 / 1, S. aureus M2, E. faecalis T2. Also, they are protective in challenge in mice against E. faecium 11236 / 1, and S. aureus MW2. Using whole cell ELISA, the inventors also detected that anti-Sc(EH)3 bound to many representatives of closely related Streptococcus sp.

[0061] Beside protecting against more pathogens, Sc(EH)3 possesses high thermal-stability and is resistant to proteolysis for prolonged storage time at 37°C. Stability of vaccines, especially during transportation, is still a great challenge (Brandau et al., 2003; Pambudi et al., 2022), as a large component of vaccination costs arises from maintaining cold chains, storage, and transport rather than the actual cost of vaccine production. In developing countries, the WHO- UNICEF Expanded Program on Immunization has the formidable task to deliver vaccines to the populations at risk, investing enormous financial and human resources to maintain a cold chain. Providing hyper-thermostable antigen formulations with prolonged expiry date could be of benefit in most demanding environments, such as tropical countries (Bulula et al., 2020). Although vaccines are not solely composed of subunit antigens, the inventors believe that soluble, highly stable multi-targeting antigen can greatly affect functionality of final formulation. Taken together, the inventor’s findings can be a step forward in a field of pan- vaccinomics, that will address AMR pathogens from the concern list of WHO (Ismail et al., 2021).

[0062] Moreover, the EH-motif epitope, particularly in the hyper stable Sc(EH)3 (SEQ ID NO: 5) configuration, shows promise as an antigen for developing a vaccine against Gram-positive ESKAPE pathogens. In conclusion, this methodology, combining bioinformatics, in vitro, and in vivo experiments, enables the identification of specific cross-reactive epitopes. It enhances vaccine formulations widening their coverage and facilitates the development of broadspectrum monoclonal antibodies.

[0063] The present invention relates to the following items:

[0064] Item 1. An antibacterial antigen comprising a continuous stretch of at least 6 amino acids of the amino acid sequence GSEEEDHDHGEEDHHHE or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to said continuous stretch of SEQ ID NO: 2, wherein the length of the antigen is between about 300 and 6 amino acids, preferably between about 50 and 6 amino acids, and most preferred about 17 amino acids.

[0065] Item 2. The antibacterial antigen according to Item 1, wherein said antigen sequence is fused to the amino acid sequence of a suitable fibrillising peptide, in particular to the amino acid sequence QQKFQFQFEQQ (SEQ ID NO: 4), optionally further comprising a linker peptide between the antigen and fibrillising peptide sequence.

[0066] Item 3. An antigenic protein construct, comprising at least one antibacterial antigen sequence according to Item 1 integrated into the DI domain of a bacterial arginine binding protein (ArgBP), without a destabilization of the protein structure, preferably wherein the at least one antibacterial antigen sequence is integrated into the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima.

[0067] Item 4. The antigenic protein construct according to Item 3, wherein the amino acid sequence is integrated in the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima between amino acids E46 and N47, G97 and G98, and at the N-terminal end of the protein.

[0068] Item 5. The antigenic protein construct according to Item 3 or 4, wherein the construct shows no substantial sign of degradation when stored for more than 30 days at 37°C.

[0069] Item 6. An antibody or antigen binding fragment thereof binding specifically to a contiguous sequence of at least six amino acids of SEQ ID NO:2, the antibacterial antigen according to Item 1 or 2 and / or the antigenic protein construct according to any one of Items 3 to 5. Item 7. The antibody according to Item 6, which is a monoclonal antibody and / or an antibody or antigen binding fragment thereof that is opsonic.

[0070] Item 8. A nucleic acid encoding the antibacterial antigen according to Item 1 or 2, the antigenic protein construct according to any one of Items 3 to 5 or the antibody or antigen binding fragment thereof according to Item 6 or 7, wherein preferably the nucleic acid is selected from DNA, RNA, stabilized RNA and combinations thereof.

[0071] Item 9. A pharmaceutical composition comprising the antibacterial antigen according to claim 1 or 2, the antigenic protein construct according to any one of Items 3 to 5 or the antibody or antigen binding fragment thereof according to Item 6 or 7 or the nucleic acid according to Item 8, and a pharmaceutically acceptable carrier, adjuvant and / or diluent, wherein said composition preferably is a vaccine.

[0072] Item 10. The pharmaceutical composition according to Item 9, wherein said vaccine is formulated for administration via intramuscular, subcutaneous, or inhalation routes.

[0073] Item 11. The antibacterial antigen according to Item 1 or 2, the antibody or antigen binding fragment thereof according to Item 6 or 7, the nucleic acid according to Item 8 or the pharmaceutical composition according to Item 9 or 10 for use in the prophylaxis or treatment of diseases, in particular for the prophylactic or therapeutic treatment of a disease or condition caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.

[0074] Item 12. A method for treating or preventing a disease or condition in a subject that is caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof, comprising administering to the subject an effective amount of the antibacterial antigen according to Item 1 or 2, the antibody or antigen binding fragment thereof according to Item 6 or 7, the nucleic acid according to claim 8 or the pharmaceutical composition according to Item 9 or 10.

[0075] The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.

[0076] In the accompanying figures and the accompanying sequence listing,

[0077] Figure 1 shows the opsonophagocytic killing activity (OPA) of anti-AdcA serum against different S. aureus strains. (A,B) A schematic representation of rabbit immunization and mechanism of OPA. (C) OPA against S. aureus MW2, (D) LAC, and (E) Reynolds strains were tested with sera raised against the recombinant AdcA from E. faecium, used at concentrations ranging from 1 to 0,25 mg / mL. The effectiveness of opsonophagocytic killing by the anti-AdcA rabbit sera obtained from terminal immune sera (MW2, LAC and Reynolds are drawn in black, light grey and dark grey, respectively) was compared to that by the pre-immune rabbit sera (white bars). Statistical significance was tested by the unpaired two-tailed T-test with a 95% confidence interval using pre-immune and terminal immune sera at the same concentration. Bars and whiskers denote mean values ± standard errors of the mean. NS, not significant (P > 0.05). *P < 0.05, ** P <0.01, *** P<0,001. (F) Schematic representation of an OPIA assay. (G) OPIA of anti-AdcA rabbit serum performed by incubation of serum against S. aureus MW2 with recombinant AdcA protein. Antibodies raised against the AdcA at 0,5mg / mL were pre-incubated with different amounts of AdcA (black bars). Pre-immune serum (P) at 0,5 mg / mL was used as a negative control, and anti-AdcA sera (C) without incubation with AdcA was used as a positive control. Bars and whiskers denote mean values ± standard errors of the mean. NS, not significant (P > 0.05). *P < 0.05, ** P <0.01, *** P<0,001.

[0078] Figure 2 shows structural information on AdcA from E. faecium and epitope identification. (A) Domain composition of AdcA based on PFAM analysis. (B) Cartoon representation of the homology model of full-length AdcA. The ZnuA domain, formed by Lobe 1 (light grey) and Lobe 2 (light blue) is connected to the ZinT domain (violet) through a loop, represented in green. The two zoom panels report putative Zn-binding histidines, resporesented in stick, in each domain. Histidine residues likely involved in Zn uptake are represented in stick and Zn ions as grey balls. (C) epitope prediction using BepiPred3 (top) and Discotope (bottom). (D) cartoon and stick representation of the most antigenic region, here named as EH-motif, located in Lobe 1 of the ZnuA domain.

[0079] Figure 3 shows the (A) immuno-reactivity towards the EH-peptides detected by enzyme-linked immunosorbent assays (ELISA). Either the N-biot-EH (red) or C-biot-EH (black) were added to streptavidin coated plates and tested by ELISA with pre-AdcA sera (empty circles) or anti- AdcA sera (solid circles). (B-E) Inhibition of opsonophagocytic killing activity of anti-AdcA 0.5 mg / mL rabbit serum against S. aureus MW2 and (F-I) E. faecium 11236 / 1 by addition of EH-peptides. Antibodies raised against the recombinant AdcA were pre-incubated with the different inhibitors (in the concentration range 0.14 to 3.56 pM), including recombinant AdcA (white), ZnuA domain (grey), C-biot-EH (light blue) or N-biot-EH (blue). As positive control (C) the inventors used sera anti-AdcA 0.5mg / mL with no inhibitor (black). Statistical significance was performed by the one-way analysis of variances test, followed by the Dunnett’s multiple comparison post hoc test. Bars and whiskers denote mean values ± standard errors of the mean. NS, not significant (P > 0.05). *P < 0.05, ** P <0.01, *** P<0,001.

[0080] Figure 4 shows computational analysis of (A) EH-motif conservation in different bacterial species, and (B) pairwise alignment of consensus sequences of each dataset in (A) with the EH- motif of AdcA of E. faecium. Only regions with the highest Waterman-Eggert score are shown (computed with Lalign).

[0081] Figure 5 shows the design, validation, production and characterization of a hyper stable multipresenting antigen. (A) Molecular model of Sc(EH)s (SEQ ID NO: 5) after energy minimization. (B) DiscoTope predictions of Sc(EH)s, showing predicted antigenicity solely for the inserted EH-motifs. (C) Root-mean-square deviations (RMSD) computed on Ca atoms, reported for either the core or the EH-motifs residues for the three MD simulations at three increasing velocities in black, red, and green. (D) Root-mean-square fluctuations (RMSF) on Ca atoms showing high flexibility solely for the three EH-motifs. (E) Far-UV CD spectra were measured at 0.2 mg mL-1 in 20 mM sodium phosphate buffer (pH 7.4) at 20°C (light gray), 37°C (dark gray) and 100°C (black). The thermal denaturation curves monitored at 222 nm are reported in the inset. Black and red curves refer to experiments in the absence and the presence of 3M GuHCl denaturant, respectively. (F) Resistance to storage at 37°C for 30 days of Sc(EH)s compared to AdcA, as shown by SDS-gel electrophoresis. Degradation bands of AdcA are highlighted by arrows.

[0082] Figure 6 shows the OPA and OPIA assays against E. faecium 11236 / 1, S.aureus MW2 and E. faecalis T2. (A, B, C) OPA assays against E. faecium 11236 / 1, S.aureus MW2 and E. faecalis T2, respectively. OPA killing percentages mediated by anti-Sc(EH)3 sera (light grey bars E. faecium 11236 / 1, white bars S. aureus MW2 and dark grey bars in E. faecalis T2) are compared to those mediated by anti-AdcA (black bars). The statistical significance was tested by the unpaired two-tailed T-test with a 95% confidence interval using anti-AdcA and anti-Sc(EH)3 sera at the same concentration. Bars and whiskers denote mean values ± standard errors of the mean. *P < 0.05, ** P <0.01, *** P<0,001. (D-F) OPIA assay with 0.4 mg / mL anti-AdcA or anti-Sc(EH)3 sera against E. faecium 11236 / 1 (D) S.aureus MW2 (E) and E. faecalis T2 (F) by addition of different inhibitors. As a killing control (0) for each plot, the inventors used sera at 0.4 mg / mL anti-AdcA (dark blue bar) and anti-Sc(EH)3 (black bars) without inhibitors. Anti- AdcA and anti-Sc(EH)3 were pre-incubated with different concentrations of inhibitors, including recombinant AdcA (light blue bars), ZnuA domain (light grey bars), and recombinant SC(EH)3 (dark gray bars). The final inhibitor concentration is shown below the x-axis expressed as pM. Anti-AdcA sera incubated with AdcA were used as positive controls. Statistical significance of inhibition was performed by the one-way analysis of variances test, followed by the Dunnett’s multiple comparison post hoc test. Bars and whiskers denote mean values ± standard errors of the mean. NS, not significant (P > 0.05). *P < 0.05, ** P <0.01, *** P<0,001.

[0083] Figure 7 shows immunoreactivity towards various streptococci as determined by whole cells ELISA assay. Bars represent differences of absorbance at 405 nm obtained with incubation of preimmune sera and terminal bleeds of rabbits immunized with Sc(EH)3, at the same dilutions (Relative absorbance). Several Streptococcus pyogenes strains - Group A Streptococcus -, 6459-1 (light gray), 6476-1 (dark gray), 6781-1 (gray) and 6792-1 (gray), as well as Streptococcus agalactiae - Group B Streptococcus -, 50081-1 (dark gray) and 6164-1 (light gray), have been tested for increase of adsorption after immunization. Bars represent the mean values ± standard deviations.

[0084] Figure 8 shows the protective effect in mice of anti-Sc(EH)3 polyclonal antibodies against Enterococcus faecium. The percentage of survival mice during the protective assay (A). The continuous dark line represents the protected group treated with the anti-Sc(EH)3 (n=9), and the discontinuous line represents the negative control group with mice receiving the normal rabbit serum (NRS) (n=9). Differences between groups were performed using Log Rank (Mantel-cox test), ## - p <0.001. Plot representing a mean clinical score of groups during the protective assay (B). The clinical score was taken at 24 and 48h of the experiment on the survival mice, as described in methods. Gray color represents control group receiving BRC, dark gray color represents anti-Sc(EH)3 protected group. Statistical significance of the differences between groups was established using one way analysis of variances test, followed by post-hoc Dunnett’s multiple comparison, Bars and whiskers denote mean values ± standard errors of the mean. NS, not significant (P > 0.05). *P < 0.05, ** P <0.01, *** P<0,001. C) Bars representing CFU per organ of bacteria recovered from liver (dark gray) and spleen (gray) after 48 hours post infection. The inoculum at baseline (dark bar) is the same for the two groups. Difference between groups for each organ were performed using two tailed T test, ns: p>0.05, ****, p<0.0001.

[0085] Figure 9 shows (A) a scheme of the polyQ-EH fibrillisation process, and (B) respective Dynamic Light scattering (DLS).

[0086] Figure 10 shows the fluorescence spectrum of ThT 400pM before (light gray) and after incubation with polyQ-EH according to the invention.

[0087] Figure 11 shows the results of OPA assays against E. faecium 11236 / 1 (A) and S. aureus MW2 (B). OPA killing percentages mediated by anti-polyQ-EH are compared to those mediated by anti-AdcA, as indicated.

[0088] Figure 12 shows the results of OPIA assays against E. faecium 11236 / 1 (A) and S. aureus MW2 (B), as indicated.

[0089] SEQ ID No: 1 shows the amino acid sequence of AdcA as isolated from the Enterococcus faecium according to the NCBI Accession number WP_130587550.

[0090] SEQ ID No: 2 shows the amino acid sequence of the EH-motif in the AdcA protein of Enterococcus faecium according to the present invention. SEQ ID No: 3 shows the amino acid sequence of a preferred EH-motif construct according to the present invention.

[0091] SEQ ID No: 4 shows the amino acid sequence of a fibrillising pepti de as used in the invention.

[0092] SEQ ID No: 5 shows the amino acid sequence of the preferred Sc(EH)3-motif construct according to the present invention..

[0093] Examples

[0094] Materials and Methods:

[0095] Bioinformatic analysis - Homology modelling of AdcA from E. faecium was obtained using MODELLER (Webb and Sali, 2021) after consensus-based sequence alignment using HHpred (Soding et al., 2005). The best template was the crystal structure of AdcA from S. pneumoniae (PDB:7JJ9, sequence identity 65,33%).

[0096] AdcA homology model and AdcA sequences were used for epitope predictions, using a panel of the software. Among these, VaxiJen is an artificial intelligence software that was trained on known antigens and properties of amino acids in antigenic proteins (Doytchinova and Flower, 2007). BepiPred2 was used to predict the presence of linear B cell epitopes (Jespersen et al., 2017). BepiPred2 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. Discotope was used for structure-based epitope predictions (Haste Andersen et al., 2006).

[0097] Allergenicity was computed using AllerTOP v2.0 (Dimitrov et al., 2013) and AllergenFP v.1.0 (Dimitrov et al., 2014), which describe amino acids in given sequences using five E-descriptors: hydrophobicity, molecular size, helix-forming propensity, a relative abundance of amino acids, and P-strand forming propensity. Proteins are classified as allergenic or non-allergenic based on the k-nearest neighbor algorithm (kNN, k = 1), to which training a set containing 2427 known allergens from different species and 2427 non-allergens was used. Toxicity of the antigen was computed with ToxinPred (Gupta et al., 2013), a protein scanning tool, based on machine learning techniques and quantitative matrix through the recognition of motifs detected in toxins. Homology searches were performed using BlastP in the non-redundant protein sequences (nr) database. To improve the inventor’s search, the inventors used the ZnuA domain of AdcA with a query subrange for peptide G125-H141. Different thresholds were used to obtain primary datasets, which further were uploaded into EMBL-EBI software Clustal Omega (McWilliam et al., 2013) and analyzed. For multiple sequence alignment visualization and manipulation, the inventors used Jalview 2 (Waterhouse et al., 2009). Sequences with additions or deletions in EH rich region were manually removed and subtracted from the dataset used for consensus sequence. It should be noted that during analysis the inventors did not exclude sequences that were identical, neither clustered them together. Alignment data, raw datasets, and final datasets can be found in the supplementary data.

[0098] Design of Sc(EH)3 (SEQ ID NO: 5)and Molecular Dynamics analysis

[0099] The inventors used domain DI of Arginine Binding Protein from Thermatoga maritima (TmArgBP) (PDB 6gpc) (Ruggiero et al., 2014), where the inventors introduced the identified highly antigenic sequence of AdcA, the EH-motif, at specific locations, guided by molecular modeling. This model was used for further computational analysis, using Molecular Dynamics (MD). MD simulations were run with GROMACS 2020.3, in an octahedron box using a TIP3P water model (Van Der Spoel et al., 2005). Ions were added to reach neutrality. Periodic Boundary Conditions were employed and the LINCS algorithm was used to constrain all bond lengths. An integration time step of 2 fs was used. The particle mesh Ewald method was used to treat electrostatics, a non-bonded cut-off was applied for the Lennard-Jones potential. Temperature (T = 300 K) and pressure (p = 1 atm) were controlled using the v-rescale and the Parrinello-Rahman(Parrinello and Rahman, 1981) algorithms, respectively. Water molecules were relaxed by energy minimization and followed by 10 ps MD at 300 K, harmonically restraining the atomic positions. The systems were heated up gradually to 300 K in a six-step process, from 50 K to 300 K. Three short 5 ps-long equilibrations at 300 K at NPT were run changing the velocity seeds and finally, three production simulations were run under NPT conditions without restraints for 500ns each. The last 400 ns of the three velocity simulations were concatenated excluding the first 100 ns of each to ignore the equilibration phase. The analyses of the trajectories were done with GROMACS, PyMOL(“The PyMOL Molecular Graphics System, Version 1.2r3pre,” 2010), and VMD (http: / / www.ks.uiuc.edu / ). The simulation frames were clustered and the structures exhibiting the lowest RMSD relative to the other members of the most populated cluster were selected as MD representatives. Antigen production

[0100] For production and vector propagation, strains of Escherichia coli TOPIO (DE3) and E.coli Rosetta 2(DE3) were routinely grown in Luria-Bertani (LB) broth with shaking and on LB agar solid medium at 37 °C with dedicated antibiotics. The synthetic gene of Sc(EH)s (EUROFINS GENOMICS ITALY S.R.L) was subcloned into the pETM-13 vector (EMBL) using Ncol / Xhol restriction enzymes, adding not removable his-tag on the C -terminus. Vector was introduced via heat shock and multiplied in Escherichia coli TOPIO (DE3) and further purified with a Qiagen kit. Production was carried out in E.coli Rosetta (DE3) 2 strain. For production, 10 mL of overnight E.coli Rosetta 2(DE3) with Sc(EH)3 plasmid were added to IL of LB broth with chloramphenicol and kanamycin. The culture was kept at 37°C with agitation at 180rpm until OD600 reached 0.8. After this, the culture was induced with 0.2 mM isopropyl P- d-1- thiogalactopyranoside (IPTG) and transferred to 22°C with agitation 180rpm for overnight protein production. For harvesting culture was centrifuged for 15 min at 4°C, at 7000 rpm. Pellet was dissolved in lysis buffer (300 mM NaCl, 50mM Tris-HCl, 2.5% glycerol, pH 7.4) containing protease inhibitor cocktail (Roche) and further sonicated on ice for 15 minutes (10- sec sonication followed by 10-sec rest). Solution centrifuged at 4°C for 45 min, 14200 rpm. The supernatant was transferred onto the nickel column, where washing steps were performed with buffer A (300 mM NaCl, 50mM Tris-HCl, 2.5% glycerol, pH 7.4). Elution of the protein was performed at a final concentration of 200 mM imidazole and concentrated on Amicons with 10k Da cut-off. Then, Sc(EH)s was loaded on Superdex 200 increase 10 / 300 GL preequilibrated in a buffer containing Tris-HCl, NaCl 150 mM, and 5% glycerol, pH 7.4. Protein was re-concentrated on Amicons with 10k Da cut-off. For protein quantification, the inventors used the Bradford reaction with a ready-to-use reagent (Sigma-Aldrich) according to manufacturer instructions.

[0101] Circular dichroism spectroscopy

[0102] Circular dichroism (CD) spectra were measured using a Jasco J-810 spectropolarimeter with a Peltier temperature control system (Model PTC-423-S, Jasco Europe, Cremella (LC), Italy). Far-UV measurements at a protein concentration of 0.2 g / L were carried out in 10 mM phosphate buffer, at 20 °C, using a 0.1 cm optical path length cell. The range of the reported wavelengths 196-260 nm was fine-tuned as a function of the observed HT voltage. The spectra, recorded with a time constant of 4 s, a 2 nm bandwidth, and a scan rate of 10 nm min-1, were signal averaged over at least two scans. The final spectra were expressed as molar ellipticity [0] (deg cm2dmoU1) per residue. The temperature of the transition midpoint (Ta) was investigated by monitoring the change in ellipticity at 222 nm while increasing the temperature from 20 °C to 100 °C. To facilitate denaturation 3 M GuHCl was added. The reversibility of the transition was checked by lowering the temperature to 20 °C and re-scanning the sample without the denaturation agent. The curve was registered using a 0.1 cm path length cell, a protein concentration of 0.2 g / L, and a scan rate of 1.0 °C min-1.

[0103] Light scattering studies

[0104] Purified protein was analyzed by size-exclusion chromatography connected to a triple-angle light scattering detector equipped with a QELS module (quasi-elastic light scattering). Protein samples of 500 pg were loaded on a Superdex 200 increase 10 / 300 GL column, equilibrated in 50 mM Tris-HCl, 150 mM NaCl, 5% (v / v) glycerol, pH 7.4. A flow rate of 0.5 mL / min was applied. Elution profiles were detected by a Shodex interferometric refractometer and analyzed using a miniDawn TREOS light scattering system (Wyatt Instrument Technology Corp.). Data processing was carried out using the Astra 5.3.4.14 software package.

[0105] Sera

[0106] Rabbit polyclonal sera against Sc(EH)3 antigen were raised as follows: two New Zealand white rabbits for each antigen were selected before starting the immunizations, 10 mL of sera from each animal were collected to be used as negative controls. An initial immunization was performed via intramuscular injection (i.m.) with 73.2 nM of protein without any adjuvant. Three i.m. boosts were performed two, fourth and sixth weeks after the initial immunization with 14.6 nM of protein without any adjuvant. One final boost was given to the rabbits on week eight using 14.6 nM of protein combined with a proprietary adjuvant from Biogenes, admini stered through the same route as previous inj ections. The terminal bleeding was collected in the tenth week. All collected sera were heat inactivated at 56°C for 30 min. Before being used, sera were frozen and kept at ””20°C. For OPA and OPIA assays sera from the same time points were mixed 1 : 1 to have the average immune response of the rabbits. Anti-AdcA polyclonal serum was collected before as described elsewhere (Romero-Saavedra et al., 2015) and further stored in aliquots at -20°C and thawed when needed.

[0107] Immunoreactivity of the EH-motif tested by ELISA

[0108] Either anti-AdcA or anti-Sc(EH)3 rabbit serum specificity against the different biotinylated peptides (i.e. C-biot-EH and N-biot-EH) was tested by ELISA using high-capacity streptavidin- coated plates as described elsewhere(Laverde et al., 2020). Briefly, wells were washed three times with 200 pL of tween-B SA buffer (TBT: 1 *TBS, 0.1% BSA, and 0.05% Tween20). Then, the plates were coated with 0.5 pg of the corresponding antigen dissolved in TBT per well and incubated for 2 h at 4 °C. Wells were washed as described above. Then, 100 pL of anti-rabbit serum in triplicate for each dilution (ranging between 1 :250 to 1 : 160.000) were added to the plates and incubated for 2 h at RT with gentle shaking. Again, wells were washed three as described above and incubated for 60 min at RT with a 100 pL of alkaline-phosphatase- conjugated anti-rabbit IgG produced in goats at a 1 :1000 dilution. Finally, wells were washed four times with 200pL of TBT, and 100 pL of p-nitro phenyl phosphate at 1 mg / mL in glycine buffer was added to each well as the substrate. Plates were incubated at RT in the dark for 60 min for color development. The reaction was then stopped by adding 50 pL of 3 M sodium hydroxide, and the absorbance was measured at 405 nm.

[0109] IgG quantification

[0110] The concentration of IgG antibodies was determined using the sandwich ELISA method (Salauze et al., 1994). Plates were coated overnight with anti-rabbit IgG at 1 pg / mL concentration in 0.2 M sodium carbonate / bicarbonate buffer, pH 9.4 and kept at 4°C. Blocking was carried out using 3% BSA in PBS solution for Ih at RT. Investigated sera and rabbit IgG used for calibration curve were diluted in 1% BSA, 0.5% Tween20, PBS solution and incubated on the plate for 2h at RT. For detection was used anti-rabbit IgG Ab conjugated to alkaline phosphatase, for this step the inventors used likewise 2h incubation at RT. All the steps were separated by washing three times each well with 0.9% NaCl solution with 0.1% Tween20. For detection was used disodium p-nitrophenyl phosphate at final concentration 1 mg / mL in 0.1 M glycine buffer with ImM MgCh and ImM ZnCh, pH 10.4. Incubation in the dark was performed for 30 minutes after which the reaction was stopped by adding 3M NaOH. Absorbance was measured at 405nm wavelength and IgG concentration was calculated using an IgG calibration curve.

[0111] Opsonophagocytic Killing Assay (OPA) and Opsonophagocytic Inhibition Assay (OPIA)

[0112] For OPA and OPIA assays, the inventors used E. faecium 11236 / 1, S. aureus MW2 and E. faecalis Type 2, which were grown in tryptic soy agar (TSA) and broth (TSB) at 37 °C. The enterococcal strains were cultivated without agitation whereas the S. aureus strains were grown with agitation. OPA and OPIA are widely used to present op sonophagocytic-dep endent bacteria-killing with the usage of chosen agent and the specificity of this action. Full protocols of both assays were previously described elsewhere (Kropec A, Sava IG, Vonend C, Sakinc T, Grohmann E, Huebner J. Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections. Microbiology 2011; 157:3429-34). Briefly, four components were prepared: (a) baby rabbit absorbed with the target bacterial strain as a source of complement, (b) rabbit sera before and after immunization with the different antigens (c) polymorphonuclear neutrophils (PMNs) freshly prepared from human blood collected from healthy adult volunteers, and (d) the bacterial strains grown to OD650 nm=0.4 in tryptic soy broth (TSB). For the OPA, the four components were mixed: 100 pL of PMNs (2.5x 104 pL-1); 100 pL of the appropriate serum concentration, 100 pL ofcomplement (l :15 dilutions for the enterococcal strains, and 1 :30 for S. aureus strains and 100 pL of an appropriate dilution of bacteria. All the needed dilutions were made with RPMI + 15% FBS (RPMI-F). The mixture was incubated on a rotor rack at 37°C for 90 minutes, after this time samples were diluted in TSB, and plated on TSA plates in quadruplicate. The percentage of killing was calculated by comparing the colony counts at 90 min of a control (no PMNs) to the colony counts of a tube that contained all four components of the assay using the following formula:

[0113] {[(mean CFU control at 90 min) - (mean CFU at 90 min)] / (mean CFU control at 90 min) x 100%}

[0114] For OPIA protocol corresponds with OPA’s one, but used sera were diluted to final concentration of 0.4mg / mL and pre-incubated overnight at 4°C after mixing with an equal volume of RPMI-F with proteins carrying the epitope. Inhibition assays were performed at serum dilutions yielding 50-60% killing of the inoculum without the addition of the inhibitor. The inventors compared the percentage of inhibition of opsonophagocytic killing to controls without inhibitors and for the killing, the inventors used the previously mentioned equation. For the controls the inventors used i) bacteria incubated in RPMI-F, ii) bacteria incubated in RPMI- F with complement at the corresponding dilution, iii) bacteria incubated in RPMI-F with PMN’s, and iv) bacteria incubated in RPMI with complement and PMN’s. As a positive control in OPIA, the inventors used sera raised against AdcA. To show that the response is immunization dependent, sera before administration of antigen were used as negative controls to show a lack of minimal natural response in rabbits. Whole cell ELISA

[0115] Streptococci isolated from patients for this study were grown in 50 ml of BHI media until OD650 of 0.4 and harvested by centrifugation. Cells washed twice with PBS and resuspended in 8 % paraformaldehyde in PBS. After a 2-hour incubation, bacteria were washed with PBS and resuspended in 10 ml of coating buffer containing 0.2 M Sodium carbonate / bicarbonate buffer, pH 9.4. Nunc-immuno Maxisorp 96-well plates were coated overnight at 4°C with 100 pl of the bacterial suspension. The following day, plates were washed three times with PBS with 0.1 % Tween20 (PBST), and blocked with 3 % BSA solution overnight at 4°C. After three consecutive washes, plates were incubated at RT for 1 h with anti-Sc(EH)3 sera diluted in 3 % BSA solution at 1 :50 and 1 :200. Plates were washed thrice with PBST and incubated with secondary anti-rabbit IgG goat antibodies conjugated with Alkaline Phosphatase at RT for 1 h, after washing, colorimetric reaction was carried out using disodium p-nitrophenyl phosphate in glycine buffer at final concentration of 1 mg / mL. Absorbance at 405 nm was measured after 360 minutes.

[0116] Mice model

[0117] Six weeks old female BALB / cJRJ were obtained from Janvier Labs (Janvier Laboratories, Lyon, Saint Berthevin, France) and were housed in a controlled temperature and pressure environment at the Imagine Institute-SFR Necker (Paris, France). Mice were adapted to new environmental conditions for one week before experimental procedure beginning. The animals were house kept in cages with water and food ad libitum enriched with cardboard house with cotton squares for nests. The experimental protocol was approved by the animal Experimentation Ethics Committee under reference number 202201311143868 and all animals handling and procedures were performed in accordance with the French Decree N° 2013-118, 7 February 2013 and with European law and in agreement with animal research. A total of forty- four mice were used, twenty-six for the median lethal dose (LDso) determination and eighteen for the protection assay. At the end of the experiment, all animals were euthanized by cervical dislocation.

[0118] Determination of LDso of E. f aecium

[0119] An approximate LDso was initially determined using five different mice group (3-4 mice per group) with increased dose of bacteria ranging from 107to 1011CFU / ml of fresh bacterial culture in stationary phase (OD=1). The mice received the different bacterial doses by intraperitoneal (i.p) injection. Protection assay of anti-Sc(EH)3 against E. faecium

[0120] Eighteen mice were divided into two groups (n=9). A negative control group and a protected group, which received the heat inactivated Baby Rabbit Complement (BRC) sera (Cedarlane laboratories, Canada) and rabbit sera raised against anti-Sc(EH)3, respectively. The protection was performed with i.p injection at 24 hours and 4 hours before the bacterial challenge and at 4 hours after the bacterial challenge. The mice were followed for 48 hours and clinical scores were established. At the end of the experiment, the mice were euthanized by cervical dislocation and the liver and spleen were recovered for bacterial counting. Organs were homogenized with 1ml of PBS and serial dilutions were plated on LB agar to count CFUs.

[0121] Clinical score assignment

[0122] A clinical score ranged from 0 to 3 was assigned as follows: 0= none, 1= slight, 2= moderate, 3= severe. This score is assigned by monitoring weight, feces condition, pelage, posture, response to stimuli, locomotive activity, eyes and ears of mice.

[0123] Quantification and statistical analysis

[0124] In vitro data were analyzed using Prism 7 (GraphPad Software, La Jolla, CA). Plots show mean ± SEM. (*P < 0.05, ** P <0.01, *** P<0,001). Animal model data were analyzed using Prism 9.2 (GraphPad Software, La Jolla, CA). The results were expressed as mean ± SD.

[0125] Table 1. Prediction of the antigenicity, allergenicity, and toxicity of AcdA portions

[0126] Anti-AdcA serum mediates Opsonophagocytic killing of different S. aureus strains

[0127] Given the crucial role of the surface-exposed lipoprotein AdcA in S. aureus (which shares with AdcA of E. faecium a sequence identity of 66%) and the important challenge of addressing vaccine development against MRSA (Bacterial vaccines in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO, n.d.), the inventors tested whether antibodies raised against the AdcA from E. faecium were also able to mediate op sonophagocytosis in three different S. aureus strains MW2, LAC, and Reynolds. To obtain antibodies, New Zealand rabbits were previously immunized via intramuscular injection with AdcA from E. faecium and sera containing anti- AdcA antibodies were collected as described (Fig. 1A,B). In opsonophagocytic killing assay (OPA) the inventors observed that anti-AdcA are, indeed, opsonic against all tested S. aureus strains, with concentration-dependent percentages of opsonophagocytic killing mediated by the anti-AdcA serum in the range 40-60%, when compared with the pre-immune sera (Fig. 1C-E). A similar concentration-dependent yield of opsonophagocytic killing was observed for all strains. To assess whether antibodies that mediate the opsonic killing of S. aureus are specific towards AdcA, the inventors incubated the anti-AdcA sera with different amounts of recombinant AdcA (opsonophagocytic killing inhibition assay, OPIA) (Fig. IF). As shown in Fig. 1G, the opsonophagocytic killing of S. aureus MW2 mediated by the anti-AdcA serum is inhibited in a dose-dependent manner by purified AdcA.

[0128] Structure-based identification of AdcA epitope

[0129] The observed ability of E. faecium AdcA to act as an antigen against both E. faecium and S. aureus, suggesting that AdcA is a cross-reactive antigen that could be used against more pathogens, prompted the inventors to investigate the molecular determinants responsible for antigenicity of this protein. As shown in Fig. 2A, three distinct regions can be identified in AdcA sequence: a signal peptide at the N-terminus, a periplasmic Zinc-uptake complex component A (ZnuA) and a lipocalin-like zinc-recruitment domain (ZinT), as detected by PF AM analysis (Mistry et al., 2021) (Fig. 2A). Aimed at structure-based epitope identification, the inventors homology modelled the structure of AdcA, after consensus-based sequence alignment, using HHPred (Sbding et al., 2005). The entire AdcA structure was built using the program MODELLER (Webb and Sali, 2021) and the structure of the homologous AdcA from S. pneumoniae (pdb code: 7jj9, sequence identity 65%) as a template (Fig. 3B). In the AdcA structure, the N-terminal ZnuA domain and the C-terminal ZinT domain are connected by a long loop region, embedded between residues E310 and G328. The ZnuA domain presents a two-lobed organisation, formed by two domains adopting (p / a)4 folds, connected by a long a- helix (residues 169-199, Fig. 2B), whereas the ZinT domain adopts an 8-stranded P-barrel structure terminating with a helical region of four short helices (Fig. 2B). Both domains present a cluster of His-residues which is putatively involved in Zn binding (Fig. 2B). Consistently, they were both shown to bind Zn in the homolog AdcA from S. pneumoniae,' albeit being the ZnuA domain necessary and sufficient for Zn acquisition (Luo et al., 2021).

[0130] The inventors used the obtained model of AdcA for B-cell epitope prediction, using a variety of bioinformatics tools. Preliminary sequence-based B-cell epitope prediction, using Bepipred3 (Larsen et al., 2006), identified the strongest immunogenic regions on the ZnuA domain. Structure-based epitope prediction using Discotope-2.0 (Kringelum et al., 2012) (Fig. 2C) and ElliPro (“ElliPro: a new structure-based tool for the prediction of antibody epitopes" - BMC Bioinformatics 2008, 9:514doi:10.1186 / 1471-2105-9-514) confirmed this region as the most immunogenic one of the AdcA structure. The best-predicted antigen (detected with epitope threshold 0.65), is located on a large and flexible loop between a4 and P4 secondary structure elements of the ZnuA domain (Fig 2D). Antigen prediction was further analyzed, using VaxiJen v2.0, also for the whole AdcA and isolated domains, together with their toxicity and allergenicity (Table 1). All protein portions were predicted to be immunogenic (default threshold 0.4), with the strongest score corresponding to the 125-GSEEEDHDHGEEDHHHE- 141 (SEQ ID NO: 2) region, which is predicted to be non-allergenic and non-toxic (Table 1). Due to the abundance of glutamic acid (E) and histidine (H) in this loop sequence, the inventors denoted it as EH-motif (Fig. 2D).

[0131] The EH-motif sequence reacts with anti-AdcA antibodies and partially inhibits the opsonic killing elicited by the anti-AdcA serum

[0132] After the bioinformatic prediction of the EH-motif epitope, the inventors tested whether the antibodies raised against AdcA were able to bind this specific peptide sequence. To this end, the inventors obtained two synthetic versions of the epitope, one with biotinylation in the N- terminal part of the peptide named N-biot-EH (K(Biot)GSEEEDHDHGEEDHHHE) (SEQ ID NO: 2) and one with biotinylation in the C-terminal part of the peptide named C-biot-EH (GSEEEDHDHGEEDHHHEK(Biot)) (SEQ ID NO: 2). Both peptides were loaded on streptavidin-coated plates for ELISA assays. As shown in Fig. 3A, both peptides were recognized by the antibodies raised against AdcA, with C-biot-EH showing a higher affinity for the anti-AdcA serum, compared with the N-biot-EH (Fig. 3 A).

[0133] Further, the inventors tested if these peptides could inhibit the opsonic killing mediated by anti- AdcA antibodies in both E. faecium and S. aureus. To assess selectivity of antibodies the inventors performed OPIA experiments, where the inventors preincubated the anti-AdcA serum with peptides C-biot-EH and N-biot-EH and with the two positive controls full-length recombinant AdcA and the peptide corresponding to the full ZnuA domain. The inventors observed that both peptides were able to inhibit the opsonic activity of the anti-AdcA antibodies against the S. aureus MW2 (3B-E) and E.faecium 11236 / 1 (3F-I) in a concentration dependent manner (Fig. 3). As expected, the observed inhibition of opsonic killing due to EH-peptides was lower than that observed for the full-length AdcA protein, since the tested serum was originally raised against full-length AdcA (Fig. 3).

[0134] The best AdcA epitope, the EH-motif, is highly conserved among multiple species and suitable as a cross-reactive antigenic sequence

[0135] To design a cross-reactive antigen, the inventors performed a thorough sequence analysis to evaluate the level of antigen conservation among different strains of Enterococcus sp, and among different pathogenic bacteria from WHO high threat list. Sequence datasets and following alignment were performed using Blast? (Madden, 2003) and Clustal Omega EMBL- EBI (McWilliam et al., 2013) software, by searching homologues of the ZnuA domain of AdcA among multiple bacterial species. First, the inventors concentrated on investigating the conservation of the EH-motif among homologs in different serotypes of E. faecium (taxid: 1352). The inventors found 157 sequences with sequence identity of ZnuA over 60%. Of these, 89 present an almost fully conserved EH-motif (sequence identity 99%, Fig. 4). This value remains high also when all sequences are taken into account (sequence identity 80%). hen, the inventors broadened the inventor’s spectrum to Enterococcus sp. (taxid: 1350), after excluding E.faecium. This analysis identified 318 sequences, 303 of which presented EH-motif with small or no variations (sequence identity 87%, Fig. 4). Data also suggest that an EH-motif exists also in clinically relevant E. faecalis. Based on the promiscuity of EH-motif in ZnuA-like domains of Zn-acquiring proteins in Enterococcus sp., the inventors decided to broaden the inventor’s investigation to include closely related streptococci sp. Using the same threshold of 60% in Streptococcus sp. (taxid: 1301), the inventors observed that 548 out of 549 sequences had a highly conserved EH-motif (sequence identity 81%, Fig. 4).

[0136] The same analysis conducted on ZnuA domains of S. aureus (taxid: 1280) shows that a lower albeit significant sequence identity exists with ZnuA of E. faecium AdcA. Therefore, the inventors decided to lower the threshold to sequence identity of 30%, a value that still identify similar structural folds. Interestingly, among almost 627 sequences with sequence identity of 30%, the inventors identified 404 sequences embedding a conserved EH-motif (sequence identity 97.5%, Fig. 4). These sequences contain the strain MW2, LAC and Reynolds, for which the inventors have experimentally proven an opsonic activity of anti-AdcA antibodies (Fig. 1).

[0137] To evaluate the similarity of the EH-motifs of each bacterial species with that of AdcA, the inventors computed the consensus sequence of each species, using the Jalview, and then aligned this consensus with the sequence of AcdA EH-motif. As shown in Fig. 4B, the EH-motif sequence shares 100% sequence identity with the consensus computed for E. faecium, consistent with the high conservation of this loop in E. faecium family. Almost full conservation of the EH-motif is also observed with the consensus sequence of streptococci (sequence identity 93.3%), Fig. 3B. Lower sequence identities, still higher than 60%, are observed with S. aureus (Fig. 4B). Full datasets, a list of bacteria and alignments are available in supplementary data. These data predict a good efficacy of the EH-motif sequence as an antigen not only against multiple E. faecium strains, but against all the described bacterial species.

[0138] Design of a hyperstable, multi-presenting, and cross-reactive scaffolded antigen

[0139] Once the inventors had established that EH-motif is strongly immunogenic and promiscuous among different pathogens, the inventors focused on designing a hyper stable protein embedding multiple copies of this antigen. As a vehiculating molecule, the inventors chose the domain DI of the highly stable arginine binding protein (ArgBP) from T. maritima (PDB code 6gpc). Molecular modelling sessions were performed to identify the proper locations of the EH- motif to prevent the destabilization of the protein structure. A chimeric protein, here denoted as Sc(EH)s, was designed to contain the EH-motif in the DI domain between E46 and N47, G97 and G98 and at the N-terminal side of the protein (Fig. 5A). The model was minimized using GROMACS (Abraham et al., 2015) and used for molecular dynamics (MD) both to validate the designed model and to analyze its structural and dynamic features. DiscoTope analysis (Haste Andersen et al., 2006) on the minimized model shows that the sole predicted antigenic regions of Sc(EH)s are the inserted EH-motifs (Fig. 5B). As for MD studies, three MD trajectories with different starting velocities were run to improve the conformational ensemble search and extensively explore the dynamic features of the protein upon insertion of EH-motifs. The rootmean-square deviation (RMSD) profiles of Sc(EH)3, computed on Ca atoms with the respect to the starting model, suggests that the simulated system consistently reaches an equilibrium phase in about 100 ns in the three parallel runs, showing comparable mean and standard deviations of RMSD values. To dissect conformational changes of the accepting scaffold from those of the inserted EH-motifs, the inventors computed RMSD values of these isolated protein core and inserted EH-motifs (Fig. 5C). It appears clear that Sc(EH)3 presents as a compact and stable core with low and stable RMSD values, whereas the EH-motifs show large conformational variations (Fig. 5C). The high dynamic nature of EH-motifs is also confirmed by the root mean squared fluctuations (RMSF) of the relative residues (Fig. 5D), which show values in the range from 5 to 15 A, while the RMSF of all other residues remains below 5 A. The MD analyses suggest that the EH-motifs are a region of peculiar flexibility, whereas the stability of the protein core region is well preserved, with no distortion in the connecting regions. The high flexibility in antigenic sites is a good premise, since flexible epitopes can elicit a diverse antibody response through their conformational adaptation during the binding event to multiple antibodies (Fedechkin et al., 2020). After validation of Sc(EH)s model, the inventors sub-cloned the synthetic gene encoding for its amino acid sequence into pETM-13, expressed and purified in high yields, as monitored by SDS-PAGE electrophoresis. Light Scattering studies confirmed that Sc(EH)s is a monomer in solution, with a weight-average molar mass of22.04±0.1 kDa.

[0140] Of note for vaccine development, Sc(EH)s is extremely thermo-stable and highly resistant to storage at 37°C. Indeed, circular dichroism (CD) spectroscopy shows a fully conserved spectrum of the protein at 37°C, compared to that at 20°C (Fig. 5E). Also, Sc(EH)s is still endowed with a secondary structure content at 100°C (Fig. 5E). Consistently, thermal unfolding, analyzed by recording the CD signal at 222 nm, shows that the denaturation transition is not complete at 100°C (Fig. 5E, inset). Full unfolding could be achieved in the temperature range 20-100°C only in the presence of a chemical denaturant, specifically 3M GuHCl, with a melting temperature (Tm) of 52°C (Fig. 5E). As shown in Fig. 5F, Sc(EH)s is also highly resistant to proteolysis, as it can be stored for at least 30 days at 37°C with no sign of degradation. In the same conditions, AdcA exhibits a visible degradation pattern, with the accumulation of two major degradation bands in the SDS-PAGE gels (Fig. 5F).

[0141] SC(EH)3 elicits antibodies that generate strong and specific opsonic killing against E. faecium, S. aureus and E. faecalis and recognize Streptococcus pyogenes and Streptococcus agalactiae

[0142] SC(EH)3 protein was used to immunize two New Zealand white rabbits, which were exsanguinated two weeks after the last injection (see Methods). To generate a serum that accounts for an average immune response, the terminal bleeds of each rabbit were mixed in equal volumes, generating the anti-Sc(EH)3 serum. The same procedure was used for the pre- immune sera, used as a negative control. As a preliminary test, the inventors evaluated the ability of the anti-Sc(EH)3 antibodies to specifically recognize C-biot-EH by ELISA. The inventors found that the anti-Sc(EH)3 could specifically bind to the epitope up to a dilution of 1 :4000 whereas the pre-Sc(EH)3 did not show any binding at dilutions as high as 1 :250 (data not shown). This confirmed that the anti-Sc(EH)3 serum contained antibodies directed against the EH-motif. Thus, the relatively small motif, vehiculated in a hyperthermostable small protein Sc(EH)3 in multiple copies (3-fold) was able to elicit opsonophagocytic antibodies in rabbits, active against E. faecium, E. faecalis and S. aureus.

[0143] Then, the inventors used OPA to test if the anti-Sc(EH)3 antibodies can mediate the opsonic killing of E. faecium 11236 / 1, 5. aureus MW2 and E. faecalis T2. As shown in Fig. 6A, the anti-Sc(EH)3 antibodies in the concentration of 0.4 mg / mL were significantly better at inducing op sonophagocytic-dep endent E. faecium 11236 / 1 killing, compared to AdcA. The inventors observed an average opsonic killing of 67% at 0.4 mg / mL IgG dilution, compared to 46% of sera raised against full-length AdcA. Importantly, Sc(EH)3 was also able to induce opsonic killing of S. aureus MW2 with similar efficacy as anti-AdcA, (49% compared to 46%) at the highest antibody concentration used, 0.4 mg / mL (Fig. 6B). However, significantly higher killing activity than anti-AdcA was observed against S. aureus MW2 at lower concentrations of anti-Sc(EH)3 sera, with 48% killing of anti-Sc(EH)3 at 0.2 mg / mL compared to 32% in the case of anti-AdcA. Even at 0.05 mg / mL, anti-Sc(EH)3 sera were significantly more effective (21% killing), compared anti-AdcA sera (6% killing) (Fig. 6B). As for E. faecalis T2, sera raised by both AdcA and Sc(EH)3 were able to kill E. faecalis T2, with a maximum killing of about 70% at the maximum antibody concentration of 0.4 mg / mL (Fig. 6C). At lower antibody concentration (0.2 mg / mL), the inventors observed higher killing for anti-Sc(EH)3 (44%) compared to anti-AdcA (30%) (Fig. 6C).

[0144] To verify specificity of opsonizing antibodies against the EH-motifs of Sc(EH)3, opsonophagocytic inhibition assays were carried out by pre-incubating the anti-Sc(EH)3 sera with different inhibitors at different concentrations, including Sc(EH)3, AdcA, or ZnuA. Anti- AdcA sera incubated with AdcA protein were used as a control (Fig. 6D-F). In all cases, the inventors observed a concentration dependent reduction of killing induced by sera, indicating specificity of antibodies against each added antigen. Specifically, for E. faecium 11236 / 1 the inventors observed a reduction in the killing of anti-Sc(EH)3 antibodies, at the maximum concentration used, by 54%, 41%, and 53% upon incubation with AdcA, ZnuA, and Sc(EH)3, respectively (Fig. 6D). In the case of 5. aureus MW2, killing reduction at the highest inhibitor concentration was 49%, 51%, and 65% for AdcA, ZnuA and Sc(EH)3, respectively (Fig. 6E). Similar OPIA results were obtained for E. faecalis T2, showing that sera-mediated killing was significantly inhibited by all proteins in a concentration dependent manner (Fig. 7F), thus confirming the specific recognition of antibodies for the EH-motif, which is common to all inhibitors.

[0145] Given the similarity of the EH-motif in Streptococcus sp., that the inventors detected by bioinformatic analysis (Fig. 4), the inventors also tested the ability of anti-Sc(EH)3 antibodies to bind Streptococci. The inventors used whole-cell ELISA, due to limitations of the OPA assay for streptococci since; IgG that were able to recognize epitopes on representants of Streptococcus sp. were detected using secondary anti-rabbit antibodies. To avoid nonvaccination dependent reactions, the inventors performed assay using anti-Sc(EH)3 sera and pre-immune sara from the same rabbits. Further absorbance detected for pre-immune sera was subtracted from this post-vaccination respecting the dilution factor. As a result, the inventors observed that anti-Sc(EH)3 antibodies recognize representatives of both groups A and B - S. agalactiae AGA and AGC and S. pyogenes serotypes PYB, PYE, PYC, PYF (Fig. 7).

[0146] Anti-Sc(EH)3 antibodies present protective effect in E. faecium 11236 / 1 infected mice

[0147] To investigate the in vivo protective effect of the polyclonal antibodies anti-Sc(EH)3 against A. faecium, the inventors firstly defined the LD50 of bacteria in mice. Based on mortality in each group LD50 was set at approximately 5 x 109CFU / ml. During the protection assay of the anti- SC(EH)3 against the E. faecium LD50, after administration of sera and bacterial challenge, the mice were monitored and the mortality rate was evaluated. As shown in Fig. 8A, after 48h, a statistically decreased rate of mortality was observed in the Sc(EH)3 protected group (22.2%) compared to 88.9% of mortality in the negative control group (p=0.001). The survived mice were followed clinically and scored at 24 and 48 hours. As shown in Fig. 8B, the protected group showed a statistically better clinical score (between slight and moderate) after 48 hours compared to the control group where the clinical score was severe (p=0.001). In fact, seven out of nine mice within the anti-Sc(EH)3 protected group survived after the bacterial challenge, with one out of those mice showing a complete clearance in these organs, whereas only one mouse of the control group survived. After euthanasia, liver and spleen of survived mice were collected and homogenized to count the CFU per organ 48 hours post-infection. As shown in Fig. 8C, the protected group showed a statistically lower CFU of bacteria recovered the liver and the spleen, compared to the negative control group.

[0148] Design of multi-presenting peptide molecule embedding the EH-motif

[0149] Furthermore, the inventors studied the effect of antigen multi -presentation on its efficacy by designing and synthesizing a peptide molecule embedding the present EH-motif which can polymerize in a way to present the EH-motif antigen in a large number of copies per molecule. To this aim, the inventors fused the sequence of the EH-motif to that of a P-sheet selfassembling peptide, known to fibrillize in solution.

[0150] Based on the identification of the EH-motif epitope belonging to the lipoprotein AdcA of E. faecium as above, the inventors then opted for the multiple presentation of this epitope. For vehiculation, the inventors decided to add the EH antigen to the sequence of a fibrillizing peptide, with sequence QQKFQFQFEQQ (SEQ ID NO: 4). The inventors included a small linker between the fibrillizing peptide and the epitope, to provide optimal exposure of the epitope for antibody recognition. To keep the neutrality of terminal ends, the inventors added acetylation and amidation of the N- and C-terminal ends, respectively.

[0151] Antigen synthesis polyQ-EH peptide was synthetized through manual solid-phase peptide synthesis (20 pmol scale) following the Fmoc strategy and using standard Fmoc-derivatized amino acids and RINK AMIDE resin (substitution 0.3 mmol / g) as solid support. Activation of amino acids was carried out using HATU and DIEA, whereas Fmoc deprotection with a 20% (v / v) piperidine solution in DMF. All couplings were performed for 20 minutes and deprotections for 10 minutes.

[0152] After assembly, a small amount of crude product was detached from the resin by a treatment with a TFA:TIS:H2O (90:5:5 v / v / v) mixture for 1.5h at room temperature, then it was precipitated in cold ether, dissolved in a H2O / CH3CN mixture and analyzed by LC-MS analysis. The main peak corresponded to 3780.40 a.m.u. in perfect agreement with the theoretical one 3779.69 a. m. u.

[0153] Then, the peptide was acetylated, detached from the resin (TFA:TIS:H2O (90:5:5 v / v / v), for 3h) and lyophilized. It was purified by preparative RP-HPLC, applying a linear gradient of 0.1% TFA CH3CN in 0.1% TFA water from 5-70% over 13 min with a semipreparative 2.2 x 5 cm Cl 8 column at a flow rate of 20 mL / min, using a UV detector set at a wavelength of 210 nm. The purity of collected fractions was evaluated by LC-MS analysis.

[0154] Opsonophagocytic Killing Assay and Opsonophagocytic Inhibition Assay

[0155] For OPA and OPIA assays, the inventors used E. faecium 11231 / 6, and for cross-reactivity S. aureus MW2, which were grown in tryptic soy agar (TSA) and broth (TSB) at 37 °C. In contrast to S. aureus MW2, in TSB E. faecium was cultivated without agitation.

[0156] OPA and OPIA are widely used to present op sonophagocytic-dep endent bacteria-killing with the usage of chosen agent and the specificity of this action. In brief, four components were prepared: (a) baby rabbit absorbed with the target bacterial strain as a source of complement, (b) rabbit sera before and after immunization with scAdcA and against native AdcA, (c) polymorphonuclear neutrophils (PMNs) freshly prepared from human blood collected from healthy adult volunteers, and (d) the bacterial strains grown to OD650 nm=0.4 in tryptic soy broth (TSB). For the OPA, the four components were mixed: 100 pL of PMNs (2.5x 104 pL-1); 100 pL of the appropriate serum concentration, 100 pL of complement (1 :15 dilutions for the E. faecium strain, and 1 :30 for S. aureus), and 100 pL of an appropriate dilution of bacteria (1 :200 E. faecium and 1 :75 S. aureus). Needed dilutions were made with RPMI. The mixture was incubated on a rotor rack at 37°C for 90 minutes, and after this time samples were diluted in TSB, and plated on TSA plates in quadruplicate. The percentage of killing was calculated by comparing the colony counts at 90 min of a control (no PMNs) to the colony counts of a tube that contained all four components of the assay using the following formula:

[0157] {[(mean CFU control at 90 min) - (mean CFU at 90 min)] / (mean CFU control at 90 min) x 100%}

[0158] For OPIA protocol corresponds with OPA’s one, but used sera were diluted to final concentration of 0.4mg / mL and pre-incubated overnight at 4°C after mixing with an equal volume of RPMI with proteins carrying epitope - AdcAfin, ZnuAOl, and polyQ-EH. Inhibition assays were performed at serum dilutions yielding 50-60% killing of the inoculum without the addition of the inhibitor. The inventors compared the percentage of inhibition of opsonophagocytic killing to controls without inhibitors and for the killing, the inventors used the previously mentioned equation. For the controls the inventors used i) bacteria incubated in RPMI, ii) bacteria incubated in RPMI with complement, iii) bacteria incubated in RPMI with PMN’s, and iv) bacteria incubated in RPMI with complement and PMN’s. As a positive control in OPIA, the inventors used sera raised against AdcA and Intratect (a human mixed IgG solution). To show that the response is immunization-dependent, sera before administration of antigen were used as negative controls to show a lack of minimal natural response in rabbits.

[0159] Quantification and statistical analysis

[0160] Data were analyzed using Prism 7 (GraphPad Software). Statistical significance was determined using either an unpaired or paired t-test. Plots show mean ± SEM. (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).

[0161] Dynamic light scattering experiments

[0162] Dynamic light scattering (DLS) measurements were performed using a Malvern NanoZetasizer (Malvern, UK). Poly Q was diluted in Phosphate-Buffered Saline (PBS) at pH 7.4 to a concentration of 1 pM from a 10 mM stock solution. Afterwards, the sample was sonicated and then incubated at 4°C with agitation. The rate of aggregation was monitored from time 0 to 6 days of incubation. For the analysis of the oligomeric state, the sample was loaded into a disposable cuvette and maintained at 20°C during analysis. Spectra were recorded three times with 12 sub-runs using the multimodal mode. Only monodisperse peaks (% polydispersity lower than 25%) were considered for analysis. The Z-average diameter of the monodisperse peak was calculated from the correlation function using the Malvern technology software.

[0163] Results polyQ-EH forms nanometric fibril assemblies

[0164] Fibrilling polyQ peptides are known to form nanometric fibril structures with variable size, through the formation of long P-sheet structures (Fig. 9A). Using Dynamic light scattering, the inventors observed that after the fibrillisation procedure, polyQ-EH presents is mono-disperse and presents a molecular size of 229.3 ±38.3 nm (Fig 9B). The fluorescence spectrum of Thioflavin T (ThT) upon reaction with polyQ-EH confirms the P-sheet nature of the fibrillised peptide (Fig. 10). polyQ-EH elicits strong opsonophagocytic effect polyQ-EH was used to immunize two New Zealand white rabbits, which were exsanguinated two weeks after the last injection (see Methods above). To generate a serum that accounts for an average immune response, the terminal bleeds of each rabbit were mixed in equal volumes, generating the anti-polyQ-EH serum. The same procedure was used for the preimmune sera, used as a negative control.

[0165] Then, the inventors used OPA to test if anti-polyQ-EH antibodies can mediate the opsonic killing of E. faecium 11231 / 6 and S. aureus MW2. The anti-polyQ-EH antibodies in the concentration of 0.4 mg / mL were significantly better at inducing opsonophagocytic-dependent E. faecium 11231 / 6 killing, compared to AdcA. Indeed, the inventors observed an average opsonic killing of 67% at 0.4 mg / mL IgG dilution, compared to 46% of sera raised against full- length AdcA (Fig. 11 A). Importantly, anti-polyQ-EH was also able to induce opsonic killing of S. aureus MW2 with similar efficacy as anti -AdcA, (49% compared to 46%) at the highest antibody concentration used, 0.4 mg / mL (Fig. 11B).

[0166] In order to verify the specificity of opsonizing antibodies against the EH-motifs of anti-polyQ- EH, opsonophagocytic inhibition assays were carried out by pre-incubating the anti-polyQ-EH sera with different inhibitors. Anti-AdcA sera incubated with AdcA protein were used as a control. For each serum, either the anti-polyQ-EH, AdcA, or ZnuA were used at different concentrations. In all cases, the inventors observed a concentration dependent reduction of killing induced by sera, indicating specificity of antibodies against the added antigen. For E. faecium 11231 / 6, results showed a reduction in the killing of anti-polyQ-EH antibodies, at the maximum concentration used, by 54%, 41%, and 53% upon incubation with AdcA, ZnuA, and Sc(EH)3 itself, respectively (Fig. 12A). In the case of S. aureus MW2, killing reduction of 49%, 51%, and 65% was observed upon incubation with AdcA, ZnuA and Sc(EH)3 itself (Fig. 12B).

[0167] Literature as cited

[0168] Abraham, M.J., Murtola, T., Schulz, R., Pall, S., Smith, J.C., Hess, B., Lindahl, E., 2015. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1-2, 19-25. https: / / doi.Org / 10.1016 / j.softx.2015.06.001 Andreini, C., Banci, L., Bertini, I., Rosato, A., 2006. Zinc through the Three Domains of Life. J. Proteome Res. 5, 3173-3178. https: / / doi.org / 10.1021 / pr0603699

[0169] Bacterial vaccines in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization; 2022. Licence: CC BY-NC-SA 3.0 IGO, n.d.

[0170] Bayle, L., Chimalapati, S., Schoehn, G., Brown, J., Vemet, T., Durmort, C., 2011. Zinc uptake by Streptococcus pneumoniae depends on both AdcA and AdcAII and is essential for normal bacterial morphology and virulence. Mol. Microbiol. 82, 904-916. https: / / doi.Org / 10. llll / j.1365-2958.2011.07862.x Ben Braiek, O., Smaoui, S., 2019. Enterococci: Between Emerging Pathogens and Potential Probiotics. BioMed Res. Int. 2019, e5938210. https: / / doi.org / 10.1155 / 2019 / 5938210

[0171] Brandau, D., Jones, L., Wiethoff, C., Rexroad, J., Middaugh, C., 2003. Thermal Stability of Vaccines. J. Pharm. Sci. 92, 218-31. https: / / doi.org / 10.1002 / jps.10296

[0172] Bulula, N., Mwiru, D.P, Swalehe, O., Thomas Mori, A., 2020. Vaccine storage and distribution between expanded program on immunization and medical store department in Tanzania: a costminimization analysis. Vaccine 38, 8130-8135. https: / / doi.Org / 10.1016 / j.vaccine.2020.10.088

[0173] Burcham, L.R., Le Breton, Y, Radin, J.N., Spencer, B.L., Deng, L., Hiron, A., Ransom, M.R., Mendonga, J. da C., Belew, A.T., El-Sayed, N.M., McIver, K.S., Kehl-Fie, T.E., Doran, K.S., 2020. Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress. mBio 11, e02302-20. https: / / doi.org / 10.1128 / mBio.02302-20

[0174] Capdevila, D.A., Wang, J., Giedroc, D.P., 2016. Bacterial Strategies to Maintain Zinc Metallostasis at the Host-Pathogen Interface. J. Biol. Chem. 291, 20858-20868. https: / / doi.org / 10.1074 / jbc.R116.742023

[0175] Centers for Disease Control and Prevention (U.S.), 2019. Antibiotic resistance threats in the United States, 2019. Centers for Disease Control and Prevention (U.S.). https: / / doi.Org / 10.15620 / cdc:82532

[0176] Chiiambi, G.S., Nordstrom, H.R., Evans, D.R., Ferrolino, J. A., Hayden, R.T., Maron, G.M., Vo, A.N., Gilmore, M.S., Wolf, J., Rosch, J.W., Van Tyne, D., 2020. Evolution of vancomycin- resistant Enterococcus faecium during colonization and infection in immunocompromised pediatric patients. Proc. Natl. Acad. Sci. U. S. A. 117, 11703-11714. https: / / doi.org / 10.1073 / pnas.1917130117

[0177] Ch’ng, J.-H., Chong, K.K.L., Lam, L.N., Wong, J. J., Kline, K.A., 2019. Biofilm-associated infection by enterococci. Nat. Rev. Microbiol. 17, 82-94. https: / / doi.org / 10.1038 / s41579-018- 0107-z

[0178] Clegg, J., Soldaini, E., McLoughlin, R.M., Rittenhouse, S., Bagnoli, F., Phogat, S., 2021. Staphylococcus aureus Vaccine Research and Development: The Past, Present and Future, Including Novel Therapeutic Strategies. Front. Immunol. 12.

[0179] Dimitrov, I., Flower, D.R., Doytchinova, I., 2013. AllerTOP - a server for in silico prediction of allergens. BMC Bioinformatics 14, S4. https: / / doi.org / 10.1186 / 1471-2105-14-S6-S4

[0180] Dimitrov, I., Naneva, L., Doytchinova, I., Bangov, I., 2014. AllergenFP: allergenicity prediction by descriptor fingerprints. Bioinformatics 30, 846-851. https: / / doi.org / 10.1093 / bioinformatics / btt619

[0181] Doytchinova, I. A., Flower, D.R., 2007. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 8, 4. https: / / doi.org / 10.1186 / 1471- 2105-8-4

[0182] ElliPro: a new structure-based tool for the prediction of antibody epitopes - PubMed [WWW Document], n.d. URL https: / / pubmed.ncbi.nlm.nih.gov / 19055730 / (accessed 2.10.23). Fedechkin, S.O., George, N.L., Nunez Castrejon, A.M., Dillen, J.R., Kauvar, L.M., DuBois, R.M., 2020. Conformational Flexibility in Respiratory Syncytial Virus GNeutralizing Epitopes. J. Virol. 94, e01879-19. https: / / doi.org / 10.1128 / JVI.01879-19

[0183] Gao, H., Dai, W., Zhao, L., Min, J., Wang, F., 2018. The Role of Zinc and Zinc Homeostasis in Macrophage Function. J. Immunol. Res. 2018, 6872621. https: / / doi.org / 10.1155 / 2018 / 6872621

[0184] Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis, 2022. . Lancet Lond. Engl. 399, 629-655. https: / / doi.org / 10.1016 / S0140-6736(21)02724-0

[0185] Grim, K.P, San Francisco, B., Radin, J.N., Brazel, E.B., Kelliher, J.L., Parraga Solorzano, P.K., Kim, P.C., McDevitt, C.A., Kehl-Fie, T.E., 2017. The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity. mBio 8, e01281-17. https: / / doi.org / 10.1128 / mBio.01281-17

[0186] Gupta, S., Kapoor, P, Chaudhary, K., Gautam, A., Kumar, R., Raghava, G.P.S., 2013. In Silico Approach for Predicting Toxicity of Peptides and Proteins. PLoS ONE 8, e73957. https: / / doi.org / 10.1371 / joumal.pone.0073957

[0187] Haste Andersen, P., Nielsen, M., Lund, O., 2006. Prediction of residues in discontinuous B-cell epitopes using protein 3D structures. Protein Sci. Publ. Protein Soc. 15, 2558-2567. https: / / doi.org / 10.1110 / ps.062405906

[0188] Ismail, S., Shahid, F., Khan, A., Bhatti, S., Ahmad, S., Naz, A., Almatroudi, A., Tahir ul Qamar, M., 2021. Pan-vaccinomics approach towards a universal vaccine candidate against WHO priority pathogens to address growing global antibiotic resistance. Comput. Biol. Med. 136, 104705. https: / / doi.Org / 10.1016 / j .compbiomed.2021.104705

[0189] Jabbari Shiadeh, S.M., Pormohammad, A., Hashemi, A., Lak, P, 2019. Global prevalence of antibiotic resistance in blood-isolated Enterococcus faecalis and Enterococcus faecium: a systematic review and meta-analysis. Infect. Drug Resist. 12, 2713-2725. https: / / doi.org / 10.2147 / IDR.S206084

[0190] Jahantigh, H.R., Faezi, S., Habibi, M., Mahdavi, M., Stufano, A., Lovreglio, P., Ahmadi, K., 2022. The Candidate Antigens to Achieving an Effective Vaccine against Staphylococcus aureus. Vaccines 10, 199. https: / / doi.org / 10.3390 / vaccinesl0020199

[0191] Jespersen, M.C., Peters, B., Nielsen, M., Marcatili, P, 2017. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes. Nucleic Acids Res. 45, W24-W29. https: / / doi.org / 10.1093 / nar / gkx346

[0192] J.P Jung, A.K. Nagaraj, E.K. Fox, J.S. Rudra, J.M. Devgun, J.H. Collier, Co-assembling peptides as defined matrices for endothelial cells, Biomaterials 30(12) (2009) 2400-10.

[0193] Kalfopoulou, E., Huebner, J., 2020. Advances and Prospects in Vaccine Development against Enterococci. Cells 9, 2397. https: / / doi.org / 10.3390 / cells9112397

[0194] Kazemian, H., Pourmand, M.R., Siadat, S.D., Mahdavi, M., Yazdi, M.H., Avakh Majelan, P, Afshar, D., Yaseri, M., Davari, M., Getso, M.I., 2019. Molecular Cloning and Immunogenicity Evaluation of PpiC, GelE, and VS87 01105 Proteins of Enterococcus faecalis as Vaccine Candidates. Iran. Biomed. J. 23, 344-353. https: / / doi.Org / 10.29252 / .23.5.344 Kodali, S., Vinogradov, E., Lin, F., Khoury, N., Hao, L., Pavliak, V, Jones, C.H., Laverde, D., Huebner, J., Jansen, K.U., Anderson, A.S., Donald, R.G.K., 2015. A Vaccine Approach for the Prevention of Infections by Multi drug-resistant Enterococcus faecium. J. Biol. Chem. 290, 19512-19526. https: / / doi.org / 10.1074 / jbc.M115.655852

[0195] Krawczyk, B., Wityk, P, Gal^cka, M., Michalik, M., 2021. The Many Faces of Enterococcus spp. — Commensal, Probiotic and Opportunistic Pathogen. Microorganisms 9, 1900. https : / / doi . org / 10.3390 / microorgani sms9091900

[0196] Kringelum, J.V, Lundegaard, C., Lund, O., Nielsen, M., 2012. Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput. Biol. 8, el002829. https: / / doi.org / 10.1371 / journal.pcbi.1002829

[0197] Kropec, A., Sava, I.G., Vonend, C., Sakinc, T., Grohmann, E., Huebner, J.Y 2011, n.d. Identification of SagA as a novel vaccine target for the prevention of Enterococcus faecium infections. Microbiology 157, 3429-3434. https: / / doi.Org / 10.1099 / mic.0.053207-0

[0198] Lam, L.N., Brunson, D.N., Molina, J. J., Flores-Mireles, A.L., Lemos, J. A., 2022. The AdcACB / AdcAII system is essential for zinc homeostasis and an important contributor of Enterococcus faecalis virulence. Virulence 13, 592-608. https: / / doi.org / 10.1080 / 21505594.2022.2056965

[0199] Larsen, J.E.P., Lund, O., Nielsen, M., 2006. Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2. https: / / doi.org / 10.1186 / 1745-7580-2-2

[0200] Laverde, D., Romero- Saavedra, F., Argunov, D.A., Enotarpi, J., Krylov, V.B., Kalfopoulou, E., Martini, C., Torelli, R., van der Marel, G.A., Sanguinetti, M., Codee, J.D.C., Nifantiev, N.E., Huebner, J., 2020. Synthetic Oligomers Mimicking Capsular Polysaccharide Diheteroglycan are Potential Vaccine Candidates against Encapsulated Enterococcal Infections. ACS Infect. Dis. 6, 1816-1826. https: / / doi.org / 10.1021 / acsinfecdis.0c00063

[0201] Lebreton, F., Willems, R.J.L., Gilmore, M.S., 2014. Enterococcus Diversity, Origins in Nature, and Gut Colonization, in: Gilmore, M.S., Clewell, D.B., Ike, Y, Shankar, N. (Eds.), Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Massachusetts Eye and Ear Infirmary, Boston.

[0202] Luo, Z., Morey, J.R., Deplazes, E., Motygullina, A., Tan, A., Ganio, K., Neville, S.L., Eleftheriadis, N., Isselstein, M., Pederick, V.G., Paton, J.C., Cordes, T., Harmer, J.R., Kobe, B., McDevitt, C.A., 2021. A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA. mBio. https: / / doi.org / 10.1128 / mBio.01958-20

[0203] Madden, T., 2003. The BLAST Sequence Analysis Tool, The NCBI Handbook [Internet], National Center for Biotechnology Information (US).

[0204] Makthal, N., Do, H., Wendel, B.M., Olsen, R.J., Heimann, J.D., Musser, JM., Kumaraswami, M., 2020. Group A Streptococcus AdcRRegulon Participates in Bacterial Defense against Host- Mediated Zinc Sequestration and Contributes to Virulence. Infect. Immun. 88, e00097-20. https: / / doi.org / 10.1128 / IAI.00097-20

[0205] McWilliam, H., Li, W., Uludag, M., Squizzato, S., Park, Y.M., Buso, N., Cowley, A.P., Lopez, R., 2013. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 41, W597- W600. https: / / doi.org / 10.1093 / nar / gkt376 Miller, L.S., Fowler, V.G., Jr, Shukla, S.K., Rose, W.E., Proctor, R.A., 2020. Development of a vaccine against Staphylococcus aureus invasive infections: Evidence based on human immunity, genetics and bacterial evasion mechanisms. FEMS Microbiol. Rev. 44, 123-153. https: / / doi.org / 10.1093 / femsre / fuz030

[0206] Mistry, J., Chuguransky, S., Williams, L., Qureshi, M., Salazar, G.A., Sonnhammer, E.L.L., Tosatto, S.C.E., Paladin, L., Raj, S., Richardson, L.J., Finn, R.D., Bateman, A., 2021. Pfam: The protein families database in 2021. Nucleic Acids Res. 49, D412-D419. http s: / / doi.org / 10.1093 / nar / gkaa913

[0207] Nallapareddy, S.R., Singh, K.V., Sillanpaa, J., Zhao, M., Murray, B.E., 2011. Relative contributions of Ebp Pili and the collagen adhesin ace to host extracellular matrix protein adherence and experimental urinary tract infection by Enterococcus faecalis OG1RF. Infect. Immun. 79, 2901-2910. https: / / doi.org / 10.1128 / IAI.00038-ll

[0208] Ong, C.-L.Y, Berking, O., Walker, M.J., McEwan, A.G., 2018. New Insights into the Role of Zinc Acquisition and Zinc Tolerance in Group A Streptococcal Infection. Infect. Immun. 86, e00048-18. https: / / doi.org / 10.1128 / IAI.00048-18

[0209] Pambudi, N.A., Sarifudin, A., Gandidi, I.M., Romadhon, R., 2022. Vaccine cold chain management and cold storage technology to address the challenges of vaccination programs. Energy Rep. 8, 955-972. https: / / doi.Org / 10.1016 / j.egyr.2021.12.039

[0210] Parrinello, M., Rahman, A., 1981. Polymorphic transitions in single crystals: A new molecular dynamics method. J. Appl. Phys. 52, 7182-7190. https: / / doi.org / 10.1063 / E328693

[0211] Romero- Saavedra, F., Laverde, D., Budin- Verneuil, A., Muller, C., Bernay, B., Benachour, A., Hartke, A., Huebner, J., 2015. Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections. PLoS ONE 10, e0136625. https: / / doi.org / 10.1371 / joumal.pone.0136625

[0212] Romero- Saavedra, F., Laverde, D., Wobser, D., Michaux, C., Budin- Verneuil, A., Bernay, B., Benachour, A., Hartke, A., Huebner, J., 2014. Identification of Peptidoglycan-Associated Proteins as Vaccine Candidates for Enterococcal Infections. PLOS ONE 9, el 11880. https: / / doi.org / 10.1371 / joumal.pone.0111880

[0213] Ruggiero, A., Dattelbaum, J.D., Staiano, M., Berisio, R., D’Auria, S., Vitagliano, L., 2014. A Loose Domain Swapping Organization Confers a Remarkable Stability to the Dimeric Structure of the Arginine Binding Protein from Thermotoga maritima. PLoS ONE 9, e96560. https: / / doi.org / 10.1371 / joumal.pone.0096560

[0214] Salauze, D., Serre, V, Perrin, C., 1994. Quantification of total IgM and IgG levels in rat sera by a sandwich ELISA technique. Comp. Haematol. Int. 4, 30-33. https: / / doi.org / 10.1007 / BF00368263

[0215] Salazar, N., Yamamoto, B.B., Souza, M.C.L. de, Silva, L.B. da, Areas, A.P.M., Barbosa, A.S., 2022. The C-Terminal Domain of Staphylococcus aureus Zinc Transport Protein AdcA Binds Plasminogen and Factor H In Vitro. Pathog. Basel Switz. 11, 240. https: / / doi.org / 10.3390 / pathogensll020240

[0216] Sbding, J., Biegert, A., Lupas, A.N., 2005. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244-W248. https: / / doi.org / 10.1093 / nar / gki408 Tacconelli, E., Carrara, E., Savoldi, A., Harbarth, S., Mendelson, M., Monnet, D.L., Pulcini, C., Kahlmeter, G., Kluytmans, J., Carmeli, Y, Ouellette, M., Outterson, K., Patel, J., Cavaleri, M., Cox, EM., Houchens, C.R., Grayson, M.L., Hansen, P, Singh, N., Theuretzbacher, U., Magrini, N., WHO Pathogens Priority List Working Group, 2018. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318-327. https: / / doi.org / 10.1016 / S1473-3099(17)30753-3

[0217] The PyMOL Molecular Graphics System, Version 1.2r3pre, 2010.

[0218] Theilacker, C., Kropec, A., Hammer, F., Sava, I., Wobser, D., Sakinc, T., Codee, J.D.C., Hogendorf, W.F.J., van der Marel, G.A., Huebner, J., 2012. Protection Against Staphylococcus aureus by Antibody to the Polyglycerolphosphate Backbone of Heterologous Lipoteichoic Acid. J. Infect. Dis. 205, 1076-1085. https: / / doi.org / 10.1093 / infdis / jis022

[0219] Van Der Spoel, D., Lindahl, E., Hess, B., Groenhof, G., Mark, A.E., Berendsen, H.J.C., 2005. GROMACS: Fast, flexible, and free. J. Comput. Chem. 26, 1701-1718. https: / / doi.org / 10.1002 / jcc.20291

[0220] Vekemans, J., Hasso-Agopsowicz, M., Kang, G., Hausdorff, W.P, Fiore, A., Tayler, E., Klemm, E.J., Laxminarayan, R., Srikantiah, P., Friede, M., Lipsitch, M., 2021. Leveraging Vaccines to Reduce Antibiotic Use and Prevent Antimicrobial Resistance: A World Health Organization Action Framework. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 73, el011-el017. https: / / doi.org / 10.1093 / cid / ciab062

[0221] Wan, L.Y.M., Chen, Z.J., Shah, N.P, El-Nezami, H., 2016. Modulation of Intestinal Epithelial Defense Responses by Probiotic Bacteria. Crit. Rev. Food Sci. Nutr. 56, 2628-2641. https: / / doi.org / 10.1080 / 10408398.2014.905450

[0222] Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M., Barton, G.J., 2009. Jalview Version 2 — a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189-1191. https: / / doi.org / 10.1093 / bioinformatics / btp033

[0223] Webb, B., Sali, A., 2021. Protein Structure Modeling with MODELLER. Methods Mol. Biol. Clifton NJ 2199, 239-255. https: / / doi.org / 10.1007 / 978-l-0716-0892-0_14

[0224] Weiner, L.M., Webb, A.K., Limbago, B., Dudeck, M.A., Patel, J., Kallen, A. J., Edwards, J.R., Sievert, D.M., 2016. Antimicrobial -Resistant Pathogens Associated With Healthcare- Associated Infections: Summary of Data Reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2011-2014. Infect. Control Hosp. Epidemiol. 37, 1288-1301. https: / / doi.org / 10.1017 / ice.2016.174

[0225] World Health Organization, 2022. Bacterial vaccines in clinical and preclinical development 2021 [WWW Document], URL https: / / www.who.int / publications-detail- redirect / 9789240052451 (accessed 3.6.23).

[0226] Xia, P, Lian, S., Wu, Y, Yan, L., Quan, G., Zhu, G., 2021. Zinc is an important inter-kingdom signal between the host and microbe. Vet. Res. 52, 39. https: / / doi.org / 10.1186 / sl3567-021- 00913-1

[0227] Zhou, X., Willems, R.J.L., Friedrich, A.W., Rossen, J.W.A., Bathoom, E., 2020. Enterococcus faecium: from microbiological insights to practical recommendations for infection control and diagnostics. Antimicrob. Resist. Infect. Control 9, 130. https: / / doi.org / 10.1186 / sl3756-020- 00770-1

Claims

Claims1. An antibacterial antigen comprising a continuous stretch of at least 6 amino acids of the amino acid sequence GSEEEDHDHGEEDHHHE (SEQ ID NO: 2) or an amino acid sequence being at least 90%, preferably 95%, and more preferably 98% identical to said continuous stretch of SEQ ID NO: 2, wherein the length of the antigen is between about 300 and 6 amino acids, preferably between about 50 and 6 amino acids, and most preferred about 17 amino acids.

2. The antibacterial antigen according to claim 1, wherein said antigen sequence is fused to the amino acid sequence of a suitable fibrillising peptide, in particular to the amino acid sequence QQKFQFQFEQQ (SEQ ID NO: 4), optionally further comprising a linker peptide between the antigen and fibrillising peptide sequence.

3. An antigenic protein construct, comprising at least one antibacterial antigen sequence according to claim 1 integrated into the DI domain of a bacterial arginine binding protein (ArgBP), without a destabilization of the protein structure, preferably wherein the at least one antibacterial antigen sequence is integrated into the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima.

4. The antigenic protein construct according to claim 3, wherein the amino acid sequence is integrated in the DI domain of the highly stable arginine binding protein (ArgBP) from T. maritima between amino acids E46 and N47, G97 and G98, and at the N-terminal end of the protein.

5. The antigenic protein construct according to claim 3 or 4, wherein the construct shows no substantial sign of degradation when stored for more than 30 days at 37°C.

6. An antibody or antigen binding fragment thereof binding specifically to a contiguous sequence of at least six amino acids of SEQ ID NO: 2, the antibacterial antigen according to claim 1 or 2 and / or the antigenic protein construct according to any one of claims 3 to 5.

7. The antibody according to claim 6, which is a monoclonal antibody and / or an antibody or antigen binding fragment thereof that is opsonic.

8. A nucleic acid encoding the antibacterial antigen according to claim 1 or 2, the antigenic protein construct according to any one of claims 3 to 5 or the antibody or antigen binding fragment thereof according to claim 6 or 7, wherein preferably the nucleic acid is selected from DNA, RNA, stabilized RNA and combinations thereof.

9. A pharmaceutical composition comprising the antibacterial antigen according to claim 1 or 2, the antigenic protein construct according to any one of claims 3 to 5 or the antibody or antigen binding fragment thereof according to claim 6 or 7 or the nucleic acid according to claim 8, and a pharmaceutically acceptable carrier, adjuvant and / or diluent, wherein said composition preferably is a vaccine.

10. The pharmaceutical composition according to claim 9, wherein said vaccine is formulated for administration via intramuscular, subcutaneous, or inhalation routes.

11. The antibacterial antigen according to claim 1 or 2, the antibody or antigen binding fragment thereof according to claim 6 or 7, the nucleic acid according to claim 8 or the pharmaceutical composition according to claim 9 or 10 for use in the prophylaxis or treatment of diseases, in particular for the prophylactic or therapeutic treatment of a disease or condition caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof.

12. A method for treating or preventing a disease or condition in a subject that is caused by a bacterium, such as, for example, enterococcal infection, urinary tract infections, bacteremia, bacterial endocarditis, peritonitis, wound and soft tissue infections, and meningitis, or pneumonia, wherein preferably said bacterium is selected from Gram-positive ESKAPE pathogens, enterococci, staphylococci or streptococci, such as, for example, E. faecium, E. faecalis, S. aureus, coagulase-negative staphylococci or S. pyogenes, and in particular antibiotic-resistant strains thereof, comprising administering to the subject an effective amount of the antibacterial antigen according to claim 1 or 2, the antibody or antigen binding fragmentthereof according to claim 6 or 7, the nucleic acid according to claim 8 or the pharmaceutical composition according to claim 9 or 10.

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