Novel gardnerella endolysin and use thereof

Abstract

The present invention relates to novel species-selective bacteriophage endolysins and their use for the treatment of bacterial vaginosis (BV). The present invention provides a recombinant endolysin, i.e., a domain-exchanged endolysin. The invention also relates to the use of said endolysin in the treatment of a disease or condition, such as a bacterial infection, in particular BV. The present invention further relates to polynucleotides encoding said endolysin. The polynucleotides may also be used to treat such diseases or disorders. The invention also provides pharmaceutical compositions comprising the endolysin of the invention for the treatment of such diseases or conditions. The endolysin, polynucleotide and pharmaceutical composition may be administered topically, in particular in the vagina.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080033977.7 (International Application No. PCT / EP2020 / 062645) with the filing date of May 7, 2020 and the invention title of "Novel Gardnerella endolysin and its uses".

[0002] The present invention relates to novel species-selective bacteriophage endolysins and their use in the treatment of bacterial vaginosis (BV). The present invention provides recombinant endolysins, namely domain-swapped endolysins. The present invention also relates to the use of said endolysins for the treatment of diseases or disorders such as bacterial infections, particularly BV. The present invention further relates to polynucleotides encoding said endolysins. Said polynucleotides can also be used for the treatment of such diseases or disorders. The present invention also provides pharmaceutical compositions comprising the endolysins of the present invention for the treatment of such diseases or disorders. The endolysins, polynucleotides and pharmaceutical compositions can be administered locally, particularly locally to the vagina.

[0003] Bacterial vaginosis (BV), also known in the literature as bacterial vaginitis, nonspecific vaginitis and nonspecific vaginosis, is the most common vaginal infection worldwide and is associated with serious adverse consequences, including preterm birth, postpartum endometritis and increased risk of HIV infection. This is a vaginal dysbiosis in which the symbiotic Lactobacilli are replaced by a polymicrobial biofilm, the pH increases from the natural 3.5 - 4.5 to 5.5 and a foul-smelling fluid is formed. Depending on the population studied, reported prevalence ranges from 10 - 40%. However, suboptimal diagnostic methods and a high proportion of asymptomatic patients make it difficult to determine the true prevalence of BV. Gardnerella vaginalis (G. vaginalis) is a bacterial species associated with BV.

[0004] The pathogenesis of BV is still poorly understood. It is generally defined as a pathological state characterized by the loss of the normal vaginal flora (especially Lactobacillus producing H 2 O 2 ) and the overgrowth of anaerobic bacteria, including Gardnerella vaginalis, Mobiluncus and Mycoplasma hominis. However, recent data suggest a major role for Gardnerella vaginalis as a specific and sexually transmitted pathogen in BV (Muzny et al., 2016, J. of Infect. Dis. 214 Suppl. 1., S1).

[0005] In the 1950s, large numbers of small, pleomorphic Gram-variable bacilli were observed in the genital tracts of women with BV. This organism was initially called Haemophilus vaginalis and was repeatedly renamed as more information about its characteristics became available. It is now classified as Gardnerella vaginalis, which until 2018 was considered the only member of the genus Gardnerella. However, in early 2019, it was shown that the genus Gardnerella actually contains at least 13 species, and the most common species were renamed G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661).

[0006] Bacteria of the genus Gardnerella are unusual in that they are Gram-variable, i.e., they do not form the outer membrane that defines Gram-negative bacterial species. The cell wall is usually very thin, and the peptidoglycan content is only 10% or less, which is why the crystal violet dye used for Gram staining does not always produce the deep purple typical of Gram-positive species. More precisely, Gardnerella cells can appear both Gram-positive and Gram-negative in a Gram stain. Phylogenetic analysis based on 16S rRNA places Gardnerella within the Gram-positive order Bifidobacteriales.

[0007] During BV, the epithelial surface is covered with dense biofilms of Gardnerella vaginalis that are often recalcitrant to treatment. Biofilms are microbial adhesion communities bound together by a polymeric matrix composed of polysaccharides, proteins, and / or nucleic acids. Unique gene expression patterns, as well as the physical structure of the biofilm, increase bacterial resistance to many negative stimuli, including chemical disinfectants, extreme pH values, host immune defenses, and antibiotics. The standard treatments for BV are the antibiotics metronidazole and clindamycin, but these often fail to eradicate the biofilm, resulting in a recurrence rate of up to 60% within 6 months. In addition, antibiotic treatment clears the vaginal microbiota, although some residual viable biofilm remains, opening up this ecological niche for other pathogens (e.g., fungi). Thus, a common consequence of BV treatment is candidiasis. Probiotics have also been tried to treat BV, particularly using beneficial Lactobacilli to recolonize the vagina. However, several clinical trials have failed to show a benefit.

[0008] Accordingly, there is a great need for new methods and compositions for treating Gardnerella vaginalis infections, particularly BV, e.g., by selectively killing bacterial cells of the genus Gardnerella, preferably without harming beneficial Lactobacilli while they recolonize the vagina. Thus, the fundamental technical problem of the present invention is to provide new means and methods for treating BV.

[0009] This technical problem is solved by providing the embodiments characterized in the claims.

[0010] The present invention is based on the preparation of novel recombinant Gardnerella prophage endolysins having unexpected properties and structures that render them particularly suitable for various uses and methods, especially for treating, purifying or detecting bacterial infections and disorders, particularly those related to Gardnerella.

[0011] A first aspect of the present invention provides an endolysin comprising or consisting of

[0012] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0013] (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

[0014] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0015] wherein the endolysin has killing activity against Gardnerella.

[0016] In one aspect of the present invention, the N-terminal catalytic domain is from a first native endolysin, the linker region and the C-terminal cell wall binding region are from a second native endolysin, and the first and second native endolysins are encoded by different genomes from different prophages. Thus, the present invention provides a recombinant endolysin comprising or consisting of

[0017] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0018] (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

[0019] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0020] wherein the N-terminal catalytic domain is from a first native endolysin, the linker region and the C-terminal cell wall binding domain are from a second native endolysin, and the first and second native endolysins are encoded by different genomes from different prophages, and

[0021] wherein the recombinant endolysin has bactericidal activity against Gardnerella bacteria.

[0022] Gardnerella bacteria are special in that they are Gram-variable species: they do not form an outer membrane that defines true Gram-negative species. The cell wall is usually very thin, and the peptidoglycan content is only 10% or less. This indicates that peptidoglycan-degrading enzymes, such as endolysin proteins, cannot effectively lyse the bacterial cell wall of Gardnerella bacteria. However, in the case of the present invention, new recombinant endolysins have been identified that have the advantageous property of effectively killing Gardnerella species and can therefore be used as a new therapy for treating BV.

[0023] Three major Lactobacillus species predominate in a healthy vagina: Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. They maintain an acidic pH of 3.5 - 4.5 by producing lactic acid and maintain a protective oxidative environment by producing H 2 O 2 Recovery from BV is accompanied by the restoration of the population of these Lactobacillus in the vagina. However, the drawback of antibiotics (routinely used to treat BV) is that they interfere with the process of restoration of the Lactobacillus population in the vagina. In contrast, the new recombinant endolysin of the present invention advantageously has species-selective bactericidal activity against Gardnerella bacteria and does not harm Lactobacillus. In addition, the attached examples show that all tested Gardnerella strains are less sensitive to metronidazole and clindamycin, which are routinely used to treat BV. This can explain the high recurrence rate of BV. This also demonstrates that the endolysin of the present invention is superior to antibiotics in treating BV. Therefore, treatment of BV with the endolysin of the present invention is far superior to currently available treatments, such as treatment with the antibiotics metronidazole and clindamycin.

[0024] As used herein, the term "from a first native lysin" refers to a moiety (i.e., the N-terminal catalytic domain) that is identical to or a functional variant of a first native lysin. 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 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 the respective moiety (i.e., the N-terminal catalytic domain) of the first native lysin and that produces a functional lysin, wherein the function includes killing activity against Gardnerella bacteria. The amino acid sequences of several native lysins are provided below and summarized in Table 7.

[0025] Accordingly, the term "from a second native lysin" refers to a moiety (i.e., the linker region and the C-terminal cell wall binding domain) that is identical to or a functional variant of a second native lysin, which is a lysin different from the first native lysin. 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 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 the respective moiety (i.e., the linker region and the C-terminal cell wall binding domain) of the second native lysin and that produces a functional lysin, wherein the function includes killing activity against Gardnerella bacteria.

[0026] As used 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 lysin (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 domains together represent the so-called "B-region". 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, and so on.

[0027] The present invention further provides a lysin comprising or consisting of

[0028] (i) An N-terminal catalytic domain composed of a polypeptide, said polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 1 to 5, 7 or 10 to 12, or a functional variant thereof having at least 80% identity with the amino acid sequence of any one of SEQ ID NO: 1 to 5, 7 or 10 to 12;

[0029] (ii) A C-terminal cell wall binding region comprising or consisting of at least one cell wall binding domain, said at least one cell wall binding domain independently selected from polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 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, 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 any one of SEQ ID NO: 15 to 24 and 26 to 33, respectively; and

[0030] (iii) A linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0031] wherein the endolysin has killing activity against Gardnerella bacteria.

[0032] As shown in the appended claims, the most active N-terminal catalytic domain (also referred to as the "H-domain") is H2 (SEQ ID NO: 2), followed by H7 (SEQ ID NO: 7), H10 (SEQ ID NO: 10) and H5 (SEQ ID NO: 5).

[0033] Thus, in a preferred aspect of the present invention, the N-terminal catalytic domain is composed of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 2, 7, 10 and 5, 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, 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 any one of SEQ ID NO: 2, 7, 10 and 5;

[0034] Thus, the endolysin is functional, and the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0035] Thus, in a preferred aspect of the present invention, the N-terminal catalytic domain consists of a polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO:5, 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, 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 SEQ ID NO:5; or more preferably comprises or consists of the amino acid sequence of SEQ ID NO:10, 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, 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 SEQ ID NO:10; or even more preferably comprises or consists of the amino acid sequence of SEQ ID NO:7, 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, 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 SEQ ID NO:7; or even more preferably comprises or consists 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, 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 SEQ ID NO:2;

[0036] Thus, the endolysin is functional, and the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0037] In the attached embodiments, it is also shown that B-region B10 (containing the cell wall binding domains of SEQ ID NO: 28 and 29) is the most active, followed by B11 (containing the cell wall binding domains of SEQ ID NO: 30 and 31), B12 (containing the cell wall domains of SEQ ID NO: 32 and 33), and B3 (containing the cell wall domains of SEQ ID NO: 19 and 20).

[0038] Thus, in a preferred aspect of the present invention, the cell wall binding domain is selected from polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 19, 20, and 28 - 33, or having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, 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 any one of SEQ ID NO: 19, 20, and 28 - 33; any functional variant thereof;

[0039] Thus, the endolysin is functional, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0040] The endolysin of the present invention preferably comprises two cell wall binding domains. In one aspect of the present invention, the cell wall binding domain (B-domain) of the endolysin of the present invention consists of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 19, 20, and 28 - 33, or having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, 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 any one of SEQ ID NO: 19, 20, and 28 - 33; any functional variant thereof;

[0041] Thus, the endolysin is functional, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0042] In a preferred aspect of the present invention, the endolysin comprises a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from SEQ ID NO: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and the second cell wall binding domain is selected from SEQ ID NO: 16, 18, 20, 22, 24, 27, 29, 31 and 33. Preferably, the first cell wall binding domain is at the N-terminus of the second cell wall binding domain.

[0043] In a more preferred aspect of the present invention, the endolysin comprises the native endolysin EL10 (SEQ ID NO: 28 and 29), the native endolysin EL11 (SEQ ID NO: 30 and 31), the native endolysin EL12 (SEQ ID NO: 32 and 33) or the native endolysin EL3 (SEQ ID NO: 19 and 20), even more preferably the two cell wall binding domains (B-domains) of the native endolysin EL10 (SEQ ID NO: 28 and 29); or a functional variant thereof. The functional variant may also be a set of two B-domains that has at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, 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 cell wall binding domains (B-domains) of the native endolysin EL10 (SEQ ID NO: 28 and 29), the native endolysin EL11 (SEQ ID NO: 30 and 31), the native endolysin EL12 (SEQ ID NO: 32 and 33) or the native endolysin EL3 (SEQ ID NO: 19 and 20), even more preferably the two cell wall binding domains (B-domains) of the native endolysin EL10 (SEQ ID NO: 28 and 29);

[0044] Thereby the endolysin is functional, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0045] In one aspect of the present invention, the cell wall binding domain (B-domain) comprises or consists of: SEQ ID NO:19 and / or 20, 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, 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 comprises or consists of: SEQ ID NO:32 and / or 33, 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, 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:32 and / or 33; even more preferably comprises or consists of: SEQ ID NO:30 and / or 31, 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, 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:30 and / or 31; or even more preferably comprises or consists 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, 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;

[0046] Thus, the endolysin is functional, and the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0047] The preferred sequences VNELL or VNKLL, more preferably VNELL, are located at the C-terminus of the B-domain. In cases where multiple B-domains are present within the B-region, the sequences VNELL or VNKLL, more preferably VNELL, are also preferably located at the C-terminus of each B-domain.

[0048] Surprisingly and unexpectedly, it has been found that, within the context of the present invention, several recombinant endolysins have stronger activity than native endolysins, particularly when observed in all 4 Gardnerella strains tested (i.e., Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii). In particular, H2B10, H2B11, H2B12, and H7B3 are each more active than all of the native endolysins tested. Thus, the recombinant endolysins according to the present invention surprisingly exhibit higher activity than native endolysins.

[0049] Thus, preferably, the "Gardnerella-killing activity" of the recombinant endolysins of the present invention is enhanced as compared to the killing activity of native endolysins (e.g., native endolysins EL1-EL12 having the amino acid sequences shown in Table 7).

[0050] Based on the quite high activity of endolysins H2B10, H2B11, H2B12, and H7B3, these endolysins (or their functional variants) are preferred in the present invention. Thus, the endolysins of the present invention preferably:

[0051] (i) an N-terminal catalytic domain composed of a polypeptide that comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 7, 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, 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 SEQ ID NO: 2;

[0052] (ii) C-terminal cell wall binding region, comprising at least one (preferably two) cell wall binding domains or consisting of at least one (preferably two) cell wall binding domains, said cell wall binding domains independently selected from or consisting of polypeptides comprising the amino acid sequence of any one of SEQ ID NO: 19, 20, and 28 to 33, 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, 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 NO: 19, 20, and 28 to 33; and

[0053] (iii) A linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region, consisting of a polypeptide comprising the amino acid sequence X 1 X 2 GLNGX 3 X 4 NGGS or consisting of the amino acid sequence X 1 X 2 GLNGX 3 X 4 NGGS, where X 1 is N or K, preferably N, X 2 is A, X 3 is Y and X 4 is K or Q,

[0054] wherein the endolysin has bactericidal activity against Gardnerella. Regarding the linker region, as described below, it is pointed out that the linker region may also consist of a polypeptide comprising the amino acid sequence (XXX) n or consisting of the amino acid sequence (XXX) n wherein each X is independently G, A, or S, preferably wherein the amino acid sequence (XXX) n is (GGS) n where n corresponds to the number of repetitions of the sequence XXX, preferably wherein n is 2, 3, 4, 5, or 6.

[0055] In the attached examples, the recombinant endolysin H2B10 showed the highest activity. Therefore, in the present invention, the most preferred endolysin of the present invention is H2B10 (or its functional variant). Therefore, the endolysin of the present invention is most preferably:

[0056] (i) An N-terminal catalytic domain composed of a polypeptide, said 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, 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 SEQ ID NO:2;

[0057] (ii) A C-terminal cell wall binding region, comprising or consisting of two cell wall binding domains, said cell wall binding domains being composed of polypeptides, said polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO:28 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, 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 SEQ ID NO:28 or 29; and

[0058] (iii) A linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region, composed of a polypeptide, said polypeptide comprising the amino acid sequence X 1 X 2 GLNGX 3 X 4 NGGS or consisting of the amino acid sequence X 1 X 2 GLNGX 3 X 4 NGGS, where X 1 is N, X 2 is A, X 3 is Y and X 4 is K,

[0059] wherein the endolysin has killing activity against Gardnerella bacteria. As described above, preferably compared with a native endolysin, such as native endolysins EL1-EL12 (having the amino acid sequences shown in Table 7), the "killing activity against Gardnerella bacteria" of the recombinant endolysin of the present invention is enhanced.

[0060] Regarding the linker region, as described below, it is pointed out that the linker region may also be composed of a polypeptide, said polypeptide comprising the amino acid sequence (XXX) n or consisting of the amino acid sequence (XXX)n comprises, wherein each X is independently 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.

[0061] In the recombinant endolysin of the present invention, the C-terminal cell wall binding region may comprise one, two or three cell wall binding domains or consist of one, two or three cell wall binding domains. The one, two or three cell wall binding domains may independently be selected from or consist of polypeptides comprising, respectively, the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, and, respectively, any 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, 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 to 24 and 26 to 33, whereby the polypeptide is functional, wherein the function includes the ability to bind to the cell wall of Gardnerella. Preferably, the C-terminal cell wall binding region consists of two cell wall binding domains. The preferred C-terminal cell wall binding region is as defined in context.

[0062] The endolysin of the present 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.

[0063] 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 the following amino acid sequence or consisting of the following amino acid sequence: (i) (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, or (ii) X 1 X 2 GLNGX 3 X 4 NGGS, wherein X 1 is N or K, X 2 is A or V, X 3 is Y or C and X 4is K or Q. As described above, in one aspect of the lysin of the present invention, the N-terminal catalytic domain is the same as or derived from a first native lysin, the linker region and the C-terminal cell wall binding domain are the same as or derived from a second native lysin, and the first and second native lysins are encoded by different genomes from different bacteriophages.

[0064] The recombinant lysin of the present invention has bactericidal activity against Gardnerella bacteria. For example, the lysin of the present invention can have bactericidal activity against Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii, preferably against all of them. The bactericidal activity of the lysin of the present invention against Gardnerella bacteria as described above is preferably genus-selective bactericidal activity against Gardnerella bacteria. As used herein, "genus-selective bactericidal activity against Gardnerella bacteria" means that the lysin of the present invention does not have bactericidal activity against general bacteria. Preferably, the lysin of the present invention has bactericidal activity against Gardnerella bacteria but not against Lactobacilli. In particular, it is preferred that the lysin does not have bactericidal activity against Lactobacilli crispatus, Lactobacilli gasseri, and / or Lactobacilli jensenii. More preferably, the lysin does not have bactericidal activity against all of these Lactobacilli, namely Lactobacilli crispatus, Lactobacilli gasseri, and Lactobacilli jensenii.

[0065] The present invention also relates to a polynucleotide molecule encoding the lysin as described above. The nucleic acid molecule can be DNA, for example, cDNA or RNA. As used herein, the terms "polynucleotide" or "polynucleotide molecule" are used synonymously with the term "nucleic acid molecule".

[0066] The present invention also relates to a vector containing the polynucleotide molecule of the present invention. In one embodiment, the vector is an expression vector. Any suitable vector known in the art can be used, such as vectors of the pET series and all T7-based vectors. For example, the vector can be a plasmid. Thus, one aspect of the present invention relates to a plasmid containing the polynucleotide of the present invention. Those skilled in the art will recognize that the choice of the expression vector can be determined by the choice of the host cell.

[0067] The present invention also provides a host cell comprising a polynucleotide according to the present invention or a vector / plasmid according to the present invention. In one embodiment, the host cell is a microbial cell, for example, a bacterial cell. Preferably, the host cell is non-pathogenic. Most preferably, the host cell is Escherichia coli. Thus, one aspect of the present invention relates to a bacterial host cell comprising a plasmid of the present invention, preferably wherein the bacterial host cell is an Escherichia coli cell.

[0068] The present invention also encompasses a method for producing an endolysin of the present invention, comprising culturing a population of host cells comprising a polynucleotide molecule according to the present invention or a vector / plasmid according to the present invention under conditions in which the endolysin is expressed.

[0069] Another aspect of the present invention provides a pharmaceutical composition comprising

[0070] (a) an endolysin according to the present invention;

[0071] (b) a polynucleotide molecule according to the present invention;

[0072] (c) a vector / plasmid according to the present invention;

[0073] (d) a host according to the present invention; and / or

[0074] (e) a bacteriophage capable of expressing an endolysin according to the present invention

[0075] and a pharmaceutically acceptable carrier, diluent or excipient. For example, the pharmaceutical composition of the present invention may comprise an endolysin of the present invention, a polynucleotide molecule of the present invention and a pharmaceutically acceptable carrier and / or diluent.

[0076] Another aspect of the present invention relates to

[0077] (a) an endolysin according to the present invention;

[0078] (b) a polynucleotide molecule according to the present invention;

[0079] (c) a vector / plasmid according to the present invention;

[0080] (d) a host according to the present invention;

[0081] (e) a bacteriophage capable of expressing an endolysin according to the present invention; and / or

[0082] (f) a pharmaceutical composition according to the present invention

[0083] For treating a disease or disorder. For example, the present invention provides an endolysin according to the present invention, a polynucleotide molecule according to the present invention, or a pharmaceutical composition according to the present invention for 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 sensustricto, Gardnerella leopoldii, Gardnerella piotii, and / or Gardnerella swidsinskii.

[0084] In one aspect of the present invention, the recombinant endolysin of the present invention, the polynucleotide molecule of the present invention, or the pharmaceutical composition of the present invention can be administered locally, preferably locally into the vagina of a subject. Thus, in one aspect of the present invention, the recombinant endolysin of the present invention, the polynucleotide or the pharmaceutical composition of the present invention is administered into the vagina of a subject.

[0085] The appended examples show that the activity of the recombinant endolysin of the present invention is particularly high at a pH of about 5. Thus, one aspect of the present invention relates to the recombinant endolysin of the present invention, the polynucleotide molecule of the present invention, or the pharmaceutical composition of the present invention, wherein the recombinant endolysin, polynucleotide, or pharmaceutical composition is co-administered with a compound or composition that adjusts the pH of the vagina to 4.0 - 6.0, preferably to 4.5 - 5.5, more preferably to about 5. Suitable compounds or compositions for adjusting the vaginal pH include, but are not limited to, phosphates, lactic acid (e.g., a natural acidifying substance secreted by lactobacilli to establish an acidic environment), or other organic acids, e.g., carboxyl-substituted polymers.

[0086] Another aspect of the present invention relates to

[0087] (a) the endolysin of the present invention;

[0088] (b) the polynucleotide molecule of the present invention;

[0089] (c) the vector / plasmid of the present invention;

[0090] (d) the host of the present invention;

[0091] (e) a bacteriophage capable of expressing the endolysin of the present invention; and / or

[0092] (f) the pharmaceutical composition of the present invention

[0093] for use as a medicament.

[0094] Another aspect of the present invention relates to the use of the following substances in the manufacture of a medicament for treating bacterial infections and disorders

[0095] (a) the endolysin of the present invention;

[0096] (b) The polynucleotide molecule of the present invention;

[0097] (c) The vector / plasmid of the present invention;

[0098] (d) The host of the present invention;

[0099] (e) A phage capable of expressing the polypeptide of the present invention; and / or

[0100] (f) The pharmaceutical composition of the present invention.

[0101] Another aspect of the present invention provides a method for treating bacterial infections and conditions (such as BV), comprising administering a therapeutically effective amount of the following substances to a subject in need thereof

[0102] (a) The endolysin of the present invention;

[0103] (b) The polynucleotide molecule of the present invention;

[0104] (c) The vector / plasmid of the present invention;

[0105] (d) The host of the present invention;

[0106] (e) A phage capable of expressing the polypeptide of the present invention; and / or

[0107] (f) The pharmaceutical composition of the present invention.

[0108] In some embodiments, the therapeutically effective amount is a dose of 10 to 100 μg of endolysin, optionally administered several times a day.

[0109] Another aspect of the present invention provides a kit comprising an endolysin as described herein and instructions for use, particularly for treating a disease or condition, preferably BV as defined above. The kit may also comprise a compound or composition for adjusting the vaginal pH to 4.0 - 6.0, preferably to 4.5 - 5.5, more preferably to about 5. The definitions and preferred aspects defined herein for the endolysin of the present invention apply mutatis mutandis to the polynucleotide molecule, vector / plasmid, host cell, pharmaceutical composition, treatment method and kit of the present invention.

[0110] Another aspect of the present invention provides an in vitro method for diagnosing a disease or condition that can be treated with an endolysin according to the present invention, the method comprising the steps:

[0111] (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of the C-terminal cell wall binding region of an endolysin according to the invention and optionally the 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 labeled;

[0112] (ii) testing whether the polypeptide binds to and / or lyses the microbial cells of the sample; and

[0113] (iii) if the polypeptide binds to and / or lyses the microbial cells, determining that the disease or disorder can be treated with an endolysin according to the invention.

[0114] The microbial cells can be Gardnerella cells, preferably cells of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or other species of the genus Gardnerella.

[0115] Other features and advantages of the invention will be apparent from the following detailed description. Brief Description of the Drawings

[0117] Figure 1 A sequence alignment (CLUSTALO (1.2.4) multiple sequence alignment) of the native Gardnerella prophage endolysins of the present disclosure is shown. Most endolysins have 306 residues, except for two that have 251 residues.

[0118] Figure 2 A phylogenetic tree of the native Gardnerella prophage endolysins of the present disclosure is shown. There are no identical pairs among the endolysins, even though they are highly homologous.

[0119] Figure 3 The domain structure of the Gardnerella prophage endolysins of the present disclosure determined using InterPro (Mitchell et al., 2019, Nucleic Acids Res. 47, D351-D360) is shown. Due to its homology to glycoside hydrolase family 25, the N-terminal portion of 196 residues of the endolysin was determined to be the catalytic domain. Following the catalytic domain is a linker region and two domains that were determined to be two cell binding domains due to their homology to the C-terminal domain (CW_7 domain) of lysozyme Cpl-7. According to the nomenclature of the present application, the catalytic domain is designated as the hydrolase or "H-domain", while the linker region and the cell wall binding domains together are designated as the binding or "B-region".

[0120] Figures 4A to 4CThree enzyme activity assays are shown, in which the enzyme activity of the native Gardnerella phage endolysin of the present disclosure is measured by detecting the turbidity change of Gardnerella cell suspensions. In Figure 4A the enzyme activity of the endolysin was measured by detecting the turbidity change of the G. leopoldii strain Gv_10 suspension at pH 6.0. In Figure 4B the enzyme activity of the endolysin was measured by detecting the turbidity change of the G. piotii strain Gv_17 suspension at pH 7.0. In Figure 4C the enzyme activity of the endolysin was measured by detecting the turbidity change of the G. swidsinskii strain Gv_23 suspension at pH 7.4. Treatments were carried out in a medium adjusted to the appropriate pH value and relative to the buffer in a photometric cuvette. Then, the change in turbidity was evaluated by measuring the optical density (OD) at 600 nm. Therefore, the turbidity of the endolysin-treated group decreased more significantly compared to the buffer, indicating enzyme activity.

[0121] Figure 5 A quantitative reduction in Colony Forming Units (CFU) assay is shown, which compares untreated cells of G. vaginalis sensu stricto strain Gv_9 incubated in media with or without imidazole at different pH values. 5 x 10 7 CFU / ml cells were incubated for 5 hours under anaerobic conditions at 37 °C under the conditions shown in the figure below, and then the surviving CFU / ml was determined by quantitative plating. The results showed that under the test conditions, the survival of Gardnerella vaginalis Gv_9 was highly dependent on the absence of imidazole and low pH values.

[0122] Figure 6 A quantitative reduction in Colony Forming Units (CFU) assay is shown, comparing cells of G. vaginalis sensu stricto strain Gv_9 treated with an eluate containing recombinant endolysin H10B1 and 250 mM imidazole at different pH values or treated with a control containing 250 mM imidazole at different pH values. 5 x 10 7 CFU / ml cells were incubated for 5 hours under anaerobic conditions at 37 °C under the conditions shown in the figure below, and then the surviving CFU / ml was determined by quantitative plating. The column labeled imidazole control depicts the same data as in Figure 5 The results showed that the enzyme activity of H10B1 (as an example of all endolysins of the present invention) was higher at low pH values, with the strongest activity shown at pH 5.5 and pH 5.0.

[0123] Figures 7A to 7DShows four quantitative decreases in the colony-forming unit (CFU) assay that measures the killing activity of the native and recombinant Gardnerella phage endolysins of the present disclosure against four major Gardnerella species. In Figure 7A , 7B , 7C, and 7D, the killing activity of the endolysin was measured by detecting viable CFUs in suspensions of G. vaginalis sensu strict strain Gv_9, G. leopoldii strain Gv_11, G. piotii strain Gv_17, and G. swidsinskii strain Gv_23, respectively. 90 ul of the indicated strain at 5e7 CFU / ml was incubated with 10 ul of the endolysin solution for 5 hours under anaerobic conditions at pH 5.0 (if possible, adjusted to a concentration of 0.2 mg / ml, see Table 4). The logarithmic Y-axis represents the count of viable cells. The dashed line represents the limit of detection (LOD) obtained by plating 2 ul of the reaction mixture (500 CFU / ml). The results show that the endolysins of the present invention have the ability to lyse four major Gardnerella species. The results also indicate that some of the recombinant endolysins of the present invention have higher killing activity than the native endolysins of the present invention.

[0124] Figure 8 Shows a phylogenetic tree (amino acid level) of the H-domain formed using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539).

[0125] Figure 9 Shows a phylogenetic tree (amino acid level) of the B-region formed using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539).

[0126] Figure 10 Shows a sequence alignment of the cell wall binding domain (also known as the B-domain) within the B-region of the native endolysin of the present 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 denoted with the _N suffix (Bx_N), and the C-terminal cell wall binding domain is denoted with the _C suffix (Bx_C). For example, B3_C represents the second (C-terminal) B-domain of B3.

[0127] Figure 11 Shows a phylogenetic tree of individual B-domains using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539).

[0128] Figure 12Shows three quantitative decreases in the viable colony-forming unit (CFU) assay that measures 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. The results indicate that the endolysin of the present invention is ineffective against beneficial Lactobacillus strains.

[0129] Figure 13 Shows the MIC microbroth dilution activity assay measuring the effect of metronidazole and clindamycin (obtained as an injectable solution from Ratiopharm, 300 mg / 2 ml) on the growth of suspensions of four major Gardnerella species. Gardnerella suspensions at 2.5x10 7 CFU / ml were incubated with the antibiotic concentrations shown on the x-axis of each figure and incubated anaerobically at 37 °C for 48 h. Before and after incubation, cell growth was evaluated by measuring the optical density at 610 nm (OD(610)) to determine the minimum inhibitory concentration (MIC). The results indicate that all Gardnerella strains are resistant to metronidazole and clindamycin (obtained as an injectable solution from Ratiopharm, 300 mg / 2 ml), with MICs of 64 to <128 μg / ml and 16 μg / ml, respectively ( Figure 13 ).

[0130] Figure 14 Shows the MIC microbroth dilution activity assay measuring the effect of metronidazole and clindamycin hydrochloride (obtained from Sigma-Aldrich) on 1x10 5 -1x10 6 CFU / ml Gardnerella suspensions. This time the results indicate that the MIC of metronidazole for all tested Gardnerella strains is between 8 and 128 μg / ml, while clindamycin hydrochloride powder (obtained from Sigma-Aldrich (C5269-10MG)) shows MICs between 0.25 and 5 μg / ml.

[0131] Figure 15 Shows the MIC microbroth dilution activity assay measuring the effect of H2B10 (a representative of the domain-exchanged endolysin described herein) on the growth of three major Gardnerella species. Gardnerella suspensions at 1x10 5 -1x10 6 CFU / ml were incubated with H2B10 at the concentrations shown on the x-axis of each figure and incubated anaerobically at 37 °C for 48 h. Before and after incubation, cell growth was evaluated by OD(610) measurement to determine the minimum inhibitory concentration (MIC). MIC values between 1 and 4 μg / ml were obtained, indicating that all Gardnerella strains are highly sensitive to the domain-exchanged endolysin H2B10.

[0132] Detailed description

[0133] Definitions

[0134] The term "lysine" refers to a cell wall-lysing enzyme encoded by a bacteriophage (endolysin) or a bacterium (autolysin), which can hydrolyze the cell wall of a target bacterium when added exogenously (lysis-from-without). Compared with classical antibiotics, this new type of antibacterial agent has important advantages. For example, a new mode of action; a narrow range of susceptible bacteria; rapid killing of stationary and exponentially growing bacteria; activity against mucous membranes and bacterial biofilms; a low probability of developing resistance; and a reduced impact on the normal microbiota. These unique properties have increased the interest in the biotechnological and pharmacological development of lysins and have included them among the current preferred alternatives to combat antibiotic resistance. Lysins from Gram-positive bacteria and their bacteriophages 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 bacteriophages contain only catalytic domains, although modular endolysins have also been reported. The catalytic unit determines the type of peptidoglycan (PG) bond to be cleaved, while the cell wall-binding domain mainly determines the lysis spectrum through specific recognition of cell wall elements distributed in a genus- or species / strain-specific manner.

[0135] In the context of the present disclosure, the term "native endolysin" refers to an endolysin encoded by a prophage sequence within the bacterial genome, particularly within the Gardnerella cell genome. In the context of the present disclosure, the term "native endolysin" thus refers to an endolysin without domain swapping. The native endolysin can be unmodified, which means that the amino acid sequence of the endolysin corresponds to the native sequence. Alternatively, the native endolysin can be modified, which means that the amino acid sequence of the endolysin contains at least one mutation compared with the native sequence. The amino acid sequences of native endolysins E1-E14 are shown in Table 7 below. The NCBI accession number WP_014554482 (version WP_014554482.1 of May 27, 2013) also provides an example of the sequence of a known 1,4-β-N-acetylmuramidase (native endolysin EL1).

[0136] In the context of the present disclosure, the term "recombinant lysin" refers to a lysin that has been domain-swapped. In the context of the present disclosure, the term "domain-swapped lysin" refers to a lysin having an N-terminal catalytic domain from a first native lysin and at least one cell wall binding domain from a second native lysin, wherein the first and second native lysins are encoded by different genomes from different prophages. The recombinant lysin of the present invention may comprise or consist of the following: an N-terminal catalytic domain from a first native lysin and two cell wall binding domains from a second native lysin, wherein the first and second native lysins are encoded by different genomes from different prophages. Alternatively, the recombinant lysin of the present invention may comprise or consist of the following: an N-terminal catalytic domain from a first native lysin, a first (N-terminal) cell wall binding domain from a second native lysin, and a second (C-terminal) cell wall binding domain from a third native lysin, wherein the first and second native lysins are encoded by different genomes from different prophages, and wherein the third native lysin is optionally encoded by a different genome from a prophage different from the first and second native lysins. The recombinant lysin may be unmodified, meaning that the amino acid sequence of the lysin corresponds to the native sequence of the respective domains that make up the lysin. Alternatively, the recombinant lysin may be modified, meaning that the amino acid sequence of the lysin contains at least one mutation compared to the native sequence of the respective domains that make up the lysin. According to this definition, it will be readily understood by those skilled in the art that a "domain-swapped" or "recombinant" lysin as described herein is a non-naturally occurring lysin. That is, the recombinant lysin of the present invention has been manually modified and by definition excludes native lysins, i.e., because the latter can be found naturally in nature. The appended examples provide suitable methods for producing the artificial lysins of the present invention.

[0137] The term "catalytic domain" or "enzymatic domain" refers to the portion of a protein chain that contains the region where the catalytic chemical reaction occurs. In the context of the present disclosure, the term "H-domain" refers to the portion of the lysin of the present invention that contains the catalytic domain.

[0138] In the context of the present disclosure, the term "B-region" refers to the portion of the lysin of the present invention that comprises or consists of a polypeptide having cell wall binding activity. In a preferred embodiment, the B-region comprises a linker region and one, two, or three cell wall binding domains or "B-domains" or consists of a linker region and one, two, or three cell wall binding domains.

[0139] In the context of the present invention, the term "B-domain" refers to the cell wall binding domain contained within the B-region.

[0140] In the context of the present disclosure, the term "CW_7 domain" refers to the cell wall binding domain of the protein Cpl-7, i.e., the endolysin encoded by Streptococcus pneumoniae Cp-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 consisting of 3 contiguous CW_7 domains. Each CW_7 domain consists of a 38-amino acid-long similar amino acid sequence, called the "CW_7 motif", defined by Interpro (Mitchell et al., 2019, Nucleic Acids Res. 47, D351-D360), and consists of the amino acid sequence

[0141] TVANEVIQGLWGNGQERYDSLANAGYDPQAVQD KVNEXL, 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 linkers of 9 residues between CW_7 motif No:1 and No:2 and between CW motif No:2 and No:3, so that the total repeat sequence is 47 residues long. In contrast, the repeat sequence of the native endolysin of the present invention is 49 residues long.

[0142] The term "minimum inhibitory concentration" or "MIC" refers to the lowest concentration of a chemical substance (usually a drug) that prevents visible growth of bacteria. The MIC is defined in the present application as the minimum concentration of an antibiotic that shows no detectable growth after 48 h as measured by OD.

[0143] The term "minimum bactericidal concentration" or "MBC" refers to the lowest concentration of an antimicrobial agent required to kill a specific bacterium. Typically, the MBC90 is measured, i.e., the concentration of an antibiotic that kills 90% of the cells within a specified time, and the MBC is defined in the present application as the minimum concentration that completely eliminates a 2.5x10 7 CFU / ml suspension. While the MIC is the lowest concentration of an antimicrobial agent required to inhibit visible growth, the MBC is the lowest concentration of an antimicrobial agent that causes the death of all bacteria in the suspension.

[0144] The terms "peptide", "polypeptide", "protein" and variants of these terms refer respectively to a peptide, oligopeptide, oligomer or protein, including fusion proteins, comprising at least two amino acids linked to each other by normal or modified peptide bonds, as in the case of isopeptides. These terms also include herein "peptide mimetics", which are defined as peptide analogues containing non-peptide structural elements that are capable of mimicking or antagonizing the biological action of the native parent peptide. Peptide mimetics lack classical peptide features such as enzymatically 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 equally be composed of amino acids modified by natural processes (such as post-translational maturation processes) or chemical processes well known to those skilled in the art. Such modifications are described in sufficient detail in the literature. These modifications may occur anywhere in the polypeptide: in the peptide backbone, in the amino acid chain or even at the carboxyl or amino terminus. A peptide or polypeptide may be branched after ubiquitination or may be cyclic, with or without branches. 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, peptide or polypeptide modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation (including pegylation), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid addition, such as arginylation or ubiquitination. Such modifications are described in sufficient detail in the literature and are well known to those skilled in the art.

[0145] 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 the genus Gardnerella selected from Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii and other Gardnerella species. Bacterial infections and disorders include, but are not limited to, bacterial vaginosis (BV).

[0146] As defined herein, the "killing activity" of an endolysin against a specific bacterium refers to the reduction in the number of viable bacterial cells caused by the lytic activity of the endolysin. The killing activity of an endolysin against the bacterium can be complete, meaning that 100% of the bacterial cells are lysed, or 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 are lysed. The killing activity can be determined by measuring the decrease in the optical density of a bacterial cell suspension at 610 - 620 nm and / or the decrease in colony-forming units (CFU) per milliliter of the bacterial cell suspension after exposure to the endolysin to be tested.

[0147] As defined herein, the "binding ability" of an endolysin to the cell wall of a specific bacterium refers to the ability of the endolysin to specifically interact with and adhere to the cell wall of the bacterium. The binding ability of an endolysin to the cell wall of a bacterium can be determined by methods known in the art.

[0148] As used herein, "treatment" and "treating" generally mean obtaining the desired pharmacological and physiological effects. In terms of preventing or partially preventing a disease, its symptoms or disorders, the effects can be prophylactic, and / or in terms of partially or completely curing a disease, disorder, condition or the adverse effects attributable to the disease, the effects can be therapeutic. The term "treatment" as used herein encompasses any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in a subject who may be predisposed to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or alleviating the disease, i.e., causing the regression of the disease and / or its symptoms or disorders, such as the improvement or remediation of the impairment. In particular, the treatment of a bacterial infection includes preventing, reducing or even eradicating the infection, for example, by killing the bacteria and thus controlling, reducing or inhibiting bacterial proliferation and reducing the number of viable bacterial cells. In this context, it is preferred that a disease (e.g., BV) be treated in terms of partially or completely curing the disease or symptoms.

[0149] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, domestic animals such as cows, sheep, pigs, horses, laboratory rodents, etc. Preferably, the subject is a human.

[0150] As used herein, the term "effective amount" refers to the amount of at least one endolysin, a composition or a pharmaceutical formulation thereof according to the present invention that elicits a biological or medical response in a tissue, system, animal, or human being sought. In one embodiment, the effective amount is a "therapeutically effective amount" for alleviating the symptoms of a disease or disorder to be treated. In another embodiment, the effective amount is a "preventively effective amount" for preventing the symptoms of a disease or disorder to be prevented. The term also includes herein the amount of an active polypeptide sufficient to reduce disease progression, particularly to reduce or inhibit a disorder or infection and thereby elicit the sought response (i.e., "inhibitory effective amount").

[0151] The "efficacy" of a treatment according to the present invention can be measured based on changes in the disease process in response to the use or method according to the present invention. The efficacy of preventing an infectious disease is ultimately evaluated through epidemiological studies of populations, which are generally correlated with the titer of neutralizing antibodies in serum and the induction of multifunctional pathogen-specific T cell responses. Preclinical evaluations can include resistance to infection after challenge with an infectious pathogen. The treatment of an infectious disease can be measured by inhibiting the growth of the pathogen or eliminating the pathogen (and thus, no pathogen is detected), which is correlated with pathogen-specific antibody and / or T cell immune responses.

[0152] The term "biomaterial" refers to any material or sample obtained from the body of a subject. This includes, for example, whole blood, serum, plasma, urine, sputum, saliva, vaginal swabs, or cerebrospinal fluid samples.

[0153] The term "inanimate material or surface" includes solutions, media, devices, objects, floors, table surfaces.

[0154] The term "medium" includes water, air, or food.

[0155] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation in a form such that the biological activity of the active ingredient is clearly effective and does not contain additional ingredients that are toxic to the subject to whom the formulation will be administered.

[0156] The term "pharmaceutically acceptable" refers to a carrier consisting of materials that are not biologically or otherwise undesirable.

[0157] The term "carrier" refers to any component present in a pharmaceutical formulation other than the active agent and thus includes diluents, binders, lubricants, disintegrants, fillers, colorants, wetting agents or emulsifiers, pH buffers, preservatives, etc.

[0158] The term "variant" refers to a polypeptide that includes non-conservative or preferably conservative insertions, deletions, and / or substitutions relative to a native amino acid sequence. For example, a polypeptide can comprise an amino acid sequence 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 a native amino acid sequence. The percent identity can be determined by methods well known in the art using suitable computer programs such as MatGAT 2.0 (Myers and Miller, CABIOS (1989)). Preferably, the % identity is determined over the full length of the sequences to be compared. It will be understood that the percent identity is calculated relative to a polypeptide whose sequence has been optimally aligned. Fragments and variants of amino acid sequences can be prepared using any of the protein engineering, directed evolution, and / or site-directed mutagenesis methods well known in the art (e.g., see Molecular Cloning: a Laboratory Manual, 3rd ed., Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). Those skilled in the art will understand that a polypeptide according to the invention, or a fragment, variant, or fusion thereof, can comprise derivatives of a native amino acid sequence, or fragments or variants thereof, or be composed of derivatives of a native amino acid sequence, or fragments or variants thereof. Chemical derivatives of one or more amino acids can be achieved by reacting with functional side groups. Such derivative molecules include, for example, those in which the free amino acid group has been derivatized to form an amine hydrochloride, tosyl, carboxyphenoxy, t-butoxycarbonyl, chloroacetyl, or formyl group. The free carboxyl can be derivatized to form salts, methyl esters, and ethyl esters or other types of esters and hydrazides. The free hydroxyl can be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides that contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine and ornithine can replace lysine. Derivatives also include peptides that contain one or more additions or deletions, provided that the necessary activity is maintained. Other included modifications are terminal modifications such as amidation, amino-terminal acylation (e.g., acetylation or mercaptoacetamide acylation), terminal carboxyl amidation (e.g., with ammonia or methylamine), etc. Those skilled in the art will further understand that peptidomimetic compounds can 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 desired characteristics of a particular polypeptide as a therapeutic agent.

[0159] Endolysin according to the invention

[0160] The endolysin of the present invention has antibacterial activity against Gardnerella strains. The optimal pH at which the endolysin according to the present invention exhibits antibacterial activity includes a pH of about 4 to 6, preferably about 5. The endolysin of the present invention comprises or consists of the following:

[0161] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0162] (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

[0163] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0164] and has killing activity against Gardnerella cells.

[0165] In some embodiments, the N-terminal catalytic domain is from a first native endolysin, the linker region and the C-terminal cell wall binding region are from a second native endolysin, and the first and second native endolysins are encoded by different genomes from different prophages. It is contemplated that the killing activity of the endolysin of the present invention against Gardnerella is species-selective killing activity against the genus Gardnerella.

[0166] The N-terminal catalytic domain is a functional polypeptide, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria. The N-terminal catalytic domain can be N-acetylmuramidase, N-acetylmuramoyl-L-alanine amidase, L-propionyl-D-glutamate endopeptidase, interpeptide bridge endopeptidase, or N-acetyl-β-D-glucosaminidase. Preferably, the N-terminal catalytic domain is N-acetylmuramidase, and most preferably 1,4-β-N-acetylmuramidase. For example, the N-terminal catalytic domain can be a polypeptide comprising or consisting of the amino acids of any one of SEQ ID NO: 1 to 5, 7, or 10 to 12 or any 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 NO: 1 to 5, 7, or 10 to 12, whereby the polypeptide is functional, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria. Preferably, the N-terminal catalytic domain is a polypeptide comprising the amino acids of SEQ ID NO: 2 or 7 or any 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 or 7, whereby the polypeptide is functional, wherein the function includes the ability to lyse the cell wall of Gardnerella bacteria.

[0167] According to the present invention, the C-terminal cell wall-binding region is a functional polypeptide, wherein the function includes the ability to bind to the cell wall of Gardnerella bacteria. The C-terminal cell wall-binding region may comprise one, two, three or more cell wall-binding domains or consist of one, two, three or more cell wall-binding domains. For example, one, two, three or more cell-binding domains may independently be selected from or consist of polypeptides comprising the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, and any 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) with the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, whereby the polypeptide is functional, wherein the function includes the ability to bind to the cell wall of Gardnerella bacteria. Preferably, one, two, three or more cell wall-binding domains are independently selected from polypeptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33 or any 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) with the amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33, whereby the polypeptide is functional, wherein the function includes the ability to bind to the cell wall of Gardnerella bacteria. More preferably, one, two, three or more cell wall-binding domains are independently selected from polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 28-33 or any 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) with the amino acid sequence of any one of SEQ ID NOs: 28-33, whereby the polypeptide is functional, wherein the function includes the ability to bind to the cell wall of Gardnerella bacteria.

[0168] Most preferably, the C-terminal cell wall binding region comprises a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain is selected from SEQ ID NO: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and the second cell wall binding domain is selected from SEQ ID NO: 16, 18, 20, 22, 24, 27, 29, 31 and 33. In a preferred embodiment, the first cell wall binding domain is at the N-terminus of the second cell wall binding domain.

[0169] In one aspect of the invention, the linker region consists of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, and even more preferably 12 amino acids. Preferably, the linker region consists of a polypeptide comprising or consisting of the following amino acid sequence: (i) (XXX) n , where each X can 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, where X 1 is N or K, X 2 is A or V, X 3 is Y or C and X 4 is K or Q. The fragment containing the linker may be absent. The fragment containing the linker may also be present and may enhance the cell wall binding and / or lytic activity of the polypeptides of the invention.

[0170] The invention further provides an endolysin having the anti-Gardnerella killing activity as described above for 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 sensu stricto, G. leopoldii, G. piotii and / or G. swidsinskii, or other species of the genus Gardnerella.

[0171] The endolysin of the invention is preferably capable of specifically binding to and / or lysing the cells of Gardnerella for use in a method of treating Gardnerella infections (such as BV).

[0172] As described above, it has been well established that many phage lysins are composed of two distinct domains (e.g., see Sheehan et al., 1996, FEMS Microbiology Letters 140:23-28). One is the catalytic domain responsible for cell wall degradation and these are known to exist in several forms. The other domain is the cell wall binding domain that recognizes cell surface motifs and allows the lysin to attach to the cell wall of the target cell. The precise pattern recognition involved in the latter provides specificity. The enzymatic domain can be identified by its amino acid homology to other similar regions of lytic enzymes with the same type of lytic activity. In the case of the novel native Gardnerella prophage lysin discovered by the present inventors, it has been determined that the domain arrangement consists of an N-terminal domain of 196 residues, followed by a linker region of 12 residues and two repeat domains of 49 residues each, except for EL6 and EL9, in which there is only one incomplete domain of 43 residues. The native amino acid sequences of these novel lysins are summarized in Table 7. The present inventors determined that the N-terminal domain is the catalytic domain because it has homology to glycoside hydrolase family 25, and the two repeat domains are two cell wall binding domains because they have homology to the C-terminal domain of the lysozyme Cpl-7 (see Example 2 and Figure 3 ).

[0173] In some embodiments, the fragment containing the enzymatic domain is unmodified, i.e., corresponds to the native amino acid sequence. In alternative embodiments, the fragment containing the enzymatic domain may contain alterations such as substitutions, deletions, insertions of amino acids or any combination of their alterations. In some embodiments, the fragment containing the enzymatic domain is a variant fragment having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, even more preferably at least 99.5%, even more preferably at least 99.7% and most preferably 100% identity to the amino acid sequence of any one of SEQ ID NO:1 to 5, 7 or 10 to 12.

[0174] 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 enzymatic domain can contain alterations such as amino acid substitutions, deletions, insertions, or any combination of such alterations. In some embodiments, the fragment comprising the cell wall binding domain has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, even more preferably at least 99.5%, even more preferably at least 99.7%, and most preferably 100% identity with the amino acid sequence of any one of SEQ ID NOs: 15 to 24 and 26 to 33.

[0175] In yet another aspect of the invention, the endolysin comprises a fusion of polypeptides, or a fragment, variant or derivative thereof, or consists of a fusion of polypeptides, or a fragment, variant or derivative thereof. For example, the polypeptide can contain one or more additional amino acids inserted internally and / or at its N- and / or C-terminus in the amino acid sequence of the endolysin according to the invention, or a fragment, variant or derivative thereof.

[0176] Thus, as described above, in one embodiment, the endolysin of the first aspect of the invention comprises a fragment consisting of one or more cell wall binding domains, each of which comprises the amino acid sequence of any one of SEQ ID NOs: 15 to 24 and 26 to 33 (or a variant of such a domain sequence that retains its cell wall binding activity) or consists of the amino acid sequence of any one of SEQ ID NOs: 15 to 24 and 26 to 33 (or a variant of such a domain sequence that retains its cell wall binding activity), with an enzymatic domain from a different source fused thereto. Examples of other suitable enzymatic domains include, but are not limited to, L-alanyl-D-glutamate endopeptidase, D-glutamyl-m-DAP endopeptidase, interpeptide bridge-specific endopeptidase, V-acetyl-β-D-glucosaminidase (muramidase), N-acetyl-β-D-muramidase (lysozyme), or lytic transglycosylase. In addition, N-acetylmuramoyl-L-alanine amidase from other sources can also be utilized.

[0177] In one aspect of the present invention, the endolysin can be fused with a polypeptide or protein to facilitate the purification of the endolysin. Examples of such fusions are well known to those skilled in the art. Similarly, the endolysin can be fused with an oligohistidine tag (such as His6) or an epitope recognized by an antibody (such as the well-known Myc tag epitope). Fusions with any fragment variants or derivatives of the endolysin according to the present invention are also included within the scope of the present invention. It will be appreciated that fusions (or their variants or derivatives) that preferably retain the desired properties (i.e., endolysin activity) are preferred. If the fusion is suitable for use in the methods described herein, it is also particularly preferred. For example, the fusion can comprise a further part that confers the desired characteristics on the endolysin of the present invention; for example, the part can be useful in detecting or isolating the endolysin, facilitating cellular uptake of the endolysin, or directing protein secretion from cells. As is well known to those skilled in the art, the part can be, for example, a biotin part, a radioactive part, a fluorescent part, for example, a small fluorophore or a green fluorescent protein (GFP) fluorophore. The part can be an immunogenic tag, for example, a Myc tag, as is known to those skilled in the art, or can be a lipophilic molecule or polypeptide domain that is capable of facilitating cellular uptake of the endolysin, as is known to those skilled in the art.

[0178] An essential characteristic of the endolysin of the present invention is the ability to lyse cells of the genus Gardnerella. Preferably, the endolysin is capable of lysing cells of multiple strains of Gardnerella. Most preferably, the endolysin is capable of lysing all strains of the genus Gardnerella, including G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Microbiol. 898661). In one embodiment, the endolysin of the present invention is substantially or completely unable to lyse bacteria that are symbiotic members of the healthy vaginal microbiota (and are known not to cause adverse effects on the host). For example, it is advantageous if the endolysin does not lyse cells of the genus Lactobacillus. Most preferably, the endolysin of the present invention is substantially or completely unable to lyse cells of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. Optionally, the endolysin of the present invention does not lyse cells of Lactobacillus iners. Advantageously, the endolysin is capable of selectively lysing cells of pathogenic bacteria, i.e., to a greater extent than cells of non-pathogenic bacteria.

[0179] The killing activity of the lysin according to the invention against specific microorganisms can be determined by standard procedures in the art, including those based on the determination of the minimum inhibitory concentration (MIC) of the antimicrobial agent, which is defined as the lowest concentration of the antimicrobial agent that inhibits visible growth of the microorganism after overnight incubation, as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16 or "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 the Clinical and Laboratory Standards Institute. Another suitable method for determining the killing activity of the lysin according to the invention is described in the Examples section of the present application and includes measuring the decrease in the optical density of a bacterial suspension measured at 610 - 620 nm, the susceptibility of which bacteria will be tested in an in vitro turbidity assay in the presence of the purified lysin according to the invention. According to another embodiment, in the in vitro turbidity test as described herein, the lysin according to the invention will reduce the OD (610 - 620 nm) of a suspension of at least one Gardnerella strain 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%.

[0180] Methods for producing endolysins or fragments, variants, fusions or derivatives thereof for use according to the present invention are well known in the art. Conveniently, the endolysin or fragments, variants, fusions or derivatives thereof are or comprise recombinant endolysins. Endolysins according to the present invention can be produced by standard techniques of genetic engineering, including the use of recombinant vectors comprising polynucleotides encoding endolysins as described herein. A variety of expression systems can be used, including bacterial plasmids and derived vectors, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses (such as baculoviruses, papillomaviruses (such as SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retroviruses), cosmids or phagemid derivatives. Nucleotide sequences can be inserted into recombinant expression vectors by methods well known to those skilled in the art, such as those described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. Recombinant vectors can include nucleotide sequences that control the regulation, expression, transcription and / or translation of polynucleotides encoding endolysins, which are selected according to the host cell used. Recombinant vectors can further include nucleotide sequences such as those encoding His tags, for facilitating purification steps. Subsequently, such recombinant vectors can be 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 Edition, McGraw-Hill Professional Publishing, 1995 and MOLECULAR CLONING: A LABORATORY MANUAL, supra, such as by calcium phosphate transfection, by DEAE-dextran transfection, transfection, microinjection, by cationic lipid transfection, electroporation, transduction or infection. Host cells can be, for example, bacterial cells such as Escherichia coli, fungal cells such as yeast cells or cells of Aspergillus, Streptomyces, 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 Escherichia coli. The host cells are then cultured under appropriate conditions to produce the endolysins described herein, which can be further purified from the culture medium or from the host cell lysate by any standard purification method, including immobilized metal affinity chromatography (IMAC) (Block et al., 2008, Protein Expr. Purif. 27, 244-254).

[0181] Composition according to the invention

[0182] In yet another aspect of the present invention, there is provided an antibacterial composition comprising an endolysin according to the first aspect of the present invention, a nucleic acid according to the second aspect of the present invention, a vector / plasmid according to the third aspect of the present invention, a host cell according to the fourth aspect of the present invention, or a bacteriophage capable of expressing the endolysin according to the first aspect of the present invention, in a specific pharmaceutical composition.

[0183] As used herein, "pharmaceutical composition" refers to a therapeutically effective formulation for use in the methods of the present invention. As used herein, "therapeutically effective amount", or "effective amount" or "therapeutically effective" means an amount that provides a therapeutic effect for a given condition and dosing regimen. This is a predetermined amount of active material calculated in combination with the required additives and diluents (i.e., carriers or delivery vehicles) to produce the desired therapeutic effect. Additionally, it means an amount sufficient to reduce and most preferably prevent clinically significant deficiencies in the activity, function, and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause a clinically significant improvement in the condition of the host. As will be understood by those skilled in the art, the amount of a compound can vary depending on its specific activity. A suitable dose can comprise a predetermined amount of an active composition calculated to produce the desired therapeutic effect in relation to the required diluent. In the methods and uses for making the compositions of the present invention, a therapeutically effective amount of the active ingredient is provided. The therapeutically effective amount can be determined by a person of ordinary skill or a veterinary worker based on patient characteristics, such as those well known in the art, e.g., age, weight, gender, condition, complications, other diseases, etc. In one embodiment of the present invention, the pharmaceutical composition comprises an endolysin according to the first aspect of the present invention. Thus, the pharmaceutical formulation can comprise an amount of an endolysin or a fragment, variant, fusion, or derivative thereof sufficient to at least partially inhibit the growth of Gardnerella cells in a patient infected or susceptible to infection by such cells. Preferably, the pharmaceutical formulation comprises an amount of an endolysin or a fragment, variant, fusion, or derivative thereof sufficient to kill Gardnerella cells in the patient. Those skilled in the art will understand that the endolysins of the present invention are generally administered in admixture with suitable pharmaceutical excipients, diluents, or carriers selected according to the intended route of administration and standard pharmaceutical practice (e.g., see Remington: The Science and Practice of Pharmacy, 19th Edition, 1995, edited by Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA). For example, the endolysin can be administered topically, i.e., topically into the vagina of a female subject, and / or into or onto the glans penis, foreskin, or urethral orifice of a male subject. The term "into or onto the glans penis" as used herein also includes "into and onto the glans penis". Consistent with this, the term "into or onto the glans penis, foreskin, or urethral orifice of a male subject" also includes "into and onto the glans penis and onto the foreskin and onto the urethral orifice of a male subject". In another embodiment, the endolysin can be co-administered with a compound or composition that modulates the vaginal pH. In some embodiments, the compound or composition adjusts the vaginal pH to a pH of 4.0 to 6.0, preferably to a pH of 5.0.

[0184] In alternative embodiments of the invention, the pharmaceutical composition does not contain the lysin itself, but instead contains a nucleic acid molecule capable of expressing said lysin. Suitable nucleic acid molecules, expression vectors and host cells have been described in detail above. For example, recombinant probiotics (LAB strains, such as Lactococcus lactis or Lactobacillus sp.) can be used. In yet another embodiment of the invention, the pharmaceutical composition contains a phage capable of expressing the lysin according to the first aspect of the invention. Methods for performing such phage-based therapies are well known in the art (e.g., see, Watanabe et al., 2007, Antimicrobial Agents & Chemotherapy 51:446-452). Thus, for the treatment of the bacterial infections described herein, the lysin of the invention can be administered as a homologous protein, as a nucleic acid construct, vector or host cell expressing the homologous protein, as part of a living organism (including a phage) expressing the homologous protein, or by any other convenient method known in the art, to effect contact of the lysin with its bacterial target, whether the target is a pathogenic bacterium, such as Gardnerella vaginalis, or another pathogen or potential pathogen, as further described herein.

[0185] The compositions of the invention can contain one or more lysin polypeptides. In this embodiment, the lysin polypeptides can exist as separate polypeptides or as fusion proteins containing the lysin polypeptides or fragments thereof.

[0186] The pharmaceutical compositions of the invention can further contain one or more other pharmaceutically acceptable ingredients, such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, etc. Preferably, the pharmaceutical compositions of the invention do not contain imidazole.

[0187] The lysin of the present invention, together with commonly used adjuvants, carriers, diluents or excipients, can be made into pharmaceutical compositions and their unit dosage forms, and can be used in solid form, such as tablets or filled capsules, or in liquid form, such as solutions, suspensions, aerosols, emulsions, elixirs or capsules containing them, all for oral administration, or in the form of a sterile injectable solution for parenteral (including subcutaneous) use. Such pharmaceutical compositions and their unit dosage forms may contain conventional proportions of ingredients, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the expected daily dosage range to be used. The compositions of the present invention may also be liquid preparations, including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The compositions may also be formulated as dry products for reconstitution with water or other suitable excipients before use. Such liquid preparations may contain additives, including but not limited to suspending agents, emulsifying agents, non-aqueous carriers and preservatives. Suspending agents include but are not limited to sorbitol syrup, methylcellulose, glucose / 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 carriers include but are not limited to edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol and ethanol. Preservatives include but are not limited to methyl or propyl p-hydroxybenzoate and sorbic acid. Other materials, as well as processing techniques, etc. are listed in Part 5 of Remington’s “The Science and Practice of Pharmacy”, 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins.

[0188] The solid compositions of the present invention can be in the form of tablets or lozenges configured in a conventional manner. Tablets can be coated according to methods well known in the art. Injectable compositions are generally based on injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art.

[0189] The compositions of the present invention can 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 can also be formulated as transdermal preparations containing aqueous or non-aqueous carriers, including but not limited to creams, ointments, lotions, pastes, plasters, patches or films.

[0190] The compositions of the present invention can also be formulated for parenteral administration, including but not limited to, administration by injection or continuous infusion. Preparations for injection can be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents including but not limited to, suspending, stabilizing and dispersing agents. The compositions can also be provided in powder form for reconstitution with a suitable carrier including but not limited to sterile, pyrogen-free water.

[0191] The compositions of the present invention can also be formulated as depot preparations which can be administered by implantation or by intramuscular injection. The compositions can be formulated with suitable polymeric or hydrophobic materials (such as, for example, as an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (such as, for example, as a sparingly soluble salt).

[0192] The compounds of the present invention can also be administered in a sustained release form or from a sustained release drug system. Descriptions of representative sustained release materials can also be found in Remington's “The Science and Practice of Pharmacy”.

[0193] Method of administration

[0194] The compositions of the present invention are preferably administered topically into the vagina of female subjects and / or onto or into the glans penis, foreskin or urethral orifice of male subjects. However, these compositions can also be administered in any manner including intravenous injection, intraarterial, intraperitoneal injection, subcutaneous injection, intramuscular, intrathecal, oral route (including sublingual or buccal administration), topical, cutaneous application, direct tissue perfusion during surgery or combinations thereof.

[0195] In a preferred embodiment, the lysins, polynucleotides or pharmaceutical compositions of the present invention as described herein are administered topically. In a further preferred embodiment, the lysins, polynucleotides or pharmaceutical compositions of the present invention as described herein are administered into the vagina of female subjects and / or onto or into the glans penis, foreskin or urethral orifice of male subjects. In a further preferred embodiment, the lysins, polynucleotides or pharmaceutical compositions of the present invention as described herein are administered topically into the vagina of a subject.

[0196] The dose administered to an individual as a single or multiple dose will vary depending on various factors including pharmacokinetic properties, patient condition and characteristics (gender, age, weight, health, body size), degree of symptoms, concurrent treatment, frequency of treatment and desired effect.

[0197] According to one aspect, the compositions of the present invention can be administered to a patient in a prophylactic manner prior to sexual intercourse.

[0198] Combination

[0199] According to the present invention, the lysin can be administered alone or in combination with co-agents useful in the prevention and / or treatment of Gardnerella infections or disorders, including those caused by Gardnerella vaginalis sensustricto, Gardnerella leopoldii, Gardnerella piotii, Gardnerella swidsinskii, and / or other species of the genus Gardnerella.

[0200] The lysin according to the present invention can be administered in combination with the following:

[0201] (a) One or more conventional antibiotic therapies. Such antibiotics can include clindamycin, metronidazole, or any other suitable antibiotics known to those skilled in the art;

[0202] (b) One or more other lysins, or nucleic acid molecules, vectors, host cells, or phages capable of expressing the lysin;

[0203] (c) Compounds or compositions that modulate the vaginal pH. In some embodiments, the compound or composition adjusts the vaginal pH to a pH of 4.0 to 6.0, preferably to a pH of 5.0. Suitable pH-modulating compounds can include phosphates, lactic acid (e.g., the natural acidifying substance secreted by Lactobacilli to establish an acidic environment), or other organic acids, such as carboxyl-substituted polymers;

[0204] (d) Treatments that neutralize toxins released upon bacterial lysis of Gardnerella cells in the vagina. Suitable neutralizing treatments can include antibodies (see, Babcock et al., 2006, Infect. Immun.

[0205] 74:6339-6347) and toxin absorbers, such as tolevamer (see, Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534);

[0206] (e) Probiotics.

[0207] Use and method according to the invention

[0208] Another aspect of the present invention provides the use of an endolysin according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, a host cell according to the present invention, a bacteriophage capable of expressing an endolysin according to the present invention, or a pharmaceutical composition according to the present invention in medicine. Thus, the endolysin of the present invention can be used in methods for treating the human or animal body by surgery or therapy and / or in diagnostic methods practiced on the human or animal body. In particular, the present invention provides an endolysin according to 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 the endolysin of the present invention, or a pharmaceutical composition of the present invention, for use in methods for treating a disease or disorder.

[0209] Another aspect of the present invention provides an endolysin according to the present invention, a nucleic acid according to the present invention, a vector / plasmid according to the present invention, a host cell according to the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition according to the present invention, for use as a medicine.

[0210] Another aspect of the present invention provides the use of an endolysin according to the present invention, a nucleic acid according to the present invention, a vector / plasmid according to the present invention, a host cell according to the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition according to the present invention in the preparation of a medicament for killing and / or inhibiting / preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis by use of the endolysin. In particular, the present invention provides the use of an endolysin according to 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 the endolysin of the present invention, or a pharmaceutical composition of the present invention in the manufacture of a medicament for treating bacterial infections and disorders.

[0211] Another aspect of the present invention provides an endolysin according to the present invention, a nucleic acid according to the present invention, a vector / plasmid according to the present invention, a host cell according to the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition according to the present invention for killing and / or inhibiting / preventing the growth of microbial cells in a patient, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis by use of the endolysin.

[0212] Another aspect of the present invention provides a method for killing and / or inhibiting / preventing the growth of microbial cells in a patient, the method comprising administering to the patient an endolysin according to the present invention, a nucleic acid according to the present invention, a vector / plasmid according to the present invention, a host cell according to the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition according to the present invention, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis by use of the endolysin.

[0213] Another aspect of the present invention provides the use of the lysin of the present invention, the nucleic acid of the present invention, the vector / plasmid of the present invention, the host cell of the present invention, the phage capable of expressing the lysin of the present invention, or the pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing a disease or disorder associated with microbial cells in a patient, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the lysin.

[0214] Another aspect of the present invention provides the use of the lysin of the present invention, the nucleic acid of the present invention, the vector / plasmid of the present invention, the host cell of the present invention, the phage capable of expressing the lysin of the present invention, or the pharmaceutical composition of the present invention for treating or preventing a disease or disorder associated with microbial cells in a patient, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the lysin of the present invention.

[0215] Another aspect of the present invention provides a method for treating or preventing a disease or disorder associated with microbial cells in a patient in need of such treatment, the method comprising administering to the patient the lysin of the present invention, the nucleic acid of the present invention, the vector / plasmid of the present invention, the host cell of the present invention, the phage capable of expressing the lysin of the present invention, or the pharmaceutical composition of the present invention, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the lysin of the present invention.

[0216] "Diseases or disorders associated with microbial cells in a patient" include diseases or disorders caused by or against which a patient is infected with Gardnerella. Such diseases and disorders include BV.

[0217] "Treatment" includes therapeutic and prophylactic treatment of a subject (or patient). In one embodiment, the lysin of the present invention, the nucleic acid of the present invention, the vector / plasmid of the present invention, the host cell of the present invention, the phage capable of expressing the lysin of the present invention, or the pharmaceutical composition of the present invention, the use and method of the present invention are used to treat existing diseases or disorders. Alternatively or additionally, the use and method of the present invention can be used for prevention. The term "prophylactic" or "prevention" is used to include the use of the lysin or composition described herein to prevent or reduce the likelihood of Gardnerella infection in a patient or subject. Prevention can be primary prevention (i.e., preventing 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). Preferably, the methods and processes provided herein are used to treat existing diseases or disorders, particularly for treating existing BV.

[0218] As described above, the term "effective amount" is used herein to describe the concentration or amount of an endolysin according to the invention that can be used to effect a beneficial change in a disease or disorder being treated, whether the change is a remission, a beneficial physiological outcome, a reversal or alleviation of the disease or disorder being treated, a prevention or reduction in the likelihood of an ongoing disease or disorder, depending on the disease or disorder 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 / plasmid according to the third aspect of the invention, the host cell according to the fourth aspect of the invention, the phage capable of expressing the endolysin according to the first aspect of the invention or the pharmaceutical composition according to the sixth aspect of the invention are administered as a single dose. Alternatively, the endolysin, nucleic acid, vector / plasmid, host cell, phage or pharmaceutical 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, phage or pharmaceutical composition is preferably administered at a frequency sufficient to maintain the persistence of the endolysin according to the first aspect of the invention in the vagina of the subject. Preferably, the dose and frequency of administration are sufficient to prevent the occurrence or recurrence of a disease or disorder associated with microbial cells (e.g., Gardnerella) in the subject. Preferably, the dose and frequency of administration are sufficient to prevent the occurrence or recurrence of growth impedance associated with microbial cells (e.g., Gardnerella) in the subject.

[0219] In one embodiment, the uses and methods, host cells or pharmaceutical compositions comprising host cells of the invention are used to deliver the endolysin according to the first aspect of the invention (preferably host cells).

[0220] It will be appreciated that the drugs described herein can be administered to a subject in combination with one or more other therapeutic agents. For example, the drugs described herein can be administered to a subject in combination with:

[0221] (a) one or more conventional antibiotic therapies. Such antibiotics can include clindamycin, metronidazole or any other suitable antibiotic known to those skilled in the art;

[0222] (b) one or more other endolysins, or nucleic acid molecules, vectors, host cells or phages capable of expressing said endolysin;

[0223] (c) compounds or compositions that condition the vaginal pH, preferably to a pH of 4.0 to 6.0, more preferably to about pH 5.0. Such pH conditioning compounds can include phosphates, lactic acid (e.g., the natural acidifying substance secreted by Lactobacilli to establish an acidic environment) or other organic acids, e.g., carboxyl-substituted polymers;

[0224] (d) Treatments that neutralize toxins released upon bacterial lysis of Gardnerella vaginalis cells in the vagina. Suitable neutralizing treatments can include antibodies (see, Babcock et al., 2006, Infect. Immun.

[0225] 74:6339 - 6347) and toxin absorbers such as tolevamer (see, Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525 - 1534);

[0226] (e) Probiotics.

[0227] Another aspect of the present invention provides the use of an endolysin having cell - lysing activity against Gardnerella or a nucleic acid molecule, vector / plasmid, host cell or phage 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 Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin of the present invention. For example, an endolysin having such activity can be used to clean surfaces such as those in hospitals, kitchens, etc., which may be susceptible to contamination by such cell bacteria. Preferably, the microbial cells comprise cells of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or other species of the genus Gardnerella or consist of cells of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or other species of the genus Gardnerella.

[0228] Another aspect of the present invention provides a kit. The kit comprises the endolysin described herein and instructions for use, particularly for treating a disease or disorder, preferably BV. The kit can be used for therapeutic or prophylactic purposes and can further comprise a compound or composition for adjusting the vaginal pH to 4.0 - 6.0, preferably to 4.5 - 5.5, more preferably to about 5. However, the kit of the present invention can also be used to detect the presence of microbial cells in a sample, the kit comprising a polypeptide having cell - lysing activity and / or cell - binding specificity of the endolysin according to the present invention or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing it, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin.

[0229] Related aspects of the present invention provide:

[0230] (a) A polypeptide having the cell wall binding activity and / or cell lysis activity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing the same, for use in the preparation of a diagnostic agent for a disease or disorder associated with microbial cells, said microbial cells being selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin;

[0231] (b) A polypeptide having the cell wall binding activity and / or cell lysis activity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing the same, for diagnosing a disease or disorder associated with microbial cells, said microbial cells being selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin;

[0232] (c) A polypeptide having the cell wall binding activity and / or cell lysis activity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing the same, for use in the in vitro and / or ex vivo detection of the presence of microbial cells in a sample, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin; and

[0233] (d) An in vitro method for diagnosing a disease or disorder treatable with an endolysin according to the present invention, the method comprising the steps of: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of the C-terminal cell wall binding region 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 comprises microbial cells, and wherein the C-terminal cell wall binding region of the endolysin is optionally labeled; (ii) testing whether the polypeptide binds and / or lyses the microbial cells of the sample; and (iii) if the polypeptide binds and / or lyses the microbial cells, determining that the disease or disorder is treatable with an endolysin according to the present invention. The microbial cells can be Gardnerella cells, preferably cells of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or other species of the genus Gardnerella. Accordingly, the present invention provides an in vitro method for diagnosing a disease or disorder treatable with an endolysin of the present invention in a subject, the method comprising contacting a cell sample obtained from the subject with a polypeptide having cell wall binding activity and / or cell lysing activity of an endolysin according to the present invention, or a nucleic acid molecule, vector / plasmid, host cell or prophage expressing the same, and determining whether the cells in the sample have thereby been lysed, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin. Preferably, the microbial cells comprise or consist of cells of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or other species of the genus Gardnerella. In such diagnostic uses and methods, methods well known in the art can be used to detect cell lysis. For example, the ATP level can be measured as an indicator of cell lysis.

[0234] In an alternative embodiment of the uses and methods of the present invention defined above, the polypeptide comprises or consists of the cell wall binding domain of an endolysin according to the present invention. To allow detection, such a polypeptide can be fused to magnetic beads or used as a fusion protein comprising a suitable reporter or label (e.g., green fluorescent protein or a color-forming enzyme such as HRP). Such diagnostic methods are well established for endolysins from other systems, such as Listeria endolysin (e.g., see, Loessner et al., 2002, Mol Microbiol 44, 335-49; Kretzer et al., 2007, Applied Environ. Microbiol. 73:1992-2000).

[0235] The illustrative embodiments of the present invention are described in the following non - limiting examples with reference to the following drawings. Example

[0236] Example 1 - Identification of Natural Endolysins in Gardnerella Genomes

[0237] Endolysins are hydrolases produced by bacteriophages to lyse the host cell wall at the final stage of the lytic cycle. They are capable of targeting one of five bonds in peptidoglycan (murein), the major component of the bacterial cell wall, which allows the release of progeny virus particles from the lysed cells. To date, no bacteriophages with lytic activity against Gardnerella have been isolated. Therefore, it is also unknown whether endolysins from phage sources with lytic activity against Gardnerella can be successfully identified. The present inventors investigated whether endolysins encoded by prophage sequences could be identified on various Gardnerella genomes. A prophage is a phage genome that has inserted and integrated into the circular bacterial DNA chromosome or exists as an extrachromosomal plasmid. This is a latent form of the phage where the viral genes are present in the bacterium without disrupting the bacterial cell. Identification of prophage sequences within bacterial genomes and plasmids can be carried out using web - based tools known to 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), etc. The inventors successfully identified sequences on 14 Gardnerella genomes predicted to constitute complete or partial prophages. These sequences were discovered by identifying DNA regions predicted to be of viral origin by gene clusters. Also included were viral gene clusters predicted to be only partial prophages rather than complete prophages.

[0238] Then, the putative prophage sequences were annotated by blast - predicted coding sequences to identify putative endolysins. Specifically, protein sequences homologous to enzymes capable of cleaving any of the key chemical bonds that construct peptidoglycan were searched. In particular, protein sequences homologous to N - acetylmuramidase, N - acetylmuramic acid - L - alanine amidase, L - alanyl - D - glutamyl endopeptidase, interpeptide - bridge endopeptidase, or N - acetyl - β - D - glucosaminidase were searched. On each individual prophage or partial prophage analyzed, the inventors found coding sequences for proteins homologous to 1,4 - β - N - acetylmuramidase and named them EL1 to EL14. The names of the source genomes are assigned as shown in Table 1.

[0239] [Table 1]

[0240] Name of the putative endolysin Name of the strain from which the putative endolysin was identified EL 1 Strain HMP9231 EL 2 Strain Gv18-4 EL 3 Strain Gv18-4 EL 4 Strain Gv5-1 EL 5 Strain JCP7276 EL 6 Strain 1400E EL 7 Strain AMD EL 8 Strain JCP7719 EL 9 Strain 0288E EL 10 Strain G30-4 EL 11 Strain JCP8017A EL 12 Strain 3549624 EL13 Strain Gv37_1 EL 14 Strain Gv37_2

[0241] In each case, only one copy of each prophage was found and no other coding sequences for enzymes predicted to lyse the bacterial cell wall were detected. The putative 1,4-β-N-acetylmuramidase was aligned to understand homology and domain structure (see Figure 1 ). As can be seen from Figure 1 , even though from different prophages on different genomes, most endolysins happen to have 306 residues. The two exceptions are EL6 and EL9. EL6 is truncated by a frameshift at the C-terminus. EL9 ends at exactly the same position as EL6, but in this case, the entire contig ends. There are no exact pairs among the endolysins, even though they are highly homologous, as Figure 2 shows.

[0242] Example 2 - Determination of the domain structure of natural Gardnerella prophage endolysins

[0243] The domain structure of the newly discovered endolysins was determined using InterPro (Mitchell et al., 2019 Nucleic Acids Res. 47, D351 - D360). Briefly, InterPro is a database of protein families, domains, and functional sites where recognizable features found in known proteins can be applied to new protein sequences in order to functionally characterize them. The content of InterPro consists of diagnostic features and proteins with significant matches. Features are composed of models, such as simple types like regular expressions or more complex types like Hidden Markov models that describe protein families, domains, or sites. From Figure 3As can be seen, all endolysins with 306 residues have the same domain arrangement. The 196-residue N-terminal domain was identified as the catalytic domain because it is homologous to glycoside hydrolase family 25. The catalytic domain is followed by a linker region and two cell-binding domains homologous to the C-terminal domain of lysozyme Cpl-7 (also known as the CW_7 domain (García et al., 1990 Gene 86, 81-88; Lopez and García, 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 catalytic domain identified above represents the "N-terminal catalytic domain" or "H-domain", 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 containing one or more cell wall-binding domains or "B-domains" or consisting of one or more cell wall-binding domains or "B-domains", together represent 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, and so on.

[0244] Example 3 - Determination of the Enzymatic Activity of Native Gardnerella Prophage Endolysins against Gardnerella Cells

[0245] The inventors studied the enzymatic activity of the newly discovered endolysins against Gardnerella. It is not possible to predict on a computer whether the identified sequences homologous to 1,4-β-N-acetylmuramidase are active. As is well known in the art, bacteria can mutate their prophage sequences and the corresponding prophages may thus lose their reproductive activity. In addition, even if the newly discovered phage-encoded peptidoglycan hydrolases are indeed active proteins, it has not been demonstrated that the proteins can enzymatically degrade the specific peptidoglycan layer of Gardnerella. In fact, Gardnerella is special in that it is a Gram-variable species: it does not form an outer membrane that defines true Gram-negative species. Its cell wall is usually very thin and the peptidoglycan content is only 10% or less. Therefore, those skilled in the art would think that peptidoglycan-degrading enzymes, such as endolysin proteins, cannot effectively lyse the bacterial cell wall of Gardnerella.

[0246] Fourteen identified endolysins EL1 to EL14 were cloned with His-tags, expressed in Escherichia coli and purified by a one-step Ni-NTA column using the method described in Reference Example 1. The assignment of endolysin names to the source genomes is shown in Table 1. The Gardnerella strains used are shown in Table 2.

[0247] [Table 2]

[0248] Name Gardnerella strains (new nomenclature according to (Vaneechoutte et al., 2019)) Gv_1 UGent 09.07, strain of G.vaginalis sensu stricto Gv_8 UGent 25.49, strain of G.vaginalis sensu stricto Gv_9 ATCC 14018, type strain of G.vaginalis sensu stricto Gv_10 UGent 06.41, type strain of G.leopoldii Gv_11 UGent 09.48, type strain of G.leopoldii Gv_17 UGent 18.01, type strain of G.piotii Gv_23 GS10234 (FC2), type strain of G.swidsinskii

[0249] To test the activity of the purified endolysin, the turbidity change of the Gardnerella suspension (see Table 2) was measured at 610 - 620 nm, essentially using the method described in Reference Example 2, where a bacterial suspension in 95 ul of Hardy Broth at the indicated pH value was mixed with 5 ul of the 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 live cell suspension can be used in a spectrophotometer to assay the activity of peptidoglycan hydrolases. The decrease in optical density over time (minutes) can be used to calculate the hydrolysis rate. The results were compared to a control preparation of "enzyme-free, buffer-only" that had been treated identically and for the same period of time. In this way, the specific activity of the enzyme preparation can be reported as ΔOD / time / ul of endolysin protein. From Figures 4A to 4C It can be seen that the turbidity decrease in the endolysin-treated group was more significant than that of the buffer, indicating enzyme activity. Surprisingly, the inventors thus found that the newly discovered endolysins EL1, EL2, EL3, EL4, EL5, EL7, EL10, EL11, and EL12 are active proteins with the ability to lyse the Gardnerella cell wall. As described above, due to the low content of peptidoglycan in the cell wall, the fact that peptidoglycan-degrading enzymes (such as the identified endolysins) can effectively lyse the bacterial cell wall is an unexpected and surprising finding.

[0250] Example 4 - Identification of the Most Active Domains of Endolysins with Artificial Domain Exchanges

[0251] The inventors then evaluated whether different N-terminal enzymatic domains could have different lytic activities and whether the B region (including the linker and cell wall binding domains) could mediate specificity for different strains. To this end, domain-exchanged endolysins were artificially generated.

[0252] 4.1 - Endolysin Constructs

[0253] To artificially generate domain-exchanged endolysins, the first 196 residues of the N-terminus of the first native endolysin containing the catalytic domain were exchanged as a block relative to the complete C-terminal region of 110 residues of the second native endolysin containing the linker region and two cell wall binding domains. The domain-exchanged proteins were prepared by performing the following method. The original construct EL1-14 was ordered from GeneWiz as a synthetic gene codon-optimized for Escherichia coli. These constructs were cloned into the pETM14_ccdB vector by GeneWiz using the recognition sites of the NcoI and NotI enzymes by the restriction / ligation method.

[0254] Table 3 below summarizes the primers used. For domain swapping of the 10 selected constructs, each H-domain was amplified with PhusionFlash polymerase (Thermo, F-548L) together with the T7 promoter using a common forward primer (no 2) and construct-specific reverse primers (no 3-12). Similarly, each B-region was amplified with construct-specific internal primers (no 13-21) and a common reverse primer (no 1) including the T7 terminator. All primers contained extensions with BsaI recognition sites to make the outer ends compatible with the pETM14-derived vector backbone pETMdest. The overhangs between domains were designed as the sequence "GGCT" within two amino acids GL of the linker sequence. Thus, for the purposes of this experiment, these 2 amino acids represent the exact boundary between domains.

[0255] Therefore, the amplified and gel-purified domains (GeneJet Gel Purification Kit, Thermo, K0692) were combined into 90 new expression constructs using the GoldenGate cloning strategy via BsaI restriction (BsaI-HFv2, NEB, R3733S) / ligation (T4 DNA ligase, Thermo, EL0011) cycling reactions.

[0256] For transformation purposes, the NEB10β E. coli strain (NEB, C3019) was used and plasmids were purified using the GeneGet Plasmid Miniprep Kit (Thermo, K0502).

[0257] [Table 3]

[0258]

[0259]

[0260] For reference purposes, the domain combinations of the engineered endolysins are designated by an H-code for the N-terminal catalytic domain (hereinafter referred to as the H-domain) and a B-code for the portion containing the C-terminal cell wall binding region and the linker region (hereinafter referred to as the B-region). For example, H2B10 refers to an endolysin with a domain swap having the N-terminal domain from native endolysin EL2 and the linker region and C-terminal cell wall binding region from native endolysin EL10. In other words, H2B10 refers to a domain-swapped endolysin composed of 196 N-terminal residues (SEQ ID NO:2) of native endolysin EL2 and 110 C-terminal residues of native endolysin EL10. In this example, the B-region B10 corresponding to the 110 C-terminal residues of native endolysin EL10, in order from the C-terminus to the N-terminus, contains the C-terminal cell wall binding domain “B10_C” (SEQ ID NO:29), the N-terminal cell wall binding domain “B10_N” (SEQ ID NO:28), and the linker region “L10” (NAGLNGYKNGGS). The nomenclature and corresponding amino acid sequences are shown in detail in Table 7 below.

[0261] Thus, according to the above nomenclature, native endolysins, for example, EL3, can be interchangeably defined as H3B3 or H3-L3(B3_N)(B3_C). Similarly, recombinant endolysins, for example, H2B10, can also be interchangeably defined as H2-L1-(B10_N)(B10_C).

[0262] Those skilled in the art will appreciate that in other embodiments the elements “Lx”, “(Bx_N)”, and “(Bx_C)” can also be independently swapped. The nomenclature is further shown in Table 7 below.

[0263] 4.2 - Optimization of assay parameters

[0264] The dependence of activity on three potentially critical parameters was analyzed, namely pH, anaerobic / microaerophilic / aerobic conditions, and absence / presence of imidazole. The reasons for choosing these three evaluation criteria are as follows:

[0265] - pH: The killing activity of the endolysins of the present invention has been successfully demonstrated by experiments conducted at a pH of approximately 7. However, the pH in a healthy vagina is approximately 3.5, while the pH in a BV vagina can be as high as approximately 5.5 or even higher. Therefore, the pH-dependence of endolysin activity was investigated.

[0266] - Oxygen: In the literature, Gardnerella has been described as anaerobic or microaerophilic. Therefore, it has been investigated whether more untreated cells survive under anaerobic, microaerophilic, or aerobic conditions during the incubation period of the experiment (usually 5 hours).

[0267] -Imidazole: According to the method described in Reference Example 1, the lysin of the present invention was purified by a one-step Ni-NTA column, and the buffer used to elute the lysin from the Ni-NTA matrix contained imidazole. Thus, without further dialysis of the sample, the obtained eluate contained 250 mM imidazole. In this regard, the effect of imidazole on Gardnerella has been studied.

[0268] First, the sensitivity of vaginal G. vaginalis Gv_9 survival to incubation in media with or without imidazole at different pH values was evaluated (see Figure 5 ). 5×10 7 CFU / ml cells were incubated under anaerobic conditions at 37 °C for 5 hours under the conditions shown below the figure. Subsequently, the surviving CFU / ml was determined by quantitative plating. As Figure 5 shown, at pH 6.0, the median numbers of 1×10 7 and 1×10 6 cells survived without and with imidazole incubation, respectively. At pH 7.0, only the median numbers of 2×10 6 and 3×10 4 cells survived without and with imidazole, respectively. In the untreated control at pH 5.0, 1e7 cells survived in this procedure, while the median survival of imidazole-treated at pH 7 was 3e4, which was 3 logarithmic orders lower than the former. Therefore, the survival of vaginal G. vaginalis Gv_9 is highly dependent on the absence of imidazole, especially at pH > 6.0 and low pH.

[0269] Second, the sensitivity of vaginal G. vaginalis Gv_9 to treatment with recombinant lysin H10B1 relative to an imidazole-containing control at different pH values was evaluated (see Figure 6 ). 5×10 7 CFU / ml cells were incubated under anaerobic conditions at 37 °C for 5 hours under the conditions shown below the figure. Subsequently, the surviving CFU / ml was determined by quantitative plating. The column labeled imidazole control depicts the same data as in Figure 5 . As Figure 6As shown, endolysin has high activity at as low as pH 5.0. Even at this low pH, the relative reduction compared to the control is more obvious, where the viable CFU is reduced by 2.5 logarithmic orders. Although at pH 7.0, the difference in survival between H10B1-treated and untreated cells is less than 1 log10, at pH 5.0, the difference is 2 log10 units. In the presence of imidazole, the survival of cells not treated with H10B1 did not increase at pH values below 6.0. Therefore, the activity of endolysin H10B1 is highly pH-dependent. When similar experiments were conducted under aerobic conditions, the survival of control cells decreased by several log10 units compared to anaerobic conditions (data not shown).

[0270] Therefore, it has been concluded that the optimal parameters for experiments with the endolysin of the present invention are under anaerobic conditions, pH 5.0, and with a step of removing imidazole from the endolysin eluate.

[0271] 4.3 - Expression level

[0272] Table 4 describes an overview of the concentrations of all endolysin constructs. Each construct with a concentration higher than 0.2 mg / ml after removing imidazole was adjusted to a concentration of 0.2 mg / ml by dilution. Constructs with lower concentrations were left as they were and their activities were tested. The concentration of native endolysin EL6 (not shown in Table 4) was lower than 0.2 mg / ml. H4, H11, and H12 seem to confer low solubility and expression levels, as most constructs were below the 0.2 mg / ml threshold. Similarly for H1, some constructs had low concentrations.

[0273] [Table 4]

[0274] Overview of expression levels (quantitative)

[0275]

[0276] 4.4 - Quantitative evaluation of the lysis of four major Gardnerella species by endolysin in suspension under optimized conditions

[0277] The lysis activity against four major Gardnerella species of 91 constructs (native and domain-exchanged endolysins) was quantitatively evaluated using the method described in Reference Example 2. Briefly, 90 ul of 5e7 CFU / ml of the designated strain was incubated for 5 hours under anaerobic conditions at pH 5.0 with 10 ul of endolysin (concentration adjusted to 0.2 mg / ml as much as possible, see Table 4).

[0278] The results are shown together with Tables 5A to 5C in Figures 7A to 7D In Figures 7A to 7DIn it, the logarithmic Y-axis depicts the count of viable cells. The dashed line represents the limit of detection (LOD) given by spotting 2 μl of the reaction mixture (500 CFU / ml). Each combination of 10 native H-domains and 9 native B-domains was evaluated, including native endolysins (H1B1, H2B2, H3B3, etc.), as well as H6B6. Other constructs with B-domain B6 were not tested because B6 inactivated all H-domains fused to it, as determined by OD measurement (data not shown). The activity of each construct against each of the 4 major Gardnerella species, G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinki, was measured. Table 5 summarizes the log10 reduction of the resulting CFUs, organized by H-domain and B-region. All conditions were measured in triplicate. Survival after incubation with endolysin vs. buffer for 5 hours at pH 5.0 under anaerobic conditions was measured by quantitative spotting. These values represent the log10 of the ratio of viable CFUs of treated vs. untreated cells. The mean of three replicate measurements was used. In Tables 5A and 5B, high negative log10 values, such as -6.7, -5.5, -4.8, etc., are associated with high enzyme activity, while log10 values close to zero or even positive log10 values are associated with low or no enzyme activity. For example, if the average CFU of 3 control treatment measurements for Gv_9 is 1.0 x 10 7 CFU / ml, and the average of 3 samples treated with H2B10 is 2.5 x 10 3 CFU / ml, then the log10 value of the CFU reduction of Gv_9 by H2B10 will be log10(2 x 10 3 / 10 7 ) = -3.7. Conversely, a reduction value of -3.7 means that the viable CFUs in the treated sample are reduced by 10 3.7 -fold ( = 5012-fold) compared to the untreated control. In the case of an inactive endolysin, for example, H4B3 against Gv_9, the treated Gv_9 CFU will be equal to the CFU measured in the control treatment sample, and the ratio of the two CFU values will be 1. Thus, the reduction value of H4B3 against Gv_9 is log10(1) = 0.0.

[0279] [Table 5A]

[0280]

[0281] Table 5B below describes the same data as Table 5A, but shown as the mean log10 activity of each construct against four Gardnerella strains. On the right and at the bottom, the mean of each native H-domain relative to all native B-regions (except B6) and the mean of each native B-region (except B6) relative to all native H-domains, as well as the activity rankings of the respective native H-domains and native B-regions, are shown.

[0282] [Table 5B]

[0283]

[0284] Table 5C depicts the activity rankings of all endolysins, based on the data in Table 5B [Table 5C]

[0285]

[0286] Table 5D depicts the mean log10 lysis of each Gardnerella strain used. The mean was calculated for the log10 activity of all tested constructs.

[0287] [Table 5D]

[0288] Mean log10 activity of all constructs against strains

[0289]

[0290] Tables 5A to 5C show that the activity of endolysins is highly specifically dependent on the H-domain / B-region combination and the bacterial strain against which it is tested. Each construct was assayed against four Gardnerella strains (see Table 5A). On average, the H-domain with the highest activity was H2, with an average 3.1 log10 unit reduction in CFU for all B-regions (except B6), followed by H7, H10, and H5 (see Table 5B). Among the B-regions, B10 had the highest activity, with an average 2.7 log10 unit reduction in CFU, followed by B11, B12, and B3.

[0291] Surprisingly and unexpectedly, the inventors found that several recombinant endolysins had stronger activity than any native endolysin (H1B1 to H12B12), especially when observed in all 4 Gardnerella strains tested (see Figures 7A to 7D)。In particular, H2B10, H2B11, and H2B12 have activity rankings 1, 2, and 3 respectively, and each is more active than any native endolysin (see Table 5C). The overall ranking of H7B3 is 4th (see Table 5C), and it is more active than any other native endolysin included in the experiment. In fact, the 6th most active native endolysin is H10B10, and the 13th most active native endolysin is H3B3. In summary, the recombinant intracellular endolysins according to the present disclosure may exhibit significantly higher activity than native endolysins.

[0292] Without being bound by a particular theory, the unexpected increase in Gardnerella - killing activity observed for the endolysins of the invention by domain swapping can be explained as follows. The native endolysins on prophages undergo a Darwinian evolutionary process in which the propagation of the entire prophage is optimized. However, the likelihood of mutations that simultaneously increase the catalytic activity of the endolysin and at the same time expand its cross - species host range only within the genus Gardnerella, resulting in higher propagation of the corresponding prophage, is very low. Instead, some Gardnerella prophages must have evolved the N - terminal catalytic domain of the endolysin to its highest activity, while some other prophages must have optimized the C - terminal region of the endolysin to obtain the broadest cross - Gardnerella - species activity. Thus, by combining the highly evolved N - terminal catalytic domain of one endolysin of the invention with the highly evolved C - terminal region of another endolysin of the invention encoded by a different genome from a different prophage, a recombinant endolysin with higher optimized Gardnerella - species killing activity than the native endolysins of the invention can be obtained. This is achieved, for example, by the recombinant endolysins H2B10, H2B11, H2B12, and H7B3 of the invention, all of which have higher killing activity than the native endolysin EL2 or any other native endolysin and span all Gardnerella species (see Figures 7A to 7D and Table 5A).

[0293] Furthermore, the activity of most constructs against Gv_23 (G. swidsinskii) is much higher than against the other three test strains (see Table 5A). The average activity of all constructs against each Gardnerella strain (see Table 5D) confirmed that Gv_23 is the most susceptible to endolysins, followed by Gv_9 and Gv_11, while Gv_17 (G. piotii) is the least susceptible. This order of susceptibility is mainly the case for endolysin constructs. Although Gv_23 is the most susceptible strain, there are differences of several log10 units for many constructs, and sometimes Gv_17 is more susceptible than Gv_9 or Gv_11 (e.g., for H2B10, the overall most active endolysin). Without being bound by a particular theory, the differences in susceptibility can be explained by structural defects (such as a weaker / thinner / more accessible cell wall in Gv_23) or stronger enzymatic activity of the tested endolysins against Gv_23.

[0294] In addition, it can be concluded that the concentration of endolysin in solution is crucial for its activity in the assay. The low-concentration constructs described in Table 4 generally also have low activity in the activity assay - especially the constructs with H-domains H4, H11, and H12 (which have low solubility in the B-region). There are some surprising cases, such as H12B11, which has a very low expression level but relatively high activity.

[0295] 4.5 - Activity pattern analysis

[0296] As Figure 8 shown in the dendrogram, the sequences of the native H-domains were aligned and compared to correlate with the activity pattern. The H-domains with the highest expected activity are also most closely related to each other. However, surprisingly, the most active N-terminal domain H2 has the highest homology with H6, but H6 has the lowest activity. The second most active H-domains H7 and H10 are also relatively distant from each other and from H2. The fourth most active H-domain H5 is most closely related to H7. In addition, the combination with the B-region that results in the highest recombinant endolysin activity does not lead to a predictable pattern. H2 has the highest binding activity with B10, B11, and B12. However, the second most active H-domain H7 has the highest binding activity with B3, as does its closest homolog H5.

[0297] The B-regions were also aligned to check for homology with the activity pattern, as Figure 9 shown in the dendrogram. The most active B-regions in the analysis of Tables 5A to 5C are B10, B11, B12, followed by B3, all with an average CFU reduction value higher than 2 log10 units. Contrary to the pattern of the H-domains, these 4 most active B-regions are the 4 closest homologs within the group of B-regions tested. Interestingly, the B5 and B7 regions are identical (see Figure 9 ). As can be seen from Figures 7A to 7D , H2B10, H2B11, and H2B12 obtained the best overall results.

[0298] As explained in Example 2, each native B-region contains two B-domains, namely the N-terminal cell wall region and the C-terminal cell wall domain. As Figure 10 and 11As shown, the sequences of each native B-domain within the B-region were also aligned and compared. 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 two repeat motifs within each B-region. The C-terminus of all B-domains is a conserved sequence (VNELL or VNKLL), and a homologous sequence can also be found at the C-terminus of the CW_7 motif (VNELL or VNEIL) of the protein Cpl-7, thus defining the boundaries of the two B-domains within each B-region. As an exception, B6 has only one truncated B-domain, which is likely the reason for the complete inactivity of EL6.

[0299] The conclusion is that a specific combination of H-domains and B-regions has been shown to be crucial, and each H / B combination resulting in an endolysin with higher killing activity compared to native endolysins is a surprising and unpredictable finding.

[0300] Example 5 - Activity Assay Against Beneficial Lactobacilli

[0301] A healthy vagina is mainly colonized by three Lactobacillus species: Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. They maintain an acidic pH of 3.5 - 4.5 by producing lactic acid and maintain a protective oxidative environment by producing H 2 O 2 . Recovery from BV is associated with the re-proliferation of these Lactobacilli within the vagina, and drugs against BV should advantageously not interfere with this process. Antibiotics clearly interfere with this process, which is why there is still a strong 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 those endolysins could lyse strains of the three most common Lactobacillus species in a healthy vagina. The experiment was conducted under anaerobic conditions at pH 5.0 using the method described in Reference Example 2. As Figure 12As shown, the recombinant lysins tested did not exhibit any killing activity against the three beneficial Lactobacillus species used, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. Although the lysin of the present invention exhibits high killing activity against Gardnerella, it is ineffective against the most common beneficial Lactobacillus species. Thus, these results confirm the genus-selective activity of the lysin of the present invention and its status as a candidate for an innovative drug against BV. In this regard, treatment of BV with the lysin of the present invention is far superior to currently available treatments, such as treatment with the antibiotics metronidazole and clindamycin.

[0302] Example 6 - Determination of the Activity of the Standard Treating Antibiotics Metronidazole and Clindamycin against Gardnerella Strains Growing in Suspension

[0303] One of the major drawbacks of BV treatment is the high recurrence rate in many women, which leads to repeated administration of antibiotics and is accompanied by instability of the microbiome and other side effects. Then, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Gardnerella cells of the strains also used for the lysin activity assay were measured using the methods described in Reference Examples 6 and 7. Briefly, the measurement protocol had to be substantially adapted from the international standard, which is not applicable to the measurement of MIC and MBC for Gardnerella. The main parameters to be changed were the growth medium (Gardnerella does not grow in Mueller-Hinton broth commonly used for MIC measurement), anaerobic conditions, incubation time, and the initial bacterial concentration in the first round of experiments. The initial concentration was changed from the standard 5×10 5 CFU / ml to 2.5×10 7 CFU / ml, mainly because in the vagina of BV patients, the cells are also very concentrated, and the effect of the antibiotic should be measured under conditions where the cell density is more comparable to those used for the lysin activity assay. The effects of metronidazole (obtained from Gatt-Koller) and clindamycin (obtained from Ratiopharm) on the suspended growth of Gardnerella strains were evaluated. Basically, a Gardnerella suspension of 2.5×10 7 CFU / ml was incubated with the specified concentration of the antibiotic and incubated under anaerobic conditions at 37°C for 48 hours. The MIC was defined as the minimum antibiotic concentration at which no growth was detected by OD measurement after 48 hours. The OD (610) was measured at the start and end of the experiment. At the end of the experiment, 2 μl of each reaction mixture was spotted on agar to determine the MBC. Table 6A summarizes the Figure 13 experimental results described therein. Resistance (R) was defined as > = 32 μg / ml for metronidazole and > 8 mg / ml for clindamycin. According to the international standard, sensitivity (S) was defined as <= 8 μg / ml for metronidazole and <= 2 μg / ml for clindamycin.

[0304] [Table 6A]

[0305]

[0306] [Table 6B]

[0307]

[0308] According to Figure 13 the results shown, all Gardnerella strains have low susceptibility to metronidazole and clindamycin. The measurement conditions for MIC and MBC are more stringent than the standard. For example, MBC is usually measured 90 , i.e., the antibiotic concentration that kills 90% of the cells within a defined time, while MBC is defined in this application as the minimum concentration that completely eliminates a 2.5×10 7 CFU / ml suspension. Nevertheless, these conditions are more comparable to those found in the vagina of BV patients. The high MIC and MBC values measured under these conditions can explain the high recurrence rate of BV. In contrast, the determined endolysins are bactericidal by definition because they cause the complete lysis of bacterial cells. Therefore, these results indicate that the intracellular endolysins of the present invention are superior to antibiotics in the treatment of BV.

[0309] A second round of MIC and MBC experiments was conducted, with some experimental parameters changed: (i) according to the CLSI (Clinical and Laboratory Standards Institute) standard, the starting cell number is now 1×10 5 -1×10 6 , (ii) powder of clindamycin hydrochloride (Sigma Aldrich, catalog number no. C5269) was used, (iii) NYC-III broth was used instead of Hardy broth, and (iv) 96-well plates were used instead of 384-well plates. As Figure 14 and shown in Table 6B, all Gardnerella strains still have very low MICs (8 - 128 μg / ml) to metronidazole, while clindamycin hydrochloride - contrary to Figure 13 the clindamycin presented in

[0310] Example 7 - Activity assay of the representative domain-swapped endolysin (H2B10) against the suspended growth of different Gardnerella strains.

[0311] To analyze the MIC and MBC of endolysin H2B10, a cell suspension of 1×10 5 -1×10 6 was used. H2B10 showed MICs in the low μg / ml range (0.5 - 4 μg / ml), indicating a high sensitivity of Gardnerella cells to the endolysin( Figure 15, Table 7). The conditions for measuring MBC are more stringent than the standard. For example, MBC is usually measured 90 , that is, the antimicrobial concentration that kills 90% of the cells within a defined time, while MBC is defined in the present application as the dose that reduces the initial cell count by at least 99.5%. H2B10, as a representative of the endolysins claimed herein, shows MIC and MBC far superior to the standard therapeutic antibiotic metronidazole, which is ineffective against many Gardnerella strains due to resistance formation. However, clindamycin gave inconsistent results. According to international standards, all four Gardnerella strains should be resistant to clindamycin (obtained from Ratiopharm) (MIC > 8 μg / ml) ( Figure 13 ), while clindamycin hydrochloride (Sigma Aldrich) has a much better bactericidal effect at a concentration ≤ 1 μg / ml ( Figure 15 ). In general, it is known that antibiotics alone cannot sufficiently eradicate the Gardnerella biofilm that is a hallmark of BV, which is a suspected reason for the very high reported recurrence rate of BV. In addition to leaving residual viable biofilm, antibiotics will eliminate some beneficial organisms of the vaginal microbiota and then open ecological niches for other pathogens (e.g., fungi). Therefore, endolysin-based therapy can selectively eradicate bacterial cells of the genus Gardnerella and presumably the biofilm without harming beneficial Lactobacilli, which is considered superior to the standard antibiotic treatment for BV.

[0312] Table 7

[0313]

[0314] Reference Example 1 - Cloning, Expression, and Purification of Endolysin

[0315] Materials :

[0316] · 96-well Multiscreen HTS Durapore 96-well Filterplatten, PS (Labshop catalog number no. 44.MSGVS22)

[0317] · PD MiniTrap desalting column with Sephadex G-25 (GE Lifescience, catalog number no. 28918007)

[0318] · Slide-A-Lyzer TM MINI dialysis device, 10K MWCO, 2 mL (Thermo Scientific, catalog number no. 88404)

[0319] · Fastbreak reagent (Promega, catalog number no. V8571)

[0320] · Lysis buffer: 50 mM phosphate pH 6, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP, 1x FastBreak, Benzonase

[0321] · Wash buffer I: 50 mM phosphate pH 6, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP (1.5 ml)

[0322] · Wash buffer II: 50 mM phosphate pH 6, 150 mM NaCl, 40 mM imidazole, 1 mM TCEP (1.5 ml)

[0323] · Elution buffer: 50 mM phosphate pH 6, 150 mM NaCl, 250 mM imidazole, 1 mM TCEP (1.1 ml).

[0324] Methods :

[0325] Transform the expression construct into Escherichia coli strain Bl21(DE3) and select using the appropriate antibiotic. Resuspend the cells from 2 ml of culture (TB + lactose, 25 °C, O / N) in 1.5 ml of lysis buffer and lyse by FastBreak reagent (Promega). Separate the intracellular soluble fraction by centrifugation at 15,000 g for 30 min at 4 °C. Load the soluble protein fraction onto 100 μL of nickel affinity matrix, wash with 15 column volumes (CV) each of wash buffer I and II, and then elute with 10 CV of elution buffer. Then, the eluate buffer can be changed to 20 mM phosphate pH 6.0, 150 mM NaCl using a desalting column to remove imidazole. After elution (or appropriate buffer change), adjust the concentration of the purified protein to 0.2 mg / ml, and then filter-sterilize the solution using a 96-well filter plate.

[0326] Refer to Example 2 - Activity assay in bacterial suspension

[0327] Materials :

[0328] 1. Hardy broth, autoclaved at 121 °C for 20 min:

[0329] · 12.0 g of tryptic digest of casein (Sigma Aldrich, catalog number no. 70172-100G)

[0330] · 10.0 g peptone (Sigma Aldrich, catalog no. 82450-100G)

[0331] · 5.0 g peptide digest of animal tissue (Sigma Aldrich, catalog no. 70174-100G)

[0332] · 5.0 g sodium chloride (Carl Roth, catalog no. 3957.1)

[0333] · 3.0 g beef extract (Sigma Aldrich, catalog no. B4888-50G)

[0334] · 3.0 g yeast extract (Sigma Aldrich, catalog no. Y1625-250G)

[0335] · 1.0 g soluble starch (Sigma Aldrich, catalog no. S9765-250G)

[0336] · Deionized H 2 O to 1 liter (produced using Millipore RiOs Essential16 in the PhagoMed laboratory)

[0337] 2. Hardy broth agar, autoclaved at 121 °C for 20 min: same as Hardy broth, but using 15 g Agar Bacteriogical (OXOID catalog no. #LP0011)

[0338] 3. Hardy broth top agar, autoclaved at 121 °C for 20 min: same as Hardy broth, but using 7 g Agar Bacteriogical (OXOID catalog no. #LP0011)

[0339] 4. NYC-III medium, pH 5.0, autoclaved at 121 °C for 20 min, then horse serum added thereafter (NYC-III-HS-5.0)

[0340] · 12 g HEPES (Sigma Aldrich, catalog no. H4034-100G)

[0341] · 7.5 g peptone No. 3 (BD, catalog no. 211693)

[0342] · 1.9 g yeast extract (Sigma Aldrich, catalog no. Y1625-250G)

[0343] · 2.5 g of sodium chloride (Sigma Aldrich, catalog number S9888-1kg-M)

[0344] · 2.5 g of glucose (MW 180.16 g / mol) (Sigma Aldrich, catalog number no. G6152-1KG)

[0345] · Deionized water to a total volume of 450 ml

[0346] · 50 ml of horse serum (HS), heat-inactivated 100 ml (Thermo Fisher Scientifi, catalog number no. 26050070), added after autoclaving

[0347] 5. General materials:

[0348] · BD chocolate agar plates for Gardnerella (BD, catalog number no. 254060)

[0349] · BD Schaedler / 5% sheep blood plates for Lactobacillus (BD, catalog number no. 24042)

[0350] · Isovitalex (BD, catalog number no. 211876)

[0351] · Hardy broth + Isovitalex (see above), adjusted to the indicated pH

[0352] · Hardy agar + Isovitalex (see above)

[0353] · Hardy top agar + Isovitalex (see above)

[0354] · 96-U-well plates (Sigma Aldrich, catalog number no. M2311-100EA)

[0355] · 96 flat-bottom plates with lids (Labshop, catalog number no. 44.781662)

[0356] · Greiner 384-well plates (Sigma Aldrich, catalog number no. M1937-32EA)

[0357] · Anaerobic agent bags (Sigma-Aldrich, catalog number no. 68061-10SACHETS-F)

[0358] · Anaerobic indicator test (Sigma-Aldrich, catalog number no. 59886-1PAK-F)

[0359] · Anaerobic jar (Sigma - Aldrich, catalog number no. 28029 - 1EA - F) or a plastic lunch box sealed with a rubber gasket, purchased from a local home appliance store.

[0360] 6. Bacterial strains:

[0361] Gardnerella strains:

[0362] · Gv_1: UGent 09.07

[0363] · Gv_8: UGent 25.49

[0364] · Gv_9: ATCC 14018

[0365] · Gv_10: UGent 06.41

[0366] · Gv_11: UGent 09.48

[0367] · Gv_17: UGent 18.01

[0368] · Gv_23: GS10234(FC2)

[0369] Lactobacillus strains:

[0370] · Lactobacillus jensenii PB2003 - 013 - T2 - 2

[0371] · Lactobacillus gasseri 020566

[0372] · Lactobacillus crispatus LAB117

[0373] Methods :

[0374] Gardnerella cells were recovered from the frozen stock by plating on chocolate agar plates (Beckton Dickinson) and incubating anaerobically at 37 °C for 48 h. For Lactobacillus, BD Schaedler / 5% sheep blood agar plates were used instead. Colonies were scraped from the plates, resuspended in Hardy broth or NYC-III-HS-5.0 at the indicated pH values, and the suspension was adjusted to an OD (610 or 620 nm as indicated) of 0.1. It is important to note that two Tecan microplate readers (with 610 nm or 620 nm filters respectively) were interchangeable in the experiments. Although it made no difference to the experiment, the exact wavelength used was specified in each example. If not otherwise stated, for different species / endolysin combinations, 90 μl of the cell suspension was mixed with 10 μl of the endolysin solution in a 384-well plate. OD (610 - 620 nm as indicated) was measured at the start and end of the reaction, either as continuous kinetics or two measurement points in a Tecan F200 microplate reader. The reaction was incubated at 37 °C for 5 h (or other specified time) under the indicated anaerobic, microaerophilic or aerobic conditions. Anaerobic conditions mean complete depletion of oxygen from the container (Sigma-Aldrich anaerobic jar or sealed lunch box), where bacteria were incubated with an anaerobic bag and anoxia was confirmed by an anaerobic indicator inside the container. In the case of the indicated microaerophilic conditions, the candle-in-jar method was used (lighting a tea candle in a suitable sealable container, which reduces the oxygen content until the flame goes out). Then each well was diluted in a 96-U well bottom plate in 5 steps (10 -1 to 10 -5 ), and 2 μl of each dilution of each reaction mixture was inoculated onto BD chocolate agar plates or BD Schaedler / 5% sheep blood agar plates, for Gardnerella and Lactobacillus respectively, for detection and quantification of viable CFU. The detection plates were incubated at 37 °C for 48 h under anaerobic conditions.

[0375] Refer to Examples 6 and 7 - MIC and MBC measurements

[0376] Materials :

[0377] General materials:

[0378] · Metronidazole (Gatt-Koller, Metronidazolum mikronisiert, 10 g, 606293914)

[0379] · Clindamycin (Ratiopharm, 300 mg / 2 ml ampoules 5x)

[0380] · Clindamycin Hydrochloride (Sigma Aldrich, 10 mg, catalog number no. C5269)

[0381] · Lysin H2B10 [530 μg / ml] in MES buffer (50 mM MES, 100 mM NaCl, 8 mM MgSO 4 , pH = 5.5)

[0382] · BD Chocolate Plate for Gardnerella (BD, catalog number no. 254060)

[0383] · BD Schaedler / 5% Sheep Blood Agar Plate for Lactobacillus (BD, catalog number no. 254042)

[0384] · Isovitalex (BD, catalog number no. 211876)

[0385] · Hardy Broth + Isovitalex (see above), adjusted to the indicated pH

[0386] · NYC-III + HS Broth, pH 5.5 NYC-III-HS Agar Plate: NYC-III + HS medium as described above, but with 1.5% agar added before autoclaving

[0387] · Hardy Agar + Isovitalex (see above)

[0388] · Hardy Top Agar + Isovitalex (see above)

[0389] · 96-U-Well Plate (Sigma Aldrich, catalog number no. M2311-100EA)

[0390] · 96 Flat-Bottom Plate with Lid (Labshop, catalog number no. 44.781662)

[0391] · Greiner 384-Well Plate (Sigma Aldrich, catalog number no. M1937-32EA)

[0392] · Anaerobic Agent Pouch (Sigma-Aldrich, catalog number no. 68061-10SACHETS-F)

[0393] · Anaerobic Indicator Test (Sigma-Aldrich, catalog number no. 59886-1PAK-F)

[0394] · An anaerobic jar (Sigma-Aldrich, catalog no. 28029-1EA-F) or a plastic lunch box sealed with a rubber gasket, purchased from a local home appliance store.

[0395] Methods :

[0396] Bacteria were streaked from the frozen stock onto BD Choc agar plates (for Gardnerella) and incubated at 37 °C under anaerobic conditions for 48 h. Colonies were scraped from the plates, resuspended in Hardy broth or NYC-III-HS-pH 5.0, and the suspension was adjusted to an OD (at 610 or 620 nm as indicated) of 0.05. It should be noted that two Tecan microplate readers (each with a 610 nm or 620 nm filter) were interchangeable in the experiment. Although it made no difference to the experiment, the exact wavelength used was specified in each example. For each desired final concentration, the antibiotic was made as a 20× stock solution. In a 384-well plate, 95 μl of the cell suspension was mixed with 5 μl of the antibiotic dilution. The OD (at 610 - 620 as indicated) was measured at the start of the reaction, and then the plate was incubated at 37 °C under anaerobic conditions for 48 h. After that, the OD (at 610 - 620 as indicated) was measured again for MIC determination, where the MIC was defined as the lowest concentration of the antibiotic at which the OD (at 610 - 620 as indicated) was not higher than the level measured at the start of the experiment. After measuring the OD, 2 μl of each well was spotted onto a NYC-III+HS agar plate. Then the plate was incubated at 37 °C under anaerobic conditions for another 48 h. After incubation, the cell growth at each spot was evaluated, and the MBC was defined as the lowest antibiotic concentration at which no bacterial growth was observed on the plate. For each condition, the experiment was performed in triplicate.

[0397] In the second round of MIC and MBC experiments using antibiotics, the Gardnerella cell suspension was adjusted to McFarland standard 0.5 (approx. OD(610) 0.07) and subsequently diluted 1:75 according to CLSI (Clinical and Laboratory Standards Institute) standards. The antibiotics were prepared according to CLSI standards and 50 μl of the cell suspension was mixed with 50 μl of the antibiotic in a 96-well plate. Additionally, the MIC and MBC were determined as described above.

[0398] For MIC and MBC determination of the domain-swapped endolysin H2B10, 50 μl of the Gardnerella cell suspension was mixed with 50 μl of the solution containing H2B10 in a 96-well plate, which was serially diluted 1:1. The OD was measured at the start of the reaction 610, and then the plate was incubated at 37 °C under anaerobic conditions for 48 h. Thereafter, OD(610) was measured again for MIC determination, where MIC was defined as the lowest concentration of H2B10 at which the OD was not higher or only slightly higher than the level measured at the start of the experiment.

[0399] [Table 7]

[0400]

[0401] *Row 1 = Name according to this application, if applicable; Row 2 = Corresponding natural amino acid sequence

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

Claims

1. A recombinant lysin, comprising: (i) an N-terminal catalytic domain having an amino acid sequence as shown in SEQ ID NO:2; (ii) a C-terminal cell wall binding region comprising a first cell wall binding domain and a second cell wall binding domain, wherein the first cell wall binding domain comprises an amino acid sequence that is at least 94% identical to the amino acid sequence of any one of SEQ ID NO:28, 30, 32, and 19, and the second cell wall binding domain comprises an amino acid sequence that is at least 96% identical to the amino acid sequence of any one of SEQ ID NO:29, 31, 33, and 20, and (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region.

2. The recombinant lysin according to claim 1, wherein the first cell wall binding domain comprises the amino acid sequence of any one of SEQ ID NO:28, 30, 32, and 19, or comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:28, 30, 32, and 19, and / or the second cell wall binding domain comprises the amino acid sequence of any one of SEQ ID NO:29, 31, 33, and 20, or comprises an amino acid sequence that is at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:29, 31, 33, and 20.

3. The recombinant lysin according to claim 1, wherein the first cell wall binding domain is at the N-terminus of the second cell wall binding domain.

4. The recombinant lysin according to any one of claims 1-3, wherein the N-terminal catalytic domain comprises the amino acid sequence as shown in SEQ ID NO:2, the first cell wall binding domain comprises the amino acid sequence as shown in SEQ ID NO:28, and the second cell wall binding domain comprises the amino acid sequence as shown in SEQ ID NO:

31.

5. A recombinant lysin, comprising: (i) an N-terminal catalytic domain having an amino acid sequence of any one of SEQ ID NO:7, 1 to 5, and 10 to 12, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:7, 1 to 5, and 10 to 12; (ii) A C-terminal cell wall binding region, which comprises at least one cell wall binding domain, wherein said cell wall binding domain comprises the amino acid sequence of any one of SEQ ID NO: 19, 20, 15 to 18, 21 to 24 and 24 to 33, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 19, 20, 15 to 18, 21 to 24 and 24 to 33, and (iii) A linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region; wherein the N-terminal catalytic domain is from a first native endolysin, at least one cell wall binding domain is from a second native endolysin, and wherein the first and second native endolysins are encoded by different genomes from different prophages.

6. The recombinant endolysin according to claim 5, wherein said 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 comprises the amino acid sequence of any one of SEQ ID NO: 19, 15, 17, 21, 23, 26, 28, 30 and 32, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 19, 15, 17, 21, 23, 26, 28, 30 and 32, and said second cell wall binding domain comprises the amino acid sequence of any one of SEQ ID NO: 20, 16, 18, 22, 24, 27, 29, 31 and 33, or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 20, 16, 18, 22, 24, 27, 29, 31 and 33.

7. The recombinant endolysin of claim 5 or 6, which has killing activity against Gardnerella.

8. The recombinant endolysin according to any one of claims 1-7, wherein the linker region consists of the following amino acid sequence consisting of: (i)X 1 X 2 GLNGX 3 X 4 NGGS, where X 1 is N or K, X 2 is A or V, X 3 is Y or C and X 4 is K or Q; or (ii)(XXX) n , where each X is independently G, A, or S, and where n corresponds to the number of repeats of the sequence XXX.

9. A polynucleotide encoding the recombinant endolysin according to any one of claims 1-8.

10. A pharmaceutical composition comprising the recombinant endolysin according to any one of claims 1-8 or the polynucleotide of claim 9 and further comprising a pharmaceutically acceptable carrier and / or diluent.

11. Use of the recombinant endolysin as defined in any one of claims 1-8, or the polynucleotide as defined in claim 9, or the pharmaceutical composition as defined in claim 10 in the preparation of a drug for the treatment of a disease or disorder, wherein the disease or disorder is bacterial vaginosis.

12. Use according to claim 11, wherein the bacterial vaginitis is caused by Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii and / or Gardnerella swidsinskii.

13. Use according to claim 11 or 12, wherein the recombinant lysin, polynucleotide or pharmaceutical composition is administered locally.

14. Use according to claim 11 or 12, wherein the recombinant lysin, polynucleotide or pharmaceutical composition is administered into the vagina of a female subject and / or into or onto the glans penis, foreskin or urethral orifice of a male subject.

15. Use according to claim 11 or 12, wherein the recombinant lysin, polynucleotide or pharmaceutical composition is co-administered with a compound or composition that adjusts the vaginal pH to 4.0 - 6.

0.

16. A plasmid comprising the polynucleotide of claim 9.

17. A bacterial host cell comprising the plasmid of claim 16.

18. The bacterial host cell of claim 17, which is an Escherichia coli cell.

19. Use of the lysin of any one of claims 1 - 8 in the preparation of a diagnostic kit for an in vitro method of diagnosing a disease or disorder treatable with the lysin of any one of claims 1 - 8, the method comprising the steps of: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of : the C-terminal cell wall binding region of the lysin of any one of claims 1 - 8, and optionally the N-terminal catalytic domain of the lysin of any one of claims 1 - 8, wherein the sample comprises microbial cells and wherein the C-terminal cell wall binding region of the lysin is optionally labeled; (ii) testing whether the polypeptide binds and / or lyses the microbial cells of the sample; and (iii) if the polypeptide binds and / or lyses the microbial cells, determining that the disease or disorder is treatable with the lysin of any one of claims 1 - 8.

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