New Gardnerella endolysin and its use

Abstract

The present invention relates to novel species-selective bacteriophage endolysins and their use in treating bacterial vaginosis (BV). The present invention provides recombinant endolysins, i.e., domain-swapped endolysins. The present invention also relates to the use of the endolysins in treating diseases or conditions, such as bacterial infections, in particular BV. The present invention further relates to polynucleotides encoding the endolysins. The polynucleotides may also be used to treat such diseases or conditions. The present invention also provides pharmaceutical compositions comprising the endolysins of the present invention for treating such diseases or conditions. The endolysins, polynucleotides and pharmaceutical compositions may be administered topically, in particular topically in the vagina.

Description

[0001] The present invention relates to novel species-selective bacteriophage endolysins and their use in treating bacterial vaginosis (BV). The present invention provides recombinant endolysins, i.e. domain-swapped endolysins. The present invention also relates to the use of the endolysins in treating diseases or conditions, such as bacterial infections, in particular BV. The present invention further relates to polynucleotides encoding the endolysins. The polynucleotides may also be used to treat such diseases or conditions. The present invention also provides pharmaceutical compositions comprising the endolysins of the present invention for treating such diseases or conditions. The endolysins, polynucleotides and pharmaceutical compositions may be administered topically, in particular topically into the vagina.

[0002] Bacterial vaginosis (BV), also referred to in the literature as bacterial vaginitis, nonspecific vaginosis, and nonspecific vaginitis, is the most common vaginal infection worldwide and is associated with serious adverse outcomes, including premature birth, postpartum endometritis, and increased risk of HIV infection. This is a dysbiosis of the vagina in which commensal Lactobacilli are replaced by polymicrobial biofilms, the pH increases from a natural 3.5-4.5 to 5.5, and a foul-smelling fluid is formed. Reported prevalence rates range from 10-40%, depending on the population studied. However, suboptimal diagnostic methods and a high proportion of asymptomatic patients make the true prevalence of BV difficult to determine. Gardnerella vaginalis (G. vaginalis) is a bacterial species associated with BV.

[0003] The pathogenesis of BV remains poorly understood. It is generally defined as a pathological state characterized by the loss of normal vaginal flora (especially H2O2-producing Lactobacillus) and the overgrowth of anaerobic bacteria (including Gardnerella vaginalis, Mobiluncus, and Mycoplasma hominis). However, recent data suggest the major role of Gardnerella vaginalis as a specific and sexually transmitted pathogen in BV (Muzny et al., 2016, J. of Infect. Dis. 214 Suppl. 1., SI).

[0004] In the 1950s, large numbers of small, pleomorphic, Gram-adventitious rod-shaped bacteria were observed in the reproductive tracts of women with BV. This organism was originally called Haemophilus vaginalis, and has been repeatedly renamed as more information about its characteristics became available, and 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, with the most common species being renamed G. vaginalis sensu stricto, G. leopoldii, G. pioti, and G. swidsinskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661).

[0005] Gardnerella bacteria are special in that they are Gram-indeterminate, i.e. they do not form an outer membrane that defines Gram-negative 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 dark purple color typical of Gram-positive species. Rather, Gardnerella cells can appear both Gram-positive and Gram-negative in Gram staining. Phylogenetic analysis based on 16S rRNA places Gardnerella in the Gram-positive Bifidobacteriales.

[0006] During BV, epithelial surfaces are covered with dense Gardnerella vaginalis in biofilms that are often stubborn and difficult to treat. Biofilms are adherent communities of microorganisms held together by a polymer matrix composed of polysaccharides, proteins, and / or nucleic acids. The unique gene expression patterns and physical structure of the biofilm increase the resistance of the bacteria to many negative stimuli, including chemical disinfectants, extreme pH values, host immune defenses, and antibiotics. The standard of care for BV is the antibiotics metronidazole and clindamycin, but they often fail to eradicate the biofilm, resulting in recurrence rates as high as 60% within 6 months. In addition, although some residual viable biofilms are left behind, treatment with antibiotics clears the vaginal microbiota, which opens this ecological niche for other pathogens (e.g., fungi). Therefore, a common effect 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 benefit.

[0007] Therefore, there is a great need for new methods and compositions for treating vaginal Gardnerella infections, in particular BV, for example, by selectively killing bacterial cells of the genus Gardnerella, preferably without harming the beneficial Lactobacilli which repopulate the vagina. Therefore, the technical problem underlying the present invention is to provide new means and methods for treating BV.

[0008] This technical problem is solved by providing the embodiments characterized in the claims.

[0009] The present invention is based on the preparation of novel recombinant Gardnerella prophage endolysins having unexpected properties and structures which make them particularly suitable for a variety of uses and methods, particularly for the treatment, decontamination or detection of bacterial infections and disorders, particularly those associated with Gardnerella.

[0010] A first aspect of the present invention provides an endolysin comprising or consisting of

[0011] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0012] (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

[0013] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0014] Among them, endolysin has anti-Gardnerella killing activity.

[0015] In one aspect of the invention, the N-terminal catalytic domain is from a first natural endolysin, the linker region and the C-terminal cell wall binding region are from a second natural endolysin, and the first and second natural endolysins are encoded by different genomes from different prophages. Therefore, the present invention provides a recombinant endolysin comprising or consisting of

[0016] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0017] (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

[0018] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0019] wherein 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, and

[0020] The recombinant endolysin has anti-Gardnerella killing activity.

[0021] Gardnerella is unique in that it is a Gram-indeterminate species: it does not form an outer membrane that defines a true Gram-negative species. The cell wall is typically very thin and contains only 10% or less peptidoglycan. This suggests that peptidoglycan-degrading enzymes, such as endolysin proteins, are not effective in lysing the bacterial cell wall of Gardnerella. However, in the case of the present invention, new recombinant endolysins have been identified that have the favorable properties of being effective in killing Gardnerella species and can therefore be used as new therapeutics for the treatment of BV.

[0022] There are mainly three lactobacillus species in the 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 H2O2. Recovery from BV is accompanied by the recovery of the population of these lactobacilli in the vagina. However, the disadvantage of antibiotics (which are conventionally used to treat BV) is that they interfere with the population recovery process of lactobacilli in the vagina. In contrast, the new recombinant endolysin of the present invention advantageously has species-selective killing activity against Gardnerella and does not harm lactobacilli. In addition, the attached examples show that all tested Gardner strains have low sensitivity to metronidazole and clindamycin, which are conventionally used to treat BV. This can explain the high recurrence rate of BV. This also proves that the endolysin of the present invention is superior to antibiotics in the treatment of BV. Therefore, the treatment of BV with the endolysin of the present invention is far superior to currently available treatments, such as treatment with antibiotics metronidazole and clindamycin.

[0023] The term "from the first natural endolysin" herein means that the respective part (i.e., the N-terminal catalytic domain) is identical to the first natural endolysin or is a functional variant of the first natural endolysin. As defined herein, a functional variant is a polypeptide having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity and most preferably at least 99.7% identity) with the amino acid sequence of the respective part (i.e., the N-terminal catalytic domain) of the first natural endolysin and producing a functional endolysin, wherein the function includes killing activity against Gardnerella. The amino acid sequences of several natural endolysins are provided below and summarized in Table 7.

[0024] According to this, the term "from a second natural endolysin" means that the respective part (i.e., the linker region and the C-terminal cell wall binding region) is identical to the second natural endolysin or is a functional variant of the second natural endolysin, i.e., an endolysin that is different from the first natural endolysin. As defined herein, a functional variant is a polypeptide that has 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) with the amino acid sequence of the respective part (i.e., the linker region and the C-terminal cell wall binding region) of the second natural endolysin and produces a functional endolysin, wherein the function includes anti-Gardnerella killing activity.

[0025] 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 natural endolysin (EL) 2. "C-terminal cell wall binding region" refers to one or more cell wall binding domains. The linker and cell wall binding domain together represent the so-called "B-region". For example, B10 refers to the B-region of natural EL10. Similarly, B11_N refers to the N-terminal cell wall binding domain of natural EL11, B12_C refers to the C-terminal cell wall binding domain of natural EL12, and so on.

[0026] The present invention further provides an endolysin comprising or consisting of

[0027] (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 5, 7 or 10 to 12, or a functional variant thereof having at least 80% identity with an amino acid sequence of any one of SEQ ID NOs: 1 to 5, 7 or 10 to 12;

[0028] (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 being independently selected from a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 15 to 24 and 26 to 33, respectively, and any functional variants thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, 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: 15 to 24 and 26 to 33, respectively; and

[0029] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0030] The endolysin has anti-Gardnerella killing activity.

[0031] As shown in the appended claims, the most active N-terminal catalytic domain (also referred to as "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).

[0032] Thus, in a preferred aspect of the invention, the N-terminal catalytic domain consists of a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 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 an amino acid sequence of any one of SEQ ID NOs: 2, 7, 10 and 5;

[0033] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0034] Thus, in a preferred aspect of the invention, the N-terminal catalytic domain consists of a polypeptide comprising or consisting of: an 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 comprising or consisting of an 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: NO:10 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) any functional variant thereof; or even more preferably comprises or consists of the amino acid sequence of SEQ ID NO:7, or 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) any functional variant thereof 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:7. 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;

[0035] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0036] It is also shown in the accompanying Examples that B-region B10 (comprising the cell wall binding domains of SEQ ID NOs: 28 and 29) is the most active, followed by B11 (comprising the cell wall binding domains of SEQ ID NOs: 30 and 31), B12 (comprising the cell wall domains of SEQ ID NOs: 32 and 33) and B3 (comprising the cell wall domains of SEQ ID NOs: 19 and 20).

[0037] Thus, in a preferred aspect of the invention, the cell wall binding domain is selected from a polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33, 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) with the amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33;

[0038] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0039] 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: an amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33, 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) with an amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33;

[0040] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0041] In a preferred aspect of the 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 the group consisting of SEQ ID NO: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and the second cell wall binding domain is selected from the group consisting of SEQ ID 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.

[0042] In a more preferred aspect of the present invention, the endolysin comprises two cell wall binding domains (B-domains) of natural endolysin EL10 (SEQ ID NOs: 28 and 29), natural endolysin EL11 (SEQ ID NOs: 30 and 31), natural endolysin EL12 (SEQ ID NOs: 32 and 33) or natural endolysin EL3 (SEQ ID NOs: 19 and 20), even more preferably natural endolysin EL10 (SEQ ID NOs: 28 and 29); or a functional variant thereof. The functional variant may also be a set of two B-domains, which have 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 two cell wall binding domains (B-domains) of natural endolysin EL10 (SEQ ID NOs: 28 and 29), natural endolysin EL11 (SEQ ID NOs: 30 and 31), natural endolysin EL12 (SEQ ID NOs: 32 and 33) or natural endolysin EL3 (SEQ ID NOs: 19 and 20), even more preferably natural endolysin EL10 (SEQ ID NOs: 28 and 29);

[0043] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0044] In one aspect of the 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) with 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 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: 19 and / or 20; NO:32 and / or 33 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, 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) any functional variant thereof; even more preferably comprising or consisting of: SEQ ID NO:30 and / or 31, or with SEQ ID NO:32 and / or 33; NO:30 and / or 31 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, 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) any functional variant thereof; or even more preferably comprising or consisting 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) with the amino acid sequence of SEQ ID NO:28 and / or 29;

[0045] The endolysin is thus functional, wherein said function comprises the ability to lyse the cell wall of Gardnerella.

[0046] Preferably the sequence VNELL or VNKLL, more preferably VNELL, is located at the C-terminus of the B-domain. In case there are multiple B-domains within the B-region, it is also preferred that the sequence VNELL or VNKLL, more preferably VNELL, is located at the C-terminus of each B-domain.

[0047] Surprisingly and unexpectedly, it has been found that in the context of the present invention, several recombinant endolysins have stronger activity than natural endolysins, especially 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 natural endolysins tested. Therefore, the recombinant endolysins according to the present invention surprisingly exhibit an activity higher than that of natural endolysins.

[0048] Therefore, it is preferred that the "anti-Gardnerella killing activity" of the recombinant endolysin of the present invention is enhanced compared to the killing activity of natural endolysins (eg, natural endolysins EL1-EL12 (having the amino acid sequences shown in Table 7)).

[0049] Based on the relatively high activity of endolysins H2B10, H2B11, H2B12 and H7B3, these endolysins (or their functional variants) are preferred in the present invention. Therefore, the endolysins of the present invention are preferably:

[0050] (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or 7, or any 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;

[0051] (ii) a C-terminal cell wall binding region comprising or consisting of at least one (preferably two) cell wall binding domains independently selected from a polypeptide comprising or consisting of an amino acid sequence of SEQ ID NOs: 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) with the amino acid sequence of SEQ ID NOs: 19, 20 and 28 to 33, respectively; and

[0052] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region consisting of a polypeptide comprising or consisting of the amino acid sequence X1X2GLNGX3X4NGGS, wherein X1 is N or K, preferably N, X2 is A, X3 is Y and X4 is K or Q,

[0053] Wherein the endolysin has anti-Gardnerella killing activity. Regarding the linker region, as described below, it is pointed out that the linker region can also be composed of a polypeptide, and the polypeptide comprises the amino acid sequence (XXX) n Or by amino acid sequence (XXX) n wherein each X is independently G, A or S, preferably wherein the amino acid sequence (XXX) n Yes (GGS) n , wherein n corresponds to the number of repetitions of sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6.

[0054] In the attached examples, the recombinant endolysin H2B10 was shown to have 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:

[0055] (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, 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;

[0056] (ii) a C-terminal cell wall binding region comprising or consisting of two cell wall binding domains, said cell wall binding domain consisting of a polypeptide 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

[0057] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region consisting of a polypeptide comprising or consisting of the amino acid sequence X1X2GLNGX3X4NGGS, wherein X1 is N, X2 is A, X3 is Y and X4 is K,

[0058] Wherein the endolysin has anti-Gardnerella killing activity. As described above, preferably compared with natural endolysin, such as natural endolysin EL1-EL12 (having the amino acid sequence shown in Table 7), the "anti-Gardnerella killing activity" of the recombinant endolysin of the present invention is enhanced.

[0059] With regard to the linker region, as described below, it is noted that the linker region may also consist of a polypeptide comprising the amino acid sequence (XXX) n Or by amino acid sequence (XXX) n wherein each X is independently G, A or S, preferably wherein the amino acid sequence (XXX) n Yes (GGS) n , wherein n corresponds to the number of repetitions of sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6.

[0060] 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 be independently selected from a polypeptide comprising or consisting of: the amino acid sequences of SEQ ID NOs: 15 to 24 and 26 to 33, respectively, and 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, 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, respectively, whereby the polypeptide is functional, wherein the function comprises 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. Preferred C-terminal cell wall binding regions are as defined above and below.

[0061] The endolysin of the present invention preferably does not comprise the H-domain or B-region of the natural endolysin EL6. The amino acid sequences of the H-domain and B-region of the natural endolysin EL6 are shown in Table 7.

[0062] The linker region may consist of a polypeptide having a length of 6 to 18 amino acids, preferably a length of 9 to 15 amino acids, and even more preferably a length of 12 amino acids. Preferably, the linker region may consist of a polypeptide comprising or consisting of the following amino acid sequence: (i) (XXX) n , wherein each X can independently be G, A or S, preferably wherein the amino acid sequence (XXX) n Yes (GGS) n , wherein n corresponds to the number of repetitions of the sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6, or (ii) XXGLNGXXNGGS, wherein X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q. As described above, in one aspect of the endolysin of the present invention, the N-terminal catalyst domain is identical to or derived from a first natural endolysin, the linker region and the C-terminal cell wall binding region are identical to or derived from a second natural endolysin, and the first and second natural endolysins are encoded by different genomes from different bacteriophages.

[0063] The recombinant endolysin of the present invention has killing activity against Gardnerella. For example, the endolysin of the present invention may have killing activity against Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii, preferably against all. The killing activity of the endolysin of the present invention as described above against Gardnerella is preferably a genus-selective killing activity against Gardnerella. Herein, "genus-selective killing activity against Gardnerella" means that the endolysin of the present invention does not have killing activity against general bacteria. Preferably, the endolysin of the present invention has killing activity against Gardnerella, but not against lactobacillus. In particular, it is preferred that the endolysin does not have killing activity against Lactobacilli crispatus, Lactobacilli gasseri and / or Lactobacilli jensenii. More preferably, the endolysin has no killing activity against all of these lactobacilli, namely Lactobacilli crispatus, Lactobacilli gasseri and Lactobacilli jensenii.

[0064] The present invention also relates to polynucleotide molecules encoding endolysins as described above. The nucleic acid molecule may be DNA, for example, cDNA or RNA. Herein, the term "polynucleotide" or "polynucleotide molecule" is used synonymously with the term "nucleic acid molecule".

[0065] The present invention also relates to vectors comprising polynucleotide molecules 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. Therefore, one aspect of the present invention relates to a plasmid comprising a polynucleotide of the present invention. Those skilled in the art will recognize that the selection of expression vectors can be determined by the selection of host cells.

[0066] The present invention also provides host cells comprising polynucleotides according to the present invention or vectors / plasmids 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. Therefore, one aspect of the present invention relates to bacterial host cells comprising plasmids according to the present invention, preferably wherein the bacterial host cell is an Escherichia coli cell.

[0067] 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 wherein the endolysin is expressed.

[0068] Another aspect of the present invention provides a pharmaceutical composition comprising

[0069] (a) an endolysin according to the present invention;

[0070] (b) a polynucleotide molecule according to the present invention;

[0071] (c) a vector / plasmid according to the invention;

[0072] (d) a host according to the invention; and / or

[0073] (e) Bacteriophage capable of expressing the endolysin according to the present invention

[0074] and a pharmaceutically acceptable carrier, diluent or excipient. For example, the pharmaceutical composition of the present invention may comprise the endolysin of the present invention, the polynucleotide molecule of the present invention and a pharmaceutically acceptable carrier and / or diluent.

[0075] Another aspect of the present invention relates to

[0076] (a) an endolysin according to the present invention;

[0077] (b) a polynucleotide molecule according to the present invention;

[0078] (c) a vector / plasmid according to the invention;

[0079] (d) a host according to the present invention;

[0080] (e) a bacteriophage capable of expressing an endolysin according to the invention; and / or

[0081] (f) Pharmaceutical composition according to the present invention

[0082] For treating a disease or condition. For example, the invention provides an endolysin according to the invention, a polynucleotide molecule according to the invention or a pharmaceutical composition according to the invention for treating a disease or condition. The disease or condition 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.

[0083] In one aspect of the invention, the recombinant endolysin of the invention, the polynucleotide molecule of the invention or the pharmaceutical composition of the invention can be administered topically, preferably topically to the vagina of a subject. Therefore, in one aspect of the invention, the recombinant endolysin of the invention, the polynucleotide molecule of the invention or the pharmaceutical composition of the invention is administered to the vagina of a subject.

[0084] The accompanying examples show that the activity of the recombinant endolysin of the invention is particularly high at a pH of about pH 5. Therefore, one aspect of the invention relates to a recombinant endolysin of the invention, a polynucleotide molecule of the invention or a pharmaceutical composition of the invention, wherein 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 that adjust the vaginal pH include, but are not limited to, phosphates, lactic acid (e.g., a natural acidifying substance secreted by lactobacilli to create an acidic environment) or other organic acids, for example, carboxyl-substituted polymers.

[0085] Another aspect of the present invention relates to

[0086] (a) an endolysin of the present invention;

[0087] (b) a polynucleotide molecule of the present invention;

[0088] (c) vectors / plasmids of the present invention;

[0089] (d) a host of the present invention;

[0090] (e) a bacteriophage capable of expressing an endolysin of the present invention; and / or

[0091] (f) Pharmaceutical composition of the present invention

[0092] Used as medicine.

[0093] 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

[0094] (a) an endolysin of the present invention;

[0095] (b) a polynucleotide molecule of the present invention;

[0096] (c) vectors / plasmids of the present invention;

[0097] (d) a host of the present invention;

[0098] (e) a bacteriophage capable of expressing a polypeptide of the present invention; and / or

[0099] (f) The pharmaceutical composition of the present invention.

[0100] Another aspect of the present invention provides a method for treating bacterial infections and conditions (such as BV) comprising administering to a subject in need thereof a therapeutically effective amount of

[0101] (a) an endolysin of the present invention;

[0102] (b) a polynucleotide molecule of the present invention;

[0103] (c) vectors / plasmids of the present invention;

[0104] (d) a host of the present invention;

[0105] (e) a bacteriophage capable of expressing a polypeptide of the present invention; and / or

[0106] (f) The pharmaceutical composition of the present invention.

[0107] In some embodiments, the therapeutically effective amount is a dose of 10 to 100 ug of endolysin, optionally administered several times per day.

[0108] A further aspect of the invention provides a kit comprising an endolysin as described herein and instructions for use, in particular for use in treating a disease or condition, preferably BV as defined above. The kit may also comprise a compound or composition for adjusting the pH of the vagina to 4.0-6.0, preferably to 4.5-5.5, more preferably to about 5. The definitions and preferences defined above and below for the endolysin of the invention also apply mutatis mutandis to the polynucleotide molecules, vectors / plasmids, host cells, pharmaceutical compositions, methods of treatment and kits of the invention.

[0109] A further aspect of the invention provides an in vitro method for diagnosing a disease or condition that can be treated with an endolysin according to the invention, the method comprising the steps of:

[0110] (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of a C-terminal cell wall binding region of an endolysin according to the invention and optionally an N-terminal catalytic domain of an endolysin according to the invention, wherein the sample comprises microbial cells, and wherein the C-terminal cell wall binding region of the endolysin is optionally labeled;

[0111] (ii) testing whether the polypeptide binds to and / or lyses microbial cells in the sample; and

[0112] (iii) If the polypeptide binds to, and / or lyses microbial cells, it is determined that the disease or disorder can be treated with an endolysin according to the invention.

[0113] The microbial cell may be a Gardnerella cell, preferably a cell of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or another species of the genus Gardnerella.

[0114] Other features and advantages of the present invention will be apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 A sequence alignment of the natural Gardnerella prophage endolysins of the present disclosure is shown (CLUSTALO (1.2.4) multiple sequence alignment). Most endolysins have 306 residues, except for two that have 251 residues.

[0117] Figure 2 A phylogenetic tree of the natural Gardnerella prophage endolysins of the present disclosure is shown. There are no identical pairs between the endolysins, even though they are highly homologous.

[0118] Figure 3 The domain structure of the Gardnerella prophage endolysin disclosed herein as determined by InterPro is shown (Mitchell et al., 2019, Nucleic Acids Res.47, D351-D360). Due to its homology with glycoside hydrolase family 25, the N-terminal portion of 196 residues of endolysin is determined to be the catalytic domain. The catalytic domain is followed by a linker region and two domains, which are determined to be two cell binding domains due to their homology with the C-terminal domain (CW_7 domain) of lysozyme Cpl-7. According to the nomenclature of the present application, the catalytic domain represents a hydrolase or "H-domain", while the linker region and the cell wall binding domain collectively represent binding or "B-region".

[0119] Figures 4A to 4C Three enzyme activity assays are shown in which the enzyme activity of the natural Gardnerella prophage endolysin of the present disclosure is measured by detecting the turbidity change of Gardnerella cell suspension. Figure 4A In the present study, the enzyme activity of endolysin was measured by detecting the turbidity change of a suspension of G.leopoldii strain Gv_10 at pH 6.0. Figure 4B In the present study, the enzyme activity of endolysin was measured by detecting the turbidity change of the suspension of G.piotii strain Gv_17 at pH 7.0. Figure 4CIn, the enzyme activity of endolysin was measured by detecting the turbidity change of G.swidsinskii strain Gv_23 suspension under pH 7.4. It was processed in the culture medium adjusted to the appropriate pH value, and the relative buffer was carried out in the photometric cuvette. Then, the change of turbidity was evaluated by measuring the optical density (OD) at 600nm. Therefore, compared with the buffer, the turbidity decline of the endolysin treatment group was more obvious, indicating enzyme activity.

[0120] Figure 5 Quantitative reduction in colony forming units (CFU) assay comparing untreated cells of G. vaginalis sensu stricto strain Gv-9 incubated in medium with or without imidazole at different pH values ​​is shown. 7 CFU / ml cells were incubated under the conditions indicated below the graph at 37°C under anaerobic conditions for 5 hours and then the surviving CFU / ml was determined by quantitative plating. The results show that under the experimental conditions, the survival of G. vaginalis Gv_9 is highly dependent on the absence of imidazole and low pH.

[0121] Figure 6 Quantitative reductions in colony forming units (CFU) assays are shown comparing cells of G. vaginalis sensu stricto strain Gv-9 treated with eluates containing recombinant endolysin H10B1 and 250 mM imidazole at different pH values ​​or with controls containing 250 mM imidazole at different pH values. 7 CFU / ml Cells were incubated under the conditions indicated below the graph at 37°C under anaerobes for 5 hours and then viable CFU / ml was determined by quantitative plating. The column labeled Imidazole Control depicts the Figure 5 The results show that the enzyme activity of H10B1 (as an example of all endolytic enzymes of the present invention) is higher at low pH values, with pH 5.5 and pH 5.0 showing the strongest activity.

[0122] Figures 7A to 7D Four quantitative reductions in colony forming unit (CFU) assays measuring the killing activity of the natural and recombinant Gardnerella prophage endolysins of the present disclosure against four major Gardnerella species are shown. Fig. 7A , 7B, 7C and 7D, respectively, by detecting the live CFU in the suspension of G.vaginalis sensu strict strain Gv_9, G.leopoldii strain Gv_11, G.piotii strain Gv_17 and G.swidsinskii strain Gv_23 to measure the killing activity of endolysin. The bacterial strain shown in 90ul 5e7 CFU / ml was incubated for 5 hours under anaerobic conditions of pH 5.0 with 10ul endolysin solution (if possible, the concentration was adjusted to 0.2mg / ml, see Table 4). The logarithmic Y axis represents the count of viable cells. The dotted line represents the detection limit (LOD) obtained by coating 2ul reaction mixture (500CFU / ml). The results show that the endolysin of the present invention has the ability to crack four main Gardnerella species. The results also point out that some recombinant endolysins of the present invention have a higher killing activity than natural endolysins of the present invention.

[0123] Figure 8 A phylogenetic tree of H-domains (amino acid level) generated using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539) is shown.

[0124] Fig. 9 A phylogenetic tree (amino acid level) of the B-region generated using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539) is shown.

[0125] Fig.10 The sequence alignment of the cell wall binding domain (also referred to as B-domain) within the B-region of the natural endolysin of the present invention using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539) is shown. For each B-region, the N-terminal cell wall binding domain is indicated by the _N suffix (Bx_N), and the C-terminal cell wall binding domain is indicated by the _C suffix (Bx_C). For example, B3_C represents the second (C-terminal) B-domain of B3.

[0126] Fig.11 A phylogenetic tree of individual B-domains using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539) is shown.

[0127] Fig.12Three quantitative reductions in the viable colony forming unit (CFU) assay, which 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, are shown. The results indicate that the endolysin of the present invention is ineffective against beneficial Lactobacillus strains.

[0128] Fig.13 Shown is a MIC microbroth dilution activity assay measuring the effect of metronidazole and clindamycin (obtained from Ratiopharm as a solution for injection, 300 mg / 2 ml) on the growth of four major Gardnerella species suspensions. 7 Suspensions of Gardnerella at CFU / ml were incubated with the antibiotic concentrations indicated on the x-axis of each graph and incubated at 37°C under anaerobic conditions for 48 h. Cell growth was assessed by optical density measurement at 610 nm (OD(610)) before and after incubation to determine the minimum inhibitory concentration (MIC). The results showed that all Gardnerella strains were resistant to metronidazole and clindamycin (obtained from Ratiopharm as a solution for injection, 300 mg / 2 ml) with MICs of 64 to <128 μg / ml and 16 μg / ml, respectively. Fig.13 ).

[0129] Fig.14 Shown is the measurement of metronidazole and clindamycin hydrochloride (obtained from Sigma-Aldrich) on 1x10 5 -1x10 6 The results showed that metronidazole had an MIC between 8 and 128 μg / ml against all tested Gardnerella strains, while clindamycin hydrochloride powder (obtained from Sigma-Aldrich (C5269-10MG)) exhibited an MIC between 0.25 and 5 μg / ml.

[0130] Fig.15 Shown is a MIC microculture dilution activity assay measuring the effect of H2B10 (a representative of the domain-swapped endolysins described herein) on the growth of three major Gardnerella species. 5 -1x10 6 CFU / ml suspensions of Gardnerella were incubated with H2B10 at the concentrations indicated on the x-axis of each graph and incubated for 48 h at 37°C under anaerobic conditions. Cell growth was assessed by OD (610) measurement before and after incubation to determine the minimum inhibitory concentration (MIC). MIC values ​​between 1 and 4 μg / ml were obtained, indicating that all Gardnerella strains were highly sensitive to the domain-swapped endolysin H2B10.

[0131] Details

[0132] definition

[0133] The term "lysin" refers to a cell wall lytic enzyme encoded by a bacteriophage (endolysin) or a bacterium (autolysin) that is capable of hydrolyzing the cell wall of the target bacteria when added exogenously (lysis-from-without). This new class of antimicrobial agents has important advantages over classical antibiotics, such as a new mode of action; a narrow range of susceptible bacteria; rapid killing of both quiescent and exponentially growing bacteria; activity against mucosa and bacterial biofilms; a low probability of developing resistance; and a reduced impact on the normal microbiota. These unique properties have increased interest in the biotechnological and pharmacological development of lysins and have led to their inclusion in the current list of preferred alternatives to combat antibiotic resistance. Lysins from Gram-positive bacteria and their phages typically contain at least one catalytic domain and one or more cell wall binding domains. In contrast, many lysins produced by Gram-negative species or their phages contain only a catalytic domain, although modular endolysins have also been reported. The catalytic unit determines the type of peptidoglycan (PG) bond to be cleaved, whereas the cell wall binding domain determines the cleavage spectrum mainly through specific recognition of cell wall elements distributed in a genus or species / strain-specific manner.

[0134] In the content of the present disclosure, the term "natural endolysin" refers to an endolysin encoded by a prophage sequence in a bacterial genome, particularly in a Gardnerella cell genome. In the content of the present disclosure, the term "natural endolysin" therefore refers to an endolysin without domain exchange. Natural endolysin can be unmodified, which means that the amino acid sequence of the endolysin corresponds to the native sequence. Alternatively, the natural endolysin can be modified, which means that the amino acid sequence of the endolysin comprises at least one mutation compared to the native sequence. The amino acid sequence of natural endolysin E1-E14 is shown in Table 7 below. NCBI accession number WP_014554482 (May 27, 2013 WP_014554482.1 version) also provides an example of a known 1,4-β-N-acetyl wall amidase (natural endolysin EL1) sequence.

[0135] In the context of the present disclosure, the term "recombinant endolysin" refers to an endolysin that has been domain-swapped. In the context of the present disclosure, the term "domain-swapped endolysin" refers to an endolysin having an N-terminal catalytic domain from a first natural endolysin and at least one cell wall binding domain from a second natural endolysin, wherein the first and second natural endolysins are encoded by different genomes from different prophages. The recombinant endolysin of the present invention may comprise or consist of: an N-terminal catalytic domain from a first natural endolysin and two cell wall binding domains from a second natural endolysin, wherein the first and second natural endolysins are encoded by different genomes from different prophages. Alternatively, the recombinant endolysin of the present invention may comprise or consist of the following: an N-terminal catalytic domain from a first natural endolysin, a first (N-terminal) cell wall binding domain from a second natural endolysin, and a second (C-terminal) cell wall binding domain from a third natural endolysin, wherein the first and second natural endolysins are encoded by different genomes from different prophages, and wherein the third natural endolysin is optionally encoded by different genomes from prophages different from the first and second natural endolysins. The recombinant endolysin may be unmodified, meaning that the amino acid sequence of the endolysin corresponds to the native sequence of the respective domains constituting the endolysin. Alternatively, the recombinant endolysin may be modified, meaning that the amino acid sequence of the endolysin comprises at least one mutation compared to the native sequence of the respective domains constituting the endolysin. According to this definition, it is readily understood by those skilled in the art that a "domain-swapped" or "recombinant" endolysin as described herein is a non-naturally produced endolysin. That is, the recombinant endolysin of the present invention has been manually modified and excludes natural endolysins by definition, i.e., because the latter can be found naturally in nature. The accompanying examples provide suitable methods for producing the artificial endolysins of the present invention.

[0136] The term "catalytic domain" or "enzymatic domain" refers to the portion of a protein chain containing the region where the catalyzed chemical reaction occurs. In the context of the present disclosure, the term "H-domain" refers to the portion of an endolysin of the present invention that contains the catalytic domain.

[0137] In the context of the present disclosure, the term "B-region" refers to a portion of an endolysin of the present invention that comprises or consists of a polypeptide having cell wall binding activity. In a preferred embodiment, the B-region comprises or consists of a linker region and one, two or three cell wall binding domains or "B-domains".

[0138] In the context of the present invention, the term "B-domain" refers to the cell wall binding domain contained within the B-region.

[0139] 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). In short, the Cpl-7 protein has a C-terminal cell wall binding region consisting of 3 consecutive CW_7 domains. Each CW_7 domain consists of a similar amino acid sequence of 38 amino acids long, called a "CW_7 motif", defined by Interpro (Mitchell et al., 2019, Nucleic Acids Res. 47, D351-D360), consisting of an amino acid sequence

[0140] TVANEVIQGLWGNGQERYDSLANAGYDPQAVQD KVNEXL, wherein X is I in CW_7 motif No:1 (amino acids 207-245) and No:2 (amino acids 255-293), and wherein X is L in CW_7 motif No:3 (amino acids 303-341). In the Cpl-7 protein, there is a short linker of 9 residues between CW_7 motif No:1 and No:2 and between CW_ motif No:2 and No:3, so the total repeat sequence is 47 residues long. In contrast, the repeat sequence of the natural endolysin of the present invention is 49 residues long.

[0141] The term "minimum inhibitory concentration" or "MIC" refers to the lowest concentration of a chemical (usually a drug) that prevents visible growth of bacteria. MIC is defined in this application as the lowest concentration of an antibiotic that results in no detectable growth after 48 h as measured by OD.

[0142] 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, which is the concentration of antibiotic that kills 90% of cells within a specified time, while the MBC is defined in this application as the complete elimination of 2.5x10 7 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 bacterial death of all cells in the suspension.

[0143] The terms "peptide", "polypeptide", "protein" and variants of these terms refer to peptides, oligopeptides, oligomers or proteins, including fusion proteins, comprising at least two amino acids connected to each other by normal or modified peptide bonds, such as in the case of isosteric peptides. These terms also include "peptide mimetics" herein, which are defined as peptide analogs containing non-peptide structural elements that can mimic or antagonize the biological effects of natural parent peptides. 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 also be composed of amino acids modified by natural processes (such as post-translational maturation processes) or chemical processes known to those skilled in the art. Such modifications are described in sufficient detail in the literature. These modifications may occur anywhere in a polypeptide: in a peptide backbone, in an amino acid chain or even at a 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 a natural or synthetic post-translational process known to those skilled in the art. For example, peptide or polypeptide modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositols, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, demethylation, glycosylation (including pegylation), hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, amino acid additions 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.

[0144] As used herein, "bacterial infections and conditions" refers to infections and conditions caused by bacteria, particularly infections and conditions caused by at least one strain of Gardnerella selected from Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella piotii and Gardnerella swidsinskii and other Gardnerella species. Bacterial infections and conditions include, but are not limited to, bacterial vaginosis (BV).

[0145] As defined herein, the term "killing activity" of an endolysin against a particular bacterium refers to a 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 may 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. Killing activity can be determined by measuring the decrease in optical density at 610-620 nm of a bacterial cell suspension after exposure to the endolysin to be tested and / or the decrease in colony forming units (CFU) per milliliter of bacterial cell suspension.

[0146] As defined herein, the term "binding ability" of an endolysin to a 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 a cell wall of a bacterium can be determined by methods known in the art.

[0147] As used herein, "treatment" and "treating" and the like generally refer to obtaining the desired pharmacological and physiological effects. The effect may be preventive in terms of preventing or partially preventing a disease, its symptoms or conditions, and / or may be therapeutic in terms of partially or completely curing a disease, condition, disorder or adverse effects attributable to the disease. The term "treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; or alleviating the disease, i.e., causing regression of the disease and / or its symptoms or conditions, such as improvement or remediation of damage. In particular, treatment of bacterial infections includes preventing, reducing or even eradicating the infection, for example, by killing bacteria and thereby controlling, reducing or inhibiting bacterial proliferation and reducing the number of viable bacterial cells. In this article, it is preferred that the disease (e.g., BV) is treated in terms of partially or completely curing the disease or symptoms.

[0148] 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 cattle, sheep, pigs, horses, laboratory rodents, etc. Preferably, the subject is a human.

[0149] As used herein, the term "effective amount" refers to the amount of at least one endolysin, composition or pharmaceutical preparation thereof according to the present invention that elicits a biological or medical response in the tissue, system, animal or human being sought. In one embodiment, the effective amount is a "therapeutically effective amount" for alleviating the symptoms of the disease or condition to be treated. In another embodiment, the effective amount is a "prophylactically effective amount" for preventing the symptoms of the disease or condition to be prevented. The term also includes in this article an amount of active polypeptide sufficient to reduce disease progression, particularly to reduce or inhibit a condition or infection and thereby elicit the response sought (i.e., an "inhibitory effective amount").

[0150] The term "efficacy" of the 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 infectious diseases is ultimately evaluated by epidemiological studies of the population, which is generally associated with the titer of neutralizing antibodies in serum and the induction of multifunctional pathogen-specific T cell responses. Preclinical evaluation can include resistance to infection after infection pathogen attack. The treatment of infectious diseases can be measured by inhibiting the growth of pathogens or eliminating pathogens (and therefore, no pathogens are detected), which is associated with pathogen-specific antibodies and / or T cell immune responses.

[0151] The term "biological material" refers to any material or sample obtained from a subject's body. This includes, for example, whole blood, serum, plasma, urine, sputum, saliva, vaginal swab, or spinal fluid samples.

[0152] The term "inanimate material or surface" includes solutions, media, equipment, objects, floors, table surfaces.

[0153] The term "medium" includes water, air or food.

[0154] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in such a form that the biological activity of the active ingredient is clearly effective and that contains no additional ingredients that would be toxic to a subject to which the formulation would be administered.

[0155] The term "pharmaceutically acceptable" refers to a carrier that is composed of a material that is not biologically or otherwise undesirable.

[0156] 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 or emulsifying agents, pH buffering agents, preservatives and the like.

[0157] The term "variant" refers to a polypeptide comprising non-conservative or preferably conservative insertions, deletions and / or substitutions relative to a native amino acid sequence. For example, a polypeptide may 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. Percent identity may be determined by methods known in the art using a suitable computer program, such as MatGAT 2.0 (Myers and Miller, CABIOS (1989)). Preferably, % identity is identified over the full length of the sequence to be compared. It will be understood that percentage identity is calculated relative to the polypeptide of the best alignment of its sequence. Fragments and variants of amino acid sequences can be prepared using any protein engineering, directed evolution and / or site-directed mutagenesis methods known in the art (e.g., see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). It will be appreciated by those skilled in the art that polypeptides, or fragments, variants or fusions thereof, according to the present invention may contain derivatives of natural amino acid sequences, or fragments or variants thereof, or consist of derivatives of natural amino acid sequences, or fragments or variants thereof. Chemical derivatives of one or more amino acids may be achieved by reacting with functional side groups. Such derivative molecules include, for example, those molecules in which free amino acid groups have been derived to form amine hydrochlorides, p-toluenesulfonyl, carboxyphenoxy, tert-butoxycarbonyl, chloroacetyl or formyl groups. Free carboxyl groups may be derived to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derived to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides containing naturally occurring amino acid derivatives of 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 containing one or more additions or deletions, as long as the necessary activity is maintained. Other modifications included are terminal modifications such as amidation, amino terminal acylation (such as acetylation or thioglycolic acid amidation), terminal carboxyl amidation (such as with ammonia or methylamine). Those skilled in the art will further understand that peptide mimetic compounds can also be useful. Therefore, by "polypeptide", we include peptide mimetic compounds that exhibit endolysin activity. The term "peptide mimetic" refers to a compound that mimics the conformation and desired characteristics of a specific polypeptide as a therapeutic agent.

[0158] Endolysin according to the invention

[0159] The endolysin of the present invention has antibacterial activity against Gardnerella strains. The optimal pH for the endolysin of the present invention to exhibit 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:

[0160] (i) an N-terminal catalytic domain, or a functional variant thereof;

[0161] (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

[0162] (iii) a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region,

[0163] It also has anti-Gardnerella cell-killing activity.

[0164] 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 anti-Gardenella killing activity of the endolysins of the invention is species-selective anti-Gardenella killing activity.

[0165] The N-terminal catalytic domain is a functional polypeptide, wherein the function includes the ability to lyse the cell wall of Gardnerella. The N-terminal catalytic domain can be N-acetylmuramidase, N-acetylmuramidase-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, most preferably 1,4-β-N-acetylmuramidase. For example, the N-terminal catalytic domain may be a polypeptide comprising or consisting of an amino acid of any one of SEQ ID NOs: 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 NOs: 1 to 5, 7 or 10 to 12, whereby the polypeptide is functional, wherein the function comprises the ability to lyse the cell wall of Gardnerella. Preferably, the N-terminal catalytic domain 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 comprises the ability to lyse the cell wall of Gardnerella.

[0166] 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. 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 be independently selected from polypeptides comprising or consisting of the following, respectively: SEQ ID NOs: 15 to 24 and 26 to 33 amino acid sequences, 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), whereby the polypeptide is functional, wherein the function includes the ability to bind to the cell wall of Gardnerella. Preferably, one, two, three or more cell wall binding domains are independently selected from a polypeptide comprising or consisting of an 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) to an amino acid sequence of any one of SEQ ID NOs: 19, 20 and 28-33, whereby the polypeptide is functional, wherein the function comprises the ability to bind to a Gardnerella cell wall. More preferably, one, two, three or more cell wall binding domains are independently selected from a polypeptide comprising an 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) to an amino acid sequence of any one of SEQ ID NOs: 28-33, whereby the polypeptide is functional, wherein the function comprises the ability to bind to a Gardnerella cell wall.

[0167] 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 the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 26, 28, 30 and 32, and the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, 27, 29, 31 and 33. In a preferred embodiment, the first cell wall binding domain is at the N-terminus of the second cell wall binding domain.

[0168] In one aspect of the invention, the linker region consists of a polypeptide having a length of 6 to 18 amino acids, preferably a length of 9 to 15 amino acids, even more preferably a length of 12 amino acids. Preferably, the linker region consists of a polypeptide comprising or consisting of the following amino acid sequence: (i) (XXX) n , wherein each X can independently be G, A or S, preferably wherein the amino acid sequence (XXX) n Yes (GGS) n , wherein n corresponds to the number of repetitions of the sequence XXX, preferably wherein n is 2, 3, 4, 5 or 6, or (ii) XXGLNGXXNGGS, wherein X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q. The fragment comprising a linker may not be present. The fragment comprising a linker may also be present and may enhance the cell wall binding and / or lytic activity of the polypeptide of the present invention.

[0169] The present invention further provides an endolysin having anti-Gardnerella killing activity as described above for use in treating a disease or condition. The disease or condition 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.

[0170] The endolysin of the present invention is preferably capable of specifically binding to and / or lysing Gardnerella cells and is used in a method for treating Gardnerella infection (such as BV).

[0171] As mentioned above, it is fully confirmed that many phage endolysins are composed of two different domains (for example, see Sheehan et al., 1996, FEMS Microbiology Letters 140:23-28). One is the catalytic domain responsible for cell wall degradation and it is known that these exist in several forms. Another domain is to recognize cell surface motifs and allow endolysin to attach to the cell wall binding domain of target cells. The latter is related to precise pattern recognition and provides specificity. The enzymatic domain can be identified by its amino acid homology with other similar regions of the lytic enzyme with the same type of lytic activity. In the case of the natural Gardnerella prophage endolysin newly found by the inventor, it has been determined that the domain arrangement is composed of an N-terminal domain of 196 residues, followed by a linker region of 12 residues and two repeat domains of 49 residues respectively, except EL6 and EL9, in which there is only an incomplete domain of 43 residues. The natural amino acid sequences of these newly found endolysins are summarized in Table 7. The inventors determined that the N-terminal domain is the catalytic domain because it has homology with glycoside hydrolase family 25, and the two repeat domains are two cell wall binding domains because they have homology with the C-terminal domain of lysozyme Cpl-7 (see Examples 2 and Figure 3 ).

[0172] In some embodiments, the fragment comprising the enzymatic domain is unmodified, i.e., corresponds to the native amino acid sequence. In alternative embodiments, the fragment comprising the enzymatic domain may comprise changes, such as amino acid replacement, deletion, insertion or any combination of its changes. In some embodiments, the fragment comprising the enzymatic domain is a variant fragment with SEQ ID NO: 1 to 5, 7 or 10 to 12 amino acid sequences having at least 80%, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, even more preferably at least 99.7% identity and most preferably 100% identity.

[0173] 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 may comprise changes, such as substitutions, deletions, insertions of amino acids or any combination of changes thereof. In some embodiments, the fragment comprising the cell wall binding domain is a variant fragment having at least 80%, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, even more preferably at least 99.7% identity and most preferably 100% identity with the amino acid sequence of any one of SEQ ID NOs: 15 to 24 and 26 to 33.

[0174] In another aspect of the present invention, the endolysin comprises or consists of a fusion of a polypeptide, or a fragment, variant or derivative thereof. For example, the polypeptide may comprise one or more other amino acids inserted into the interior of the amino acid sequence of the endolysin according to the present invention, or a fragment, variant or derivative thereof and / or at its N- and / or C-terminus.

[0175] Therefore, as described above, in one embodiment, the endolysin of the first aspect of the present invention comprises a fragment consisting of one or more cell wall binding domains, the cell wall binding domains respectively comprising the amino acid sequence of any one of SEQ ID NO: 15 to 24 and 26 to 33 (or such a domain sequence retains its cell wall binding activity variant) or consisting of the amino acid sequence of any one of SEQ ID NO: 15 to 24 and 26 to 33 (or such a domain sequence retains its cell wall binding activity variant), and enzymatic domains from different sources are 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-aminoglucosidase (muramyl hydrolase), N-acetyl-β-D-muramic acid enzyme (lysozyme) or cleavage transglycosylase. In addition, N-acetylmuramoyl-L-alanine amidase from other sources can also be used.

[0176] In one aspect of the present invention, 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, 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 variant or derivative of the endolysin according to the present invention are also included in the scope of the present invention. It will be recognized that the fusion (or its variant or derivative) preferably retains the desired properties (i.e., endolysin activity). If the fusion is a fusion suitable for use in the methods described herein, it is also particularly preferred. For example, the fusion may include another part that gives the desired features of the endolysin of the present invention; for example, the part may be useful in detecting or separating endolysin, promoting cellular absorption of endolysin, or directing protein secretion from cells. As known to those skilled in the art, the part may be, for example, a biotin part, a radioactive part, a fluorescent part, for example, a small fluorophore or a green fluorescent protein (GFP) fluorophore. This part may be an immunogenic tag, for example, a Myc tag, as known to those skilled in the art, or may be a lipophilic molecule or polypeptide domain capable of promoting cellular uptake of the endolysin, as known to those skilled in the art.

[0177] The essential feature 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. Biol. 898661). In one embodiment, the endolysin of the present invention is substantially or completely unable to lyse bacteria that are symbiotic members of healthy vaginal microbiota (and are known to have no adverse effects on the host). For example, if the endolysin does not understand cells of the genus Lactobacillus, it is advantageous. 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 invention does not lyse cells of L. iners. Advantageously, the endolysin is capable of selectively lysing cells of pathogenic bacteria, ie to a greater extent than cells of non-pathogenic bacteria.

[0178] The killing activity of the endolysin according to the invention against a particular microorganism can be determined by standard procedures in the art, including those based on the determination of the minimum inhibitory concentration (MIC) of an antimicrobial agent, which is defined as the lowest concentration of the antimicrobial agent that inhibits visible growth of a microorganism after overnight incubation, as described in Andrews, 2001, J Antimicrobial Chemotherapy, 48, Suppl. SI, 5-16 or in "Document M7-A7, Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standards, 7th edition, January 2006, vol. 26, No. 2" published by Clinical and Laboratory Standards Institute. Another suitable method for determining the killing activity of the endolysin according to the invention is described in the Examples section of the present application and comprises determining the decrease in the optical density of a bacterial suspension measured at 610-620 nm, the susceptibility of the bacteria being tested in an in vitro turbidity assay performed in the presence of a purified endolysin according to the invention. According to another embodiment, the endolysin according to the present invention reduces 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% in an in vitro turbidity test as described herein.

[0179] Methods for producing the endolysin or its fragment, variant, fusion or derivative used according to the present invention are well known in the art. Conveniently, the endolysin or its fragment, variant, fusion or derivative is or comprises a recombinant endolysin. The endolysin according to the present invention can be produced by standard techniques of genetic engineering, including the use of a recombinant vector comprising a polynucleotide encoding an endolysin as described herein. A variety of expression systems can be used, including bacterial plasmids and derived vectors, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses (such as baculovirus, papillomavirus (such as SV40), vaccinia virus, adenovirus, fox poxvirus, pseudorabies virus, retrovirus), clay 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, NY, 2001. The recombinant vector can include the nucleotide sequence of the regulation, expression, transcription and / or translation of the polynucleotide controlling the coding endolysin, and these sequences are selected according to the host cell used. The recombinant vector can further include the nucleotide sequence such as encoding His tag, for promoting the purification step. Subsequently, such recombinant vector can be introduced into the host cell according to methods well known to those skilled in the art, such as BASIC METHODS INMOLECULAR BIOLOGY, Davis etc., the 2nd edition, McGraw-Hill Professional Publishing, 1995 and MOLECULAR CLONING:A LABORATORY MANUAL, those described above, such as by calcium phosphate transfection, by DEAE dextran transfection, transfection, microinjection, by cationic lipid transfection, electroporation, transduction or infection. The host cell can be, for example, a bacterial cell, such as E. coli, a fungal cell, such as a yeast cell or a cell of Aspergillus, Streptomyces, an insect cell, a Chinese hamster ovary cell (CHO), a C127 mouse cell line, a BHK cell line of a Syrian hamster cell, a human embryonic kidney 293 (HEK293) cell. Preferably, the host cell is E. coli. The host cell is then cultured under appropriate conditions to produce the endolysin 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).

[0180] Compositions according to the invention

[0181] In another aspect of the present invention, an antibacterial composition is provided which comprises 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 phage capable of expressing an endolysin according to the first aspect of the present invention in a specific pharmaceutical composition.

[0182] As used herein, "pharmaceutical composition" means 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" refers to 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 dosing vehicles) to produce the desired therapeutic effect. In addition, it means an amount sufficient to reduce and most preferably prevent clinically significant defects in the activity, function and response of the host. Alternatively, the therapeutically effective amount is sufficient to cause a clinically significant improvement in the condition of the host. As will be appreciated by those skilled in the art, the amount of the compound may vary according to its specific activity. A suitable dose may contain a predetermined amount of an active composition that is calculated to produce the desired therapeutic effect associated with the required diluent. In the methods and uses for making the compositions of the present invention, a therapeutically effective amount of an active ingredient is provided. The therapeutically effective amount may be determined by a skilled person or veterinary practitioner based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as known to those skilled in the art. In one embodiment of the invention, the pharmaceutical composition comprises an endolysin according to the first aspect of the invention. Thus, the pharmaceutical preparation may comprise an amount of endolysin or a fragment, variant, fusion or derivative thereof sufficient to at least partially inhibit the growth of Gardnerella cells in patients infected or susceptible to said cells. Preferably, the pharmaceutical preparation comprises an amount of endolysin or a fragment, variant, fusion or derivative thereof sufficient to kill Gardnerella cells in the patient. It will be appreciated by those skilled in the art that the endolysin of the present invention is typically administered in admixture with a suitable pharmaceutical excipient, diluent or carrier 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, editor Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA). For example, the endolysin may be administered topically, i.e., topically in the vagina of a female subject, and / or in or on the glans penis, foreskin or urethral entrance of a male subject. The term "(administered) in or on the glans penis" herein also includes "(administered) in and on the glans penis". In line with this, the term "(administered) in or on the glans penis, foreskin or urethral entrance of a male subject" also includes "(administered) in and on the glans penis and on the foreskin and on the urethral entrance of a male subject". In another embodiment, the endolysin can be co-administered with a compound or composition that adjusts the pH of the vagina. In some embodiments, the compound or composition adjusts the pH of the vagina to pH 4.0 to 6.0, preferably to pH 5.0.

[0183] In an alternative embodiment of the invention, the pharmaceutical composition does not comprise the endolysin itself, but instead comprises a nucleic acid molecule capable of expressing said endolysin. Suitable nucleic acid molecules, expression vectors and host cells have been described in detail above. For example, recombinant probiotics (LAB strains, e.g., Lactococcus lactis or Lactobacillus sp.) may be used. In yet another embodiment of the invention, the pharmaceutical composition comprises a bacteriophage capable of expressing an endolysin according to the first aspect of the invention. Methods for conducting 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 bacterial infections described herein, the endolysin of the invention may be administered as a homologous protein, as a nucleic acid construct, vector or host cell expressing a homologous protein, as part of a living organism (including a bacteriophage) expressing a homologous protein, or by any other convenient method known in the art to achieve contact of the endolysin 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.

[0184] The composition of the present invention may contain one or more endolysin polypeptides. In this embodiment, the endolysin polypeptide may be present as an independent polypeptide or as a fusion protein comprising the endolysin polypeptide or a fragment thereof.

[0185] The pharmaceutical composition of the present invention may further comprise one or more other pharmaceutically acceptable ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, etc. Preferably, the pharmaceutical composition of the present invention does not comprise imidazole.

[0186] The endolysin of the present invention can be prepared into a pharmaceutical composition and a unit dosage form thereof together with conventionally used adjuvants, carriers, diluents or excipients, and can be used in solid form, such as tablets or filled capsules, or liquids, such as solutions, suspensions, aerosols, emulsions, elixirs or capsules containing them, all for oral administration, or in the form of sterile injection solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof 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 active ingredients commensurate with the expected daily dose range to be used. The composition of the present invention may also be a liquid preparation, including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The composition may also be formulated into a dry product for reconstitution with water or other suitable excipients before use. Such liquid preparations may contain additives, including but not limited to suspending agents, emulsifiers, 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. Emulsifiers 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 paraben and sorbic acid. Other materials and processing techniques are listed in Remington's "The Science and Practice of Pharmacy", 22nd edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, Part 5 of Part 5.

[0187] The solid composition 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 usually based on injection of sterile saline or phosphate buffered saline or other injectable carriers known in the art.

[0188] The composition of the present invention can also be formulated into a suppository, which can contain a suppository base, including but not limited to cocoa butter or glycerides. The composition of the present invention can also be formulated into a transdermal preparation comprising an aqueous or non-aqueous carrier, including but not limited to a cream, ointment, lotion, paste, salves, patches or membranes.

[0189] The compositions of the present invention may also be formulated for parenteral administration, including, but not limited to, administration by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions or emulsions in oily or sober carriers, and may contain preparatants, including, but not limited to, suspending agents, stabilizers and dispersants. The compositions may also be provided in powder form for reconstitution with a suitable carrier, including, but not limited to, sterile, pyrogen-free water.

[0190] 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 (e.g., as an emulsion in an acceptable oil), ion exchange resins, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0191] The compounds of the invention may also be administered in sustained release form or from a sustained release pharmaceutical system. A description of representative sustained release materials may also be found in Remington's "The Science and Practice of Pharmacy".

[0192] Dosage

[0193] The compositions of the present invention are preferably administered topically in the vagina of female subjects and / or in or on the glans penis, foreskin or urethral entrance of male subjects. However, these compositions may also be administered in any manner, including intravenous injection, intraarterial injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, intrathecal injection, oral route (including sublingual or via buccal administration), topical, skin application, direct tissue perfusion during surgery, or a combination thereof.

[0194] In a preferred embodiment, the endolysin, polynucleotide or pharmaceutical composition of the invention as described herein is administered topically. In a further preferred embodiment, the endolysin, polynucleotide or pharmaceutical composition of the invention as described herein is administered into the vagina of a female subject and / or into or onto the glans penis, foreskin or urethral entrance of a male subject. In a further preferred embodiment, the endolysin, polynucleotide or pharmaceutical composition of the invention as described herein is administered topically into the vagina of a subject.

[0195] The dosage administered to an individual as a single dose or multiple doses will vary depending on a variety of factors, including pharmacokinetic properties, patient condition and characteristics (sex, age, weight, health, size), degree of symptoms, concurrent treatment, frequency of treatment and desired effect.

[0196] According to one aspect, the compositions of the invention may be administered to a patient in a prophylactic manner prior to sexual intercourse.

[0197] joint

[0198] According to the present invention, endolysins can be administered alone or in combination with co-agents useful in preventing and / or treating Gardnerella infections or conditions, including those caused by Gardnerella vaginalis sensustricto, Gardnerella leopoldii, Gardnerella piotii, Gardnerella swidsinskii and / or other species of the genus Gardnerella.

[0199] The endolysins according to the invention can be administered in combination with:

[0200] (a) treatment with one or more conventional antibiotics. Such antibiotics may include clindamycin, metronidazole or any other suitable antibiotic known to those skilled in the art;

[0201] (b) one or more other endolysins, or nucleic acid molecules, vectors, host cells or bacteriophages capable of expressing said endolysins;

[0202] (c) Compounds or compositions that adjust vaginal pH. In some embodiments, the compound or composition adjusts vaginal pH to pH 4.0 to 6.0, preferably to pH 5.0. Suitable pH-adjusting compounds may include phosphates, lactic acid (e.g., natural acidifying substances secreted by Lactobacilli to create an acidic environment) or other organic acids, e.g., carboxyl-substituted polymers;

[0203] (d) Treatments that neutralize toxins released upon bacterial lysis of intravaginal Gardnerella cells. Suitable neutralizing treatments may include antibodies (see, Babcock et al., 2006, Infect. Immun.

[0204] 74:6339-6347) and toxin absorbers, such as tolevamers (see, Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534);

[0205] (e) Probiotics.

[0206] Use and method according to the invention

[0207] Another aspect of the present invention provides 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 phage capable of expressing an endolysin according to the present invention, or a pharmaceutical composition according to the present invention for use in a medicine. Therefore, the endolysin of the present invention can be used in a method for treating the human or animal body by surgery or therapy and / or in a diagnostic method implemented on the human or animal body. In particular, 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 phage capable of expressing an endolysin according to the present invention, or a pharmaceutical composition according to the present invention, for use in a method for treating a disease or condition.

[0208] A further aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing an endolysin of the present invention or a pharmaceutical composition of the present invention for use as a medicament.

[0209] Another aspect of the present invention provides the use of the endolysin 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 endolysin of the present invention, or the pharmaceutical composition of 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 using the endolysin. In particular, the present invention provides the use of the endolysin 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 endolysin of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for treating bacterial infections and disorders.

[0210] Another aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing the endolysin of the present invention or a pharmaceutical composition of the present invention for use in 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 using the endolysin.

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

[0212] Another aspect of the present invention provides the use of the endolysin 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 endolysin 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 condition 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 endolysin.

[0213] Another aspect of the present invention provides an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition of the present invention for use in treating or preventing a disease or condition 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 endolysin of the present invention.

[0214] Another aspect of the present invention provides a method for treating or preventing a disease or condition associated with a microbial cell in a patient in need of such treatment, the method comprising administering to the patient an endolysin of the present invention, a nucleic acid of the present invention, a vector / plasmid of the present invention, a host cell of the present invention, a bacteriophage capable of expressing the endolysin of the present invention, or a pharmaceutical composition of the present invention, wherein the microbial cell is selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin of the present invention.

[0215] "A disease or condition associated with a microbial cell in a patient" includes a disease or condition caused by or antagonistic to infection of a patient with Gardnerella. Such diseases and conditions include BV.

[0216] "Treatment" includes therapeutic and prophylactic treatment of a subject (or patient). In one embodiment, the endolysin 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 endolysin of the present invention, or the pharmaceutical composition of the present invention, the uses and methods of the present invention are used to treat an existing disease or condition. Alternatively or additionally, the uses and methods of the present invention can be used for prevention. The term "prophylactic" or "prevention" is used to include the use of the endolysin or composition described herein to prevent or reduce the possibility of Gardnerella infection in a patient or subject. Prevention can be primary prevention (i.e., preventing the disease from developing) or secondary prevention (where the disease has already developed and the patient is protected from the deterioration of this process). Preferably, the means and methods provided herein are used to treat an existing disease or condition, particularly for the treatment of an existing BV.

[0217] As described above, the term "effective amount" is used herein to describe the concentration or content of an endolysin according to the present invention that can be used to produce a favorable change in the disease or condition being treated, regardless of whether the change is relief, a favorable physiological outcome, a reversal or alleviation of the condition or condition being treated, the prevention or reduction of the possibility of an ongoing condition or condition, depending on the disease or condition being treated. In one embodiment, the endolysin according to the first aspect of the present invention, the nucleic acid according to the second aspect of the present invention, the vector / plasmid according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, the phage capable of expressing the endolysin according to the first aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention is administered in 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 continued presence 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 condition associated with a microbial cell (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 a microbial cell (e.g., Gardnerella) in the subject.

[0218] In one embodiment, the uses and methods, host cells or pharmaceutical compositions comprising host cells of the present invention are used to deliver the endolysin (preferably the host cell) of the first aspect of the present invention.

[0219] 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:

[0220] (a) one or more conventional antibiotic treatments. Such antibiotics may include clindamycin, metronidazole or any other suitable antibiotic known to those skilled in the art;

[0221] (b) one or more other endolysins, or nucleic acid molecules, vectors, host cells or bacteriophages capable of expressing said endolysins;

[0222] (c) compounds or compositions that adjust the vaginal pH, preferably to pH 4.0 to 6.0, more preferably to about pH 5.0. Such pH-adjusting compounds may include phosphates, lactic acid (e.g., natural acidifying substances secreted by Lactobacilli to create an acidic environment) or other organic acids, e.g., carboxyl-substituted polymers;

[0223] (d) Treatments that neutralize toxins released upon bacterial lysis of intravaginal Gardnerella cells. Suitable neutralizing treatments may include antibodies (see, Babcock et al., 2006, Infect. Immun.

[0224] 74:6339-6347) and toxin absorbers, such as tolevamers (see, Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534);

[0225] (e) Probiotics.

[0226] Another aspect of the present invention provides an endolysin with anti-Gardnerella cell lysis activity or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing it for use in vitro and / or in vitro killing and / or inhibiting / preventing the growth of microbial cells, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to the lysis of the endolysin of the present invention. For example, the endolysin with the activity can be used for cleaning surfaces, such as those in hospitals, kitchens, etc., which may be susceptible to the contamination of such cell bacteria. Preferably, the microbial cells include cells of other species of G.vaginalis sensu stricto, G.leopoldii, G.piotii, G.swidsinskii or Gardnerella or are composed of cells of other species of G.vaginalis sensu stricto, G.leopoldii, G.piotii, G.swidsinskii or Gardnerella.

[0227] Another aspect of the present invention provides a kit. The kit comprises an endolysin as described herein and instructions for use, in particular for treating a disease or condition, preferably BV. The kit can be used for therapeutic or preventive purposes and can further comprise a compound or composition for adjusting the pH of the vagina 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 lytic activity and / or cell binding specificity of an endolysin according to the present invention or a nucleic acid molecule, vector / plasmid, host cell or phage capable of expressing it, wherein the microbial cell is selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin.

[0228] Related aspects of the invention provide:

[0229] (a) Use of a polypeptide having cell wall binding activity and / or cell lytic activity of an endolysin according to the present invention or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing the same in the preparation of a diagnostic agent for a disease or disorder associated with a microbial cell, wherein the microbial cell is selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin;

[0230] (b) a polypeptide having cell wall binding activity and / or cell lytic activity according to the endolysin of the present invention or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing the same for use in diagnosing a disease or disorder associated with a microbial cell, wherein the microbial cell is selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin;

[0231] (c) use of a polypeptide having cell wall binding activity and / or cell lytic activity of an endolysin according to the present invention or a nucleic acid molecule, vector / plasmid, host cell or bacteriophage capable of expressing the same for detecting the presence of microbial cells in a sample 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; and

[0232] (d) An in vitro method for diagnosing a disease or condition that can be treated with an endolysin according to the invention, the method comprising the steps of: (i) contacting a sample obtained from a subject with a polypeptide comprising or consisting of: a C-terminal cell wall binding region of an endolysin according to the invention and optionally an N-terminal catalytic domain of an endolysin according to the invention, wherein the sample comprises microbial cells, and wherein the C-terminal cell wall binding region of the endolysin is optionally labeled; (ii) testing whether the polypeptide binds to and / or lyses microbial cells of the sample; and (iii) if the polypeptide binds to and / or lyses microbial cells, determining that the disease or condition can be treated with an endolysin according to the invention. The microbial cell can be a Gardnerella cell, preferably a cell of G. vaginalis sensu stricto, G. leopoldii, G. piotii, G. swidsinskii or another species of the genus Gardnerella. Therefore, 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 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 lysis 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 been lysed thereby, wherein the microbial cells are selected from Gardnerella cells and other bacterial cells susceptible to lysis using the endolysin. Preferably, the microbial cells include or consist of cells of other species of G. vaginalis sensu stricto, G. leopoldii, G. pioti, G. swidsinskii or Gardnerella. In such diagnostic uses and methods, methods known in the art can be used to detect cell lysis. For example, ATP levels can be measured as an indicator of cell lysis.

[0233] In an alternative embodiment of the uses and methods of the invention defined above, the polypeptide comprises or consists of a cell wall binding domain of an endolysin according to the invention. To allow detection, such polypeptides can be fused to magnetic beads or used as fusion proteins comprising suitable reporters or markers (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 endolysins (e.g., see, Loessner et al., 2002, Mol Microbiol 44, 335-49; Kretzer et al., 2007, Applied Environ. Microbiol. 73: 1992-2000).

[0234] Illustrative embodiments of the invention are described in the following non-limiting examples with reference to the following figures. Example

[0235] Example 1 - Identification of natural endolysins in the Gardnerella genome

[0236] Endolysins are hydrolytic enzymes produced by phages to lyse the host's cell wall in the final stage of the lysis cycle. They can target one of the five bonds in the peptidoglycan (murein), the main component of the bacterial cell wall, which allows the release of progeny virus particles from the lysed cells. So far, phages that resist Gardnerella lysis have not been isolated. Therefore, whether it is possible to successfully identify endolysins from phage sources and with lytic activity against Gardnerella is also unknown. The inventors have studied whether endolysins encoded by prophage sequences can be identified on various Gardnerella genomes. Prophages are phage genomes that are inserted and integrated into circular bacterial DNA chromosomes or exist as extrachromosomal plasmids. This is a latent form of phage, in which viral genes are present in bacteria without destroying bacterial cells. The identification of prophage sequences in bacterial genomes and plasmids can be carried out using network-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 have successfully identified sequences on 14 Gardnerella genomes that are predicted to constitute complete or partial prophages. These sequences are found by identifying DNA regions where gene clusters are predicted to be of viral origin. Also included are viral gene clusters predicted to be only partial prophages rather than complete prophages.

[0237] The putative prophage sequences were then annotated with coding sequences predicted by blast to identify putative endolysins. Specifically, protein sequences homologous to enzymes capable of cutting any key chemical bonds that build peptidoglycan were searched. In particular, protein sequences homologous to N-acetylmuramidase, N-acetylmuramic acid-L-alanine amidase, L-alanyl-D-glutamate 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 name assignments for the source genomes are shown in Table 1.

[0238] [Table 1]

[0239] Name of putative endolysin Name of strain from which 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 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

[0240] In each case, only one copy per prophage was found, and no other coding sequences were found for enzymes predicted to be able to cleave the bacterial cell wall. The putative 1,4-β-N-acetylmuramidase enzymes were aligned for homology and domain structure (see Figure 1 ).from Figure 1 As can be seen in Figure 2, most endolysins have exactly 306 residues, even if they come from different prophages on different genomes. The two exceptions are EL6 and EL9. EL6 is truncated at the C-terminus by a frameshift. EL9 ends at exactly the same position as EL6, but in this case the entire contig ends. There are no identical pairs between endolysins, even if they are highly homologous, such as Figure 2 shown.

[0241] Example 2 - Determination of the domain structure of the natural Gardnerella prophage endolysin

[0242] The domain structure of the newly discovered endolysin was determined using InterPro (Mitchell et al., 2019 Nucleic Acids Res. 47, D351-D360). In short, InterPro is a database of protein families, domains, and functional sites in which identifiable features found in known proteins can be applied to new protein sequences in order to functionally characterize them. The content of InterPro consists of significantly matching diagnostic features and proteins. Features consist 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 in Figure 1, all endolysins with 306 residues have the same domain arrangement. The N-terminal domain of 196 residues is identified as the catalytic domain because it has homology with 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., 1990Gene 86, 81-88; Lopez and García, 2004FEMS Microbiol.Rev.28, 553-580; Bustamante et al., 2010J.Biol.Chem.285, 33184-33196, 2012PLoS One 7, e46654)). In the following examples, the catalytic domain identified above represents "N-terminal catalytic domain" or "H-domain", wherein, for example, "H2" refers to the H-domain of native EL2. In the following examples, the "linker region" and the "C-terminal cell wall binding region", the latter comprising or consisting of one or more cell wall binding domains or "B-domains", are collectively referred to as the "B-region", wherein, 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.

[0243] Example 3 - Determination of the Enzyme Activity of Natural Gardnerella Prophage Endolysin on Gardnerella Cells

[0244] The inventors investigated the enzymatic activity of the newly discovered endolysin against Gardnerella. It is impossible 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 lose their reproductive activity as a result. Moreover, even if the peptidoglycan hydrolase encoded by the newly discovered phage is indeed an active protein, it has not been demonstrated that the protein is able to 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 a true Gram-negative species. Its cell wall is usually thin and has a peptidoglycan content of only 10% or less. Therefore, one skilled in the art would have expected that peptidoglycan degrading enzymes, such as endolysin proteins, would not be able to effectively lyse the bacterial cell wall of Gardnerella.

[0245] The 14 identified endolysins EL1 to EL14 were cloned with a His-tag, expressed in E. coli and purified by a single-step Ni-NTA column using the method described in Reference Example 1. The assignment of endolysin names to source genomes is shown in Table 1. The Gardnerella strains used are shown in Table 2.

[0246] [Table 2]

[0247] name Gardnerella strain (newly named according to (Vaneechoutte et al., 2019)) Gv_1 UGent 09.07, strain of G.vaginalis sensu stricto Gv_8 UGent 25.49, a strain of G.vaginalis sensu stricto Gv_9 ATCC 14018, type strain of G. vaginalis sensu stricto Gv_10 UGent 06.41, the type strain of G. leopoldii Gv_11 UGent 09.48, the type strain of G. leopoldii Gv_17 UGent 18.01, the model strain of G. pioti Gv_23 GS10234(FC2), the type strain of G. swidsinskii

[0248] To test the activity of the purified endolysin, the turbidity change of a Gardnerella suspension (see Table 2) was measured at 610-620 nm essentially using the method described in Reference Example 2, wherein 95 ul of a bacterial suspension in Hardy Broth at the indicated pH was mixed with 5 ul of an endolysin solution in a photometric cuvette under aerobic conditions at room temperature. In a turbidity reduction assay, the decrease in light scattering (i.e., decrease in turbidity) of a live cell suspension can be used in a spectrophotometer to determine the activity of a peptidoglycan hydrolase. The decrease in optical density over time (minutes) can be used to calculate the rate of hydrolysis. The results are compared to a "no enzyme added, buffer only" control preparation that has undergone the same treatment and for the same period of time. In this way, the specific activity of the enzyme preparation can be reported as ΔOD / time / ul endolysin protein. From Figures 4A to 4C As can be seen, the turbidity decline of the endolysin treatment group is more significant than that of the buffer, indicating enzyme activity. Surprisingly, the inventors therefore found that the newly discovered endolysin EL1, EL2, EL3, EL4, EL5, EL7, EL10, EL11 and EL12 are active proteins with the ability to crack the Gardnerella cell wall. As mentioned above, due to the low content of peptidoglycan in the cell wall, the fact that the peptidoglycan degrading enzyme (such as the endolysin identified) can effectively crack the bacterial cell wall is an unexpected and surprising discovery.

[0249] Example 4 - Identification of the most active domain of an endolysin with artificial domain swapping

[0250] The inventors then assessed whether different N-terminal enzymatic domains could have different cleavage activities and whether the B region (including the linker and cell wall binding domains) could mediate specificity for different strains. To this end, domain-swapped endolysins were artificially generated.

[0251] 4.1-Endolysin constructs

[0252] In order to artificially generate domain-swapped endolysins, the N-terminal 196 residues of the first natural endolysin containing the catalytic domain were exchanged as a block, relative to the complete C-terminal region of 110 residues containing the linker region and two cell wall binding domains of the second natural endolysin. The domain-swapped proteins were prepared by performing the following method. The original constructs EL1-14 were ordered from GeneWiz as synthetic genes that were 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 through a restriction / ligation method.

[0253] Table 3 below summarizes the primers used.For the domain exchange of selected 10 constructs, each H-domain was amplified by a common forward primer (no 2) and a construct-specific reverse primer (no 3-12) using PhusionFlash polymerase (Thermo, F-548L) together with a T7 promoter. Similarly, each B-region was amplified by a construct-specific internal primer (no 13-21) and a common reverse primer (no 1) including a T7 terminator. All primers included an extension with a BsaI recognition site to make the outer end compatible with the carrier backbone pETMdest derived from pETM14. The overhang between the domains was designed as a sequence "GGCT" within two amino acids GL of the linker sequence. Therefore, for the purpose of this experiment, these 2 amino acids represent the exact boundary between the domains.

[0254] Therefore, the amplified and gel-purified domains (GeneJet gel purification kit, Thermo, K0692) were subsequently combined into 90 new expression constructs using the GoldenGate cloning strategy via BsaI restriction (BsaI-HFv2, NEB, R3733S) / ligation (T4 DNA ligase, Thermo, EL0011) cycle reactions.

[0255] For transformation purposes, the NEB10β E. coli strain (NEB, C3019) was used and the plasmids were purified using the GeneGet Plasmid Miniprep Kit (Thermo, K0502).

[0256] [Table 3]

[0257]

[0258]

[0259] For ease of reference, the domain combination of the artificial endolysin is represented by the H-code of the N-terminal catalytic domain (hereinafter referred to as the H-domain) and the B-code of the portion including 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 having an N-terminal domain from the natural endolysin EL2 and a linker region and a C-terminal cell wall binding region from the natural endolysin EL10. In other words, H2B10 refers to an endolysin having a domain exchange consisting of 196 N-terminal residues of the natural endolysin EL2 (SEQ ID NO: 2) and 110 C-terminal residues of the natural endolysin EL10. In this example, the B-region B10 corresponding to the 110 C-terminal residues of the native endolysin EL10 comprises, from C-terminus to N-terminus, a C-terminal cell wall binding domain "B10_C" (SEQ ID NO: 29), an N-terminal cell wall binding domain "B10_N" (SEQ ID NO: 28) and a linker region "L10" (NAGLNGYKNGGS). The nomenclature and the corresponding amino acid sequence are shown in detail in Table 7.

[0260] Therefore, according to the above nomenclature, natural endolysin, for example, EL3, can be interchangeably defined as H3B3 or H3-L3(B3_N)(B3_C). Similarly, recombinant endolysin, for example, H2B10, can also be interchangeably defined as H2-L1-(B10_N)(B10_C).

[0261] Those skilled in the art will appreciate that in other embodiments the elements "Lx", "(Bx_N)" and "(Bx_C)" may also be independently swapped. The nomenclature is further shown in Table 7 below.

[0262] 4.2-Optimization of assay parameters

[0263] The dependence of activity on three potential key parameters was analyzed, namely pH, anaerobic / microaerobic / aerobic conditions, and absence / presence of imidazole. Three evaluation criteria were chosen for the following reasons:

[0264] - pH: The killing activity of the endolysin of the present invention has been successfully demonstrated by experiments conducted at a pH of about 7. However, the pH in a healthy vagina is about 3.5, while the pH in a BV vagina is as high as about 5.5 or even higher. Therefore, the pH dependence of the activity of the endolysin was studied.

[0265] - Oxygen: In the literature, Gardnerella is 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).

[0266] - Imidazole: The endolysin of the invention was purified by a one-step Ni-NTA column according to the method described in reference example 1, wherein the buffer used to elute the endolysin from the Ni-NTA matrix contained imidazole. Thus, without a further step of dialysis of the sample, the eluate obtained contained 250 mM imidazole. In this regard, the effect of imidazole on Gardnerella has been studied.

[0267] First, the sensitivity of G. vaginalis Gv_9 survival to incubation in medium with or without imidazole at different pH values ​​was evaluated (see Figure 5 ). 7 CFU / ml cells were incubated under anaerobic conditions at 37°C for 5 hours under the conditions shown below the graph. The viable CFU / ml was then determined by quantitative plating. Figure 5 As shown, at pH 6.0, the median is 1×10 7 and 1×10 6 At pH 7.0, only a median of 2 × 10 6 and 3×10 4 The cells survived in the absence and presence of imidazole, respectively. In the untreated control at pH 5.0, 1e7 cells survived the procedure, while the median survival of the imidazole treated at pH 7 was 3e4, i.e., 3 logs lower than the former. Therefore, the survival of G. vaginalis Gv_9 is highly dependent on the absence of imidazole, especially at pH>6.0 and low pH.

[0268] Secondly, the sensitivity of G. vaginalis Gv_9 to treatment with recombinant endolysin H10B1 at different pH values ​​was evaluated relative to imidazole-containing controls (see Figure 6 ). 7 CFU / ml cells were incubated under the conditions indicated below the graph at 37°C for 5 hours under anaerobic conditions. Surviving CFU / ml were then determined by quantitative plating. The column labeled Imidazole Control depicts the difference between the Figure 5 The same data in Figure 6As shown in, endolysin is highly active down to pH 5.0, and even at this low pH, the relative reduction compared to the control is more pronounced, with a 2.5 log reduction in viable CFU. While at pH 7.0, the survival difference between H10B1 treated and untreated cells was less than 1 log10, at pH 5.0, the difference was 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. Thus, the activity of endolysin H10B1 is highly pH dependent. When similar experiments were performed under aerobic conditions, the survival of control cells was reduced by several log10 units compared to anaerobic conditions (data not shown).

[0269] Therefore, it has been concluded that the optimal parameters for experiments with the endolysins of the present invention are under anaerobic conditions, pH 5.0, and with a step of removing the imidazole from the endolysin eluate.

[0270] 4.3-Expression level

[0271] Table 4 describes the concentration overview of all endolysin constructs. Each construct with a concentration higher than 0.2 mg / ml after removal of imidazole was adjusted to a concentration of 0.2 mg / ml by dilution. The constructs with lower concentrations were kept intact and tested for activity. The concentration of the native endolysin EL6 (not shown in Table 4) was lower than 0.2 mg / ml. H4, H11 and H12 appear to confer low solubility and expression levels, because most constructs are below the threshold of 0.2 mg / ml. Also for H1, some constructs have low concentrations.

[0272] [Table 4]

[0273] Expression level overview (quantitative)

[0274]

[0275] 4.4-Quantitative evaluation of lysis of four major Gardnerella species by endolysin action in suspension under optimized conditions

[0276] The lytic activity of four major Gardnerella species against 91 constructs (native and domain-swapped endolysins) was quantitatively evaluated using the method described in Reference Example 2. Briefly, 90ul5e7 CFU / ml of the indicated strains were incubated with 10ul of endolysin (concentration adjusted to 0.2mg / ml whenever possible, see Table 4) under anaerobic conditions at pH 5.0 for 5 hours.

[0277] The results are shown together with Tables 5A to 5C. Figures 7A to 7D In. Figures 7A to 7DIn, the logarithmic Y axis depicts the count of viable cells. The dotted line represents the limit of detection (LOD) provided by coating 2ul reaction mixture (500CFU / ml). Every combination of 10 natural H-domains and 9 natural B-domains was assessed, including natural endolysin (H1B1, H2B2, H3B3, etc.), and H6B6. Other constructs with B-domain B6 were not tested, because B6 inactivated all H-domains fused thereto, as determined by OD measurement (data not shown). Each construct was measured for each of 4 main Gardnerella species G.vaginalissensu stricto, G.leopoldii, G.piotii and G.swidsinki. Table 5 summarizes the log10 of the resulting CFU and reduces, organized by H-domain and B-district. All conditions were measured in triplicate. Survival after 5 hours of incubation with endolysin vs. buffer at pH 5.0 under anaerobic conditions was measured by quantitative plating. These values ​​represent the log10 of the ratio of viable CFU of treated vs. untreated cells. The average of three replicates 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 the 3 control treatment measurements for Gv-9 is 1.0x10 7 CFU / ml, while the average of the three samples treated with H2B10 was 2.5x10 3 CFU / ml, then the log10 value of the reduction in CFU of Gv_9 by H2B10 would be log10(2x10 3 / 10 7 ) = -3.7. Conversely, a reduction value of -3.7 means that the viable CFU in the treated sample decreased by 10 compared to the untreated control. 3.7 In the case of an inactive endolysin, for example, H4B3 on Gv_9, the Gv_9 CFU after treatment will be equal to the CFU measured in the control treated sample, and the ratio of the two CFU values ​​will be 1. Therefore, the reduction value of H4B3 on Gv_9 is log10(1)=0.0.

[0278] [Table 5A]

[0279]

[0280] Table 5B below describes the same data as Table 5A, but shows the average of the log10 activity of each construct against four Gardnerella strains. On the right and bottom, the activity rankings of each native H-domain relative to all native B-domains (except B6) and each native B-domain relative to the average of all native H-domains (except B6) and the respective native H-domains and native B-domains are shown, respectively.

[0281] [Table 5B]

[0282]

[0283] Table 5C depicts the activity ranking of all endolysins based on the data in Table 5B

[0284] [Table 5C]

[0285]

[0286] Table 5D depicts the average log 10 lysis for each Gardnerella strain used. The average was calculated for the log 10 activity of all constructs tested.

[0287] [Table 5D]

[0288]

[0289] Tables 5A to 5C show that the activity of endolysin is highly specific and depends on the H-domain / B-region combination and the bacterial strain tested. Each construct was measured for four Gardnerella strains (see Table 5A). On average, the most active H-domain is H2, and the CFU of all B-regions (except B6) is reduced by an average of 3.1 log10 units, followed by H7, H10 and H5 (see Table 5B). In the B-region, B10 is the most active, with an average CFU reduction of 2.7 log10 units, followed by B11, B12 and B3.

[0290] Surprisingly and unexpectedly, the inventors discovered that several recombinant endolysins had greater activity than any of the native endolysins (H1B1 to H12B12), especially when observed in all four 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 natural endolysin (see Table 5C). H7B3 is ranked 4th overall (see Table 5C) and is more active than any other natural endolysin included in the experiment. In fact, the top 10 most active natural endolysins are H10B10 (6th), and the second most active natural endolysin is H3B3 (13th). In short, recombinant cell endolysins according to the present disclosure may exhibit significantly higher activity than natural endolysins.

[0291] Although not limited by a particular theory, the unexpected increase in the anti-Gardnerella killing activity observed by the endolysin according to the present invention through domain exchange can be explained as follows. The natural endolysin on the prophage undergoes a Darwinian evolution process, in which the reproduction of the entire prophage is optimized. However, the possibility of mutations that lead to higher reproduction of the corresponding prophage by simultaneously improving the catalytic activity of the endolysin and simultaneously expanding its cross-species host range only in the genus Gardnerella is very low. In contrast, some Gardnerella prophages must evolve the N-terminal catalytic domain of the endolysin to the highest activity, while other prophages must optimize the C-terminal region of the endolysin to obtain the most extensive cross-Gardnerella species activity. Therefore, by combining the highly evolved N-terminal catalytic domain of one of the endolysins of the present invention with the highly evolved C-terminal region of another endolysin of the present invention encoded by different genomes from different prophages, a recombinant endolysin with a higher optimized anti-Gardnerella species killing activity than the natural endolysin of the present invention can be obtained. This is achieved, for example, by the recombinant endolysins H2B10, H2B11, H2B12 and H7B3 of the present invention, all of which have higher killing activity than the natural endolysin EL2 or any other natural endolysin and span all Gardnerella species (see Figures 7A to 7D and Table 5A).

[0292] In addition, most constructs are much more active to Gv_23 (G.swidsinskii) than to other three tested strains (see Table 5A). The average activity of all constructs of each Gardner strain (see Table 5D) confirmed that Gv_23 is most susceptible to endolysin, followed by Gv_9 and Gv_11, and Gv_17 (G.piotii) is the least susceptible. This susceptibility order is mainly the situation of endolysin constructs. Although Gv_23 is the most susceptible strain, several log10 units are poor for many constructs, and sometimes Gv_17 is more susceptible than Gv_9 or Gv_11 (for example, for H2B10, the most active endolysin on the whole). Not bound by a specific theory, susceptibility differences can be explained by structural defects (such as Gv_23 weaker / thinner / more accessible cell wall) or the endolysin tested is more strongly to the enzymatic activity of Gv_23.

[0293] Furthermore, it can be concluded that the concentration of the endolysin in solution is crucial for its activity in the assay. The low concentration constructs described in Table 4 generally also had 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 were some surprising cases, such as H12B11, which had very low expression levels but relatively high activity.

[0294] 4.5-Activity Mode Analysis

[0295] like Figure 8 As shown in the dendrogram of , the sequences of the native H-domains were aligned and compared to correlate with activity patterns. It is expected that the most active H-domains 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 is the least active. The second most active H-domains H7 and H10 are also quite distant from each other and from H2. The fourth most active H-domain H5 is closest to H7. In addition, the combination with the B-region that makes the recombinant endolysin most active 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 is the case with its closest homolog H5.

[0296] The B-region was also aligned to check homology with the activity pattern, e.g. Fig. 9 The most active B-regions in the analysis of Tables 5A to 5C were B10, B11, B12, followed by B3, all with mean CFU reduction values ​​greater than 2 log10 units. In contrast to the pattern of the H-domains, the 4 most active B-regions were the 4 closest homologs within the group of B-regions tested. Interestingly, the B5 and B7 regions were identical (see Fig. 9 ).from Figures 7A to 7D As can be seen, H2B10, H2B11, and H2B12 achieved the best overall results.

[0297] As explained in Example 2, each native B-region comprises two B-domains, an N-terminal cell wall domain and a C-terminal cell wall domain. Fig.10 and 11As shown in , the sequences of each native B-domain in the B-region are 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 the two repeated motifs in each B-region. The C-terminus of all B-domains is a conserved sequence (VNELL or VNKLL), and sequences homologous to it 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 in each B-region. As an exception, B6 has only one truncated B-domain, which is likely the reason why EL6 is completely inactive.

[0298] In conclusion, specific combinations of H-domain and B-region have been shown to be crucial and each H / B combination that resulted in an endolysin with higher killing activity compared to the native endolysin was a surprising and unpredictable finding.

[0299] Example 5 - Determination of activity against beneficial Lactobacilli

[0300] The healthy vagina is colonized primarily by three Lactobacillus species: L. crispatus, L. gasseri and L. jensenii. They maintain an acidic pH of 3.5-4.5 by producing lactic acid and a protective oxidative environment by producing H2O2. Recovery from BV is associated with the repopulation of these Lactobacilli in the vagina, and anti-BV drugs should advantageously not interfere with this process. Antibiotics obviously interfere with this process, which is why there is still a strong need for improved methods and compositions for the treatment of 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 Lactobacilli species in the healthy vagina. The experiments were performed under anaerobic conditions at pH 5.0 using the method described in Reference Example 2. As Fig.12As shown in , the tested recombinant endolysins did not exhibit any killing activity against the three beneficial lactobacilli used, Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii. Although the endolysins of the present invention exhibited high killing activity against Gardnerella, they were ineffective against the most common beneficial lactobacilli. Therefore, these results confirm the genus-selective activity of the endolysins of the present invention and their status as candidate drugs for innovative drugs against BV. In this regard, the treatment of BV with the endolysins of the present invention is far superior to currently available treatment methods, such as treatment with the antibiotics metronidazole and clindamycin.

[0301] Example 6 - Activity assay of standard therapeutic antibiotics metronidazole and clindamycin against Gardnerella strains grown in suspension

[0302] One of the major drawbacks of BV treatment is the high recurrence rate in many women, which leads to repeated administration of antibiotics with attendant instability of the microbiome and other side effects. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Gardnerella cells of the strain also used for the endolysin activity assay were then measured using the methods described in Reference Examples 6 and 7. In short, the measurement protocol had to be substantially adapted from international standards, which are not suitable for MIC and MBC measurements of Gardnerella. The main parameters to be changed were the growth medium (Gardnerella does not grow in Mueller-Hinton broth, which is typically used for MIC measurements), anaerobic conditions, incubation time, and the starting concentration of bacteria in the first round of experiments. The starting concentration was changed from the standard 5×10 5 CFU / ml was changed to 2.5×10 7 CFU / ml, primarily because in the vagina of BV patients, cells are also very concentrated and the effect of antibiotics should be measured at cell densities more comparable to those used for endolysin activity assays. The effects of metronidazole (obtained from Gatt-Koller) and clindamycin (obtained from Ratiopharm) on suspension growth of Gardnerella strains were evaluated. Basically, 2.5 × 10 7 CFU / ml suspensions of Gardnerella were incubated with the indicated concentrations of antibiotics and incubated at 37°C under anaerobic conditions for 48 hours. The MIC was defined as the minimum antibiotic concentration at which no growth was detected after 48 hours as measured by OD. OD (610) was measured at the beginning and end of the experiment. At the end of the experiment, 2 ul of each reaction mixture was spotted on agar to determine the MBC. Table 6A summarizes the Fig.13 The experimental results described in. Resistance (R) is defined as >= 32 μg / ml for metronidazole and > 8 mg / ml for clindamycin. According to international standards, sensitivity (S) is defined as <= 8 μg / ml for metronidazole and <= 2 μg / ml for clindamycin.

[0303] [Table 6A]

[0304]

[0305] [Table 6B]

[0306]

[0307] according to Fig.13 The results shown in Figure 2 show that 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 , which is the concentration of antibiotic that kills 90% of cells within a defined time, while the MBC is defined in this application as the complete elimination of 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 endolysins measured are bactericidal by definition, since they cause complete decomposition of bacterial cells. Therefore, these results show that the endolysins of the present invention are superior to antibiotics in the treatment of BV.

[0308] A second round of MIC and MBC experiments was performed, in which some experimental parameters were changed (i) the starting cell number was now 1 × 10 according to CLSI (Clinical and Laboratory Standards Institute) standards. 5 -1×10 6 , (ii) clindamycin hydrochloride powder (Sigma Aldrich, catalog no. C5269), (iii) NYC-III broth instead of Hardy broth, and (iv) 96-well plates instead of 384-well plates. Fig.14 As shown in Table 6B, all Gardnerella strains still had very low MICs to metronidazole (8-128 μg / ml), while clindamycin hydrochloride- Fig.13 In contrast to clindamycin presented in Table 6A - now there is both inhibitory and bactericidal effect at low concentrations (MIC ≤ 1 μg / ml).

[0309] Example 7 - Activity assay of a representative domain-swapped endolysin (H2B10) against different Gardnerella strains grown in suspension.

[0310] To analyze the MIC and MBC of endolysin H2B10, 1 × 10 5 -1×10 6 H2B10 showed MICs in the low μg / ml range (0.5-4 μg / ml), indicating that Gardnerella cells are highly sensitive to endolysins ( Fig.15, Table 7). The conditions for measuring MBC are stricter than the standard. For example, MBC is usually measured 90 , i.e., the antimicrobial concentration that kills 90% of the cells within a defined time, while the MBC is defined in this application as the dose that reduces the starting cell count by at least 99.5%. H2B10, as a representative of the endolysins claimed for protection herein, showed MICs and MBCs 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>8μg / ml) ( Fig.13 ), while the bactericidal effect of clindamycin hydrochloride (Sigma Aldrich) at a concentration of ≤1 μg / ml is much better ( Fig.15 ). In general, antibiotics alone are known to be inadequate in eradicating the Gardnerella biofilm that is a hallmark of BV, a suspected reason for the reportedly very high recurrence rate of BV. In addition to leaving a residual viable biofilm, antibiotics remove part of the beneficial organisms of the vaginal microbiota, which then opens up niches for other pathogens (e.g., fungi). Therefore, endolysin-based treatments that selectively eradicate bacterial cells of the genus Gardnerella and presumably eradicate biofilms without harming beneficial Lactobacilli are considered superior to standard antibiotic treatments for BV.

[0311] Table 7

[0312]

[0313] Reference Example 1 - Cloning, expression and purification of endolysin

[0314] Material :

[0315] 96-well Multiscreen HTSDurapore 96-well Filterplatten, PS (Labshop catalog no. 44.MSGVS22)

[0316] PD MiniTrap desalting column with Sephadex G-25 (GE Lifescience, catalog no. 28918007)

[0317] Slide-A-Lyzer TM MINI Dialysis Apparatus, 10K MWCO, 2 mL (Thermo Scientific, catalog no. 88404)

[0318] Fastbreak reagent (Promega, catalog no. V8571)

[0319] Lysis buffer: 50 mM phosphate pH 6, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP, 1xFastBreak, Benzonase

[0320] Wash buffer I: 50 mM phosphate pH 6, 150 mM NaCl, 20 mM imidazole, 1 mM TCEP (1,5 ml)

[0321] Wash buffer II: 50 mM phosphate pH 6, 150 mM NaCl, 40 mM imidazole, 1 mM TCEP (1,5 ml)

[0322] • Elution buffer: 50 mM phosphate pH 6, 150 mM NaCl, 250 mM imidazole, 1 mM TCEP (1,1 ml).

[0323] method :

[0324] The expression construct was transformed into Escherichia coli strain Bl21 (DE3) and selected using appropriate antibiotics. Cells from 2 ml culture (TB + lactose, 25 ° C, O / N) were resuspended in 1.5 ml lysis buffer and lysed by FastBreak reagent (Promega). The soluble fraction in the cells was separated by centrifugation at 15000g, 30min, 4 ° C. The soluble protein fraction was loaded onto 100 μL nickel affinity matrix, washed with 15 column volumes (CV) of wash buffer I and II, respectively, and then eluted in 10 CV elution buffer. Then, the eluent buffer can be replaced with 20mM phosphate pH 6.0, 150mM NaCl using a desalting column to remove imidazole. After elution (or appropriate replacement of buffer), the concentration of the purified protein was adjusted to 0.2 mg / ml, and then the solution was sterilized by filtration with a 96-well filter plate.

[0325] Reference Example 2 - Activity determination in bacterial suspension

[0326] Material :

[0327] 1. Hardy broth, autoclaved at 121°C for 20 min:

[0328] 12.0 g pancreatic digest of casein (Sigma Aldrich, catalog no. 70172-100G)

[0329] 10.0 g peptone (Sigma Aldrich, catalog number no. 82450-100G)

[0330] 5.0 g of peptide digest of animal tissue (Sigma Aldrich, catalog no. 70174-100G)

[0331] 5.0 g sodium chloride (Carl Roth, catalog number no. 3957.1)

[0332] 3.0 g beef extract (SigmaAldrich, catalog no. B4888-50G)

[0333] 3.0 g yeast extract (SigmaAldrich, catalog no. Y1625-250G)

[0334] 1.0 g soluble starch (SigmaAldrich, catalog no. S9765-250G)

[0335] Deionized H2O to 1 liter (produced in PhagoMed laboratories using Millipore RiOs Essential16)

[0336] 2. Hardy broth agar, autoclaved at 121°C for 20 min: Same as Hardy broth, but use 15 g Agar Bacteriogical (OXOID catalog number #LP0011)

[0337] 3. Hardy Broth Top Agar, autoclaved at 121°C for 20 min: Same as Hardy Broth, but using 7 g Agar Bacteriogical (OXOID Cat. #LP0011)

[0338] 4. NYC-III medium, pH 5.0, autoclaved at 121°C for 20 min, after which horse serum (NYC-III-HS-5.0) was added

[0339] 12 g HEPES (Sigma Aldrich, catalog no. H4034-100G)

[0340] 7.5 g peptone No.3 (BD, catalog number no.211693)

[0341] 1.9 g yeast extract (Sigma Aldrich, catalog no. Y1625-250G)

[0342] 2.5 g sodium chloride (Sigma Aldrich, catalog number S9888-1 kg-M)

[0343] 2.5 g glucose (MW 180.16 g / mol) (Sigma Aldrich, catalog no. G6152-1KG)

[0344] Deionized water to 450 ml total volume

[0345] 50 ml horse serum (HS), heat inactivated 100 ml (Thermo Fisher Scientific, catalog no. 26050070), autoclaved and added

[0346] 5. General Materials:

[0347] BD Chocolate Agar Plates for Gardnerella (BD, catalog no. 254060)

[0348] BD Schaedler / 5% Sheep Blood Plate for Lactobacillus (BD, catalog no. 24042)

[0349] Isovitalex (BD, catalog no. 211876)

[0350] Hardy broth + Isovitalex (see above), adjusted to pH as indicated

[0351] Hardy agar + Isovitalex (see above)

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

[0353] 96-U-well plate (Sigma Aldrich, catalog no. M2311-100EA)

[0354] 96-well plate with lid (Labshop, catalog no. 44.781662)

[0355] Greiner 384-well plate (Sigma Aldrich, catalog no. M1937-32EA)

[0356] Anaerobic agent bag (Sigma-Aldrich, catalog number no. 68061-10SACHETS-F)

[0357] Anaerobic index test (Sigma-Aldrich, catalog number no.59886-1PAK-F)

[0358] Anaerobic jars (Sigma-Aldrich, catalog no. 28029-1EA-F) or plastic lunch boxes sealed with rubber gaskets, purchased at a local appliance store.

[0359] 6. Bacterial strains:

[0360] Gardnerella strains:

[0361] ·Gv_1:UGent 09.07

[0362] ·Gv_8:UGent 25.49

[0363] ·Gv_9:ATCC 14018

[0364] ·Gv_10:UGent 06.41

[0365] ·Gv_11:UGent 09.48

[0366] ·Gv_17:UGent 18.01

[0367] ·Gv_23:GS10234(FC2)

[0368] Lactobacillus strains:

[0369] Lactobacillus jensenii PB2003-013-T2-2

[0370] · Lactobacillus gasseri (L. gasseri) 020566

[0371] Lactobacillus crispatus LAB117

[0372] method :

[0373] Gardnerella cells were recovered from frozen stocks by plating on chocolate agar plates (Beckton Dickinson) and incubating at 37°C for 48h under anaerobic conditions. For Lactobacilli, 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 620nm as indicated) of 0.1. It must be noted that two Tecan microplate readers (with 610nm or 620nm filters, respectively) were used interchangeably in the experiments. Although it did not cause any difference to the experiments, the exact wavelength used is specified in each example. If not otherwise stated, for different species / endolysin combinations, 90μl of cell suspension was mixed with 10μl of endolysin solution in a 384-well plate. OD (610-620 nm as indicated) was measured at the beginning and end of the reaction, either as a continuous kinetic or as two measurement points in a Tecan F200 microplate reader. Reactions were incubated at 37°C for 5 h (or for another specified time) under anaerobic, microaerobic or aerobic conditions as indicated. Anaerobic conditions meant complete depletion of oxygen from the container (Sigma-Aldrich anaerobic jar or sealed lunch box) with anaerobic bags in which the bacteria were incubated and confirmation of lack of oxygen by an anaerobic indicator within the container. In the case of microaerobic conditions as indicated, the candle in a jar method was used (a tea candle was lit in a suitable sealable container, which would reduce the oxygen content until the flame went out). The reaction was then performed in 5 steps (10 -1 to 10 -5 ) dilute each well and plate 2 μl of each dilution of each reaction mixture 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 assay plates were incubated at 37°C for 48 hours under anaerobic conditions.

[0374] Reference Examples 6 and 7 - MIC and MBC Measurements

[0375] Material :

[0376] General Materials:

[0377] Metronidazole (Gatt-Koller, Metronidazole, 10 g, 606293914)

[0378] Clindamycin (Ratiopharm, 300mg / 2ml ampoule 5x)

[0379] Clindamycin hydrochloride (Sigma Aldrich, 10 mg, catalog no. C5269)

[0380] Endolysin H2B10 [530 μg / ml] in MES buffer (50 mM MES, 100 mM NaCl, 8 mM MgSO4, pH = 5.5)

[0381] BD Chocolate Plates for Gardnerella (BD, catalog no. 254060)

[0382] BD Schaedler / 5% sheep blood agar plates for Lactobacillus (BD, catalog no. 254042)

[0383] Isovitalex (BD, catalog no. 211876)

[0384] Hardy broth + Isovitalex (see above), adjusted to pH as indicated

[0385] NYC-III+HS broth, pH 5.5 NYC-III-HS agar plates: NYC-III+HS medium as above, but with 1.5% agar added before autoclaving

[0386] Hardy agar + Isovitalex (see above)

[0387] Hardy top agar + Isovitalex (see above)

[0388] 96-U-well plate (Sigma Aldrich, catalog no. M2311-100EA)

[0389] 96-well plate with lid (Labshop, catalog no. 44.781662)

[0390] Greiner 384-well plate (Sigma Aldrich, catalog no. M1937-32EA)

[0391] Anaerobic agent bag (Sigma-Aldrich, catalog number no. 68061-10SACHETS-F)

[0392] Anaerobic index test (Sigma-Aldrich, catalog number no.59886-1PAK-F)

[0393] Anaerobic jars (Sigma-Aldrich, catalog no. 28029-1EA-F) or plastic lunch boxes sealed with rubber gaskets, purchased at a local appliance store.

[0394] method :

[0395] Bacteria were plated from frozen stocks onto BD Choc agar plates (Gardnerella) and incubated at 37°C for 48 h under anaerobic conditions. 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 (610 or 620 nm as indicated) of 0.05. It must be noted that two Tecan microplate readers (with 610 nm or 620 nm filters, respectively) were used interchangeably in the experiments. Although it did not make any difference to the experiments, the exact wavelength used is specified in each example. Antibiotics were made into 20× stocks for each desired final concentration. 95 μl of cell suspension was mixed with 5 μl of antibiotic dilution in a 384-well plate. The OD at the start of the reaction was measured (610-620 as indicated), and the plates were then incubated at 37°C for 48 h under anaerobic conditions. Afterwards, the OD (610-620 shown) was measured again for MIC determination, where the MIC was defined as the lowest concentration of the antibiotic at which the OD (610-620 shown) was not higher than the level measured at the beginning of the experiment. After measuring the OD, 2 μl per well was spotted on NYC-III + HS agar plates. The plates were then incubated for another 48 h at 37 ° C under anaerobic conditions. After incubation, cell growth on each spot was assessed, and the MBC was defined as the lowest antibiotic concentration at which no bacteria grew on the plate. For each condition, the experiment was performed in triplicate.

[0396] In the second round of MIC and MBC experiments using antibiotics, the Gardnerella cell suspension was adjusted to McFarland standard 0.5 (approximately OD (610) 0.07) and then diluted 1:75 according to CLSI (Clinical and Laboratory Standards Institute) standards. Antibiotics were prepared according to CLSI standards and 50 μl of cell suspension was mixed with 50 μl of antibiotics in a 96-well plate. In addition, MIC and MBC were determined as described above.

[0397] For MIC and MBC determination of domain-swapped endolysin H2B10, 50 μl of Gardnerella cell suspension was mixed with 50 μl of H2B10-containing solution, which was serially diluted 1:1, in a 96-well plate. The OD at the start of the reaction was measured. 610The plates were then incubated under anaerobic conditions at 37° C. for 48 h. Thereafter, the OD (610) was measured again for MIC determination, where the MIC was defined as the lowest concentration of H2B10 where the OD was not higher or only slightly higher than the level measured at the beginning of the experiment.

[0398] [Table 7]

[0399]

[0400] *Row 1 = Name according to this application, if applicable; Row 2 = Corresponding native amino acid sequence listing

[0401] SEQ ID NO:1

[0402] >H1

[0403] >196aa

[0404] MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0405] KTAGLDVGAYWYSYANSGFEAAEEAQSLMNMLSGKSFEYPVYFDLEEKSQ

[0406] LNRGRAFCDSLITSFCNKLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIA

[0407] QWNTHCNYQGSYGLWQYSSNGSVPGVAGRVDMDYAYVDYPSIIK

[0408] SEQ ID NO:2

[0409] >H2

[0410] >196aa

[0411] MSKRGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0412] KTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ

[0413] LNRGRAFCDSLITGFCNKLESCGYYAGFYTSLSTANNLVSAHVRNRYALWI

[0414] <h2 style=";text-align:left;direction:ltr">AQWNTHCSYQGSYGLWQYSSSSGSVPGVAGRVDMDYAYVDYPSIIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0415] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0416] <h2 style=";text-align:left;direction:ltr"> >H3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0417] <h2 style=";text-align:left;direction:ltr"> >196aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0418] <h2 style=";text-align:left;direction:ltr"> MSKKGIDVSVWQGDIDFNSVKASGVEFVIIRAGYGIGHKDKWFEENYRKA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0419] <h2 style=";text-align:left;direction:ltr"> KTAGLDVGSYWYSYASSAGEASEEAQSCVNILSGKSFEYPIYFDLEEKSQLN<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0420] <h2 style=";text-align:left;direction:ltr"> RGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0421] <h2 style=";text-align:left;direction:ltr"> WNTHCSYQGSYGLWQYSSSSGSVNGIAGRVDMDYAYVDYPSVIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0422] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0423] <h2 style=";text-align:left;direction:ltr"> >H4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0424] <h2 style=";text-align:left;direction:ltr"> >196aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0425] <h2 style=";text-align:left;direction:ltr"> MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0426] <h2 style=";text-align:left;direction:ltr"> KTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0427] <h2 style=";text-align:left;direction:ltr"> LNRGRAFCDSLITSFCSKLETYGYYAGFYTSLSVVNNLVSAHVRDRYALWIA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0428] <h2 style=";text-align:left;direction:ltr"> QWNTHCSYQGSYGLWQYSSSSGSVPGVAGRVDMDYAYVDYPSIIK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0429] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0430] <h2 style=";text-align:left;direction:ltr"> >H5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0431] <h2 style=";text-align:left;direction:ltr"> >196 aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0432] MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0433] KTAGLDVGAYWYSYANSSSEAAEEAQSCANMLSGKSFEYPVYFDLEEKSQL

[0434] NRGRAFCDSLITGFCSKLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIA

[0435] QWNTHCSYQGSYGLWQYSSNGSVPGVAGRVDMDYAYKDYPSIIK

[0436] SEQ ID NO:6

[0437] >H6

[0438] >196 aa

[0439] MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0440] KTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ

[0441] LNRGRAFCDSLITSFCSKLESCGYYAGFYTSLSTANNLVSAHVRNRYALWIA

[0442] QWNTHCDYQGSYGLWQYSSSGSVPGVAGRVDMDYAYKNYPSIIK

[0443] SEQ ID NO:7

[0444] >H7

[0445] >196aa

[0446] MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0447] KTAGLDVGAYWYSYANSASEAAEEAQSCANMLSGKSFEYPVYFDLEEKSQ

[0448] LNRGRAFCDSLITSFCSKLETYGYYAGFYTSLSTANNLVSSHVRNRYALWIA

[0449] QWNTHCSYQGSYGLWQYSSSGSVPGVAGRVDMDYAYKDYPSIIK

[0450] SEQ ID NO:8

[0451] >EL8

[0452] >306 aa

[0453] MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0454] KTAGLDVGAYWYSYANSSSEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQL

[0455] NRGRAFCDSLITSFCSKLETYGYYAGFYTSLSTANNLVSSHVRNRYALWIAQ

[0456] WNTHCSYQGSYGLWQYSSNGSVPGVAGRVDMDYAYVDYPSIIKNAGLNGY

[0457] KNGGSYTAPQTSSIDDVAREVINGAWGNGNERKQRLTQAGYDYTSVQNKV

[0458] NKLLGVKACRKSVDELAREVIRGTWGNGNERKNRLTQAGYDYDTVQKRV

[0459] NELL

[0460] SEQ ID NO:9

[0461] >EL9

[0462] >251 aa

[0463] MSKKGIDVSEWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA

[0464] KTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ

[0465] LNRGRVFCDSLITSFCNKLEACGYYAGFYTSLSTANNLVSSHVRNRYALWIA

[0466] QWNTHCDYQGSYGLWQYSSSGSVPGVAGRVDMDYAYVDYPSIIKNAGLNG

[0467] CKNGGSDQAARTSSIDEVAREVINGAWGNGSTRKQRLTSAGYDYASVAKSEQ ID NO:10

[0468] >H10

[0469] >196aa

[0470] MSKRGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEENYRKA

[0471] KTVGLDVGAYWYSYASSAGEASEEAQSCVNILSGKSFEYPVYFDLEEKSQL

[0472] NRGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQ

[0473] WNTHCSYQGSYGLWQYSSSGSVNGIAGRVDMDYAYVDYPSVIK

[0474] SEQ ID NO:11

[0475] >H11

[0476] >196aa

[0477] MSKRGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEQNYRKA

[0478] KTTGLDVGAYWYSYASSAGEAAEEAQSCVNILSGKSFEYPVYFDLEEKSQL

[0479] NRGRDFCDSLITSFCNKLETYGYYAGFYTSLSVANNLVSSHVRDRYALWIAQ

[0480] WNTHCDYQGSYGLWQYSSSGSVDGIAGRVDMDYTYVDYPSVIK

[0481] SEQ ID NO:12

[0482] >H12

[0483] >196 aa

[0484] MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGHKDKWFEENYRKA

[0485] KTAGLDVGSYWYSYASSAGEVALEAQSCVNILSGKSFEYPVYFDLEEKSQL

[0486] NRGRDFCDSLITSFCNKLEACGYYAGFYTSLSVANNLVSSHVRDRYALWIAQ

[0487] WNTHCSYQGSYGLWQYSSSGSVNGIAGRVDMDYAYVDYPSVIK

[0488] SEQ ID NO:13

[0489] >EL13

[0490] >306 aa

[0491] MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIECKDKWFEQNYRKA

[0492] KTAGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ

[0493] LNRGRAFCDSLITSFCNKLESCGYYAGFYTSLSTANNLVPAHVRNRYALWIA

[0494] QWNTHCDYQGSYGLWQYSSSGSVPGVAGRVDMDYAYVDYPSIIKNAGLNG

[0495] YKNGESHQATRTTSIDEVAREVINGAWGNGNERKQRLTQAGYDYASVQNK

[0496] VNELLGVKACRKSVDELAREVIRGTWGNGNERKNRLTSAGYDYDTVQKR

[0497] VNELL

[0498] SEQ ID NO:14

[0499] >EL14

[0500] <h2 style=";text-align:left;direction:ltr">>306 aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0501] <h2 style=";text-align:left;direction:ltr"> MSKKGIDVSVWQGDIDFNAVKASGVEFVIIRAGYGIGCKDKWFEQNYRKA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0502] <h2 style=";text-align:left;direction:ltr"> KTCGLDVGAYWYSYANSGFEAAEEAQSCVNMLSGKSFEYPVYFDLEEKSQ<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0503] <h2 style=";text-align:left;direction:ltr"> LNRGRAFCDSLITSFCNKLEACGYYAGFYTSLSTANNLVSAHVRNRYALWIA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0504] <h2 style=";text-align:left;direction:ltr"> QWNTHCSYQGSYGLWQYSSSSGSVPGVAGRVDMDYAYVDYPSIIKNAGLNG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0505] <h2 style=";text-align:left;direction:ltr"> YKNGESHQATRTTSIDEVAREVINGAWGNGNERKQRLTSAGYDYASVQNK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0506] <h2 style=";text-align:left;direction:ltr"> VNELLGVKACRKSVDELAREVIRGAWGNGSTRKQRLTSAGYDYDTVQKR<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0507] <h2 style=";text-align:left;direction:ltr"> VNELL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0508] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0509] <h2 style=";text-align:left;direction:ltr"> >B1_N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0510] <h2 style=";text-align:left;direction:ltr"> >49 aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0511] <h2 style=";text-align:left;direction:ltr"> DQAARTSSIDEVAREVINGAWGNGSTRKQRLTSAGYDYASVQNKVNELLSEQ ID NO:16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0512] <h2 style=";text-align:left;direction:ltr"> >B1_C<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0513] <h2 style=";text-align:left;direction:ltr"> >49 aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0514] <h2 style=";text-align:left;direction:ltr"> GVKACRKSVDELAREVIRGAWGNGSTRKQRLAQAGYDYDTVQKRVNELLSEQ ID NO:17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0515] <h2 style=";text-align:left;direction:ltr"> >B2_N<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0516] <h2 style=";text-align:left;direction:ltr"> >49 aa<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0517] <h2 style=";text-align:left;direction:ltr"> DQAARTSSIDEVAREVINGAWGNGNERKQRLTSAGYDYASVQNKVNELLSEQ ID NO:18<h2 style=";text-align:left;direction:ltr">

[0518] >B2_C

[0519] >49 aa

[0520] GVKACRKSVDEIAREVIRGTWGNGSTRKQRLTQAGYDYDTVQKRVNELLSEQ ID NO:19

[0521] >B3_N

[0522] >49 aa

[0523] YTAPQTSSIDEVAREVINGDWGNGNDRKNRLISAGYDYASVQNKVNELLSEQ ID NO:20

[0524] >B3_C

[0525] >49 aa

[0526] GVKAIRKSVDELAREVIRGTWGNGSMRKHRLTQAGYDYDAVQKRVNELLSEQ ID NO:21

[0527] >B4_N

[0528] >49 aa

[0529] DQATRTSSIDEVAREVINGAWGGNNERKQRLTSAGYDYASVQNKVNKLLSEQ ID NO:22

[0530] >B4_C

[0531] >49 aa

[0532] GVKAYRKSVDELAREVIRGTWGNGNERKQRLAQAGYDYDTVQKRVNELLSEQ ID NO:23

[0533] >B5_N

[0534] >49 aa

[0535] DQAARTSSIDEVAREVINGAWGGNNERKQRLTQAGYDYTSVQNKVNKLLSEQ ID NO:24

[0536] >B5_C

[0537] >49 aa

[0538] GVKACRKSVDELAREVIRGTWGGNNERKNRLTQAGYDYDTVQKRVNELLSEQ ID NO:25

[0539] >B6_N

[0540] >43 aa

[0541] NQAARTSSIDVAREVINGAWGGNNERKQRLTQAGYDYASVAK

[0542] SEQ ID NO:26

[0543] >B7_N

[0544] >49 aa

[0545] DQAARTSSIDEVAREVINGAWGGNNERKQRLTQAGYDYTSVQNKVNKLLSEQ ID NO:27

[0546] >B7_C

[0547] >49 aa

[0548] GVKACRKSVDELAREVIRGTWGGNNERKNRLTQAGYDYDTVQKRVNELLSEQ ID NO:28

[0549] >B10_N

[0550] >49 aa

[0551] YTAPQISSIDEVAREVINGDWGNGNERKQRLTSAGYDYASVQNKVNELLSEQ ID NO:29

[0552] >B10_C

[0553] >49 aa

[0554] GVKAYRKSVDELAREVIRGTWGNGSTRKQRLTQAGYDYNAVQKRVNELLSEQ ID NO:30

[0555] >B11_N

[0556] >49 aa

[0557] YTAPQTSSIDEVAREVINGDWGNGNERKNRLTSAGYDYTSVQNKVNELLSEQ ID NO:31

[0558] >B11_C

[0559] >49 aa

[0560] GVKAYRKSVDELAREVIRGTWGNGSTRKQRLTQAGYDYDAVQKRVNELLSEQ ID NO:32

[0561] >B12_N

[0562] >49 aa

[0563] YTAPQTSSIDEVAREVINGDWGNGIERKNRLTSAGYDYTSVQNKVNELLSEQ ID NO:33

[0564] >B12_C

[0565] >49 aa

[0566] GVKAYRKSVDELAREVIRGTWGNGKTRKQRLTQAGYDYNAVQKRVNELL

Claims

1. A recombinant endolysin, comprising: (i) an N-terminal catalytic domain, the amino acid sequence of which is 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 amino acid sequence of the first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 28, 30 and 32, and the amino acid sequence of the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 29, 31 and 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 of the cell wall binding domains is from a second native endolysin, and wherein the first and second native endolysins are encoded by different genomes from different prophages.

2. The recombinant endolysin of claim 1, wherein the first cell wall binding domain is at the N-terminus of the second cell wall binding domain.

3. The recombinant endolysin of claim 1, wherein the N-terminal catalytic domain consists of the amino acid sequence shown in SEQ ID NO: 2, the first cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO: 28, and the second cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO:

29.

4. The recombinant endolysin of claim 1, wherein the N-terminal catalytic domain consists of the amino acid sequence shown in SEQ ID NO: 2, the first cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO: 28, and the second cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO:

31.

5. The recombinant endolysin according to any one of claims 1 to 4, wherein the linker region is a polypeptide consisting of the following amino acid sequence: (i) (XXX) n , wherein each X is independently G, A or S, and wherein n corresponds to the number of repetitions of sequence XXX; or (ii) X1X2GLNGX3X4NGGS, wherein X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q.

6. The recombinant endolysin according to claim 5, wherein the linker region is composed of the amino acid sequence (XXX): n Peptide composition: wherein the amino acid sequence (XXX) n Yes (GGS) n , and / or n is 2, 3, 4, 5 or 6.

7. The recombinant endolysin according to any one of claims 1 to 4, wherein the linker region consists of an amino acid sequence selected from the group consisting of: NVGLNGYKNGGS, NAGLNGYKNGGS, KAGLNGYKNGGS and NAGLNGYQNGGS.

8. The recombinant endolysin of claim 2, wherein the recombinant endolysin consists of an N-terminal catalytic domain, a linker region, and a C-terminal cell wall binding region consisting of a first cell wall binding domain and a second cell wall binding domain, wherein the N-terminal catalytic domain consists of the amino acid sequence shown in SEQ ID NO: 2, the linker region consists of the amino acid sequence NVGLNGYKNGGS, the first cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO: 28, and the second cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO:

29.

9. The recombinant endolysin of claim 2, wherein the recombinant endolysin consists of an N-terminal catalytic domain, a linker region, and a C-terminal cell wall binding region consisting of a first cell wall binding domain and a second cell wall binding domain, wherein the N-terminal catalytic domain consists of the amino acid sequence shown in SEQ ID NO: 2, the linker region consists of the amino acid sequence NVGLNGYKNGGS, the first cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO: 28, and the second cell wall binding domain consists of the amino acid sequence shown in SEQ ID NO:

31.

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

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

12. Use of the recombinant endolysin defined in any one of claims 1 to 9, the polynucleotide defined in claim 10 or the pharmaceutical composition defined in claim 11 for the preparation of a medicament for treating a disease or condition, wherein the disease or condition is bacterial vaginosis.

13. The use according to claim 12, wherein the bacterial vaginosis is caused by Gardnerella vaginalis strict sense , Gardnerella leopoldii , Gardnerella piotii and / or Gardnerella swidsinskii caused by.

14. The use according to claim 12 or 13, wherein the recombinant endolysin, polynucleotide or pharmaceutical composition is administered topically.

15. The use of claim 12 or 13, wherein the recombinant endolysin, polynucleotide or pharmaceutical composition is administered into the vagina of a female subject and / or into or onto the glans penis, foreskin or urethral entrance of a male subject.

16. The use according to claim 12 or 13, wherein the recombinant endolysin, polynucleotide or pharmaceutical composition is co-administered with a compound or composition that adjusts vaginal pH to 4.0-6.

0.

17. A plasmid comprising the polynucleotide of claim 10.

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

19. The bacterial host cell of claim 18, which is an E. coli cell.

20. Use of an endolysin according to any one of claims 1 to 9 in the preparation of a diagnostic kit for use in an in vitro method for diagnosing a disease or condition treatable with an endolysin according to any one of claims 1 to 9, 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 any one of claims 1 to 9, and optionally the N-terminal catalytic domain of an endolysin according to any one of claims 1 to 9, wherein the sample comprises microbial cells, and wherein the C-terminal cell wall binding region of the endolysin is optionally labeled; (ii) testing whether the polypeptide binds to and / or lyses microbial cells of the sample; and (iii) If the polypeptide binds to and / or lyses microbial cells, it is determined that the disease or disorder can be treated with an endolysin according to any one of claims 1 to 9.

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