Novel gardnerella endolysins and uses thereof

Abstract

To provide new species-selective phage endolysins and their use to treat bacterial vaginosis (BV), to provide recombinant endolysins, i.e., domain-swapped endolysins, and also provide the endolysins for use in treating diseases or disorders such as bacterial infections, in particular BV.SOLUTION: The invention relates to polynucleotides encoding the endolysins. The polynucleotides can also be used for treating such diseases or disorders. Also provided herein is a pharmaceutical composition comprising an endolysin of the invention for use in treating such diseases or disorders. The endolysins, polynucleotides and pharmaceutical composition may be administered locally, in particular locally into the vagina.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to new species-selective phage endolysins and their use for treating bacterial vaginosis (BV). The present invention provides recombinant endolysins, i.e. domain-swapped endolysins. The present invention also relates to said endolysins for use in treating diseases or disorders such as bacterial infections, in particular BV. The present invention further relates to polynucleotides encoding said endolysins. The polynucleotides are also used for treating such diseases or disorders. Pharmaceutical compositions comprising the endolysins of the present invention for use in treating such diseases or disorders are also provided by the present invention. The endolysins, polynucleotides and pharmaceutical compositions may be administered topically, in particular locally in the vagina. [Background technology]

[0002] In the literature, bacterial vaginosis (BV), also called bacterial vaginosis, nonspecific vaginosis and nonspecific vaginitis, is the most common vaginal infection worldwide and is associated with significant adverse consequences including preterm labor and delivery, postpartum endometritis, and a high risk of HIV acquisition. It is a vaginal dysbiosis in which commensal Lactobacilli are replaced by polymicrobial biofilms, the formation of a foul-smelling fluid and an elevated pH from the natural 3.5-4.5 to 5.5. Reported prevalence ranges from 10-40% depending on the population studied. However, suboptimal diagnostic methods and a high rate of asymptomatic patients make the true prevalence of BV difficult to ascertain. Gardnerella vaginalis (G. vaginalis) is a bacterial species associated with BV.

[0003] The etiopathogenesis of BV remains poorly understood. It is most commonly defined as a pathological state characterized by the loss of normal vaginal flora, especially H2O2-producing species of Lactobacillus, and the concomitant overgrowth of anaerobic bacteria, including G. vaginalis, Mobiluncus species, and Mycoplasma hominis. However, recent data suggest a central role for G. vaginalis as a specific and sexually transmitted etiologic agent in BV (Muzny et al., 2016, J. of Infect. Dis. 214 Suppl. 1., S1).

[0004] In the 1950s, large numbers of small pleomorphic Gram-variant bacilli were observed in the genital tract of women with BV. This organism was first called Haemophilus vaginalis, was repeatedly renamed as more information on its characteristics became available, and is currently classified as G. vaginalis, which was considered the only member of the genus Gardnerella as of 2018. However, in early 2019, it was shown that the genus Gardnerella actually encompasses at least 13 species, and the most frequent ones were renamed G. vaginalis sensu stricto, G. leopoldii, G. piochii, and G. swidszynskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661).

[0005] Gardnerella bacteria are special in that they are Gram-variant, i.e., they do not form the outer membrane that defines Gram-negative species. The cell wall is generally very thin, with less than 10% peptidoglycan content, and this is why the crystal violet dye used in Gram staining does not always produce the deep purple color typical of Gram-positive species. Rather, Gardnerella cells can appear both Gram-positive and Gram-negative in a Gram stain. A phylogenetic analysis based on 16S rRNA placed Gardnerella in the Gram-positive family Bifidobacteriales.

[0006] During BV, the epithelial surface is covered by a thick volume of G. vaginalis in a biofilm that is often resistant to treatment. Biofilms are adhesive collections of microorganisms held together by a polymeric matrix composed of polysaccharides, proteins and / or nucleic acids. The unique gene expression and physical structure of the biofilm enhance the resistance of the bacteria to many negative stimuli, including chemical germicides, extreme pH, host immune defenses and antibiotics. Standard BV treatments are the antibiotics metronidazole and clindamycin, which, however, often do not completely remove the biofilm, resulting in up to 60% recurrence within 6 months. Furthermore, antibiotic treatments wipe out the vaginal microflora, albeit leaving a significant portion of the remaining available biofilm, which opens this ecological niche for other pathogens, e.g. fungi. Therefore, a common effect of BV treatment is candidiasis. Treatment of BV is also attempted with probiotics, specifically with beneficial lactobacilli that are expected to recolonize the vagina. However, several clinical trials have failed to show benefit.

[0007] Therefore, there is a great need for new methods and compositions for treating G. vaginalis infection, especially BV, by selectively killing, for example, Gardnerella genus bacterial cells, preferably without harming beneficial lactobacilli, and allowing them to repopulate the vagina.Therefore, the technical problem underlying the present invention is to provide new means and methods for treating BV. The technical problem is solved by providing the embodiments characterized in the claims. Summary of the Invention

[0008] The present invention is based on the preparation of novel recombinant Gardnerella prophage endolysins having unexpected properties and structures that make them particularly suitable for various uses and methods, in particular for treating, decontaminating or detecting bacterial infections and disorders, in particular involving Gardnerella.

[0009] A first aspect of the present invention is a method for producing a composition comprising the steps of: (i) the N-terminal catalytic domain, or a functional variant thereof; (ii) a C-terminal cell wall binding region, or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region; and providing an endolysin comprising or consisting of: Here, the endolysin has killing activity against Gardnerella.

[0010] In one embodiment of the invention, the N-terminal catalytic domain is derived from a first naturally occurring endolysin, the linker region and the C-terminal cell wall binding region are derived from a second naturally occurring endolysin, and the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages. (i) the N-terminal catalytic domain, or a functional variant thereof; (ii) a C-terminal cell wall binding region, or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region; The present invention provides a recombinant endolysin comprising or consisting of: wherein the N-terminal catalytic domain is derived from a first naturally occurring endolysin, the linker region and the C-terminal cell wall binding region are derived from a second naturally occurring endolysin, and the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages; and Here, said recombinant endolysin has killing activity against Gardnerella.

[0011] Gardnerella is unique in that it is a Gram-variant species: it does not form an outer membrane that defines a true Gram-negative species. Its cell wall is generally very thin, with less than 10% peptidoglycan content. This indicates that peptidoglycan degrading enzymes such as endolysin proteins are unable to efficiently lyse the bacterial cell wall of Gardnerella. However, in the context of the present invention, a genetically engineered endolysin has been identified that has the advantageous property of effectively killing Gardnerella species, and thus may be used as a novel therapeutic for the treatment of BV.

[0012] Healthy vagina is mainly inhabited by three types of Lactobacillus: L. crispatus, L. gasseri and L. jensenii. They maintain an acidic pH of 3.5-4.5 by producing lactic acid and a protective oxidative environment by producing H2O2. Recovery from BV is associated with vaginal repopulation by these Lactobacilli. However, antibiotics (conventionally used to treat BV) have the disadvantage of hindering the process of vaginal repopulation by Lactobacilli. In contrast, the novel recombinant endolysin of the present invention advantageously has species-selective killing activity against Gardnerella and does not harm Lactobacilli. In addition, the accompanying examples show that all tested Gardnerella strains have low susceptibility to metronidazole and clindamycin, which are conventionally used to treat BV. This could explain the high recurrence rate of BV. This also confirms that the endolysins of the invention are superior to antibiotics in the treatment of BV, and thus treating BV with the endolysins of the invention has significant advantages over currently available treatments, such as treatment with the antibiotics metronidazole and clindamycin.

[0013] As used herein, the term "derived from a first naturally occurring endolysin" means that the respective portion (i.e. the N-terminal catalytic domain) is identical to the first naturally occurring endolysin or is a functional variant thereof. As defined herein, a functional variant is a polypeptide having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the amino acid sequence of the respective portion (i.e. the N-terminal catalytic domain) of the first naturally occurring endolysin, resulting in a functional endolysin, wherein the function comprises killing activity against Gardnerella. The amino acid sequences of some naturally occurring endolysins are provided herein below and summarized in Table 7.

[0014] Thus, the term "derived from a second naturally occurring endolysin" means that the respective parts (i.e. the linker region and the C-terminal cell wall binding region) are identical to the second naturally occurring endolysin or are functional variants thereof, i.e. endolysins different from the first naturally occurring endolysin. As defined herein, a functional variant is a polypeptide having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity and most preferably at least 99.7% identity) with the amino acid sequence of the respective parts (i.e. the linker region and the C-terminal cell wall binding region) of the second naturally occurring endolysin, resulting in a functional endolysin, wherein said function comprises Gardnerellacidal activity.

[0015] Herein, the N-terminal catalytic domain is also referred to as the "H-domain". For example, the term "H2" refers to the H-domain of native endolysin (EL)2. The "C-terminal cell wall binding domain" refers to one or more cell wall binding domains. The linker and the cell wall binding domain together represent the so-called "B domain". For example, B10 refers to the B domain of native EL10. Similarly, B11_N refers to the N-terminal cell wall binding domain of native EL11, B12_C refers to the C-terminal cell wall binding domain of native EL12, etc.

[0016] The present invention further comprises the following: (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 5, 7, or 10 to 12, or a functional variant thereof having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, 7, or 10 to 12; (ii) a C-terminal cell wall-binding region comprising or consisting of at least one cell wall-binding domain independently selected from the group consisting of polypeptides comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, respectively, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, respectively; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding domain; and providing an endolysin comprising or consisting of: wherein said endolysin has killing activity against Gardnerella.

[0017] As shown in the accompanying examples, the most active N-terminal catalytic domain (also referred to as the "H-domain") is H2 (SEQ ID NO:2), followed by H7 strains (SEQ ID NO:7), H10 (SEQ ID NO:10) and H5 (SEQ ID NO:5).

[0018] Thus, in a preferred embodiment of the invention, the N-terminal catalytic domain consists of a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 2, 7, 10 and 5, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 2, 7, 10 and 5; Thereby, the endolysin is functional, where said function comprises the ability to lyse the cell wall of Gardnerella.

[0019] Thus, in a preferred embodiment of the invention, the N-terminal catalytic domain has either the amino acid sequence of SEQ ID NO:5, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO:5; or more preferably the amino acid sequence of SEQ ID NO:10, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity) to the amino acid sequence of SEQ ID NO:10. or even more preferably, the amino acid sequence of SEQ ID NO:7, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO:7.or even more preferably, a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:2 or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO:2; Thereby, the endolysin is functional, where said function comprises the ability to lyse the cell wall of Gardnerella.

[0020] It is also shown in the appended examples that B region B10 (containing the cell wall binding domains of SEQ ID NOs: 28 and 29) is the most active, followed by B11 (containing the cell wall binding domains of SEQ ID NOs: 30 and 31), B12 (containing the cell wall binding domains of SEQ ID NOs: 32 and 33) and B3 (containing the cell wall binding domains of SEQ ID NOs: 19 and 20).

[0021] Thus, in a preferred embodiment of the present invention, the cell wall binding domain(s) is(are) selected from the group consisting of polypeptides comprising or consisting of any of the amino acid sequences of any one of SEQ ID NOs: 19, 20 and 28-33, and functional variants thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 19, 20 and 28-33; Thereby, the endolysin is functional, where said function comprises the ability to lyse the cell wall of Gardnerella.

[0022] The endolysin of the invention preferably comprises two cell wall binding domains. In one embodiment of the invention, the cell wall binding domain (B domain) of the endolysin of the invention consists of a polypeptide comprising or consisting of any of the amino acid sequences of any one of SEQ ID NOs: 19, 20 and 28 to 33, as well as any of its functional variants having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity and most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 19, 20 and 28 to 33; Thereby, the endolysin is functional, where the function comprises the ability to lyse the cell wall of Gardnerella. In a preferred embodiment of the invention, the endolysin comprises a first cell wall binding domain and a second cell wall binding domain, wherein said first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and said second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 27, 29, 31 and 33. Preferably, said first cell wall binding domain is N-terminal to said second cell wall binding domain.

[0023] In a more preferred embodiment of the invention, the endolysin comprises two cell wall binding domains (B domains) of native endolysin EL10 (SEQ ID NOs: 28 and 29), native endolysin EL11 (SEQ ID NOs: 30 and 31), native endolysin EL12 (SEQ ID NOs: 32 and 33) or native endolysin EL3 (SEQ ID NOs: 19 and 20), even more preferably native endolysin EL10 (SEQ ID NOs: 28 and 29); or a functional variant thereof. Said functional variant may also comprise It may also be a set of two B-domains having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity and most preferably at least 99.7% identity) with the amino acid sequence of the two B-domains of native endolysin EL10 (SEQ ID NOs: 28 and 29), native endolysin EL11 (SEQ ID NOs: 30 and 31), native endolysin EL12 (SEQ ID NOs: 32 and 33) or native endolysin EL3 (SEQ ID NOs: 19 and 20), even more preferably at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity and most preferably at least 99.7% identity); Thereby, the endolysin is functional, where the function comprises the ability to lyse the cell wall of Gardnerella.

[0024] In one embodiment of the invention, the cell wall binding domain(s) (B domain) is / are either the amino acid sequence of SEQ ID NO: 19 and / or 20, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 19 and / or 20; more preferably the amino acid sequence of SEQ ID NO: 32 and / or 33, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 32 and / or 33. or a functional variant thereof having at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity); even more preferably, the amino acid sequence of SEQ ID NO: 30 and / or 31, or a functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.or even more preferably, comprises or consists of the amino acid sequence of SEQ ID NO: 28 and / or 29, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 28 and / or 29; Thereby, the endolysin is functional, where said function comprises the ability to lyse the cell wall of Gardnerella.

[0025] It is preferred that the sequence VNELL or VNKLL, more preferably VNELL, is located at the C-terminus of the B-domain. If there are multiple B-domains in the B region, it is also preferred that the sequence VNELL or VNKLL, more preferably VNELL, is located at the C-terminus of each B-domain.

[0026] Surprisingly and unexpectedly, it has been found in the context of the present invention that some of the recombinant endolysins have stronger activity than the native endolysins, especially when viewed across all four Gardnerella strains tested (i.e. Gardnerella vaginalis sensu strict, Gardnerella leopoldii, Gardnerella pioti and Gardnerella swidszynskii). In particular H2B10, H2B11, H2B12 and H7B3 are each more active than all the native endolysins tested. Thus, the recombinant endolysins according to the present invention show significantly higher activity than the native endolysins.

[0027] Therefore, it is preferable that the "cidal activity against Gardnerella" of the recombinant endolysin of the present invention is enhanced compared to the cidal activity of naturally occurring endolysins, such as naturally occurring endolysins EL1 to EL12 (having the amino acid sequences shown in Table 7).

[0028] Consistent with the very high activity of endolysins H2B10, H2B11, H2B12 and H7B3, these endolysins (and functional variants thereof) are preferred in the present invention. Thus, the endolysins of the invention preferably have the following structure: (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or 7, or any of its functional variants having at least 80% identity to the amino acid sequence of SEQ ID NO: 2 (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity); (ii) a C-terminal cell wall-binding region comprising or consisting of at least one (preferably two) cell wall-binding domains independently selected from the group consisting of polypeptides comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 19, 20, and 28 to 33, respectively, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NOs: 19, 20, and 28 to 33, respectively; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region consisting of a polypeptide comprising or consisting of the amino acid sequence X1X2GLNGX3X4NGGS, With regard to the linker region, as mentioned below, it is indicated that the linker region may also consist of a polypeptide comprising or consisting of an amino acid sequence (XXX)n {wherein each X may independently be G, A or S}, preferably where the amino acid sequence (XXX)n is (GGS)n {wherein n corresponds to the number of repeats of the sequence XXX, preferably where n is 2, 3, 4, 5 or 6}.

[0029] In the accompanying examples it has been shown that recombinant endolysin H2B10 has the highest activity. Therefore, in the present invention it is most preferred that the endolysin of the present invention is H2B10 (or a functional variant thereof). Thus, the endolysin of the present invention preferably has the following activity: (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:2, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO:2; (ii) a C-terminal cell wall binding region comprising or consisting of a cell wall binding domain comprising or consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 28 or 19, or any of its functional variants having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of SEQ ID NO: 28 or 19; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region consisting of a polypeptide comprising or consisting of the amino acid sequence X1X2GLNGX3X4NGGS, Here, said endolysin has Gardnerella-cidal activity. As mentioned above, the "Gardnerella-cidal activity" of the recombinant endolysin of the present invention is preferably enhanced compared to the cidal activity of natural endolysins, for example, natural endolysins EL1 to EL12 (having the amino acid sequences shown in Table 7). With regard to the linker region, as mentioned below, it is indicated that the linker region may also consist of a polypeptide comprising or consisting of an amino acid sequence (XXX)n {wherein each X may independently be G, A or S}, preferably wherein the amino acid sequence (XXX)n is (GGS)n {wherein n corresponds to the number of repeats of the sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6}.

[0030] In the recombinant endolysin of the invention, the C-terminal cell wall binding region may comprise or consist of one, two or three cell wall binding domains, which may be independently selected from the group consisting of polypeptides comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, and variants thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, whereby said polypeptides are functional, wherein the function comprises the ability to bind to the cell wall of Gardnerella. It is preferred that the C-terminal cell wall binding region consists of two cell wall binding domains. Preferred C-terminal cell wall binding regions are defined herein above and below.

[0031] An endolysin according to the invention preferably does not contain the H-domain or B-region of the native endolysin EL6. The amino acid sequences of the H-domain and B-region of the native endolysin EL6 are shown in Table 7.

[0032] The linker region may consist of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, even more preferably 12 amino acids. Preferably, the linker region may consist of a polypeptide comprising or consisting of the amino acid sequence (i) (XXX)n {wherein each X may independently be G, A or S}, preferably where the amino acid sequence (XXX)n is (GGS)n, where n corresponds to the number of repeats of the sequence XXX, preferably where n is 2, 3, 4, 5 or 6, or (ii) X1X2GLNGX3X4NGGS {wherein X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q}. As described above, in one embodiment of the endolysin of the present invention, the N-terminal catalytic domain is identical to or derived from a first naturally occurring endolysin, the linker region and the C-terminal cell wall binding region are identical to or derived from a second naturally occurring endolysin, and the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages.

[0033] The recombinant endolysin of the present invention has killing activity against Gardnerella. For example, the endolysin of the present invention may have killing activity against Gardnerella vaginalis in the narrow sense, Gardnerella leopoldii, Gardnerella pyoti, and / or Gardnerella swidszynskii, preferably against all of them. The killing activity of the endolysin of the present invention described above against Gardnerella is preferably a genus-selective killing activity against Gardnerella. In this specification, "genus-selective killing activity against Gardnerella" means that the endolysin of the present invention does not generally have killing activity against bacteria. Preferably, the endolysin of the present invention has killing activity against Gardnerella but not against Lactobacillus. In particular, it is preferred that the endolysin has no killing activity against Lactobacilli crispatus, Lactobacilli gasseri and / or Lactobacilli jensenii. More preferably, the endolysin has no killing activity against all of these lactobacilli, i.e. Lactobacilli crispatus, Lactobacilli gasseri and Lactobacilli jensenii.

[0034] The present invention also relates to a polynucleotide molecule encoding an endolysin as described above. The nucleic acid molecule may be DNA, e.g., cDNA or RNA. As used herein, the term "polynucleotide" or "polynucleotide molecule" is used synonymously with the term "nucleic acid molecule".

[0035] The present invention also relates to a vector comprising the polynucleotide molecule of the present invention. In one embodiment, the vector is an expression vector. Any suitable vector known in the art may be used, such as all of the pET series vectors and T7-based vectors. For example, the vector may be a plasmid. Thus, one aspect of the present invention relates to a plasmid comprising the polynucleotide of the present invention. It will be understood by those skilled in the art that the choice of expression vector may be determined by the choice of host cell.

[0036] A host cell comprising the polynucleotide molecule according to the invention or the vector / plasmid according to the invention is also provided by the present invention. In one embodiment, the host cell is a microbial cell, e.g. a bacterial cell. Preferably, the host cell is non-pathogenic. Most preferably, the host cell is E. coli. Thus, one aspect of the present invention relates to a bacterial host cell comprising the plasmid of the present invention, preferably wherein the bacterial host cell is an E. coli cell.

[0037] Also encompassed by the present invention is a method for producing an endolysin of the present invention, which comprises culturing a population of host cells containing a polynucleotide molecule according to the present invention or a vector / plasmid according to the present invention under conditions for expressing the endolysin, and isolating the endolysin therefrom.

[0038] Further aspects of the present invention include the following: (a) an endolysin according to the invention; (b) a polynucleotide molecule according to the invention; (c) a vector / plasmid according to the present invention; (d) a host according to the present invention; and / or (e) a bacteriophage capable of expressing an endolysin according to the invention, and a pharma- ceutical composition comprising the endolysin of the invention, a polynucleotide molecule of the invention, and a pharma- ceutical acceptable carrier, diluent or excipient. For example, a pharmaceutical composition of the invention may comprise an endolysin of the invention, a polynucleotide molecule of the invention, and a pharma- ceutical acceptable carrier and / or diluent.

[0039] Further aspects of the invention include the use of the following in treating a disease or disorder: (a) an endolysin according to the invention; (b) a polynucleotide molecule according to the invention; (c) a vector / plasmid according to the present invention; (d) a host according to the present invention; (e) a bacteriophage capable of expressing an endolysin according to the invention; and / or (f) a pharmaceutical composition according to the present invention; For example, the present invention provides an endolysin according to the invention, a polynucleotide molecule according to the invention, or a pharmaceutical composition according to the invention for use in treating a disease or disorder. The disease or disorder may be a bacterial infection, preferably bacterial vaginosis. For example, bacterial vaginosis may be caused by Gardnerella vaginalis strictus, Gardnerella leopoldii, Gardnerella pyotiii and / or Gardnerella swidszynskii.

[0040] In one embodiment of the invention, the recombinant endolysin of the invention, the polynucleotide molecule of the invention or the pharmaceutical composition of the invention should be administered topically, preferably into the vagina of a subject. Thus, in one embodiment of the invention, the recombinant endolysin of the invention, the polynucleotide molecule of the invention or the pharmaceutical composition of the invention should be administered into the vagina of a subject.

[0041] The accompanying examples show that the activity of the recombinant endolysin of the invention is particularly high at a pH around pH 5. Therefore, one aspect of the invention relates to a recombinant endolysin of the invention, a polynucleotide molecule of the invention, or a pharmaceutical composition of the invention, wherein said recombinant endolysin, polynucleotide or pharmaceutical composition should be co-administered with a compound or composition that adjusts the vaginal pH to 4.0-6.0, preferably 4.5-5.5, more preferably about 5. Suitable compounds or compositions that adjust the vaginal pH include, but are not limited to, phosphate, lactic acid (e.g., a natural acidifying agent secreted by Lactobacillus to establish an acidic environment) or other organic acids, such as carboxy-substituted polymers.

[0042] A further aspect of the present invention is the use of the following compound for use as a medicament: (a) an endolysin of the invention; (b) a polynucleotide molecule of the invention; (c) a vector / plasmid of the present invention; (d) a host of the present invention; (e) a bacteriophage capable of expressing an endolysin of the invention; and / or (f) a pharmaceutical composition of the present invention; Regarding.

[0043] A further aspect of the present invention is the use in the manufacture of a medicament for treating bacterial infections and disorders of: (a) an endolysin of the invention; (b) a polynucleotide molecule of the invention; (c) a vector / plasmid of the present invention; (d) a host of the present invention; (e) a bacteriophage capable of expressing a polypeptide of the invention; and / or (f) a pharmaceutical composition of the present invention; This relates to the use of

[0044] A further aspect of the invention is a method of treating bacterial infections and disorders, such as BV, comprising the steps of: Administering to a subject in need thereof a therapeutically effective amount of: (a) an endolysin of the invention; (b) a polynucleotide molecule of the invention; (c) a vector / plasmid of the present invention; (d) a host of the present invention; (e) a bacteriophage capable of expressing a polypeptide of the invention; and / or (f) a pharmaceutical composition of the present invention; The method includes administering In some embodiments, the therapeutically effective amount is a dose of 10-100 μg of endolysin, optionally administered several times daily.

[0045] A further aspect of the present invention provides a kit comprising an endolysin as described herein and instructions for use, in particular for treating a disease or disorder, preferably BV as defined above. Said kit may also comprise a compound or composition for adjusting the vaginal pH to between 4.0 and 6.0, preferably between 4.5 and 5.5, more preferably about 5. The definitions and preferred embodiments defined herein above and below with respect to the endolysins of the invention also apply mutatis mutandis with respect to the polynucleotide molecules, vectors / plasmids, host cells, pharmaceutical compositions, methods of treatment and kits of the invention. A further aspect of the present invention is an in vitro method for the diagnosis of a disease or condition treatable with an endolysin according to the present invention, comprising the following steps: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of a C-terminal cell wall binding region of an endolysin according to the invention and, optionally, an N-terminal catalytic domain of an endolysin according to the invention, wherein the sample comprises microbial cells and wherein the C-terminal cell wall binding region of the endolysin is optionally labelled; (ii) testing whether the polypeptide binds to and / or lyses microbial cells in the sample; and (iii) determining if the disease or condition can be treated by an endolysin according to the invention if the polypeptide binds to and / or lyses microbial cells; The present invention provides a method comprising: The microbial cell may be a Gardnerella cell, preferably a cell of G. vaginalis spe- cifically, G. leopoldii, G. pyotsi, G. swidszynskii or another species of the genus Gardnerella. Other features and advantages of the present invention will become apparent from the following detailed description. [Brief description of the drawings]

[0046] [Figure 1] Figure 1 shows a sequence alignment (CLUSTAL O(1.2.4) multiple sequence alignment) of the naturally occurring Gardnerella prophage endolysins of the present disclosure. Most of the endolysins have 306 residues, except for two that have 251 residues.

[0047] [Diagram 2] Figure 2 shows a phylogenetic tree of the naturally occurring Gardnerella prophage endolysins of the present disclosure. Although they are highly homologous, there are no homologous pairs among the endolysins.

[0048] [Diagram 3] FIG. 3 shows the domain structure of the Gardnerella prophage endolysin of the present disclosure as determined using InterPro (Mitchell et al., 2019, Nucleic Acids Res. 47, D351-D360). The 196-residue N-terminal part of the endolysin is identified as the catalytic domain due to its homology to glycoside hydrolases, family 25. The catalytic domain is followed by a linker region and two domains identified as two cell-binding domains due to their homology to the C-terminal domain of lysozyme Cpl-7 (CW_7 domain). According to the nomenclature of the present application, the catalytic domain represents the hydrolase or "H-domain", while the linker region and the cell wall binding domain together represent the binding or "B-domain".

[0049] [Figure 4] 4A-4C show three enzyme activity assays in which the enzymatic activity of the native Gardnerella prophage endolysin of the present disclosure is measured by detecting the change in turbidity of a suspension of Gardnerella cells. In FIG. 4A, the enzymatic activity of the endolysin is measured by detecting the change in turbidity of a suspension of G. leopoldii strain Gv_10 at pH 6.0. In FIG. 4B, the enzymatic activity of the endolysin is measured by detecting the change in turbidity of a suspension of G. pyotiii strain Gv_17 at pH 7.0. In FIG. 4C, the enzymatic activity of the endolysin is measured by detecting the change in turbidity of a suspension of G. swidszynskii strain Gv_23 at pH 7.4. Treatments were performed in media adjusted to the appropriate pH in photometric cuvettes against buffer. The change in turbidity was then assessed by measuring the optical density (OD) at 600 nm. As a result, the decrease in turbidity was more obvious for the endolysin treatment group than for buffer, indicating the enzymatic activity.

[0050] [Diagram 5] Figure 5 shows the quantitative reduction in viable colony forming units (CFU) assays comparing G. vaginalis stricto strain Gv_9 and untreated cells incubated in medium with or without imidazole at various pH values. 5x107 CFU / ml cells were incubated under anaerobic conditions at 37°C for 5 hours as indicated below the graph, after which the surviving CFU / ml were determined by quantitative plating. The results show that survival of G. vaginalis Gv_9 is highly dependent on the absence of imidazole and low pH under the conditions tested.

[0051] [Figure 6]Figure 6 shows the quantitative reduction in viable colony forming units (CFU) assays comparing cells from G. vaginalis strictus strain Gv_9 treated with recombinant endolysin H10B1 and an eluate solution containing 250 mM imidazole at various pH values, or with a control containing 250 mM imidazole at various pH values. 5x107 CFU / ml of cells were incubated under anaerobic conditions at 37°C for 5 hours under the conditions indicated below the graph, after which the surviving CFU / ml was measured by quantitative plating. The vertical bar labeled imidazole control shows the same data as in Figure 5. The results show that the enzymatic activity of H10B1, as an example of all endolysins of the invention, is higher at low pH values ​​and shows the strongest activity at pH 5.5 and pH 5.0.

[0052] [Figure 7] Figures 7A-7D show a quantitative reduction in viable colony forming units (CFU) assays measuring the killing activity of native and recombinant Gardnerella prophage endolysins of the present disclosure against the four major Gardnerella species. In Figures 7A, 7B, 7C and 7D, the killing activity of the endolysins is measured by detecting viable CFUs of suspensions of G. vaginalis sensu stricto strain Gv_9, G. leopoldii strain Gv_11, G. pyotsi strain Gv_17 and G. swidszynskii strain Gv_23, respectively. 90 μl of 5e7 CFU / ml of the indicated strains were incubated with 10 μl of endolysin solution for 5 hours under anaerobic conditions at pH 5.0 (where possible, the concentration was adjusted to 0.2 mg / ml, see Table 4). The logarithmic Y-axis shows the total number of viable cells. The dotted line indicates the limit of detection (LOD) produced by plating 2 μl of the reaction mixture (500 CFU / ml). The results show that the endolysins of the invention have the ability to lyse the four main Gardnerella species. The results also indicate that some of the recombinant endolysins of the invention have higher killing activity than the native endolysins of the invention.

[0053] [Figure 8]Figure 8 shows a phylogenetic tree (amino acid level) of the H-domain created using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539).

[0054] [Figure 9] FIG. 9 shows a phylogenetic tree (amino acid level) of the B region created using Clustal Omega (Siefers et al., 2011 MoL Syst. Biol 7, 539). [Figure 10] FIG. 10 shows a phylogenetic tree (amino acid level) of the B region created using Clustal Omega (Siefers et al., 2011 MoL Syst. Biol 7, 539).

[0055] [Figure 11] Figure 11 shows a sequence alignment of cell wall binding domains (also called B-domains) within the B regions of native endolysins of the invention using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539). For each B region, the N-terminal cell wall binding domain is indicated with a _N suffix (Bx_N) and the C-terminal cell wall binding domain is indicated with a _C suffix (Bx_C). As an example, B3_C indicates the second (C-terminal) B domain of B3.

[0056] [Figure 12] Figure 12 shows a phylogenetic tree of individual B-domains using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539).

[0057] [Figure 13]Figure 13 shows a viable colony forming unit (CFU) assay measuring the killing activity of the recombinant Gardnerella prophage endolysin of the present disclosure against the three most common beneficial Lactobacillus species under anaerobic conditions at pH 5.0, with three showing quantitative reductions. The results show that the endolysins of the present invention are ineffective against beneficial Lactobacillus strains.

[0058] [Figure 14] FIG. 14 shows MIC broth microdilution activity assays measuring the effect of metronidazole and clindamycin (obtained from Ratiopharm as a solution for injection, 300 mg / 2 ml) on the growth of four major Gardnerella species in suspension. Gardnerella suspensions of 2.5×107 CFU / ml were incubated with antibiotics at the concentrations shown on the x-axis of each graph and incubated at 37°C for 48 hours under anaerobic conditions. Cell growth was assessed by optical density measurements at 610 nm (OD(610)) before and after incubation to determine the minimum inhibitory concentration (MIC). The results show that all Gardnerella strains are resistant to both metronidazole and clindamycin (obtained from Ratiopharm as a solution for injection, 300 mg / 2 ml), showing minimum inhibitory concentrations of 64 to <128 μg / ml and 16 μg / ml, respectively (FIG. 14). [Figure 15] Figure 15 shows a MIC broth microdilution activity assay measuring the effect of metronidazole and clindamycin hydrochloride (obtained from Sigma Aldrich) against Gardnerella suspensions of 1x105-1x106 CFU / ml. Here, the results show that metronidazole had MICs of 8-128 μg / ml against all of the Gardnerella strains tested, and clindamycin hydrochloride powder (obtained from Sigma Aldrich (C5269-10MG)) showed minimum inhibitory concentrations of 0.25-5 μg / ml. [Figure 16]Figure 16 shows MIC broth microdilution activity assays measuring the effect of H2B10, a representative example of the domain-swapped endolysin claimed herein, on the growth of three major Gardnerella species. Gardnerella suspensions of 1x105-1x106 CFU / ml were incubated with H2B10 at the concentrations indicated on the x-axis of each graph and incubated for 48 hours at 37°C under anaerobic conditions. Cell growth was assessed by OD(610) measurements before and after incubation to determine the minimum inhibitory concentration (MIC). MIC values ​​of 1-4 μg / ml were obtained, indicating that all Gardnerella strains were highly sensitive to the domain-swapped endolysin H2B10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Detailed Description definition The term "lysin" refers to cell wall lytic enzymes encoded by bacteriophages (endolysins) or bacteria (autolysins) that have the ability to hydrolyze the cell wall of target bacteria when added exogenously (exogenous lysis). This novel class of antibacterial agents has important advantages over classical antibiotics, such as a new mechanism of action; a narrow range of susceptible bacteria; rapid killing of both stationary and exponentially growing bacteria; activity against mucosal and bacterial biofilms; low likelihood of developing resistance; and low impact on normal microbiota. These unique features have sparked interest in the biotechnological and pharmacological development of lysins and their recent inclusion among the top ten choices for combating antibiotic resistance. Lysins derived from Gram-positive bacteria and their phages usually contain at least one catalytic domain and one or more cell wall binding domains. In contrast, many lysins produced by Gram-negative species or their phages contain only a catalytic domain, although modular endolysins have also been reported. The catalytic unit determines the type of peptidoglycan (PG) bond that is cleaved, whereas the cell wall binding domain(s) primarily determines the lysis spectrum by specific recognition of cell wall elements that are compartmentalized in a genus- or species / strain-specific manner.

[0060] In the context of the present disclosure, the term "native endolysin" refers to an endolysin encoded by a prophage sequence in a bacterial genome, in particular in the genome of a Gardnerella cell. In the context of the present disclosure, the term "native endolysin" refers to an endolysin that has not been domain-swapped. It means that the native endolysin may not have been modified and that its amino acid sequence corresponds to the native sequence. Alternatively, it means that the native endolysin may have been modified and that, compared to the native sequence, its amino acid sequence comprises at least one mutation. The amino acid sequences of native endolysins E1 to E14 are shown in Table 7 below. Examples of known 1,4-β-N-acetylmuramidase (native endolysin EL1) sequences are also provided by NCBI accession number WP_014554482 (version WP_014554482.1 of May 27, 2013).

[0061] In the context of the present disclosure, the term "engineered endolysin" refers to a domain-swapped endolysin. In the context of the present disclosure, the term "domain-swapped endolysin" refers to an endolysin possessing an N-terminal catalytic domain derived from a first naturally occurring endolysin and at least one cell wall binding domain derived from a second naturally occurring endolysin, wherein the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages. The engineered endolysin of the present invention may comprise or consist of an N-terminal catalytic domain derived from a first naturally occurring endolysin and two cell wall binding domains derived from a second naturally occurring endolysin, wherein the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages. Alternatively, the genetically engineered endolysin of the invention may comprise or consist of an N-terminal catalytic domain derived from a first naturally occurring endolysin, a first (N-terminal) cell wall binding domain derived from a second naturally occurring endolysin and a second (C-terminal) cell wall binding domain derived from a third naturally occurring endolysin, wherein the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages, and wherein the third naturally occurring endolysin is optionally encoded by a genome derived from a different prophage from the first and second naturally occurring endolysins. The genetically engineered endolysin may be unmodified, meaning that the amino acid sequence of the endolysin corresponds to the native sequence of each of the domains that make up the endolysin. Alternatively, the genetically engineered endolysin may be modified, meaning that the amino acid sequence of the endolysin comprises at least one mutation compared to the native sequence of each of the domains that make up the endolysin. Consistent with this definition, one of skill in the art will readily appreciate that the "domain-swapped" or "engineered" endolysins described herein are non-naturally occurring endolysins, i.e., the engineered endolysins of the present invention have been modified by the hand of man, and therefore by definition exclude naturally occurring endolysins, i.e., as they are naturally found in nature.The accompanying examples provide suitable methods how to generate the artificial endolysin(s) of the present invention.

[0062] The term "catalytic domain" or "enzyme domain" refers to the portion of a protein chain that contains the area where the catalyzed chemical reaction occurs. In the context of the present disclosure, the term "H-domain" refers to the part of an endolysin of the invention that comprises the catalytic domain.

[0063] In the context of the present disclosure, the term "B region" refers to a portion of an endolysin of the invention that comprises or consists of a polypeptide having cell wall binding activity. In a preferred embodiment, the B region comprises or consists of a linker region and one, two or three cell wall binding domains or "B domains".

[0064] In the context of the present invention, the term "B domain" refers to the cell wall binding domain contained within the B-region.

[0065] In the context of the present disclosure, the term "CW_7 domain" refers to the cell wall binding domain of the protein Cpl-7, an endolysin encoded by the Streptococcus pneumoniae bacteriophage ceruloplasmin-7 (see Bustamante et al., 2010 J. Biol. Chem. 285, 33184-33196, 2012 PLoS One 7, e46654). Briefly, the Cpl-7 protein has a C-terminal cell wall binding region composed of three consecutive CW_7 domains. Each CW_7 domain: It is composed of a similar amino acid sequence 38 amino acids long, referred to as the "CW_7 motif" and defined by Interpro (Mitchell et al., 2019, Nucleic Acids Res. 47, D351-D360) as consisting of the amino acid sequence TVANEVIQGLWGNGQERYDSLANAGYDPQAVQDKVNEXL, where X is I in CW_7 motif no. 1 (amino acids 207-245) and no. 2 (amino acids 255-293), and where X is L in CW_7 motif no. 3 (amino acids 303-341). In the Cpl-7 protein, there are short, 9 residue linkers between CW_7 motif no. 1 and no. 2, and between CW_motif no. 2 and no. 3, making the total repeat 47 residues long. In comparison, the repeat of the native endolysin of the invention is 49 residues long.

[0066] The term "minimum inhibitory concentration" or "MIC" refers to the lowest concentration of a chemical, usually a drug, that prevents visible growth of bacteria. The MIC is defined herein as the lowest concentration of an antibiotic at which there is no detectable growth after 48 hours by OD measurement.

[0067] The term "minimum bactericidal concentration" or "MBC" refers to the lowest concentration of an antimicrobial agent required to kill a particular bacterium. 90 , i.e., the antibiotic concentration that kills 90% of cells in a specified time, is measured, whereas MBC is 2.5 × 10 7 It is defined herein as the minimum concentration that completely eradicates a suspension of CFU / ml. The MIC is the lowest concentration of an antimicrobial agent required to inhibit visible growth, whereas the MBC is the minimum concentration of an antimicrobial agent that results in bacterial kill of all cells in suspension.

[0068] The terms "peptide", "polypeptide", "protein" and variations of these terms refer to peptides, oligopeptides, oligomers or proteins, including fusion proteins, respectively, that contain at least two amino acids linked together by normal or modified peptide bonds, such as in the case of isosteric peptides. These terms also include "peptidomimetics", which are defined as peptide analogs that include non-peptidic structural elements, and which are peptides that are capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. Peptidomimetics lack classical peptide characteristics, such as enzymatically uniformly cleavable peptide bonds. A peptide or polypeptide may be composed of amino acids other than the 20 amino acids defined by the genetic code. It may be composed of L-amino acids and / or D-amino acids. A peptide or polypeptide may also be composed of amino acids that have been modified by natural processes, such as post-translational maturation processes, or by chemical processes, which are well known to those skilled in the art. Such modifications are well described in the literature. These modifications may occur anywhere in the polypeptide: within the peptide backbone, within the amino acid chain, or even at the carboxy or amino termini. Peptides or polypeptides may be branched following ubiquitination or cyclic with or without branching. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. For example, modifications of peptides or polypeptides may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfatation, arginylation or ubiquitination, or other amino acid additions.Such modifications are well described in detail in the literature and are therefore well known to those skilled in the art.

[0069] As used herein, "bacterial infections and disorders" refers to infections and disorders caused by bacteria, particularly infections and disorders caused by at least one strain of Gardnerella species selected from the group consisting of Gardnerella vaginalis sect., Gardnerella leopoldii, Gardnerella pyoti, and Gardnerella swidszynskii, and other Gardnerella species, including, but not limited to, bacterial vaginosis (BV).

[0070] When defined herewith, the term "cidal activity" of an endolysin against a particular bacterium refers to the reduction in the number of viable bacterial cells caused by the lytic activity of said endolysin. The cidal activity of an endolysin against said bacterium may be complete, meaning that 100% of the bacterial cells have been lysed, or may be partial, meaning that at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% of the bacterial cells have been lysed. The cidal activity may be measured by measuring the decrease in absorbance at 610-620 nm of a bacterial cell suspension and / or the decrease in colony forming units (CFU) per milliliter of bacterial cell suspension after exposure to the endolysin being tested.

[0071] As defined herewith, the term "binding ability" of an endolysin to a particular bacterial cell wall refers to the ability of said endolysin to specifically interact with and adhere to the bacterial cell wall. The binding ability of an endolysin to a bacterial cell wall can be measured by methods known in the art.

[0072] As used herein, "treatment" and "treating" and similar terms generally refer to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic, in terms of preventing or partially preventing a disease, its symptoms or condition, and / or may be therapeutic, in terms of partially curing or completely curing a disease, condition, symptom, or adverse effects caused by the disease. The term "treatment" as used herein includes any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from developing in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., preventive early asymptomatic intervention); (b) inhibiting the disease, i.e., arresting its progression; or relieving the disease, i.e., causing the disease and / or its symptoms or conditions to regress (e.g., ameliorating or curing damage). In particular, the treatment of bacterial infections includes preventing, relieving, or even eradicating the infection, for example, by killing the bacteria, thereby controlling, reducing or inhibiting bacterial growth, and reducing the number of viable bacterial cells. Herein, a disease, such as BV, is preferably treated therapeutically in terms of a partial or complete cure of the disease or condition.

[0073] The term "subject" as used herein refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, livestock animals such as cows, sheep, pigs, horses, laboratory rodents, etc. Preferably, the subject is a human.

[0074] The term "effective amount", as used herein, refers to an amount of at least one endolysin according to the invention, a composition or a pharmaceutical preparation thereof, that induces a biological or pharmaceutical response in a tissue, system, animal or human being under investigation. In one embodiment, the effective amount is a "therapeutically effective amount" for alleviating the symptoms of the disease or condition being treated. In another embodiment, the effective amount is a "prophylactically effective amount" for preventing the symptoms of the disease or condition being prevented. The term, as used herein, also includes an amount of active polypeptide sufficient to slow down the progression of a disease, in particular to reduce or inhibit a lesion or infection (i.e., an "inhibitory effective amount").

[0075] The term "efficacy" of the treatment according to the invention can be measured based on the change in the course of the disease that occurs in response to the use or method according to the invention. The efficacy of preventing infectious diseases is ultimately evaluated by epidemiological surveys in human populations, which correlate well with neutralizing antibody titers in serum and the induction of pathogen-specific polyfunctional T cell responses. Preclinical evaluations may include resistance to infection after exposure to infectious pathogens. The treatment of infectious diseases can be measured by the inhibition of pathogen growth or elimination of the pathogen (and therefore non-detection of the pathogen), which correlates with pathogen-specific antibody and / or T cell immune responses.

[0076] The term "biological material" refers to any material or sample obtained from the body of a subject, including, for example, a sample of whole blood, serum, plasma, urine, sputum, saliva, vaginal swab, or spinal fluid.

[0077] The term "inanimate material or surface" includes the surfaces of solutions, media, devices, objects, floors, and tables.

[0078] The term "medium" includes water, air or food.

[0079] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is demonstrably effective and which does not contain additional ingredients that are toxic to the subject to which the formulation is administered.

[0080] The term "pharmaceutical acceptable" refers to a carrier that is made of materials that are not biologically or otherwise objectionable.

[0081] The term "carrier" refers to any ingredient present in a pharmaceutical formulation other than the active substance, and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, etc.

[0082] The term "variant" refers to a polypeptide that contains insertions, deletions, and / or substitutions, either non-conservative or preferably conservative, relative to the native amino acid sequence. For example, the polypeptide may comprise an amino acid sequence that has at least 80% identity to the native amino acid sequence, preferably at least 85% identity to said amino acid sequence, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity. The percentage of identity is measured by methods well known in the art using a suitable computer program, for example MatGAT2.0 (Myers and Miller, CABIOS (1989)). Preferably, the % identity is determined over the entire length of the sequences to be compared. Of course, the percent identity is calculated with respect to the polypeptides whose sequences are optimally aligned. Fragments and variants of the amino acid sequence may be made using methods of protein engineering, directed evolution, and / or site-directed mutagenesis well known in the art (see, for example, Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). It will be appreciated by those skilled in the art that the polypeptides according to the invention, or fragments, variants or fusions thereof, may comprise or consist of derivatives of the native amino acid sequence, or fragments or variants thereof. Chemical derivatives of one or more amino acids may be obtained by reaction with a functional side group. Such derivatized molecules include, for example, those molecules in which a free amino group is derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group or a formyl group.Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include those peptides that contain natural amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides that contain one or more additions or deletions, so long as the required activity is maintained. Other modifications that are included are terminal modifications such as amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxyamidation (e.g., with ammonia or methylamine), and the like. It will be further appreciated by those skilled in the art that peptidomimetic compounds may also be useful. Thus, by "polypeptide" we include peptidomimetic compounds that exhibit endolysin activity. The term "peptidomimetic" refers to a compound that mimics the conformation and desirable characteristics of a particular polypeptide as a therapeutic agent. Endolysins according to the invention

[0083] The endolysin of the present invention has antibacterial activity against Gardnerella strains. The optimum pH at which the endolysin of the present invention exerts its antibacterial activity is comprised between about pH 4 and 6, preferably about pH 5. The endolysins of the present invention are (i) the N-terminal catalytic domain, or a functional variant thereof; (ii) a C-terminal cell wall binding region, or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of at least one cell wall binding domain; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region; and It has killing activity against Gardnerella cells. In some embodiments, the N-terminal catalytic domain is derived from a first naturally occurring endolysin, the linker region and the C-terminal cell wall binding region are derived from a second naturally occurring endolysin, and the first endolysin and the second naturally occurring endolysin are encoded by different genomes derived from different prophages. The killing activity of the endolysins of the invention against Gardnerella is expected to be a species-selective killing activity against Gardnerella.

[0084] The N-terminal catalytic domain is a functional polypeptide, where the function comprises the ability to lyse the cell wall of Gardnerella. The N-terminal catalytic domain may be N-acetylmuramidase, N-acetylmuramoyl-L-alanine amidase, L-alanoyl-D-glutamic acid endopeptidase, interpeptide cross-linking endopeptidase or N-acetyl-β-D-glucosaminidase. Preferably, the N-terminal catalytic domain is N-acetylmuramidase, most preferably 1,4-β-N-acetylmuramidase. For example, the N-terminal catalytic domain comprises a polypeptide comprising or consisting of any one of the amino acids of SEQ ID NOs: 1-5, 7 or 10-12, or a variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 1-5, 7 or 10-12, whereby the polypeptide is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. Preferably, the N-terminal catalytic domain consists of a polypeptide comprising the amino acids of SEQ ID NO: 2 or 7, or any variant thereof having at least 80% identity to the amino acid sequence of SEQ ID NO: 2 or 7 (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity), whereby the polypeptide is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella.

[0085] According to the present invention, the C-terminal cell wall binding region is a functional polypeptide, wherein the function comprises the ability to bind to the cell wall of Gardnerella. The C-terminal cell wall binding region may comprise or consist of one, two, three or more cell wall binding domains. For example, the one, two, three or more cell-binding domains may be independently selected from the group consisting of polypeptides comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, and variants thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, whereby the polypeptide is functional, wherein the function comprises the ability to bind to a cell wall of Gardnerella. Preferably, the one, two, three or more cell wall binding domains are independently selected from the group consisting of a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 19, 20 and 28-33, or a variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33, whereby the polypeptide is functional, wherein the function comprises the ability to bind to a cell wall of Gardnerella. Most preferably, the C-terminal cell wall binding region comprises a first cell wall binding domain and a second cell wall binding domain, wherein said first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and said second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 27, 29, 31 and 33. In a preferred embodiment, said first cell wall binding domain is N-terminal to the second said cell wall binding domain.

[0086] In one embodiment of the invention, the linker region may consist of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, even more preferably 12 amino acids. Preferably, the linker region consists of a polypeptide comprising or consisting of the amino acid sequence (i) (XXX)n {wherein each X can be independently G, A or S}, preferably where the amino acid sequence (XXX)n is (GGS)n, where n corresponds to the number of repeats of the sequence XXX, preferably where n is 2, 3, 4, 5 or 6, or (ii) X1X2GLNGX3X4NGGS {wherein X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q}. The linker-containing fragment may be absent. The linker-containing fragment may also be present and may enhance the cell wall binding and / or lytic activity of the polypeptide of the invention.

[0087] The present invention further provides an endolysin having Gardnerellacidal activity as described above for use in treating a disease or disorder. The disease or disorder to be treated may be a bacterial infection, preferably bacterial vaginosis. Bacterial vaginosis may be caused by G. vaginalis stricto, G. leopoldii, G. pyoti, and / or G. swidszynskii, or other species of the genus Gardnerella.

[0088] The endolysins of the invention are preferably capable of specifically binding to and / or lysing Gardnerella cells for use in methods of treating Gardnerella infections such as BV.

[0089] As mentioned above, it is well established that many bacteriophage endolysins consist of two distinct domains (see, for example, Sheehan et al., 1996, FEMS Microbiology Letters 140:23-28). One is the catalytic domain involved in cell wall degradation, which is known to exist in several forms. The other domain is the cell wall binding domain that recognizes cell surface motifs and allows the attachment of the endolysin to its target cells. The precise pattern recognition involved in the latter is what provides the specificity. The enzymatic domain can be identified by its amino acid homology to other similar regions of lytic enzymes that share the same type of lytic activity. In the case of the newly discovered native Gardnerella prophage endolysins by the inventors, the domain arrangement was identified to consist of an N-terminal domain of 196 residues, followed by two repeat domains of 49 residues each, except for EL6 and EL9, which have only a linker region of 12 residues and one incomplete domain of 43 residues. The native amino acid sequences of these newly discovered endolysins are summarized in Table 7. The inventors identified the N-terminal domain as the catalytic domain, due to its homology to glycoside hydrolases, family 25, and the two repeat domains as the two cell wall binding domains, due to their homology to the C-terminal domain of lysozyme Cpl-7 (see Example 2 and Figure 3).

[0090] In some embodiments, the fragment containing the enzyme domain is unmodified, i.e., corresponds to the native amino acid sequence. In alternative embodiments, the fragment containing the enzyme domain may contain modifications such as amino acid substitutions, deletions, insertions, or any combination of modifications. In some embodiments, the fragment containing the enzyme domain is a variant fragment having at least 80%, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, even more preferably at least 99.7% identity, and most preferably 100% identity to the amino acid sequence of any one of SEQ ID NOs: 1-5, 7, or 10-12.

[0091] In some embodiments, the fragment comprising the cell wall binding domain is unmodified, i.e., corresponds to the native amino acid sequence. In alternative embodiments, the fragment comprising the cell wall binding domain may include modifications such as amino acid substitutions, deletions, insertions, or any combination of modifications. In some embodiments, the fragment comprising the enzyme domain is a variant fragment having at least 80%, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, even more preferably at least 99.7% identity, and most preferably 100% identity to the amino acid sequence of any one of SEQ ID NOs: 15-24 and 26-33.

[0092] A "fusion" of a polypeptide includes a polypeptide fused to another polypeptide. For example, a polypeptide may contain one or more further amino acids inserted internally and / or at the N- and / or C-terminus of the amino acid sequence of an endolysin according to the invention, or a fragment, variant or derivative thereof. Thus, as mentioned above, in one embodiment, the endolysin of the first aspect of the invention comprises a fragment of one or more cell wall binding domains comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 15-24 and 26-33, respectively (or a variant of the domain sequence such that it retains its cell wall binding activity), to which an enzymatic domain of different origin has been fused. Examples of other suitable enzymatic domains include, but are not limited to: L-alanoyl-D-glutamic acid endopeptidase; D-glutamyl-m-DAP endopeptidase; interpeptide crosslink specific endopeptidase; N-acetyl-β-D-glucosaminidase (muramoyl hydrolase); N-acetyl-β-D-muramidase (lysozyme) or lytic transglycosylase. N-acetyl-β-D-alanine amidases from other sources may also be used.

[0093] In one embodiment of the invention, the endolysin may be fused to a polypeptide or protein to facilitate purification of said endolysin. Examples of such fusions are well known to the skilled artisan. Similarly, the endolysin may be fused to an oligohistidine tag, such as His6, or to an epitope recognized by an antibody, such as the well-known Myc tag epitope. Fusions to any fragment, mutant or derivative of the endolysin according to the invention are also included within the scope of the invention. Of course, fusions (or mutants or derivatives thereof) that retain the desired properties, i.e. endolysin activity, are preferred. Fusions are particularly preferred if they are suitable for use in the methods described herein. For example, the fusion may comprise a further moiety that confers a desired property to the endolysin of the invention; for example, the moiety may be useful for detecting or isolating the endolysin, facilitating intracellular uptake of the endolysin, or directing secretion of the protein from a cell. The moiety may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, such as a small molecule fluorescent probe or a green fluorescent protein (GFP) fluorescent probe, as well known to the skilled artisan. The partial structure may be an immunogenic tag known to those skilled in the art, such as a Myc tag, or it may be a lipophilic molecule or a polypeptide domain known to those skilled in the art that can promote intracellular uptake of the endolysin.

[0094] The most important feature of the endolysins of the invention is their ability to lyse cells of the genus Gardnerella. Preferably, the endolysins are capable of lysing cells of multiple strains of Gardnerella. Most preferably, the endolysins are capable of lysing all strains of the genus Gardnerella, including G. vaginalis sect., G. leopoldii, G. pyotsi and G. swidszynskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). In one embodiment, the endolysins of the invention are substantially or completely unable to lyse bacteria that are commensal members of the healthy vaginal microflora (and are not known to cause deleterious effects to the host). For example, it is advantageous if the endolysins do not lyse cells of the genus Lactobacillus. Most preferably, the endolysins of the invention are substantially or completely unable to lyse cells of L. crispatus, L. gasseri and L. jensenii. Optionally, the endolysins of the invention do not lyse cells of L. iners. Advantageously, the endolysins are capable of lysing cells of pathogenic bacteria selectively, i.e. to an extent that differs significantly from cells of non-pathogenic bacteria.

[0095] The killing activity of the endolysins according to the invention against a particular microorganism can be measured by procedures standard in the field, including those based on measuring the minimum inhibitory concentration (MIC) of an antimicrobial agent, defined as the lowest concentration of said antimicrobial agent that inhibits the visible growth of a microorganism after overnight incubation, as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16 or in “Document M7-A7, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standards, 7th Edition, January 2006, vol. 26, No. 2” published by Clinical and Laboratory Standards Institute. Another suitable method for measuring the killing activity of the endolysins according to the invention is described in the Examples section of the present application and consists in measuring the decrease in absorbance measured at 610-620 nm of a suspension of bacteria whose susceptibility is tested in an in vitro turbidity assay carried out in the presence of the purified endolysin according to the invention. According to another embodiment, in the in vitro turbidity test described herewith, the endolysin according to the invention reduces the OD (610-620 nm) of a suspension of at least one strain of Gardnerella bacteria by more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95%.

[0096] Methods for the production of endolysins, or fragments, mutants, fusions or derivatives thereof, for use according to the invention are well known in the art. Advantageously, the endolysins, or fragments, mutants, fusions or derivatives thereof, are or comprise recombinant endolysins. Endolysins according to the invention may be produced by standard techniques of genetic engineering, including the use of recombinant vectors comprising the polynucleotides encoding the endolysins described herewith. Numerous expression systems may be used, such as bacterial plasmids and derived vectors, transposons, yeast episomes, insertion factors, yeast chromosomal elements, viruses, such as baculoviruses, papillomaviruses, such as SV40, vaccinia viruses, adenoviruses, foxpox viruses, pseudorabies viruses, retroviruses, cosmids or phagemid derivatives. The nucleotide sequence may be inserted into a recombinant expression vector by methods well known to those skilled in the art, for example, the methods described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. The recombinant vector may contain nucleotide sequences that control the regulation, expression, transcription, and / or translation of the polynucleotide encoding the endolysin, these sequences being selected according to the host cell used. The recombinant vector may further contain nucleotide sequences, such as those encoding a His tag to facilitate purification steps.Such recombinant vectors are then introduced into host cells by methods well known to those skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., 2nd ed., McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, for example, calcium phosphate transfection, DEAE dextran transfection, transfection, microinjection, cationic lipid transfection, electroporation, transduction or infection. Host cells may be, for example, bacterial cells, such as E. coli, yeast cells and fungal cells, such as Aspergillus, Streptomyces cells, insect cells, Chinese hamster ovary cells (CHO), C127 mouse cell line, BHK cell line of Syrian hamster cells, human embryonic kidney 293 (HEK293) cells. Preferably, the host cell is E. coli. The host cell is then cultured under appropriate conditions to produce the endolysin described herewith, which can then be further purified from the culture medium or from host cell lysates by any standard purification method, including Immobilized-Metal Affinity Chromatography (IMAC) (Block et al. 2008, Protein Expr. Purif. 27, 244-254). Compositions according to the invention

[0097] In a further aspect of the invention there is provided an antibacterial composition, in particular a pharmaceutical composition, comprising an endolysin according to the first aspect of the invention, a nucleic acid according to the second aspect of the invention, a vector / plasmid according to the third aspect of the invention, a host cell according to the fourth aspect of the invention or a bacteriophage capable of expressing an endolysin according to the first aspect of the invention.

[0098] As used herein, a "pharmaceutical composition" refers to a therapeutically effective formulation for use in the method of the present invention. As used herein, a "therapeutically effective amount", or an "effective amount", or "therapeutically effective" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. It is a predetermined amount of an active agent calculated to provide a desired therapeutic effect in conjunction with required additives and excipients, i.e., carriers or administration vehicles. It is further intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in the activity, function and responsiveness of the host. Alternatively, a therapeutically effective amount is an amount sufficient to provide an improvement in a clinically significant condition of the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. A suitable dose may include a predetermined amount of an active composition calculated to provide a desired therapeutic effect in conjunction with required excipients. In the method for producing the composition of the present invention and the use, a therapeutically effective amount of the active ingredient is provided. Therapeutically effective amounts can be determined by a medical or veterinary practitioner of ordinary skill based on patient characteristics such as age, weight, sex, condition, comorbidities, other diseases, etc., as is well known in the art. In one embodiment of the invention, a pharmaceutical composition comprises an endolysin according to the first aspect of the invention. Thus, the pharmaceutical formulation may comprise an amount of endolysin, or a fragment, variant, fusion or derivative thereof, sufficient to at least partially inhibit Gardnerella cell proliferation in a patient infected or susceptible to infection by said cells. Preferably, the pharmaceutical formulation comprises an amount of endolysin, or a fragment, variant, fusion or derivative thereof, sufficient to kill Gardnerella cells in the patient. It will be appreciated by those skilled in the art that the polypeptides of the invention will generally be administered in admixture with suitable pharmaceutical excipients, excipients or carriers selected with respect to the given route of administration and standard pharmaceutical treatment (see, for example, Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA).For example, endolysin may be administered topically, i.e., locally in the vagina of a female subject, and / or in or on the glans penis, prepuce or urethral opening of a male subject. As used herein, the term "administration into or on the glans penis" also encompasses "administration into and on the glans penis". As such, the term "administration into or on the glans penis, prepuce or urethral opening of a male subject" also encompasses "administration into and on the glans penis, and on the prepuce and on the urethral opening of a male subject". In another embodiment, endolysin may be co-administered with a compound or composition that adjusts the pH of the vagina. In some embodiments, the compound or composition adjusts the pH of the vagina to a pH between 4.0 and 6.0, preferably pH 5.0. In an alternative embodiment of the invention, the pharmaceutical composition does not comprise the endolysin itself, but instead comprises a nucleic acid molecule capable of expressing said endolysin. Suitable nucleic acid molecules, expression vectors and hosts are described in detail above. For example, recombinant probiotics may be used (LAB strains, e.g. Lactococcus lactis or Lactobacillus species). In a further embodiment of the invention, the pharmaceutical composition comprises a bacteriophage capable of expressing the endolysin of the first aspect of the invention. Methods for carrying out such bacteriophage-based therapies are well known in the art (see, e.g., Watanabe et al., 2007, Antimicrobial Agents & Chemotherapy 51:446-452). Thus, in the treatment of bacterial infections as described herein, the endolysins of the invention may be administered as the cognate protein, as a nucleic acid construct, vector or host cell expressing the cognate protein, as part of an organism expressing the cognate protein (including a bacteriophage), or by any other convenient method known in the art to achieve contact of the endolysin with its bacterial target, whether that be a pathogenic bacterium such as G. vaginalis, or another pathogen or potential pathogen as further described herein.

[0099] The compositions of the invention may include one or more endolysin polypeptides, in this embodiment the endolysin polypeptide may be present either as an independent polypeptide or as a fusion protein comprising said endolysin polypeptide or a fragment thereof.

[0100] The pharmaceutical composition of the present invention may further comprise one or more additional pharma- ceutically acceptable components, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavorings, adjuvants, etc. The pharmaceutical composition of the present invention is preferably free of imidazole.

[0101] The endolysins of the invention, together with adjuvants, carriers, diluents or excipients normally used, may be placed into the form of pharmaceutical compositions and unit doses thereof, and in such form may be used as solids, such as tablets or filled capsules, or liquids, such as solutions, suspensions, aerosols, emulsions, elixirs, or capsules filled therewith (all for oral use), or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dose forms thereof may contain the ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dose forms may contain any suitable effective amount of the active ingredients corresponding to the intended daily dose range for which they are used. The compositions of the present invention may also be liquid preparations, such as, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. These compositions may also be formulated as a dry product to be reconstituted with water or other suitable vehicle before use. Such liquid preparations may contain additives such as, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and gum arabic. Non-aqueous vehicles include, but are not limited to, edible oils, almond oil, coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid.Further materials, as well as processing techniques, are explored in Part 5 of Remington's "The Science and Practice of Pharmacy", 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins.

[0102] The solid compositions of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. Tablets may be coated by methods well known in the art. Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable carriers known in the art.

[0103] The compositions of the present invention may also be formulated as suppositories, which may contain suppository bases, including but not limited to, cocoa butter or glycerides. The compositions of the present invention may also be formulated as transdermal formulations with aqueous or non-aqueous vehicles, including but not limited to, creams, ointments, lotions, pastes, medicated plasters, patches, or membranes.

[0104] The compositions of the present invention may also be formulated for parenteral application, including, but not limited to, injection or continuous infusion. Preparations for injection may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulatory agents, including, but not limited to, suspending, stabilizing, and dispersing agents. The compositions may also be provided in powder form for reconstitution with a suitable vehicle, including, but not limited to, sterile pyrogen-free water.

[0105] The compositions of the present invention may also be formulated as a depot preparation, which may be administered by implantation or intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (for example as a sparingly soluble salt).

[0106] The compounds of the invention can also be administered from sustained release forms or sustained release drug delivery systems. A description of representative sustained release materials can also be found in Remington's "The Science and Practice of Pharmacy." Method of administration

[0107] The compositions of the present invention are preferably administered locally into the vagina of a female subject and / or in or on the glans penis, prepuce or urethral opening of a male subject, however, the compositions may also be administered in any manner, including intravenous injection, intraarterial injection, intraperitoneal injection, subcutaneous injection, intramuscular, intrathecal, oral routes including sublingual or via buccal administration, topical, dermal administration, direct tissue perfusion during surgery, or a combination thereof.

[0108] In a preferred embodiment, the endolysin, polynucleotide or pharmaceutical composition of the invention described herein is to be administered topically. In a further preferred embodiment, the endolysin, polynucleotide or pharmaceutical composition of the invention described herein is to be administered into the vagina of a female subject, or into or on the glans penis, prepuce or urethral opening of a male subject. In a further preferred embodiment described herein, the endolysin, polynucleotide or pharmaceutical composition of the invention is to be administered topically into the vagina of a subject.

[0109] The dosage administered to an individual as a single or multiple doses will vary depending on a variety of factors, including pharmacokinetic properties, the condition and characteristics of the patient (sex, age, weight, health, size), the severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect.

[0110] According to one embodiment, the compositions of the present invention may be administered in a prophylactic manner to a patient prior to sexual intercourse. combination

[0111] According to the present invention, endolysins may be administered alone or in combination with co-agents useful in the treatment and / or prevention of Gardnerella infections or disorders, including those caused by Gardnerella vaginalis strickenorrhoeae, Gardnerella leopoldii, Gardnerella pioti, Gardnerella swidszynskii, and / or other species of the genus Gardnerella.

[0112] The endolysins according to the invention have the following structure: (a) one or more conventional antibiotic treatments. Such antibiotics may include clindamycin, metronidazole, or any other suitable antibiotic known by one of skill in the art; (b) one or more additional endolysins, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing same; (c) A compound or composition that adjusts vaginal pH. In some embodiments, the compound or composition adjusts vaginal pH to pH 4.0-6.0, preferably pH 5.0. Suitable pH adjusting compounds may include phosphates, lactic acid (e.g., a natural acidifying substance secreted by Lactobacillus to establish an acidic environment) or other organic acids, such as carboxy-substituted polymers; (d) Therapies that neutralize toxins released upon bacterial lysis of Gardnerella cells in the vagina. Suitable neutralizing therapies may include antibodies (see Babcock et al., 2006, Infect. Immun. 74:6339-6347) and toxin adsorbents such as tolevamer (see Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534); (e) probiotics, may be administered in combination with Uses and methods according to the present invention

[0113] A further aspect of the present invention provides an endolysin according to the invention, a nucleic acid according to the invention, a vector according to the invention, a host cell according to the invention, a bacteriophage capable of expressing an endolysin according to the invention or a pharmacological composition according to the invention for use in medicine. Thus, the endolysin of the present invention may be for use for the treatment of the human or animal body by a surgical or therapeutic procedure and / or a diagnostic procedure performed on the human or animal body. In particular, the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell according to the present invention, a bacteriophage capable of expressing an endolysin according to the invention or a pharmacological composition according to the present invention for use in treating a disease or disorder.

[0114] A further aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention, or a pharmacological composition according to the present invention for use as a medicament.

[0115] A further aspect of the invention provides the use of an endolysin of the invention, a nucleic acid of the invention, a vector / plasmid of the invention, a host cell of the invention, a bacteriophage capable of expressing an endolysin of the invention or a pharmacological composition of the invention in the preparation of a medicament for killing and / or inhibiting or preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin. In particular, the invention provides the use of an endolysin of the invention, a nucleic acid of the invention, a vector / plasmid of the invention, a host cell of the invention, a bacteriophage capable of expressing an endolysin of the invention or a pharmacological composition of the invention in the manufacture of a medicament for treating bacterial infections and disorders.

[0116] A further aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention or a pharmacological composition of the present invention for use in killing and / or inhibiting or preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin.

[0117] A further aspect of the present invention provides a method for killing and / or inhibiting or preventing the growth of microbial cells in a patient, comprising administering to the patient an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention or a pharmacological composition of the present invention, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin.

[0118] A further aspect of the present invention provides the use of an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention or a pharmacological composition of the present invention in the preparation of a therapeutic agent for the treatment or prevention of a disease or condition associated with microbial cells in a patient, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin.

[0119] A further aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention or a pharmacological composition of the present invention for use in the treatment or prevention of a disease or condition associated with a microbial cell in a patient, wherein the microbial cell is selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by an endolysin of the present invention.

[0120] A further aspect of the present invention provides a method for the treatment or prevention of a disease or condition associated with microbial cells in a patient in need of such treatment, comprising administering to said patient an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention or a pharmacological composition of the present invention, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by an endolysin of the present invention.

[0121] "Diseases or conditions associated with microbial cells in a patient" include diseases and conditions caused by or antagonistic to infection of a patient with Gardnerella. Such diseases and conditions include BV.

[0122] By "treatment" we include both therapeutic and prophylactic treatment of a subject (or patient). In one embodiment, the endolysin of the invention, the nucleic acid of the invention, the vector / plasmid of the invention, the host cell of the invention, the bacteriophage capable of expressing the endolysin of the invention or the pharmacological composition of the invention, the use and method of the invention are for the treatment of an existing disease or condition. Alternatively or additionally, the use and method of the invention may be for prophylaxis. The term "prophylactic" or "prevention" is used to encompass the use of the endolysin or composition described herein to prevent or reduce the likelihood of infection with Gardnerella in a patient or subject. Prevention may be primary prevention (i.e. to prevent the development of the disease) or secondary prevention (where the disease has already developed and the patient is protected against the worsening of this process). It is preferred that the means and methods provided herein are for the treatment of an existing disease or condition, in particular for the treatment of existing BV.

[0123] As mentioned above, the term "effective amount" is used herein to describe a concentration or amount of an endolysin of the invention that can be used to bring about a favorable change in the disease or condition being treated, whether that change is amelioration, a favorable physiological outcome, an improvement or attenuation of the disease state or condition being treated, or prevention or reduction in the likelihood of a causing condition or condition, depending on the disease or condition being treated. In one embodiment, the endolysin according to the first aspect of the invention, the nucleic acid according to the second aspect of the invention, the vector according to the third aspect of the invention, the host cell according to the fourth aspect of the invention, the bacteriophage capable of expressing an endolysin according to the first aspect of the invention or the pharmacological composition according to the sixth aspect of the invention is administered in a single dose. Alternatively, the endolysin, nucleic acid, vector / plasmid, host cell, bacteriophage or pharmacological composition is administered as multiple doses (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more doses). The endolysin, nucleic acid, vector / plasmid, host cell, bacteriophage or pharmacological composition is preferably administered at a frequency sufficient to maintain the residence of the endolysin according to the first aspect of the invention in the vagina of the subject. Preferably, the frequency of administration and dosage is sufficient to prevent the appearance or recurrence of a disease or condition associated with the microbial cells (e.g., Gardnerella) in the subject. Preferably, the frequency of administration and dosage is sufficient to prevent the appearance or recurrence of a growth impedance associated with the microbial cells (e.g., Gardnerella) in the subject.

[0124] In one embodiment, the uses and methods of the present invention, a host cell or a pharmaceutical composition comprising a host cell is used to deliver an endolysin according to the first aspect of the invention to a (preferably a host cell).

[0125] Of course, the agents described herein may be administered to a subject in combination with one or more additional therapeutic agents. For example, the agents described herein may be administered in combination with one or more additional therapeutic agents, such as the following: (a) one or more conventional antibiotic treatments. Such antibiotics may include clindamycin, metronidazole, or any other suitable antibiotic known by one of skill in the art; (b) one or more additional endolysins, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing same; (c) A compound or composition that adjusts the vaginal pH, preferably to a pH between 4.0 and 6.0, more preferably to about pH 5.0. Such pH adjusting compounds may include phosphates, lactic acid (e.g., a natural acidifying substance secreted by Lactobacillus to establish an acidic environment) or other organic acids, such as carboxy-substituted polymers; (d) therapies that neutralize toxins released upon bacterial lysis of G. vaginalis cells in the vagina. Suitable neutralizing therapies may include antibodies (see Babcock et al., 2006, Infect. Immun. 74:6339-6347) and toxin adsorbents such as torevamer (see Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534); (e) probiotics, may be administered to a subject in combination with

[0126] A further aspect of the invention provides the use of an endolysin having cytolytic activity against Gardnerella, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing it, for killing and / or inhibiting / preventing the growth of microbial cells in vitro and / or ex vivo, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by the endolysin of the invention. For example, the endolysin having said activity can be used to clean surfaces in hospitals, kitchens, etc. that are susceptible to contamination with such bacterial cells. Preferably, the microbial cells comprise or consist of cells of Gardnerella vaginalis sect., G. leopoldii, G. pyotsi, G. swidszynskii or other species of the genus Gardnerella.

[0127] A further aspect of the present invention provides a kit, comprising an endolysin as described herein and instructions for use, in particular for treating a disease or disorder, preferably BV. The kit may be used for therapeutic or prophylactic purposes and may further comprise a compound or composition for adjusting the vaginal pH to between 4.0 and 6.0, preferably between 4.5 and 5.5, more preferably about 5. However, the present invention also provides a kit for detecting the presence of microbial cells in a sample, comprising a polypeptide having the cell lytic activity and / or cell binding specificity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing same, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin.

[0128] Related aspects of the invention include the following: (a) the use of a polypeptide having the cell wall binding and / or cytolytic activity of an endolysin according to the invention, or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing same, in the manufacture of a diagnostic for a disease or condition associated with microbial cells selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin; (b) the use of a polypeptide having the cell wall binding and / or cytolytic activity of an endolysin according to the invention, or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing same, for diagnosing a disease or condition associated with microbial cells selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin; (c) the use of a polypeptide having the cell wall binding and / or cytolytic activity of an endolysin according to the invention, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing same, for detecting the presence of microbial cells in a sample in vitro and / or ex vivo, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin; and (d) An in vitro method for the diagnosis of a disease or condition treatable with an endolysin according to the present invention, comprising the following steps: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of the C-terminal cell wall binding domain of an endolysin according to the present invention and, optionally, the N-terminal catalytic domain of an endolysin according to the present invention, wherein the sample contains microbial cells and wherein the C-terminal cell wall binding domain of the endolysin is optionally labelled; (ii) testing whether the polypeptide binds to and / or lyses microbial cells in the sample; and (iii) determining whether the disease or condition can be treated by an endolysin according to the present invention if the polypeptide binds to and / or lyses microbial cells. Thus, the present invention relates to an in vitro method for diagnosing in a patient a disease or condition treatable with an endolysin of the present invention, comprising contacting a cell sample obtained from the subject with a polypeptide having the cell wall binding and / or cytolytic activity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or prophage capable of expressing it, and determining whether cells in the sample are lysed thereby, wherein the microbial cells are selected from the group consisting of Gardnerella cells and other bacterial cells susceptible to lysis by said endolysin. Preferably, the microbial cells comprise or consist of cells of Gardnerella vaginalis strickenorum, G. leopoldii, G. pyotsi, G. swidszynskii or other species of the genus Gardnerella. In such diagnostic uses and methods, cell lysis may be detected using methods well known in the art. For example, the level of ATP may be measured as an indicator of cell lysis.

[0129] In an alternative embodiment of the above uses and methods of the invention, the polypeptide comprises or consists of the cell wall binding domain of an endolysin according to the invention. To allow detection, such polypeptides may be used as fusion proteins fused to magnetic beads or containing suitable reporters or labels (e.g. green fluorescent protein or chromogenic enzymes such as HRP). Such diagnostic methods are well established for endolysins from other systems, such as Listeria endolysins (see for example Loessner et al., 2002, Mol Microbiol 44, 335-49; Kretzer etai, 2007, Applied Environ. Microbiol. 73:1992-2000).

[0130] Illustrative embodiments of the invention will now be described by way of the following non-limiting examples with reference to the following drawings: EXAMPLES

[0131] Example 1 – Identification of native endolysins in the Gardnerella genome

[0132] Endolysins are hydrolases produced by bacteriophages to cleave the host cell wall during the final stage of the lytic cycle. They have the ability to target one of the five bonds of the peptidoglycan (murein), the main component of the bacterial cell wall, which allows the release of progeny virus particles from the lysed cell. So far, no bacteriophage lytic factors for Gardnerella have been isolated. Therefore, it is also not known whether endolysins from a bacteriophage origin and with lytic action against Gardnerella can be successfully identified. The inventors investigated whether endolysins encoded by prophage sequences could be identified in various Gardnerella genomes. Prophages are bacteriophage genomes that are inserted and integrated into the circular bacterial DNA chromosome or exist as extrachromosomal plasmids. This is the latent form of the phage, in which the viral genes are present in the bacteria without causing the destruction of the bacterial cell. Identification of prophage sequences in bacterial genomes and plasmids can be performed using web-based tools known by those skilled in the art. For example, such tools include, but are not limited to, PHASTER (Arndt et al., 2016 Nucleic Acids Res. 44, W16-W21), PROPHINDER (Lima-Mendez et al., 2008 Bioinformatics 24, 863-865) or similar. The inventors were able to identify sequences of 14 Gardnerella genomes that are predicted to constitute complete or partial prophages. The sequences were found by identifying DNA regions whose cluster genes were predicted to be of viral origin. Viral gene clusters that were predicted to be only partial prophages as opposed to complete prophages were also included.

[0133] The putative prophage sequences were then annotated by blasting predicted coding sequences of the endolysins to identify putative endolysins. We searched for protein sequences homologous to enzymes capable of cleaving any of the major chemical bonds that make up peptidoglycan. In particular, we searched for protein sequences homologous to N-acetylmuramidases, N-acetylmuramoy-L-alanine amidases, L-alanoyl-D-glutamic acid endopeptidases, interpeptide cross-linking endopeptidases, or N-acetyl-β-D-glucosaminidases. In every individual prophage or partial prophage analyzed, we found coding sequences for proteins homologous to 1,4-β-N-acetylmuramidases, which we named EL1 to EL14. The assignment of names to the genome of origin is shown in Table 1. [Table 1] In each case, only one copy was found per prophage, and no other coding sequences were found that predicted enzymes capable of lysing bacterial cell walls. The putative 1,4-β-N-acetylmuramidases were aligned to understand the homology and domain structure (see Figure 1). As can be seen in Figure 1, the majority of endolysins have exactly 306 residues, even from different prophages on different genomes. Two exceptions are EL6 and EL9. EL6 is truncated at the C-terminus by a frameshift. However, EL9 ends in exactly the same position as EL6, in this case the entire contig end. As can be seen in Figure 2, there were no identical pairs among the endolysins, even though they are highly homologous.

[0134] Example 2 – Determination of the domain structure of the native Gardnerella prophage endolysin

[0135] The domain structures of the newly discovered endolysins were determined using InterPro (Mitchell et al., 2019 Nucleic Acids Res. 47, D351-D360). Briefly, InterPro is a database on protein families, domains and functional sites where identifiable features found in known proteins can be applied to new protein sequences to functionally characterize them. The content of InterPro consists of diagnostic characteristic features and proteins with which they significantly match. Characteristic features consist of models, e.g., simple types such as normal expression, or more complex ones such as Hidden Markov models where a protein describes a family, domain or site. As can be seen in Figure 3, all endolysins with 306 residues have the same domain arrangement. The 196-residue N-terminal domain was identified as the catalytic domain due to its homology to glycoside hydrolases, family 25. The catalytic domain is followed by a linker region and two cell-binding domains, also called CW_7 domain, homologous to the C-terminal domain of lysozyme Cpl-7 (Garcia et al., 1990 Gene 86, 81-88; Lopez and Garcia, 2004 FEMS Microbiol. Rev. 28, 553-580; Bustamante et al., 2010 J. Biol. Chem. 285, 33184-33196, 2012 PLoS One 7, e46654). In the following examples, the above-identified catalytic domains are referred to as "N-terminal catalytic domains" or "H-domains", where, for example, "H2" refers to the H-domain of native EL2. In the following examples, the "linker region" and the "C-terminal cell wall binding region", the latter comprising or consisting of one or more cell wall binding domains or "B domains", together refer to the "B region", where, for example, B10 refers to the B region of native EL10. Similarly, B11_N refers to the N-terminal cell wall binding domain of native EL11, B12_C refers to the C-terminal cell wall binding domain of native EL12, etc.

[0136] Example 3 - Measurement of enzymatic activity of native Gardnerella prophage endolysin on Gardnerella cells

[0137] The inventors investigated the enzymatic activity of the newly discovered endolysin against Gardnerella. It was not possible to predict by computer whether the identified sequence homologous to 1,4-β-N-acetylmuramidase would be active. As is well known in the art, bacteria can mutate their prophage sequences, so that the corresponding prophage may lose the ability to grow. Moreover, even if the peptidoglycan hydrolase encoded by the newly discovered phage is indeed an active protein, it has not yet been demonstrated that said protein can enzymatically degrade the specific peptidoglycan layer of Gardnerella. In fact, Gardnerella is special in that it is a Gram-indeterminate species: it does not form an outer membrane, which defines a true Gram-negative species. Its cell wall is generally very thin and has a peptidoglycan content of less than 10%. Thus, one skilled in the art would have thought that peptidoglycan-degrading enzymes such as endolysin proteins would not be able to efficiently lyse the bacterial cell wall of Gardnerella.

[0138] The 14 identified endolysins EL1 to EL14 were cloned with a His-tag, expressed in E. coli, and purified by a single-step Ni-NTA column using the method described in Reference Example 1. The assignment of endolysin names to the genome of origin is shown in Table 1. The Gardnerella strains used are shown in Table 2. [Table 2] To test the activity of the purified endolysins, the turbidity change of Gardnerella suspensions (see Table 2) was measured at 610-620 nm essentially using the method described in Reference Example 2, where 95 μl of bacterial suspension in Hardy Broth at the indicated pH was mixed with 5 μl of endolysin solution in a photometric cuvette at room temperature under aerobic conditions. In the turbidity reduction assay, the reduction in light scattering (i.e., turbidity reduction) of a suspension of live cells can be used to assay the activity of peptidoglycan hydrolases by spectrophotometry. The reduction in absorbance over time (minutes) can be used to calculate the hydrolysis reaction rate. The results are compared to a "no enzyme, buffer only" control preparation treated similarly over the same period of time. Thus, the specific activity of the enzyme preparation can be reported as ΔOD / hour / μl lysin protein. As can be seen in Figures 4A-4C, the reduction in turbidity was much more pronounced for the endolysin-treated groups than for the buffer, indicating enzyme activity. Surprisingly, as a result, the inventors have found that the newly discovered endolysins EL1, EL2, EL3, EL4, EL5, EL7, EL10, EL11 and EL12 are active proteins capable of lysing Gardnerella cell walls. As explained above, due to the low peptidoglycan content in the cell walls, the fact that peptidoglycan degrading enzymes such as the identified endolysins can efficiently lyse bacterial cell walls was an unexpected and surprising discovery.

[0139] Example 4 – Identification of the most active domains using artificial domain-swapped endolysins

[0140] We next assessed whether different N-terminal enzyme domains might have different lytic activities and whether the B region (including the linker and cell wall binding domains) might mediate specificity for different strains, by engineering domain-swapped endolysins.

[0141] 4.1 - Endolysin constructs

[0142] To engineer the domain-swapped endolysins, the N-terminal 196 residues of the first native endolysin, including the catalytic domain, were exchanged as a block against the complete C-terminal region of 110 residues of the second native endolysin, including the linker region and the two cell wall binding domains. The domain-swapped proteins were prepared by carrying out the following procedure: The prototype construct EL1-14 was ordered from GeneWiz as a synthetic gene codon-optimized for E. coli. These constructs were cloned by GeneWiz into the pETM14_ccdB vector by a restriction / ligation approach using the recognition sites for the NcoI and NotI enzymes. Table 3 below summarizes the primers used. For the domain exchange of the 10 selected constructs, each H-domain with the T7 promoter was amplified by a general forward primer (no. 2) and a construct-specific reverse primer (no. 3-12) using PhusionFlash polymerase (Thermo, F-548L). Similarly, each B region was amplified by a construct-specific internal primer (no. 13-21) and a general reverse primer (no. 1) encompassing the T7 terminator. All primers encompassed an extension carrying a BsaI recognition site and the outer ends were adapted to the pETM14-derived vector backbone pETMdest. The overhang between the domains was designed to have the sequence "GGCT" within two amino acids GL of the linker sequence. As a result, these two amino acids represented the exact boundary between the domains for the purposes of this experiment. Therefore, the amplified and gel-purified domains (GeneJet Gel Purification Kit, Thermo, K0692) were then combined into 90 new expression constructs using the GoldenGate cloning strategy by BsaI restriction (BsaI-HFv2, NEB, R3733S) / ligation (T4DNA Ligase, Thermo, EL0011) cycle reactions. For transformation purposes, the NEB10β E. coli strain was used (NEB, C3019) and plasmids were purified using the GeneGet Plasmid Miniprep Kit (Thermo, K0502). [Table 3] For ease of reference, the domain combinations of the artificial endolysins are represented using the H-code (hereafter referred to as H-domain) for the N-terminal catalytic domain and the B-code (hereafter referred to as B-domain) for the part including the C-terminal cell wall binding region and the linker region. As an example, H2B10 refers to a domain-exchanged endolysin having the N-terminal domain from the natural endolysin EL2 and the linker region and the C-terminal cell wall binding region from the natural endolysin EL10. In other words, H2B10 refers to a domain-exchanged endolysin consisting of the 196 N-terminal residues of the natural endolysin EL2 (SEQ ID NO: 2) and the 110 C-terminal residues of the natural endolysin EL10. In this example, the B region B10, which corresponds to the 110 C-terminal residues of the native endolysin EL10, comprises, from the C-terminus to the N-terminus, the C-terminal cell wall binding domain "B10_C" (SEQ ID NO: 29), the N-terminal cell wall binding domain "B10" (SEQ ID NO: 28) and the linker region "L10" (NAGLNGYKNGGS). The nomenclature and the corresponding amino acid sequences are detailed in Table 7. Consequently, according to the above nomenclature, a naturally occurring endolysin, such as EL3, can be interchangeably defined as either H3B3 or H3-L3-(B3_N)(B3_C). Similarly, a genetically engineered endolysin, such as H2B10, can also be interchangeably defined as H2-L10-(B10_N)(B10_C). Those skilled in the art will appreciate that the components "Lx", "(Bx_N)" and "(Bx_C)" may also be independently interchanged in other embodiments. The nomenclature is further illustrated in Table 7 below.

[0143] 4.2 - Optimization of assay parameters

[0144] The dependence of activity on three potentially important parameters, namely pH, anaerobic / microaerobic / aerobic conditions and the presence / absence of imidazole, was analyzed. Three criteria for evaluation were selected for the following reasons. - pH: The killing activity of the endolysin of the invention was successfully demonstrated using experiments carried out at a pH value of about 7. However, while the pH of a healthy vagina is about 3.5, in the BV vagina the pH increases to about 5.5 and even higher. Therefore, the pH dependence of the endolysin activity was investigated. - Oxygen: In the literature, Gardnerella is described as anaerobic or microaerophilic, so we investigated whether more intact cells survived under anaerobic, microaerophilic or aerobic conditions over the incubation period of the experiment (usually 5 hours). - Imidazole: The endolysin of the invention was purified by a one-step Ni-NTA column according to the method described in Reference Example 1, where the buffer used to elute the endolysin from the Ni-NTA matrix contained imidazole. Therefore, when there is no further step of dialyzing the sample, the eluted solution obtained contains 250 mM imidazole. In that respect, the effect of imidazole on Gardnerella was investigated. First, the sensitivity of survival of G. vaginalis Gv_9 to incubation in media with or without imidazole at different pH values ​​was evaluated (see FIG. 6). 7 CFU / ml of cells were incubated under anaerobic conditions at 37°C for 5 hours under the conditions indicated below the graph. Viable CFU / ml were then determined by quantitative plating. As shown in Figure 6, at pH 6.0, a median of 1 x 10 7 and 1×10 6 cells survived incubation without and with imidazole, respectively. At pH 7.0, a median of 2 × 10 6 and 3 x 10 4Only 1e7 cells survived without and with imidazole, respectively. In the untreated control at pH 5.0, 1e7 cells survived the procedure, whereas the median survival for imidazole treatment at pH 7 was 3e4, i.e., 3 logs below the former. Thus, survival of G. vaginalis Gv_9 is highly dependent on the absence of imidazole, especially at pH>6.0, and at low pH. Second, the susceptibility of G. vaginalis Gv_9 to treatment with recombinant endolysin H10B1 versus a control containing imidazole at various pH values ​​was evaluated (see FIG. 7). 7 CFU / ml of cells were incubated under anaerobic conditions at 37°C for 5 hours under the conditions indicated below the graph. Viable CFU / ml were then measured by quantitative plating. The column labeled imidazole control shows the same data as in Figure 5. As shown in Figure 7, the endolysin was highly active down to pH 5.0, and even the relative reduction to the control was very significant at this low pH, with a 2.5 log reduction in viable CFU. At pH 7.0 there was less than 1 log10 difference in survival between H10B1 treated and untreated cells, but the difference was 2 log10 units at pH 5.0. The survival of cells not treated with H10B1 was not enhanced at pH values ​​lower than 6.0 in the presence of imidazole. Thus, the activity of endolysin H10B1 is highly pH dependent. When similar experiments were performed under aerobic conditions, the survival of control cells was reduced by several log10 units compared to anaerobic conditions (data not shown). It was therefore concluded that the optimized parameters for carrying out experiments with the endolysin of the present invention were pH 5.0, under anaerobic conditions and with the step of removing imidazole from the endolysin eluate.

[0145] 4.3 - Expression Levels

[0146] Table 4 gives an overview of the concentrations of all endolysin constructs. Constructs with a concentration above 0.2 mg / ml after imidazole removal were adjusted to a concentration of 0.2 mg / ml by dilution. Constructs with lower concentrations were left as is and their activity was tested. The native endolysin EL6 (not listed in Table 4) had a concentration below 0.2 mg / ml. H4, H11 and H12 appear to give low solubility and expression levels, as most constructs fall within the 0.2 mg / ml threshold. For H1 as well, some constructs had low concentrations. [Table 4]

[0147] 4.4 - Quantitative evaluation of lysis of four major species of Gardnerella by endolysin activity in suspensions under optimized conditions

[0148] The lytic activity of 91 constructs (native and domain-swapped endolysins) against the four main species of Gardnerella was quantitatively assessed using the method described in Reference Example 2. Briefly, 90 μl of 5e7 CFU / ml of the indicated strains were incubated with 10 μl of endolysin for 5 h under anaerobic conditions at pH 5.0 (where possible, the concentration was adjusted to 0.2 mg / ml, see Table 4). The results are shown in Figures 8A-8D together with Tables 5A-5C. In Figures 8A-8D, the logarithmic Y-axis shows the count of viable cells. The dotted line shows the limit of detection (LOD) given by plating 2 μl of the reaction mixture (500 CFU / ml). Each combination of native 10H-domains and native 9B-regions was evaluated, including native endolysins (H1B1, H2B2, H3B3, etc.) and H6B6. No other constructs with B-domain B6 were tested, since B6 rendered all H-domains fused to it inactive as measured by OD measurements (data not shown). The activity of each construct was measured against each of the four main Gardnerella species: G. vaginalis sensu stricto, G. leopoldii, G. piochii, and G. swidsinkii. Table 5, organized by H-domain and B-region, summarizes the log10 reduction in CFU obtained. All conditions were measured in triplicate. Survival after 5 hours of incubation with endolysin versus buffer under anaerobic conditions at pH 5.0 was measured by quantitative plating. Values ​​indicate the log10 ratio of surviving CFU for treated versus untreated cells. The average of the triplicate measurements is used. In Tables 5A and 5B, highly negative log10 values, e.g., -6.7, -5.5, -4.8, etc., are associated with high enzyme activity, whereas log10 values ​​close to zero or even positive log10 values ​​are associated with low or no enzyme activity. For example, the average CFU of three control treated measurements for Gv_9 was 1.0 x 10 7 CFU / ml, and the average of the three samples treated with H2B10 was 2.5 x 10 3 CFU / ml, then the log10 value of CFU reduction of Gv_9 by H2B10 is log10(2×10 3 / 10 7 Conversely, a reduction of -3.7 would represent a 10 reduction in viable CFU in treated samples versus untreated controls. 3.7This means a fold (=5012-fold) reduction in the activity of H4B3 against Gv_9. In the case of an inactive endolysin, e.g., H4B3 against Gv_9, the CFU of Gv_9 after treatment would be identical to the CFU counted in the control treated sample and the ratio of the two CFU values ​​would be equal to 1. As a result, the reduction value of H4B3 against Gv_9 would be log10(1)=0.0. [Table 5] Table 5B below shows the same data as Table 5A, but expressed as the average log10 activity of each construct across the four Gardnerella strains. On the right and at the bottom, the average values ​​for each native H-domain across all native B-regions (except B6), and each native B-region across all native H-domains (except B6), respectively, are shown along with the activity ranks for each native H-domain and native B-region. [Table 6] Table 5C shows the activity ranking for all endolysins based on the data in Table 5B. [Table 7] Table 5D shows the average log10 lysis of each Gardnerella strain used. Mean values ​​were calculated for log10 activity across all constructs tested. [Table 8] Tables 5A-5C show that the activity of the endolysins is highly specific depending on the H-domain / B-region combination and the strain it is tested on. Each construct was assayed against four Gardnerella strains (see Table 5A). On average, the most active H-domain was H2 with a mean reduction in CFU of 3.1 log10 units across the B-region (excluding B6), followed by H7, H10 and H5 (see Table 5B). For the B-region, B10 was the most active, followed by B11, B12 and B3, with a mean CFU reduction of 2.7 log10 units. Surprisingly and unexpectedly, the inventors found that some of the engineered endolysins have stronger activity than any of the native endolysins (H1B1-H12B12), especially across all four Gardnerella strains tested (see Figures 8A-8D). In particular, H2B10, H2B11, and H2B12 have activity ranks 1, 2, and 3, respectively, each more active than any of the native endolysins (see Table 5C). Overall, H7B3 has a rank of 4 (see Table 5C), which is also more active than any other native endolysin included in the experiment. In fact, when limited to the ranking of native endolysins within the top 10, the most active is H10B10 (rank 6), and the next most active native endolysin is H3B3 (rank 13). In summary, the engineered endolysins according to the present disclosure may exhibit significantly higher activity than the naturally occurring endolysins. Without being limited to a particular theory, the unexpected increase in the killing activity against Gardnerella observed by domain swapping of endolysins according to the invention can be explained as follows: Natural endolysins on prophages have undergone a Darwinian evolution process, in which the propagation of the entire prophage is optimized. However, the probability of a mutation leading to a higher propagation of the respective prophage by simultaneously improving the catalytic activity of the endolysin and at the same time broadening its host range across species within the Gardnerella-limited range is very low. In contrast, some Gardnerella prophages had to evolve the N-terminal catalytic domain of the endolysin for the highest activity, and furthermore, some other prophages had to optimize the C-terminal region of the endolysin for the broadest activity across Gardnerella species. Therefore, by combining the highly evolved N-terminal catalytic domain of one of the endolysins of the invention with the highly evolved C-terminal region of another endolysin of the invention encoded by a different genome derived from a different prophage, a genetically engineered endolysin with a more highly optimized killing activity against Gardnerella species than the native endolysins of the invention can be achieved. This can be achieved, for example, by combining the highly evolved N-terminal catalytic domain of one of the endolysins of the invention with the highly evolved C-terminal region of another endolysin of the invention encoded by a different genome derived from a different prophage. This is achieved by the recombinant endolysins H2B10, H2B11, H2B12, and H7B3 of the present invention, all of which have higher killing activity than native endolysin EL2 or any other native endolysin, and across all Gardnerella species (see Figures 8A-8D and Table 5A). Moreover, most constructs are much more active against Gv_23 (G. swidszynskii) than against the other three strains tested (see Table 5A). The average activity across all constructs against each Gardnerella strain (see Table 5D) confirms that Gv_23 is the most sensitive to the endolysins, followed by Gv_9 and Gv_11, whereas Gv_17 (G. pyoti) is the least sensitive. This order of sensitivity is mostly the case across the endolysin constructs. For many constructs, Gv_23 is the most sensitive strain, by a difference of several log10 units, but there are also times when Gv_17 is more sensitive than Gv_9 or Gv_11 (e.g. for H2B10, the overall most active endolysin). Without being bound by any particular theory, the difference in sensitivity could be explained by either structural defects such as a weaker / thinner / more accessible cell wall of Gv_23 or by stronger enzymatic activity of the tested endolysins against Gv_23. Furthermore, it can be concluded that the concentration of the endolysins in solution is important for their activity in the assay. In general, constructs with low concentrations as shown in Table 4 - especially those with H-domains H4, H11 and H12 (which confer poor solubility over the B region) - also have low activity in the activity assay. It was a bit surprising, like H12B11, which has a relatively high activity despite having a very low expression level.

[0149] 4.5 - Activity Pattern Analysis

[0150] The sequences of the native H-domains were aligned and compared for related activity patterns, as shown in the dendrogram of FIG. 9. The most active H-domains were also expected to be most closely related to each other. However, to our surprise, the most active N-terminal domain, H2, is almost homologous to the least active, H6. The next most active H-domains, H7 and H10, are also more distant than expected with respect to each other and with respect to H2. H5, the four most active H-domains, is most closely related to H7. Combinations with the B-regions that make the recombinant endolysins most active also do not lead to a predictable pattern. H2 is most active in combination with B10, B11 and B12. However, the second most active H-domain, H7, is most active in combination with B3, as is the case with its closest homologue, H5. The B regions were also aligned for homology matching against activity patterns, as shown in the dendrogram in Figure 10. The most active B regions at the time of analysis in Tables 5A-5C are B10, B11, B12, followed by B3, all of which have an average CFU reduction of more than 2 log10 units. In contrast to the pattern seen for the H-domain, these four most active B regions are the four closest homologs within the group of B-regions tested. Interestingly, the B5 and B7 regions are identical (see Figure 10). As seen in Figures 8A-8D, the best overall results were obtained with H2B10, H2B11 and H2B12. As described in Example 2, each native B-region contains two B-domains, an N-terminal cell wall domain and a C-terminal cell wall domain. The sequences of each native B-domain within the B-region were also aligned and compared, as shown in Figures 11 and 12. The boundaries of the B-domains can be identified by analyzing the sequences with Interpro (Mitchell et al., 2019 Nucleic Acids Res. 47, D351-D360) and by aligning the two repeat motifs within each B-region. The C-terminus of all B-domains is a conserved sequence (VNELL or VNKLL), the homolog of which can also be found at the C-terminus of the CW_7 motif of protein Cpl-7 (VNELL or VNEIL), thereby defining the boundaries of the two B-domains of each B-region. As an exception, B6 has only one truncated B-domain, which is likely the reason for the complete inactivity of EL6. In conclusion, specific combinations of H-domains and B-regions have been shown to be important and it was a surprising and unexpected finding that each H / B combination leads to an endolysin with higher killing activity compared to the native endolysin.

[0151] Example 5 - Activity assay against beneficial lactobacilli

[0152] Three main species of Lactobacillus live in the healthy vagina: L. crispatus, L. gasseri, and L. jensenii. They maintain an acidic pH of 3.5-4.5 by producing lactic acid, and protect the oxidative environment by producing H2O2. Recovery from BV is associated with the repopulation of these Lactobacilli in the vagina, so medicines against BV should advantageously not interfere with this process. Antibiotics clearly work, and that is why there is still a strong medical need for improved methods and compositions for treating Gardnerella infections and BV. After successfully demonstrating the high activity of the endolysins of the present invention against Gardnerella, the inventors investigated whether they could lyse strains of the three most frequent Lactobacillus species in the healthy vagina. The experiments were carried out under anaerobic conditions at pH 5.0 using the method described in Reference Example 2. As shown in Figure 13, the tested recombinant endolysins do not show any killing activity against the three beneficial lactobacilli used, namely L. crispatus, L. gasseri and L. jensenii. The endolysins of the present invention show high killing activity against Gardnerella, but are ineffective against the most frequent beneficial lactobacilli. These results therefore confirm the genus-selective activity of the endolysins of the present invention and their drug candidate status as innovative medicines against BV. In that respect, the treatment of BV with the endolysins of the present invention is far more advantageous than currently available treatments, such as treatment with the antibiotics metronidazole and clindamycin.

[0153] Example 6 - Activity analysis of standard of care antibiotics metronidazole and clindamycin against Gardnerella strains grown in suspension

[0154] One of the main deficiencies in the treatment of BV is the high recurrence rate in many women, which leads to repeated antibiotic administration and destabilization of the microflora and other side effects. Next, the minimum growth inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Gardnerella cells of the strains also used in the endolysin activity assay were measured using the method described in Reference Examples 6 and 7. In short, the measurement protocol had to be heavily edited from the international standard, which does not match the MIC and MBC measurements for Gardnerella. The main parameters that were changed were the growth medium (Gardnerella does not grow in Mueller-Hinton Broth, which is usually used for MIC measurements), the anaerobic conditions, the incubation time, and the starting concentration of bacteria in the first round of the experiment. The starting concentration was changed from the standard 5 × 10 to 10 × 10 , mainly because cells are also highly concentrated in the vagina of BV patients and the effect of the antibiotic must be measured at a cell density comparable to that used for the endolysin activity assay. 5 CFU / ml to 2.5 x 10 7 The effect of metronidazole (obtained from Gatt-Koller) and clindamycin (obtained from Ratiopharm) on the growth of Gardnerella strains in suspension was evaluated. 7 Gardnerella suspensions of CFU / ml were incubated with the indicated concentrations of antibiotics and incubated for 48 h at 37°C under anaerobic conditions. The MIC was defined as the lowest concentration of antibiotic at which no growth was detectable after 48 h by OD measurements. OD(610) was measured at the beginning and end of the experiment. At the end of the experiment, 2 μl of each reaction mixture was spotted on an agar medium to measure the MBC. Table 6A summarizes the results of the experiment described in Figure 14. Resistant (R) was defined as >=32 μg / ml for metronidazole and >8 mg / ml for clindamycin. Susceptible (S) was defined as <=8 μg / ml for metronidazole and <=2 μg / ml for clindamycin according to international standards. [Table 9] [Table 10] According to the results shown in Figure 14, all Gardnerella strains have low susceptibility to both metronidazole and clindamycin. The conditions under which MIC and MBC were measured were stricter than the standard. For example, the MBC 90 , i.e., the antibiotic concentration that kills 90% of cells within a defined time, is measured, whereas MBC is 2.5 × 10 7 The concentration was defined here as the minimum concentration that eradicates the CFU / ml suspension. Nevertheless, these conditions are closer to those found in the vagina of BV patients. The high MIC and MBC values ​​measured under these conditions could explain the high recurrence rate of BV. In contrast, the endolysins assayed are bactericidal by definition, since they lead to the complete destruction of bacterial cells. Therefore, these results confirm that the endolysins of the present invention are superior to antibiotics in the treatment of BV. Some experimental parameters (i) 1×10 5 ~1×10 6 A second round of MIC and MBC experiments was performed here according to the Clinical and Laboratory Standards Institute (CLSI) standards, with the following modifications: (ii) use of clindamycin hydrochloride powder (Sigma Aldrich, cat. no. C5269), (iii) NYC-III broth instead of Hardy broth, and (iv) 96-well plates instead of 384-well plates. As shown in Figure 15 and summarized in Table 6B, all Gardnerella strains still had very low MICs to metronidazole (8-128 μg / ml), whereas - in contrast to clindamycin shown in Figure 14 and Table 6A - clindamycin hydrochloride was now inhibitory and bactericidal at low concentrations (MIC ≦ 1 μg / ml). Example 7 - Activity assay for a domain-swapped endolysin representative (H2B10) against growth in suspension of various Gardnerella strains 1×105 ~1×10 6 The MIC and MBC analyses of endolysin H2B10 using cell suspensions were used. H2B10 showed MICs in the low μg / ml range (0.5-4 μg / ml) indicating that Gardnerella cells are highly sensitive to endolysins (Figure 16, Table 7). The conditions for measuring MBC were more stringent than standard. For example, the MBC 90 , i.e. the antibiotic concentration that kills 90% of the cells within a defined time, is measured, whereas MBC is defined in this application as the dose that reduces the starting cell count by at least 99.5%. H2B10, as a representative of the endolysins claimed herein, shows a very good MIC and MBC over the standard care antibiotic metronidazole, which is ineffective against many Gardnerella strains due to resistance formation. However, clindamycin gave contradictory results. According to the international standards, all four Gardnerella strains were expected to be resistant to clindamycin (obtained from Ratiopharm (Figure 14)) (MIC>8 μg / ml), whereas clindamycin hydrochloride (Sigma Aldrich) was very effective with a bactericidal effect already at concentrations ≦1 μg / ml (Figure 16). It is generally known that antibiotics only poorly eradicate the Gardnerella biofilm, which is a hallmark of BV, and this is one of the suspected reasons for the reported high recurrence rate of BV.Although they leave the remaining viable biofilm, antibiotics wipe out some of the beneficial organisms of the vaginal microflora, and then open up the ecological niche for other pathogens, such as fungi.Therefore, it is presumed that endolysin-based treatment, which selectively eradicates Gardnerella bacterial cells and probably eradicates biofilm without harming beneficial Lactobacillus, is superior to standard antibiotic treatment for BV. [Table 11]

[0155] Reference Example 1 - Cloning, expression and purification of endolysins material: 96-well Multiscreen HTS Durapore 96-well Filterplatten, PS (Labshop cat. no. 44.MSGVS22) PD MiniTrap desalting column with Sephadex G-25 (GE Lifescience, cat. no. 28918007) ·Slide-A-Lyzer(TM) MINI Dialysis Device, 10K MWCO, 2mL (Thermo Scientific, cat. no.88404) Fastbreak reagent (Promega, cat. no. V8571) Lysis Buffer: 50mM Phosphate pH 6, 150mM NaCl, 20mM Imidazole, 1mM TCEP, 1x FastBreak, Benzonase Wash Buffer I: 50 mM phosphate pH 6, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP (1.5 ml) Wash Buffer II: 50 mM phosphate pH 6, 150 mM NaCl, 40 mM imidazole, 1 mM TCEP (1.5 ml) Elution buffer: 50 mM phosphate pH 6, 150 mM NaCl, 250 mM imidazole, 1 mM TCEP (1.1 ml) method: The expression constructs were transformed into E. coli strain Bl21(DE3) and selected using the appropriate antibiotic. Cells from 2 ml culture (TB+lactose, 25°C, O / N) were resuspended in 1.5 ml lysis buffer and lysed with FastBreak reagent (Promega). The intracellular soluble fraction was isolated by centrifugation at 15000g for 30 min at 4°C. The soluble protein fraction was loaded onto 100 μL of Nickel affinity matrix, washed with 15 column volumes (CV) each of wash buffer I and II, and eluted with 10 CV of elution buffer. The elution buffer will then be exchanged with 20 mM phosphate pH 6.0, 150 mM NaCl to remove imidazole using a desalting column. After elution (or buffer exchange as appropriate), the concentration of the purified protein was adjusted to 0.2 mg / ml and the solution was then sterile filtered using a 96-well filter plate.

[0156] Reference Example 2 - Activity assay in bacterial suspension material: 1. Hardy Broth, autoclaved at 121°C for 20 minutes: 12.0 g pancreatic digest of casein (SigmaAldrich, cat. no. 70172-100G) 10.0 g Proteose peptone (SigmaAldrich, cat. no. 82450-100G) 5.0 g peptic digest of animal tissue (SigmaAldrich, cat. no. 70174-100G) 5.0 g sodium chloride (Carl Roth, cat. no. 3957.1) 3.0 g beef extract (SigmaAldrich, cat. no. B4888-50G) 3.0 g yeast extract (SigmaAldrich, cat. no. Y1625-250G) 1.0 g soluble starch (Sigma Aldrich, cat. no. S9765-250G) Up to 1 liter of deionized H2O (produced in a PhagoMed Lab with a Millipore RiOs Essential16) 2. Hardy Broth Agar, autoclaved at 121°C for 20 minutes: Same as Hardy Broth but contains 15g of Agar Bacteriogical (OXOID Cat# LP0011) 3. Hardy Broth Top Agar, autoclaved at 121°C for 20 minutes: Same as Hardy Broth but contains 7g of Agar Bacteriogical (OXOID Cat# LP0011) 4. NYC-III medium, pH 5.0, autoclaved at 121°C for 20 minutes, then supplemented with horse serum (NYC-III-HS-5.0) ·12g of HEPES (Sigma Life Science, cat. no.H4034-100G) 7.5 g Proteose Peptone No. 3 (BD, cat. no. 211693) 1.9 g yeast extract (Sigma Aldrich, cat. no. Y1625-250G) 2.5 g sodium chloride (Sigma Aldrich, cat. no. S9888-1kg-M) 2.5 g glucose (MW 180.16 g / mol) (Sigma Aldrich, cat. no. G6152-1KG) Deionized water up to 450ml total volume 50 ml of Horse Serum (HS), heat inactivated (Thermo Fisher Scientific, cat. no. 26050070), added after autoclaving 5. Common materials: BD Chocolate Agar Plates for Gardnerella (BD, cat. no. 254060) BD Schaedler / 5% Sheep Blood Plates for Lactobacillus (BD, cat. no. 254042) ·Isovitalex(BD, cat. no.211876) Hardy Broth + Isovitalex (see above), adjusted to the pH indicated Hardy agar + Isovitalex (see above) Hardy top agar + Isovitalex (see above) 96-U-well plate (Sigma Aldrich, cat. no. M2311-100EA) 96 flat-bottom plates with lids (Labshop, cat. no. 44.781662) Greiner CELLSTAR® 384-well plates (Sigma-Aldrich, cat. no. M1937-32EA) Anaerogen sachets (Sigma-Aldrich, cat. no. 68061-10SACHETS-F) Anaerobic Bioindicator Test (Sigma-Aldrich, cat. no. 59886-1PAK-F) Anaerobic flask (Sigma-Aldrich, cat. no. 28029-1EA F) or plastic lunch box sealable with a rubber gasket, available from a local electronics store 6. Strains: Gardnerella strains: · Gv_1:UGent09.07 Gv_8:UGent25.49 Gv_9:ATCC14018 · Gv_10:UGent06.41 ·Gv_11:UGent09.48 · Gv_17:UGent18.01 · Gv_23: GS10234(FC2) Lactobacillus strains: L. JensenyiPB2003-013-T2-2 · L. Gasseri 020566 L. crispatus LAB117 method: Gardnerella cells were recovered from frozen stocks by plating on Chocolate agar plates (Beckton Dickinson) and incubating at 37°C for 48 hours under anaerobic conditions. For Lactobacillus, BD Schaedler / 5% sheep blood agar plates were used instead. Colonies were scraped off the plates, resuspended in Hardy broth or NYC-III-HS-5.0 at the indicated pH, and the suspension was adjusted to an OD of 0.1 (610nm or 620nm as indicated). It should be noted that two Tecan Microplate readers, each equipped with a 610nm or 620nm filter, were used interchangeably in the experiments. The exact wavelengths used were specified in each case, although there were no differences with respect to the experiments. Unless otherwise stated, 90μl of cell suspension was mixed with 10μl of endolysin solution for the various species / endolysin combinations in a 384-well plate. OD (610-620 nm as indicated) was measured at the beginning and end of the reaction, either as two measurement points or as a continuous run, with a Tecan F200 Microplate reader. Reactions were incubated at 37°C for 5 hours (or other times as indicated) under anaerobic, microaerobic or aerobic conditions as indicated. Anaerobic conditions were intended to completely remove oxygen from the container (Sigma-Aldrich anaerobic bottle or sealable lunchbox) in which the bacteria were incubated using anaerobic sachets, and the lack of oxygen was confirmed using an anaerobic indicator in the container. If microaerobic conditions were indicated, the candle-in-a-jar method was used (a tea candle was lit in a suitable sealable container, which reduced the oxygen concentration in the container until the flame was extinguished). Then, 96-U-well bottom plates were used to measure the concentration of oxygen in 5 steps (10 -1 From 10 -5Each well is diluted (up to 100 µl) and 2 µl of each dilution of each reaction mixture is plated onto BD Chocolate agar plates or BD Schaedler / 5% sheep blood agar plates for Gardnerella and Lactobacillus, respectively, to detect and quantitate surviving CFU. Detection plates are incubated at 37°C for 48 hours under anaerobic conditions.

[0157] Reference Examples 6 and 7 - MIC and MBC measurements material: Common materials: Metronidazole (Gatt-Koller, Metronidazolum mikronisiert, 10 g, 606293914) Clindamycin (Ratiopharm, 300mg / 2ml ampules 5x) Clindamycin hydrochloride (Sigma Aldrich, 10 mg, cat. no. C5269) Endolysin H2B10 [530 μg / ml] in MES buffer (50 mM MES, 100 mM NaCl, 8 mM MgSO4, pH = 5.5) BD Chocolate Agar Plates for Gardnerella (BD, cat. no. 254060) BD Schaedler / 5% Sheep Blood Plates for Lactobacillus (BD, cat. no. 254042) ·Isovitalex(BD, cat. no.211876) Hardy Broth + Isovitalex (see above), adjusted to the pH indicated NYC III+HS broth, pH 5.5 NYC-III-HS agar plates: NYC-III+HS medium as described above, but with 1.5% agar added before autoclaving. Hardy agar + Isovitalex (see above) Hardy top agar + Isovitalex (see above) 96-U-well plate (Sigma Aldrich, cat. no. M2311-100EA) 96 flat-bottom plates with lids (Labshop, cat. no. 44.781662) Greiner CELLSTAR® 384-well plates (Sigma-Aldrich, cat. no. M1937-32EA) Anaerogen sachets (Sigma-Aldrich, cat. no. 68061-10SACHETS-F) Anaerobic Bioindicator Test (Sigma-Aldrich, cat. no. 59886-1PAK-F) Anaerobic flask (Sigma-Aldrich, cat. no. 28029-1EA F) or plastic lunch box sealable with a rubber gasket, available from a local electronics store method: Bacteria were plated on BD Choc agar plates (Gardnerella) from frozen stocks and incubated for 48 hours at 37°C under anaerobic conditions. Colonies were scraped off the plates, resuspended in Hardy broth or NYC-III-HS-pH5.0, and the suspension was adjusted to an OD of 0.05 (610nm or 620nm as indicated). It should be noted that two Tecan Microplate readers, each equipped with a 610nm or 620nm filter, were used interchangeably in the experiments. The exact wavelengths used were specified in each case, although there were no differences with respect to the experiments. Antibiotics were prepared as 20x stock solutions of the respective required final concentrations. 95μl of cell suspension was mixed with 5μl of antibiotic dilution in a 384-well plate. At the start of the reaction, the OD (610-620 as indicated) was measured, and the plates were then incubated for 48 hours at 37°C under anaerobic conditions. The OD (610-620 as indicated) was then measured again for MIC determination, where the MIC was defined as the lowest concentration of antibiotic at which the OD (610-620 as indicated) did not exceed the level measured at the beginning of the experiment. After measuring the OD, 2 μl of each well was spotted onto a NYC-III+HS agar plate. The plates were then incubated for an additional 48 hours at 37°C under anaerobic conditions. After incubation, cell growth in each spot was assessed, and the MBC was defined as the lowest concentration of antibiotic at which no bacteria grew on the plate at all. The experiment was performed in triplicate for each condition. In the second round of MIC and MBC experiments with antibiotics, the Gardnerella cell suspension was adjusted to a McFarland standard of 0.5 (approximately OD(610) 0.07) and then diluted 1:75 according to CLSI (Clinical and Laboratory Standards Institute) standards. Antibiotics were prepared according to CLSI standards, and 50 μl of cell suspension was mixed with 50 μl of antibiotic in a 96-well plate. Otherwise, MIC and MBC were measured as described above. For MIC and MBC determination of the domain-swapped endolysin H2B10, 50 μl of Gardnerella cell suspension was mixed with 50 μl of H2B10-containing solution (serialized 1:1) in a 96-well plate. OD610 was measured at the beginning of the reaction, and the plate was then incubated at 37° C. for 48 h under anaerobic conditions. OD610 was then measured again for MIC determination, where the MIC was defined as the lowest concentration of H2B10 at which the OD did not or only slightly exceeded the level measured at the beginning of the experiment. [Table 12]

[0158] Sequence Listing SEQ ID NO:1 >H1 >196 aa MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTAGLDVGAYWYSYANSGFEAAEEAQSLMNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCNKLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIAQWNTHCNYQGSYGLWQYSSNGSVPGVAGRVDMDYAYVDYPSIIK SEQ ID NO:2 >H2 >196 aa MSKRGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITGFCNKLESCGYYAGFYTSLSTANNLVSAHVRNRYALWIAQWNTHCSYQGSYGLWQYSSSGSVPPGVAGRVDMDYAYVDYPSIIK SEQ ID NO:3 >H3 >196aa MSKKGIDVSVWQGDIDFNSVKASGVEVIIRAGYGIGHKWDKWFEENYRKAKTAGLDVGSYWYSYASSAGEASEEAQSCVNILSGKSFEYPIYFDLEEKSQLNRGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQWNTHCSYQGSYGLWQYSSSGSVNGIAGRVDMDYAYVDYPSVIK sequence number 4 >H4 >196aa MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTCGLDVGAYWYSYANSGFEAAEAAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCSKLETYGYYAGFYTSLSVVNNLVSAHVRDRYALWIAQWNTHCSYQGSYGLWQYSSSGSVPGVAGRVDMDYAYVDYPSIIK sequence number 5 >H5 >196 years MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTAGLDVGAYWYSYANSSSEAAEEAQSCANMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITGFSCLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIAQWNTHCSYQGSYGLWQYSSNGSVPGVAGRVDMDYAYKDYPSIIK sequence number 6 >H6 >196 years MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCSKLESCGYYAGFYTSLSTANNLVSAHVRNRYALWIAQWNTHCDYQGSYGLWQYSSSGSVPPGVAGRVDMDYAYKNYPSIIK SEQ ID NO:7 >H7 >196aa MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTAGLDVGAYWYSYANSASEAAEEAQSCANMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCSKLETYGYYAGFYTSLSTANNLVSSHVRNRYALWIAQWNTHCSYQGSYGLWQYSSSGSVPPGVAGRVDMDYAYKDYPSIIK SEQ ID NO:8 >EL8 >306 aa MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTAGLDVGAYWYSYANSSSEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCSKLETYGYYAGFYTSLSTANNLVSSHVRNRYALWIAQ WNTHCSYQGSYGLWQYSSNGSVPGVAGRVDMDYAYVDYPSIIKNAGLNGYKNGGSYTAPQTSSIDDVAREVINGAWGNGNERKQRLTQAGYDYTSVQNKVNKLLGVKACRKSVDELAREVIRGTWGNGNERKNRLTQAGYDYDTVQKRVNELL SEQ ID NO:9 >EL9 >251 aa MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRVFCDSLITSFCNKLEACGYY AGFYTSLSTANNLVSSHVRNRYALWIAQWNTHCDYQGSYGLWQYSSSGSVPPGVAGRVDMDYAYVDYPSIIKNAGLNGCKNGGSDQAARTSSIDEVAREVINGAWGNGSTRKQRLTSAGYDYASVAK SEQ ID NO:10 >H10 >196aa MSKRGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEENYRKAKTVGLDVGAYWYSYASSAGEASEEAQSCVNILSGKSFEYPVYFDLEEKSQLNRGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQWNTHCSYQGSYGLWQYSSSGSVNGIAGRVDMDYAYVDYPSVIK SEQ ID NO:11 >H11 >196aa MSKRGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEQNYRKAKTTGLDVGAYWYSYASSAGEAAEEAQSCVNILSGKSFEYPVYFDLEEKSQLNRGRDFCDSLITSFCNKLETYGYYAGFYTSLSVANNLVSSHVRDRYALWIAQWNTHCDYQGSYGLWQYSSSGSVDGIAGRVDMDYTYVDYPSVIK SEQ ID NO:12 >H12 >196 aa MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEENYRKAKTAGLDVGSYWYSYASSAGEVALEAQSCVNILSGKSFEYPVYFDLEEKSQLNRGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQWNTHCSYQGSYGLWQYSSSGSVNGIAGRVDMDYAYVDYPSVIK sequence number 13 >EL13 >306 aa MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIECKDKWFEQNYRKAKTAGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCNKLESCGYYAGFYTSLSTANNLVPAHVRNRYALWIAQ WNTHCDYQGSYGLWQYSSSGSVPGVAGRVDMDYAYVDYPSIIKNAGLNGYKNGESHQATRTTSIDEVAREVINGAWGGNNERKQRLTQAGYDYASVQNKVNELLGVKACRKSVDELAREVIRGTWGNGNERKNRLTSAGYDYDTVQKRVNELL sequence number 14 >EL14 >306 aa MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKAKTCGLDVGAYWYSYSANGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQLNRGRAFCDSLITSFCNKLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIAQWNTHCSYQGSYGLWQYSSSGSVPGVAGRVDMDYAYVDYPSIIKNAGLNGYKNGESHQATRTTSIDEVAREVINGAWGNGNERKQRLTSAGYDYASVQNKVNELLGVKACRKSVDELAREVIRGAWGNGSTRKQRLTSAGYDYDTVQKRVNELL SEQ ID NO:15 >B1_N >49 aa DQAARTSSIDEVAREVINGAWGNGSTRKQRLTSAGYDYASVQNKVNELL SEQ ID NO:16 >B1_C >49 aa GVKACRKSVDELAREVIRGAWGNGSTRKQRLAQAGYDYDTVQKRVNELL SEQ ID NO:17 >B2_N >49 aa DQAARTSSIDEVAREVINGAWGNGNERKQRLTSAGYDYASVQNKVNELL SEQ ID NO:18 >B2_C >49 aa GVKACRKSVDEIAREVIRGTWGNGSTRKQRLTQAGYDYDTVQKRVNELL SEQ ID NO:19 >B3_N >49 aa YTAPQTSSIDEVAREVINGDWGNGNDRKNRLISAGYDYASVQNKVNELL SEQ ID NO:20 >B3_C >49 aa GVKAYRKSVDELAREVIRGTWGNGSMRKHRLTQAGYDYDAVQKRVNELL SEQ ID NO:21 >B4_N >49 aa DQATRTSSIDEVAREVINGAWGNGNERKQRLTSAGYDYASVQNKVNKLL SEQ ID NO:22 >B4_C >49 aa GVKAYRKSVDELAREVIRGTWGNGNERKQRLAQAGYDYDTVQKRVNELL SEQ ID NO:23 >B5_N >49 aa DQAARTSSIDEVAREVINGAWGNGNERKQRLTQAGYDYTSVQNKVNKLL SEQ ID NO:24 >B5_C >49 aa GVKACRKSVDELAREVIRGTWGNGNERKNRLTQAGYDYDTVQKRVNELL SEQ ID NO:25 >B6_N >43 aa NQAARTSSIDDVAREVINGAWGNGNERKQRLTQAGYDYASVAK SEQ ID NO:26 >B7_N >49 aa DQAARTSSIDEVAREVINGAWGNGNERKQRLTQAGYDYTSVQNKVNKLL SEQ ID NO:27 >B7_C >49 aa GVKACRKSVDELAREVIRGTWGNGNERKNRLTQAGYDYDTVQKRVNELL SEQ ID NO:28 >B10_N >49 aa YTAPQISSIDEVAREVINGDWGNGNERKQRLTSAGYDYASVQNKVNELL SEQ ID NO:29 >B10_C >49 aa GVKAYRKSVDELAREVIRGTWGNGSTRKQRLTQAGYDYNAVQKRVNELL SEQ ID NO:30 >B11_N >49 aa YTAPQTSSIDEVAREVINGDWGNGNERKNRLTSAGYDYTSVQNKVNELL SEQ ID NO:31 >B11_C >49 aa GVKAYRKSVDELAREVIRGTWGNGSTRKQRLTQAGYDYDAVQKRVNELL SEQ ID NO:32 >B12_N >49 aa YTAPQTSSIDEVAREVINGDWGNGIERKNRLTSAGYDYTSVQNKVNELL SEQ ID NO:33 >B12_C >49 aa GVKAYRKSVDELAREVIRGTWGNGKTRKQRLTQAGYDYNAVQKRVNELL

Claims

1. below: (i) the N-terminal catalytic domain, or a functional variant thereof; (ii) a C-terminal cell wall binding region, or a functional variant thereof, wherein the C-terminal cell wall binding region comprises or consists of one or more cell wall binding domains; and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region; A recombinant endolysin comprising or consisting of: wherein the N-terminal catalytic domain is derived from a first naturally occurring endolysin, and the linker region and the C-terminal cell wall binding region are derived from a second naturally occurring endolysin, and wherein the first and second naturally occurring endolysins are encoded by different genomes derived from different prophages; and Here, an engineered endolysin with genus-selective killing activity against Gardnerella.

2. The recombinant endolysin of claim 1, wherein the N-terminal catalytic domain is a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-5, 7, or 10-12, or a variant thereof having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 1-5, 7, or 10-12, whereby the polypeptide is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella.

3. A recombinant endolysin according to claim 1 or 2, wherein the C-terminal cell wall binding region comprises or consists of one, two or three cell wall binding domains.

4. The recombinant endolysin of claim 3, wherein the one, two or three cell wall binding domains are independently selected from the group consisting of polypeptides comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, and variants thereof having at least 80% identity to the amino acid sequences of SEQ ID NOs: 15-24 and 26-33, respectively, whereby the polypeptide is functional, wherein the function comprises the ability to bind to the cell wall of Gardnerella.

5. The recombinant endolysin according to any one of claims 1 to 4, wherein the C-terminal cell wall binding region comprises or consists of a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 27, 29, 31 and 33.

6. The recombinant endolysin of claim 5, wherein the first cell wall binding domain is N-terminal to the second cell wall binding domain.

7. The linker region has the following amino acid sequence: (i) (XXX)n, where each X may independently be G, A or S, preferably where the amino acid sequence (XXX)n is (GGS)n, where n corresponds to the number of repeats of the sequence XXX, preferably where n is 2, 3, 4, 5 or 6; or (ii) X 1 X 2 GLNGX 3 X 4 NGGS {in the formula, X 1 is N or K, and X 2 is A or V, and X 3 is Y or C and X 4 is K or Q}, A recombinant endolysin according to any one of claims 1 to 6, which is a polypeptide comprising or consisting of:

8. The recombinant endolysin according to any one of claims 1 to 7, wherein the endolysin has killing activity against Gardnerella vaginalis strickenorthen, Gardnerella leopoldii, Gardnerella pyoti and / or Gardnerella swidszynskii or any other species of the genus Gardnerella.

9. A recombinant endolysin according to any one of claims 1 to 8, wherein the endolysin has no killing activity against Lactobacillus, preferably wherein the endolysin has no killing activity against Lactobacillus crispatus, Lactobacillus gasseri and / or Lactobacillus jensenii.

10. A polynucleotide encoding a recombinant endolysin according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising a recombinant endolysin according to any one of claims 1 to 9 or a polynucleotide according to claim 8, and further comprising a pharma- ceutically acceptable carrier and / or diluent.

12. A recombinant endolysin according to any one of claims 1 to 9, a polynucleotide according to claim 10 or a pharmaceutical composition according to claim 11 for use in treating a disease or disorder.

13. 13. A recombinant endolysin for use according to claim 12, a polynucleotide for use according to claim 12 or a pharmaceutical composition for use according to claim 12, wherein the disease or disorder is a bacterial infection.

14. A recombinant endolysin for use according to claim 13, a polynucleotide for use according to claim 13 or a pharmaceutical composition for use according to claim 13, wherein the bacterial infection is bacterial vaginosis.

15. A recombinant endolysin for use as claimed in claim 14, a polynucleotide for use as claimed in claim 14 or a pharmaceutical composition for use as claimed in claim 14, wherein the bacterial vaginosis is caused by Gardnerella vaginalis strictus, Gardnerella leopoldii, Gardnerella pyoti, and / or Gardnerella swidsinskii.

16. A recombinant endolysin for use according to any one of claims 13 to 15, a polynucleotide for use according to any one of claims 13 to 15 or a pharmaceutical composition for use according to any one of claims 13 to 15, wherein the recombinant endolysin, polynucleotide or pharmaceutical composition is to be administered locally, preferably the recombinant endolysin, polynucleotide or pharmaceutical composition is to be administered locally in the vagina of a female subject and / or in or on the glans penis, foreskin or urethral opening of a male subject.

17. A recombinant endolysin for use according to any one of claims 13 to 15, a polynucleotide for use according to any one of claims 13 to 15 or a pharmaceutical composition for use according to any one of claims 13 to 15, wherein said recombinant endolysin, polynucleotide or pharmaceutical composition is to be administered into the vagina of a female subject and / or into or on the glans penis, prepuce or urethral opening of a male subject.

18. A recombinant endolysin for use according to any one of claims 13 to 17, a polynucleotide for use according to any one of claims 13 to 17 or a pharmaceutical composition for use according to any one of claims 13 to 17, wherein said recombinant endolysin, polynucleotide or pharmaceutical composition is to be co-administered with a compound or composition which adjusts the vaginal pH to between 4.0 and 6.

0.

19. A plasmid comprising the polynucleotide of claim 10.

20. 20. A bacterial host cell comprising the plasmid of claim 19, preferably said bacterial host cell is an E. coli cell.

21. An in vitro method for the diagnosis of a disease or condition treatable with an endolysin according to any one of claims 1 to 9, comprising the following steps: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of a C-terminal cell wall binding domain of an endolysin according to any one of claims 1 to 9 and optionally an N-terminal catalytic domain of an endolysin according to any one of claims 1 to 9, wherein the sample comprises microbial cells and wherein the C-terminal cell wall binding domain of the endolysin is optionally labelled; (ii) testing whether the polypeptide binds to and / or lyses microbial cells in the sample; and (iii) determining whether a disease or condition can be treated by an endolysin according to any one of claims 1 to 9 if the polypeptide binds to and / or lyses microbial cells; The method includes:

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