Novel therapeutic uses of Gardnerella endolysins.

Abstract

The present invention relates to novel therapeutic uses of species-selective phage endolysins, particularly in the treatment of bacterial vaginosis (BV), and more particularly in the treatment of patients suffering from BV who have previously failed antibiotic treatment and / or where the infectious bacteria is resistant to antibiotic treatment. The present invention also relates to pharmaceutical compositions for use according to the invention and methods of treatment using same.

Description

[Technical field]

[0001] The present invention relates to novel therapeutic uses of species-selective phage endolysins, particularly in the treatment of bacterial vaginosis (BV), and more particularly in the treatment of patients suffering from BV who have previously failed antibiotic treatment and / or where the infectious bacteria is resistant to antibiotic treatment. The present invention also relates to pharmaceutical compositions for use according to the invention and methods of treatment using same.

[0002] Bacterial vaginosis (BV), also referred to in the literature as bacterial vaginosis, nonspecific vaginitis and nonspecific vaginitis, is the most common vaginal infection worldwide and is associated with significant adverse outcomes, including preterm labor and delivery, postpartum endometritis and increased risk of HIV acquisition. It is a vaginal dysbiosis in which commensal Lactobacilli are replaced by a polymicrobial biofilm, the pH increases from the native 3.5–4.5 to 5.5, and a foul-smelling fluid forms. It is most commonly defined as a pathological condition characterized by the loss of normal vaginal flora, especially the H2O2-producing species Lactobacilli, and the concomitant overgrowth of anaerobic bacteria, including Gardnerella vaginalis (G. vaginalis). The organism was initially called Haemophilus vaginalis, but has been repeatedly renamed as more information on its characteristics has been gained, and it is now classified as G. vaginalis, and until 2018 was considered to be the only member of the Gardnerella genus. However, as of early 2019, the genus Gardnerella actually includes at least 13 species, with the most frequent ones renamed as G. vaginalis sensu stricto, G. leopoldii, G. piotii, and G. swidsinskii (Vaneechoutte et al., 2019 Int. J. Syst. Evol. Biol. 898661). Gardnerella bacteria are special in that they are Gram-variable, i.e., they do not form the outer membrane that defines Gram-negative species. The cell walls are generally very thin, with only 10% or less peptidoglycan content, which is why the crystal violet dye used in Gram staining does not always produce the deep purple color typical of Gram-positive species. Rather, Gardnerella cells can appear both Gram-positive and negative in Gram stains. Based on 16S rRNA-based phylogenetic analysis, Gardnerella is classified as a Gram-positive bacterium in the family Bifidobacteriaceae.

[0003] During BV, the epithelial surface is covered with a dense collection of Gardnerella bacteria that form an adherent biofilm on the vaginal epithelium, while other species can proliferate, resulting in a polymicrobial biofilm that is often refractory to treatment. Biofilms are adherent communities of microorganisms held together by a polymeric matrix composed of polysaccharides, proteins and / or nucleic acids. The distinct gene expression patterns, as well as the physical structure of the biofilm, increase the bacterial resistance to many negative stimuli, including chemical disinfectants, extremes of pH, host immune defenses and antibiotics.

[0004] The recommended first-line therapy for BV is primarily antimicrobial treatment with nitroimidazole antimicrobials such as metronidazole (MDZ), tinidazole (TDZ) and secnidazole, and / or clindamycin (CLI). Metronidazole (MDZ) belongs to the group of nitroimidazoles and only gains its full activity when metabolized to its hydroxy metabolite (MDZ-OH). Antimicrobials are effective in rapidly reducing BV symptoms, but are associated with a high recurrence rate of up to 60% within 6 months of treatment. In a clinical trial in which patients with recurrent BV were treated with 0.75% MDZ vaginal gel for 16 weeks, the probability of sustained cure was 70% after 16 weeks (i.e., 30% of patients had symptoms at the end of the 16-week treatment period) and dropped to 34% 12 weeks after the end of treatment, i.e., at 28 weeks (Sobel et al., 2006, Am. J. Obstet. Gynecol. 194, 1283-1289). In addition to the possibility of reinfection from sexual partners, persistence of residual infection has been hypothesized as a reason for recurrence, potentially due to the formation of a biofilm that protects the bacteria causing BV from antibacterial therapy. Another reason could be antibiotic resistance of BV pathogens. Antibiotic resistance occurs naturally, but the misuse of antibiotics in humans and animals accelerates this process; i.e., existing treatments are unable to effectively penetrate the biofilm. Thus, cessation of antibiotic treatment leads to biofilm regrowth and recurrent symptomatology. Furthermore, antibiotic treatment, while leaving some remnants of viable biofilm, wipes out the vaginal microbiota, which opens this ecological niche to other pathogens (e.g., fungi). Thus, a frequent consequence of BV treatment is candidiasis. Thus, treatment failure and recurrent disease are common challenges with antibiotic treatment.

[0005] Thus, there is a need for new methods and compositions for treating patients suffering from BV, particularly those for whom antibiotic treatment is contraindicated and / or who have a record of adverse side effects from antibiotic treatment. There is also a need for the treatment of BV in individuals who have previously failed antibiotic treatment and / or who suffer from a bacterial infection in which the infectious bacteria is resistant to antibiotic treatment.

[0006] Therefore, the technical problem underlying the present invention is the provision of novel means and methods for the treatment of BV in difficult clinical situations, such as the patients identified herein above.

[0007] The technical problem is solved by providing the embodiments characterized in the claims.

[0008] In particular, the present invention provides a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin, for use in the treatment of bacterial vaginosis, which is administered to patients who have previously failed treatment with antibiotics, particularly nitroimidazoles and / or clindamycin.The present invention further provides a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin, for use in the treatment of bacterial vaginosis, which is administered to patients suffering from bacterial vaginosis, where the infectious bacteria are resistant to treatment with antibiotics, particularly nitroimidazoles and / or clindamycin.

[0009] Endolysins are promising alternatives to current antibiotics due to their ability to eradicate biofilms, their low tendency for the development of resistance, and their specificity for individual genera or species of bacteria. Natural and engineered Gardnerella-specific endolysins have been reported (WO 2020 / 225335 A1, Landlinger et al., 2021, Pathogens 10, 1-19, or WO 2020 / 229802 A1). WO 2020 / 225335 describes certain recombinant Gardnerella-specific endolysins (e.g., H2B10) for use in methods of treating Gardnerella infections, such as BV, caused by Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti and / or Gardnerella swidszynskii. Examples 6 and 7 of WO 2020 / 225335 demonstrate that endolysin H2B10 (as a representative example of a recombinant Gardnerella-specific endolysin) is superior to the antibiotics metronidazole and clindamycin, particularly in terms of the minimum inhibitory concentration (MIC) against the growth of Gardnerella strains in suspension. The results of WO 2020 / 225335 therefore demonstrate that (recombinant) Gardnerella-specific endolysins are generally superior to antibiotics in the treatment of BV. In other words, (recombinant) Gardnerella-specific endolysins are generally more effective against the growth of Gardnerella strains in suspension, as the endolysins generally show lower MIC values ​​across the various Gardnerella species compared to antibiotics. Attached Example 1 also demonstrates that these Gardnerella-specific endolysins inhibit the growth of Gardnerella strains in suspension (see Tables 1 and 2). Although endolysins have been shown to be a promising alternative to antibiotics for treating BV, no data are yet available demonstrating their potential to treat patients with a history of bacterial vaginosis (i.e., patients who have already experienced symptoms of BV or suffer from recurrent BV) and patients who have previously failed antibiotic treatment.Similarly, no data have yet been provided demonstrating the potential of endolysins for treating patients suffering from BV in which the infectious bacteria is resistant, preferably highly resistant, to antibiotic treatment (e.g., antibiotic resistance has been acquired after one or more, preferably three or more failed attempts at antibiotic treatment).

[0010] The present invention is based on the surprising and unexpected finding that the superiority of endolysins over antibiotics is even more pronounced in biofilm-grown Gardnerella strains (as illustrated in the accompanying Example 2). Indeed, biofilm-grown strains are more resistant to the action of the antibiotics metronidazole (MDZ) and clindamycin (CLI), but the recombinant Gardnerella-specific endolysin PM-477 is able to destroy these biofilm-grown organisms. This is clinically relevant, since Gardnerella strains in patients suffering from BV are typically in a biofilm state. The present invention is further based on the surprising and unexpected discovery that Gardnerella species rapidly develop resistance to antibiotic treatment, using the antibiotic metronidazole (MDZ) as an illustrative example (as illustrated in the accompanying Example 3). It is therefore believed that a similar rapid resistance formation to MDZ treatment occurs in BV patients during the course of antibiotic treatment using such antibiotics, causing a vicious cycle of BV therapy failure, further courses of MDZ treatment, and further increase in bacterial resistance to this antibiotic. Surprisingly, since the resistance status between the strains was found to be comparable for the antibacterial agents metronidazole (MDZ) and tinidazole (TDZ) (appendix Example 1), it was believed that similar rapid resistance development to TDZ treatment would occur in BV patients during the course of antibacterial treatment using such antibacterial agents. Furthermore, since both MDZ and TDZ belong to the class of nitroimidazole antibiotics, i.e., a class of antibiotics that share a similar chemical structure, it was believed that similar rapid resistance development to antibiotic treatment would occur in BV patients during the course of antibiotic treatment using any one of the antibiotics from the group consisting of nitroimidazole antibiotics, including MDZ, TDZ and secnidazole. Furthermore, it was surprisingly found in the present invention that both antibiotics MDZ and clindamycin (CLI) were ineffective against the majority of Gardnerella isolates tested when they were grown as biofilms (appendix Example 2).Gardnerella strains in patients suffering from BV are typically in a biofilm state, therefore CLI treatment also leads to failure of BV treatment, and it is believed that a similar rapid resistance formation to CLI treatment occurs in BV patients during the course of antibiotic treatment using such antibiotics. The present invention is further based on the surprising and unexpected discovery that, in contrast to such antibiotic treatment, a recombinant Gardnerella-specific endolysin (also interchangeably referred to as "H2B10" and exemplified as endolysin "PM-477", the amino acid sequence of which is shown in SEQ ID NO: 1) is highly active against biofilms preformed by Gardnerella (appendix Example 2), does not induce resistance formation (appendix Example 3), and is fully active against antibiotic-resistant Gardnerella strains, in particular those that are highly resistant to antibiotics and / or have acquired antibiotic resistance after one or more (failed) antibiotic treatments (appendix Example 4). The present invention is therefore based on the surprising and unexpected discovery that a (recombinant) Gardnerella-specific endolysin is useful for treating BV, in particular in patients with recurrent BV (i.e. patients with a history of bacterial vaginosis, i.e. patients who have already experienced symptoms of BV) and in patients suffering from bacterial vaginosis who have failed treatment with antibiotics, particularly nitroimidazoles (e.g. MDZ) and / or CLI, and / or in whom the infectious bacteria is resistant to antibiotic treatment (e.g. antibiotic resistance has been acquired after one or more, preferably three or more, failed treatments with antibiotics, particularly nitroimidazoles (e.g. MDZ) and / or CLI).

[0011] The present invention should be clearly distinguished from the therapeutic use described in WO 2020 / 225335 by the specific group of subjects to be treated. The new therapeutic use of the present invention is specifically intended for patients who have previously failed antibiotic treatment and / or suffer from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment, for example patients suffering from BV caused by Gardnerella strains that have acquired resistance to antibiotics after one or more failed treatments with such antibiotics. This new clinical situation is represented in the attached examples by "passaging" Gardnerella strains with MDZ (representative of the antibiotics used to treat BV). In the context of the present invention, "passaging" refers to treating bacterial strains with antibiotics in such a way that growth is impaired but not completely inhibited. This in vitro setting is therefore intended to artificially reproduce the effect of failed antibiotic treatment on patients suffering from bacterial infections (e.g., BV). As shown in the attached Example 3 and Figure 2, the resistance of Gardnerella strains to antibiotics increases significantly (as represented by an increase in MIC values) after each unsuccessful treatment (i.e., each "passage") with an antibiotic (e.g., MDZ). After several passaging rounds (e.g., 5 or more), the majority of the Gardnerella strains tested can no longer be inhibited even by the maximum concentration used, meaning that these Gardnerella strains have become highly resistant to antibiotic treatment (as defined herein below). Surprisingly and unexpectedly, the inventors show herein that this deleterious effect is not seen upon treatment with a recombinant Gardnerella-specific endolysin (as exemplified by endolysin PM-477). Indeed, the attached Example 3 and Figure 2 show that the MIC of the recombinant Gardnerella-specific endolysin only increases slightly, even after 25 passagings.Even more surprisingly and unexpectedly, it is shown herein that recombinant Gardnerella-specific endolysins (as exemplified by endolysin PM-477) still effectively reduce the growth of Gardnerella strains that have acquired resistance to antibiotics (as exemplified by MDZ as representative of antibiotics used to treat BV), even the most highly resistant strains. Thus, it is surprisingly shown herein that recombinant Gardnerella-specific endolysins are suitable for use in the treatment of patients suffering from BV, where such patients have previously failed antibiotic treatment, and / or where the infectious bacteria of such BV are (highly) resistant to antibiotic treatment, in particular where such antibiotics are nitroimidazoles and / or clindamycin.

[0012] Thus, in a first aspect of the present invention, the Gardnerella-specific endolysin described herein, preferably a recombinant Gardnerella-specific endolysin, is for use in the treatment of bacterial vaginosis, wherein the endolysin is administered to a patient who has previously failed antibiotic treatment.

[0013] As used herein, "patients who have previously failed antibiotic treatment" refers to patients who have a history of bacterial vaginosis, i.e., patients who have previously suffered (or have already experienced) symptoms of bacterial vaginosis, patients who have been treated for such bacterial vaginosis with antibiotics, and who have relapsed, i.e., patients whose symptoms have recurred. Such patients can also be referred to as patients suffering from recurrent BV. As used herein, the terms "patients with a history of bacterial vaginosis", "patients who have already experienced symptoms of bacterial vaginosis", and "patients suffering from recurrent BV" can be used interchangeably and include, but are not limited to, patients who have had one or more episodes of BV within six months, preferably two or more episodes of BV, more preferably two or more episodes of BV, even more preferably two or more episodes of BV within the past six months, or preferably three or more episodes of BV within twelve months, more preferably three or more episodes of BV, even more preferably three or more episodes of BV within the past twelve months. Methods for diagnosing BV in a patient or for proving the onset of BV are known to those skilled in the art, for example, BV can be diagnosed clinically by using clinical criteria (such as the Amsel criteria) or microscopically by determining the Nugent score from a vaginal Gram stain. Without being bound by any theory, relapse (or recurrence) of BV in a patient after treatment with antibiotics may be caused by persistence of residual infection due to resistance of the infectious bacteria to the antibiotic used. Thus, in one preferred embodiment of this first aspect of the invention, the patient suffers from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment. Thus, in one preferred aspect of the invention, the Gardnerella-specific endolysin described herein is for use in the treatment of bacterial vaginosis, the endolysin being administered to patients who have previously failed antibiotic treatment and who suffer from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment. As defined herein below, the infectious bacteria of BV treated herein are even more preferably highly resistant to antibiotic treatment.In a preferred embodiment of the therapeutic use of the present invention, the patient to be treated suffers from BV and the infectious bacteria of such BV are resistant, preferably highly resistant, to treatment with metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In a more preferred embodiment of the therapeutic use of the present invention, the patient to be treated suffers from BV and the infectious bacteria of such BV are resistant, preferably highly resistant, to treatment with metronidazole and / or clindamycin. In an even more preferred embodiment of the therapeutic use of the present invention, the patient to be treated suffers from BV and the infectious bacteria of such BV are resistant, preferably highly resistant, to treatment with metronidazole.

[0014] In a second aspect of the invention, the Gardnerella-specific endolysin described herein, preferably a recombinant Gardnerella-specific endolysin, is for use in the treatment of bacterial vaginosis, wherein the endolysin is administered to a patient suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment.

[0015] In the context of the present invention, the "resistance" of a bacterial strain (preferably a Gardnerella strain) with respect to an antibiotic refers to the ability of the strain to resist the activity of an antibiotic to which it was previously sensitive, allowing it to survive antibiotic treatment. Antibiotic resistance can occur naturally ("intrinsic resistance") or can be induced by the misuse of antibiotics in humans and animals ("acquired resistance"). Methods for determining the resistance or susceptibility of bacterial strains to antibiotics are known to those skilled in the art. As an example, the EUCAST breakpoints for Gram-positive anaerobes (v11, 2021) can be used. An alternative definition is given in Petrina et al. (2017, Anaerobe 47, 115-119), where a slightly higher resistance breakpoint is used, since topical formulations of some nitroimidazoles and clindamycin can establish concentrations in the mg / ml range in vaginal fluid, much higher than those achievable with orally delivered antibacterial agents. According to this alternative, in a stricter definition (also used in the accompanying examples), resistance (R) can be defined as a MIC value of 32 μg / ml or more for metronidazole and 8 μg / ml or more for clindamycin, while susceptibility (S) can be defined as a MIC value of 8 μg / ml or less for metronidazole and 2 μg / ml or less for clindamycin. The terms "minimum inhibitory concentration" and "MIC" are used interchangeably herein and refer to the lowest concentration of a chemical, usually a drug, that prevents visible growth of bacteria. The MIC can be defined as the lowest concentration at which no growth is detected after 48 hours by OD measurement. Thus, in one preferred embodiment of the therapeutic use of the present invention, the patient to be treated suffers from BV in which the infectious bacteria is resistant to antibiotic treatment as defined by EUCAST breakpoints. Other generally accepted resistance criteria can be used instead of and / or in addition to the EUCAST definition or alternative definitions mentioned above (Petrina et al., 2017, Anaerobe 47, 115-119) to define the resistance of infectious bacteria to antibiotic treatment in the context of the present invention.

[0016] As known to those skilled in the art, "resistance" and "susceptibility" may be defined in terms of MBC and / or MBEC values. The term "minimum bactericidal concentration" or "MBC" refers to the lowest concentration of an antimicrobial agent required to kill a particular bacterium. Usually, the MBC90 or MBC99.5 is measured, i.e., the antibiotic concentration that kills 90% or 99.5% of cells, respectively, in a specified time. The MBC is, for example, 2.5×10 7 It can be defined as the minimum concentration that completely eradicates a suspension of CFU / ml. The MIC is the lowest concentration of an antimicrobial agent required to inhibit visible growth, while the MBC is the lowest concentration of an antimicrobial agent that results in bacterial death of all cells in the suspension up to a defined detection limit, resulting in the killing of at least 90% (MBC90) or at least 99.5% (MBC99.5) of the bacteria. The term "minimum biofilm eradication concentration" or "MBEC" refers to the lowest concentration of an antimicrobial agent required to reduce the bacterial population growing as a biofilm below the detection limit. Details of the method used in this disclosure to measure MBEC are provided in the Materials and Methods section of the Examples.

[0017] Without being bound by any theory, it is possible that resistance of infectious bacteria to antibiotics may be involved in (or be partially or substantially responsible for) relapse (or recurrence) of BV in patients suffering from recurrent BV. Thus, patients suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment are more likely to fail antibiotic treatment. Thus, in one preferred embodiment of this second aspect, patients treated and suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment are patients prone to antibiotic treatment failure. As used herein, a patient "prone to antibiotic treatment failure" refers to a patient at high risk of antibiotic treatment failure, i.e., a patient who is highly likely to relapse (e.g., within 12 months) if such patient is treated with antibiotics. Methods for determining whether a patient is prone to antibiotic treatment failure are known to those skilled in the art. As an example, clinical samples can be collected and the resistance of vaginal microbiota strains can be evaluated. Similarly, a patient suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment is likely to have already (i.e., previously) failed treatment with an antibiotic (as defined above).Accordingly, in a further preferred embodiment of this second aspect, the patient treated suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment is a patient who has previously failed antibiotic treatment.

[0018] As surprisingly shown in the attached examples, the Gardnerella strains acquired more and more antibiotic resistance after each "passage" with such antibiotics, but all strains remained sensitive to the Gardnerella-specific endolysin (see attached Example 4). Indeed, as shown in Table 4 below, before passaging, the Gardnerella strains show MIC values ​​of 8 to 256 μg / mL for MDZ, while the MIC values ​​for the corresponding passaged strains are greater than 252 μg / mL or 2048 μg / mL, which means that the passaged Gardnerella strains increased after each passaging to the point where they could tolerate very high concentrations of antibiotic resistance without any loss of viability. After passaging, the (passaged) Gardnerella strains became highly resistant to antibiotic treatment. According to the above definitions of resistance, "high resistance" (HR) can be defined as MIC values ​​of 256 μg / ml or more for metronidazole and 64 μg / ml or more for clindamycin, i.e. 8 times higher than the resistance breakpoints of the alternative definition, which are already higher than the standard resistance breakpoints of the EUCAST definition. In contrast to antibiotics, there are no resistance breakpoints for the Gardnerella specific endolysins described herein (as exemplified by PM-477), and even such "passaged" or "highly resistant" Gardnerella strains remain sensitive to treatment with such endolysins. Thus, in one embodiment of the therapeutic use of the invention, the patient to be treated suffers from BV, and the infectious bacteria of such BV are resistant, preferably highly resistant, to antibiotic treatment, preferably as defined by EUCAST breakpoints. In a preferred embodiment of the therapeutic use of the invention, the patient to be treated suffers from BV, and the infectious bacteria of such BV are resistant, preferably highly resistant, to treatment with metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In one more preferred embodiment of the therapeutic use of the present invention, the patient to be treated is suffering from BV, the infectious bacterium of which is resistant, preferably highly resistant, to treatment with metronidazole and / or clindamycin.In an even more preferred embodiment of the therapeutic use of the present invention, the patient to be treated is suffering from BV, the infectious bacterium of which is resistant, preferably highly resistant, to treatment with metronidazole.

[0019] In another aspect of the present invention, the Gardnerella-specific endolysin described herein, preferably a recombinant Gardnerella-specific endolysin, is for use in the treatment of patients suffering from bacterial vaginosis, where such patients have previously failed antibiotic treatment and / or the infectious bacteria of such bacterial vaginosis are resistant to antibiotic treatment.

[0020] All of the aspects of the invention described herein are encompassed under "therapeutic uses of the invention."

[0021] As used herein, the terms "antibiotic treatment" and "antibiotic therapy" are used interchangeably and preferably refer to antibiotic treatment recommended or approved for the treatment of BV. Antibiotics currently recommended or approved for the treatment of BV include nitroimidazoles (including, but not limited to, metronidazole, tinidazole, and secnidazole), and clindamycin. Thus, in one embodiment, such "antibiotic treatment" or "antibiotic therapy" is treatment with nitroimidazoles and / or clindamycin. In a preferred embodiment, the antibiotic treatment described herein is treatment with metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In a more preferred embodiment, the antibiotic treatment described herein is treatment with metronidazole and / or clindamycin. In an even more preferred embodiment, the antibiotic treatment described herein is treatment with metronidazole.

[0022] In one embodiment of the therapeutic use of the present invention, the patient to be treated suffers from recurrent bacterial vaginosis (as defined herein above). In one embodiment of the therapeutic use of the present invention, the patient to be treated has a medical history of bacterial vaginosis. In one embodiment of the therapeutic use of the present invention, the patient to be treated has already experienced symptoms of bacterial vaginosis. In one embodiment of the therapeutic use of the present invention, the patient to be treated has had one or more episodes of BV within 6 months, preferably two or more episodes of BV, more preferably two or more episodes of BV, even more preferably two or more episodes of BV within the past 6 months, or preferably three or more episodes of BV within 12 months, more preferably three or more episodes of BV, even more preferably three or more episodes of BV within the past 12 months. Thus, in one embodiment, the Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, described herein is for use in the treatment of bacterial vaginosis, such bacterial vaginosis being recurrent bacterial vaginosis. In a preferred embodiment, the Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin as described herein, is for use in the treatment of bacterial vaginosis, where the endolysin is administered to a patient who has had two or more episodes of BV within six months, or to a patient who has had three or more episodes of BV within twelve months. In another preferred embodiment, the Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin as described herein, is for use in the treatment of a patient suffering from bacterial vaginosis, where such a patient has had two or more episodes of BV within six months, or to a patient who has had three or more episodes of BV within twelve months. In another preferred embodiment, the Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin as described herein, is for use in the treatment of a patient who has a history of bacterial vaginosis.

[0023] As used herein, "bacterial vaginosis" (BV), also referred to in the literature as bacterial vaginosis, nonspecific vaginitis and nonspecific vaginitis, refer to the most common vaginal infection worldwide. In one embodiment, BV is defined as a pathological condition characterized by the loss of normal vaginal flora, especially the H2O2-producing species Lactobacillus, and the simultaneous overgrowth of anaerobic bacteria, often including those from the genus Gardnerella. The genus Gardnerella includes at least 13 species, the most frequent of which have been renamed G. vaginalis sensu stricto, G. leopoldii, G. piochii and G. swidszynskii (Vanechoutte et al., 2019 Int. J. Syst. Evol. Microbiol. 69, 679-687). In one preferred embodiment of the therapeutic use of the present invention, the BV to be treated is a bacterial infection characterized by the presence of at least one strain of the genus Gardnerella selected from the group consisting of Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pioti, and Gardnerella swidszynskii, and any other Gardnerella species. Thus, in one embodiment, the Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin, described herein is for use in the treatment of bacterial vaginosis, such bacterial vaginosis being characterized by the presence of infectious bacteria of Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pioti, Gardnerella swidszynskii, and / or any other species of the genus Gardnerella. As used herein, bacterial vaginosis "characterized by the presence" (or "caused by") a particular bacterial species of the Gardnerella genus is understood to refer to an overgrowth of such bacteria (also referred to herein as "infectious bacteria") in the patient's vaginal microbiota, resulting in vaginal dysbiosis and / or loss of Lactobacillus dominance. Methods for determining whether bacterial vaginosis is characterized by the presence of infectious bacteria of a bacterial species of the Gardnerella genus are known to those skilled in the art. As an example, a PCR test can be used to check for the presence of Gardnerella strains to diagnose BV.

[0024] In one embodiment of the therapeutic use of the invention, the BV treated by the Gardnerella-specific endolysin described herein is characterized by the presence of Gardnerella strains (i.e., strains from the Gardnerella genus) that are resistant to one or more antibiotics. In a preferred embodiment, the one or more antibiotics are selected from the group consisting of nitroimidazole and clindamycin. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole, tinidazole, secnidazole, clindamycin, or any combination thereof. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole and / or clindamycin. In an even more preferred embodiment, the Gardnerella strain is resistant to metronidazole. In a most preferred embodiment, the Gardnerella strain is resistant or highly resistant to metronidazole.

[0025] The term "endolysin" as used herein refers to a polypeptide that is typically produced by bacteriophages to digest the host bacterial cell wall and release the bacteriophage progeny. Endolysins are cell wall lytic enzymes encoded by bacteriophages that have the ability to hydrolyze the cell wall of target bacteria (exogenous lysis) when added exogenously. This novel class of antibacterial agents has important advantages over classical antibiotics, such as a novel mode of action, a narrow spectrum of susceptible bacteria, rapid killing of bacteria in both stationary and logarithmic growth phases, activity against mucosa and bacterial biofilms, low likelihood of developing resistance, and low impact on normal microbiota. These unique features have boosted interest in the biotechnological and pharmacological applications of lysins and their recent inclusion among the most important current alternatives to combat antibiotic resistance. Endolysins often consist of two or more domains: at least one catalytic domain, such as a hydrolase domain (typically located at the N-terminus of the polypeptide) that cleaves a specific motif in the peptidoglycan layer, and one or more cell wall binding domains (classically located at the C-terminus of the polypeptide) that are often involved in the specific binding and processing of bacterial peptidoglycan. This typical structure provides a general organization of endolysin structures, but is not a clear feature of all endolysins. Endolysins from Gram-positive bacteria and their phages usually contain at least one catalytic domain and one or more cell wall binding domains. In contrast, many endolysins produced by Gram-negative species or their phages contain only a catalytic domain, although modular endolysins have also been reported. The catalytic unit determines the type of peptidoglycan (PG) bond that is cleaved, while the cell wall binding domain(s) primarily determine the lysis spectrum by specific recognition of cell wall elements that are distributed in a genus-specific or species / strain-specific manner.

[0026] The endolysin used in the therapeutic use of the present invention is preferably a recombinant endolysin that is Gardnerella specific, i.e., it specifically targets bacteria belonging to the Gardnerella genus. The endolysin used in the therapeutic use of the present invention further preferably has killing activity against species of the Gardnerella genus. For example, the endolysin used in the therapeutic use of the present invention may have killing activity against Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti and / or Gardnerella swidszynskii, preferably against all of them. The killing activity of the endolysin used in the therapeutic use of the present invention against Gardnerella is more preferably a genus-selective killing activity against Gardnerella. As used herein, "genus-selective killing activity" or "genus-specific lytic effect" means that the endolysin used in the therapeutic use of the present invention does not have killing activity or lytic effect against bacteria in general. In particular, the endolysin used in the therapeutic use of the present invention has no killing activity against bacteria other than Gardnerella species. Preferably, the endolysin used in the therapeutic use of the present invention has genus-selective killing activity against Gardnerella, but not against Lactobacillus. In particular, it is preferred that such endolysin has no killing activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus yensenii. More preferably, such endolysin has no killing activity against all of these lactobacilli, i.e., Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus yensenii.

[0027] As used herein, the "killing activity" of an endolysin against a particular bacterium can be defined as the reduction in the number of viable bacterial cells caused by the lytic activity of such endolysin. The killing activity of an endolysin against such a bacterium can be complete, meaning that 100% of the bacterial cells have been 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 have been lysed. The killing activity of an endolysin 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, defined as the lowest concentration of antimicrobial agent that inhibits visible growth of the microorganism after overnight incubation as described in Andrews, 2001 (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 an endolysin is described in the Examples section of WO 2020 / 225335 and consists of measuring the decrease in optical density at 610-620 nm and / or the decrease in colony forming units (CFU) per milliliter of bacterial cell suspension after exposure to the endolysin to be tested. The decrease in optical density measured at 610-620 nm of a suspension of bacteria whose susceptibility is to be tested can be determined in an in vitro turbidity assay carried out in the presence of purified endolysin.According to another embodiment, an endolysin has killing activity against Gardnerella if, in an in vitro turbidity test, the endolysin reduces the OD (610-620 nm) of a suspension of at least one Gardnerella bacterial 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%.

[0028] The endolysin used in the therapeutic use of the invention further preferably has killing activity against antibiotic-resistant or highly antibiotic-resistant Gardnerella strains as defined above. In one preferred embodiment, the endolysin used in the therapeutic use of the invention has killing activity against Gardnerella strains that are resistant to one or more antibiotics selected from the group consisting of nitroimidazoles and clindamycin. In one more preferred embodiment, the endolysin used in the therapeutic use of the invention has killing activity against Gardnerella strains that are resistant to metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof. In an even more preferred embodiment, the endolysin used in the therapeutic use of the invention has killing activity against Gardnerella strains that are resistant to metronidazole and / or clindamycin. In an even more preferred embodiment, the endolysin used in the therapeutic use of the invention has killing activity against Gardnerella strains that are resistant to metronidazole. In a more preferred embodiment, the endolysins used in the therapeutic uses of the present invention have killing activity against Gardnerella strains that are highly resistant to such antibiotics as mentioned above.

[0029] In the therapeutic use of the invention, the endolysin is administered orally (e.g. as a pill) or topically (e.g. as a topical gel, lotion or cream, or as a pessary, i.e. vaginal suppository). In one preferred embodiment of the therapeutic use of the invention, the endolysin is administered topically, i.e. locally in the vagina of a female subject, and / or locally in or on the glans penis, foreskin or urethral entry of a male subject. As used herein, the term "administration in or on the glans penis" also includes "administration in and on the glans penis". Accordingly, the term "administration in or on the glans penis, foreskin or urethral entry of a male subject" also includes "administration in or on the glans penis, foreskin and urethral entry of a male subject". As an illustrative example, endolysin for use in the present invention may be formulated as a topical gel, lotion or cream that is inserted into the vagina of a female subject and / or inserted and / or applied to the glans penis of a male subject. As another illustrative example, endolysin for use in the present invention may be inserted into the vagina of a female subject in the form of a pessary (vaginal suppository). As another illustrative example, endolysin for use in the present invention may also be applied onto a condom prior to sexual intercourse.

[0030] The optimum pH at which the endolysins used in the therapeutic use of the invention exhibit killing activity, preferably genus-selective killing activity, against Gardnerella is about 4-6, preferably about pH 5. Thus, in one preferred embodiment of the therapeutic use of the invention, the endolysins are co-administered with a compound or composition that adjusts the vaginal pH to 4.0-6.0, preferably 4.5-5.5, more preferably about 5. Suitable compounds or compositions that adjust the vaginal pH include, but are not limited to, phosphates, lactic acid (e.g., a natural acidifying substance secreted to establish an acidic environment) or other organic acids, such as carboxy-substituted polymers.

[0031] The endolysin used in the therapeutic use of the present invention is preferably a functional polypeptide, the function of which comprises specifically targeting bacteria from the genus Gardnerella, more preferably specifically killing bacteria from the genus Gardnerella. The endolysin used in the therapeutic use of the present invention further preferably comprises a catalytic domain or a functional fragment thereof and / or a cell wall binding domain or a functional fragment thereof. The endolysin used in the therapeutic use of the present invention may be a natural or recombinant endolysin. The endolysin used in the therapeutic use of the present invention is most preferably a recombinant endolysin. The endolysin used in the therapeutic use of the present invention is more preferably a recombinant endolysin comprising: (i) an N-terminal catalytic domain, or a functional variant thereof; (ii) a C-terminal cell wall binding region or a functional variant thereof, the C-terminal cell wall binding region comprising or consisting of at least one cell wall binding domain, and (iii) optionally comprising or consisting of a linker region between said N-terminal catalytic domain and said C-terminal cell wall binding region. It preferably has killing activity, more preferably genus-selective killing activity, against Gardnerella cells / strains.

[0032] In the context of the present disclosure, the term "recombinant endolysin" preferably refers to a domain-swapped endolysin, as defined in WO 2020 / 225335. In accordance with this definition, the skilled artisan will readily understand that the "domain-swapped" or "recombinant" endolysins described herein are non-naturally occurring endolysins. That is, recombinant endolysins for use in the present invention have been modified by the human hand and, by definition, exclude natural endolysins, i.e., those that can be found in nature. The accompanying examples as well as the teachings of WO 2020 / 225335 provide suitable method(s) for generating the artificial endolysins of the present invention.

[0033] The term "catalytic domain" or "enzyme domain" refers to the portion of a protein chain that contains the area where a catalytic chemical reaction occurs. As used herein, "catalytic domain" refers to a functional polypeptide whose function includes the ability to lyse the cell wall of Gardnerella. In particular, the catalytic domain described herein is preferably capable of modifying and / or cleaving a substrate, preferably peptidoglycan, in the Gardnerella cell wall. Preferably, the catalytic domain is capable of cleaving peptidoglycan in the Gardnerella cell wall and causing Gardnerella cell lysis. Preferably, the catalytic domain is capable of modifying and / or cleaving bonds present in the cell wall and / or peptidoglycan of Gardnerella species, such as Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti and / or Gardnerella swidszynskii, preferably all thereof. Suitably, the catalytic domain does not modify and / or cleave substrates, preferably peptidoglycan, present in the cell walls of bacteria other than Gardnerella species, preferably healthy vaginal commensal bacteria, such as Lactobacillus species, including Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus yensenii. The catalytic domain may be N-acetylmuramidase, N-acetylmuramoyl-L-alanine amidase, L-alanoyl-D-glutamic acid endopeptidase, interpeptide cross-linking endopeptidase, or N-acetyl-β-D-glucosaminidase. Preferably, the N-terminal catalytic domain is N-acetylmuramidase, most preferably 1,4-β-N-acetylmuramidase. The catalytic domain is preferably located at the N-terminus within the (recombinant) endolysin and is thereby referred to as an "N-terminal catalytic domain", and even more preferably, the N-terminal catalytic domain is located N-terminal from the C-terminal cell wall binding region within the (recombinant) endolysin.

[0034] The endolysin used in the therapeutic use of the present invention preferably comprises a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 2 to 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, 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, most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 2 to 10. As shown in WO 2020 / 225335, the most active catalytic domain is "H2" (SEQ ID NO: 3). Thus, in a preferred embodiment of the invention, the catalytic domain consists of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or any functional variant thereof having at least 80% identity to the amino acid sequence of SEQ ID NO: 3 (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, most preferably at least 99.7% identity), whereby the endolysin is functional, whose function comprises the ability to lyse the cell wall of Gardnerella. The catalytic domain is preferably located N-terminally in the (recombinant) endolysin, thereby referred to as "N-terminal catalytic domain", and even more preferably, the (recombinant) endolysin further comprises a cell wall binding region, the N-terminal catalytic domain being located N-terminally from the C-terminal cell wall binding region in the (recombinant) endolysin.

[0035] A "cell wall binding region" as used herein refers to a functional polypeptide whose function comprises the ability to bind to the cell wall of Gardnerella. A cell wall binding region may comprise or consist of one, two, three or more cell wall binding domains. A cell wall binding domain is a polypeptide that interacts with and / or binds to a bacterial cell wall and / or a specific substrate within the bacterial cell wall. In particular, the cell binding domain described herein is preferably capable of specifically binding (e.g., to peptidoglycan) to the cell wall of Gardnerella species, such as Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti and / or Gardnerella swidszynskii, preferably all thereof. The cell wall binding region is preferably located at the C-terminus within the (recombinant) endolysin, thereby referred to as a "C-terminal cell wall binding region", and even more preferably, the C-terminal cell wall binding region is located C-terminal from the N-terminal catalytic domain within the (recombinant) endolysin.

[0036] The endolysin used in the therapeutic application of the present invention preferably comprises a cell wall binding region comprising or consisting of at least one cell wall binding domain selected from the group consisting of a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 11 to 28, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, most preferably at least 99.7% identity) to any one of the amino acid sequences of SEQ ID NOs: 11 to 28. As shown in WO 2020 / 225335, the most active cell wall binding region is "B10" (comprising the cell wall binding domains of SEQ ID NOs: 23 and 24), followed by "B11" (comprising the cell wall binding domains of SEQ ID NOs: 25 and 26). Thus, in a preferred embodiment of the present invention, the cell wall binding domain is selected from the group consisting of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, and the endolysin is functional, the function of which comprises the ability to lyse the cell wall of Gardnerella.The cell wall binding region is preferably located at the C-terminus in the (recombinant) endolysin, thereby referred to as a "C-terminal cell wall binding region", and even more preferably, the (recombinant) endolysin further comprises a catalytic domain, which is located N-terminal from the C-terminal cell wall binding region in the (recombinant) endolysin.

[0037] The endolysins used in the therapeutic uses of the present invention preferably comprise two cell wall binding domains (within the cell wall binding region). In a preferred aspect of the present invention, the cell wall binding domains of the endolysins of the present invention each consist of a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, as well as any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23, 24, 25 and 26, whereby the endolysin is functional, whose function comprises the ability to lyse the cell wall of Gardnerella. In an even more preferred aspect of the invention, the endolysin comprises a first cell wall binding domain and a second cell wall binding domain, said first cell wall binding domain being selected from the group consisting of SEQ ID NOs: 23 and 25 and said second cell wall binding domain being selected from the group consisting of SEQ ID NOs: 24 and 26. Preferably, said first cell wall binding domain is located N-terminal to said second cell wall binding domain.

[0038] In one more preferred embodiment, the endolysin used in the therapeutic use of the invention is (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or any functional variant thereof having at least 80% identity to the amino acid sequence of SEQ ID NO: 3 (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity); (ii) a C-terminal cell wall binding region that comprises or consists of a first cell wall binding domain and a second cell wall binding domain. Such a first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 23 and 25, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 23 and 25; Such a first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 24 and 26, and any functional variant thereof having at least 80% identity (preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) to the amino acid sequence of any one of SEQ ID NOs: 24 and 26; Thereby, such endolysins are functional, whose function comprises the ability to lyse the cell wall of Gardnerella. Preferably, such first cell wall binding domain is located N-terminal to such second cell wall binding domain.

[0039] In one particularly preferred embodiment, the endolysin used in the therapeutic use of the invention is (i) an N-terminal catalytic domain consisting of a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:3; and (ii) a C-terminal cell wall binding region that comprises or consists of a first cell wall binding domain and a second cell wall binding domain. The first cell wall binding domain is selected from the group consisting of SEQ ID NOs: 23 and 25, and the second cell wall binding domain is selected from the group consisting of SEQ ID NOs: 24 and 26.

[0040] Preferably, such first cell wall binding domain is located N-terminal to such second cell wall binding domain.

[0041] Illustrative examples of particularly preferred endolysins for use in the therapeutic uses of the present invention are "H2B10" (from N-terminus to C-terminus: including SEQ ID NOs: 3, 23, and 24), "H2B11" (from N-terminus to C-terminus: including SEQ ID NOs: 3, 25, and 26), and "H2B10B11" (from N-terminus to C-terminus: including SEQ ID NOs: 3, 23, and 26) as defined in WO 2020 / 225335.

[0042] The endolysins used in the therapeutic uses of the invention further preferably comprise a linker region between the N-terminal catalytic domain and the C-terminal cell wall binding region. The linker region may consist of a polypeptide having a length of 6 to 18 amino acids, preferably 9 to 15 amino acids, even more preferably 12 amino acids. Preferably, the linker region comprises or consists of the amino acid sequence (i) (XXX)n, where each X can independently be G, A or S, preferably the amino acid sequence (GGS)n, where n corresponds to the number of repeats of the sequence XXX, preferably n is 2, 3, 4, 5 or 6, or (ii) X1X2GLNGX3X4NGGS (SEQ ID NO: 36), where X1 is N or K, X2 is A or V, X3 is Y or C and X4 is K or Q. Non-limiting examples of such linker regions are provided in SEQ ID NOs: 29 to 35.

[0043] In a particularly preferred embodiment, the endolysin for use in the therapeutic uses of the present invention is "H2B10", the sequence of which is set out in SEQ ID NO: 1, or "H2B10B11", the sequence of which is set out in SEQ ID NO: 37. Thus, the present invention provides a Gardnerella specific endolysin for use in the treatment of bacterial vaginosis, preferably a recombinant Gardnerella specific endolysin, to be administered to patients who have previously failed antibiotic treatment and / or who suffer from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment, which endolysin is a polypeptide having at least 80% sequence identity to the amino acid sequence provided in SEQ ID NO: 1 and which has killing activity against Gardnerella. In one more preferred embodiment, the present invention provides a Gardnerella specific endolysin, preferably a recombinant Gardnerella specific endolysin, for use in the treatment of bacterial vaginosis, to be administered to patients who have previously failed antibiotic treatment and / or who are suffering from bacterial vaginosis where the infectious bacteria are resistant to antibiotic treatment, the endolysin being a polypeptide having at least 90% sequence identity to the amino acid sequence provided in SEQ ID NO: 1, and having killing activity against Gardnerella. In one even more preferred embodiment, the present invention provides a Gardnerella specific endolysin, preferably a recombinant Gardnerella specific endolysin, for use in the treatment of bacterial vaginosis, to be administered to patients who have previously failed antibiotic treatment and / or who are suffering from bacterial vaginosis where the infectious bacteria are resistant to antibiotic treatment, the endolysin being a polypeptide having at least 95% sequence identity to the amino acid sequence provided in SEQ ID NO: 1, and having killing activity against Gardnerella. In an even further preferred embodiment, the present invention provides a Gardnerella-specific endolysin for use in the treatment of bacterial vaginosis, preferably a recombinant Gardnerella-specific endolysin, to be administered to patients who have previously failed antibiotic treatment and / or who are suffering from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment, wherein the endolysin is a polypeptide having at least 99% sequence identity to the amino acid sequence provided in SEQ ID NO:1, and has killing activity against Gardnerella.In a most preferred embodiment, the present invention provides a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin, for use in the treatment of bacterial vaginosis, which is administered to patients who have previously failed antibiotic treatment and / or suffer from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment, such endolysin comprising or consisting of the amino acid sequence provided in SEQ ID NO: 1. An endolysin comprising or consisting of the amino acid sequence provided in SEQ ID NO: 37 may also be used in the context of the present invention, i.e. in the context of the medical use of a (recombinant) Gardnerella-specific endolysin in the treatment of bacterial vaginosis, which endolysin is administered to patients who have previously failed antibiotic treatment and / or suffer from bacterial vaginosis in which the infectious bacteria are resistant to antibiotic treatment. Thus, an endolysin comprising or consisting of the amino acid sequence provided in SEQ ID NO: 37 is an alternative to the Gardnerella-specific endolysin provided in SEQ ID NO: 1.

[0044] The terms "peptide", "polypeptide", "protein" and variations of these terms refer to peptides, oligopeptides, oligomers or proteins, including fusion proteins, each of which comprises at least two amino acids linked together by normal or modified peptide bonds, as in the case of isosteric peptides. These terms also include "peptidomimetics", defined herein as peptide analogs containing non-peptide structural elements, which are capable of mimicking or antagonizing the biological action(s) of the natural parent peptide. Peptidomimetics lack classical peptide characteristics, 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 by chemical processes well known to those skilled in the art. Such modifications are fully detailed in the literature. These modifications may occur anywhere in the polypeptide, i.e. in the peptide backbone, in the amino acid chain, or even at the carboxy or amino termini. Peptides or polypeptides may be branched after ubiquitination or cyclic with or without branching. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art. For example, peptide or polypeptide modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond formation, glycosylation including demethylation, pegylation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, seneloylation, sulfation, arginylation or ubiquitination, or other amino acid additions. Such modifications are fully detailed in the literature and are well known to those skilled in the art.

[0045] As defined herein, the terms "bind" and "bind to", referring to the binding ability of an endolysin to a particular bacterial cell wall, refer to the ability of such endolysin to specifically interact with and adhere to the bacterial cell wall. The binding ability of an endolysin to a bacterial cell wall can be determined by methods known in the art.

[0046] As used herein, "treatment" and "treating" and the like generally refer to obtaining a desired pharmacological and physiological effect. The effect may be preventative, in terms of preventing or partially preventing a disease, its symptoms or condition, and / or may be therapeutic, in terms of partially or completely curing the adverse effects caused by a disease, condition, symptom or disease. The term "treating" as used herein includes any treatment of bacterial vaginosis in a mammal, particularly a human, and includes (a) preventing bacterial vaginosis from occurring in a patient who may be susceptible to bacterial vaginosis but has not yet been diagnosed as having it, and (b) inhibiting bacterial vaginosis, i.e., preventing its development, or alleviating the bacterial infection, i.e., causing the regression of the bacterial infection and / or its symptoms or condition, e.g., ameliorating or repairing damage. In particular, the treatment of bacterial vaginosis also includes preventing, reducing or eradicating the infection, for example, by killing the infectious bacteria, thus controlling, reducing or inhibiting bacterial growth, as well as reducing the number of viable bacterial cells. As used herein, the disease, ie BV, is preferably treated therapeutically with respect to partial or complete cure of the disease or symptoms.

[0047] The terms "patient" and "subject" are used interchangeably herein and refer to a mammal. For example, mammals contemplated by the present invention include humans, primates, domestic animals (e.g., cows, sheep, pigs, horses, laboratory rodents, etc.). Preferably, the patient is a human. Even more preferably, the patient is a female (also referred to herein as a "female subject").

[0048] The term "variant" refers to a polypeptide that contains non-conservative or preferably conservative insertions, deletions, and / or substitutions with respect to a native amino acid sequence. For example, a polypeptide may contain an amino acid sequence that has at least 80% identity with respect to a native amino acid sequence, preferably at least 85% identity with respect to such amino acid sequence, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 96% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity. Percent identity can be determined by methods well known in the art using a suitable computer program, for example MatGAT 2.0 (Myers and Miller, CABIOS (1989)). Preferably, the percent identity is determined over the entire length of the sequences to be compared. It is understood that the percent identity is calculated with respect to the polypeptide whose sequences are optimally aligned. Fragments and variants of amino acid sequences can be made using any of the methods of protein engineering, directed evolution and / or site-directed mutagenesis well known in the art (see, for example, Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press). Those skilled in the art will appreciate that the polypeptides according to the invention, or fragments, variants or fusions thereof, can comprise or consist of derivatives of native amino acid sequences, or fragments or variants thereof. Chemical derivatization of one or more amino acids can be achieved by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which a free amino acid group has been derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxybenzoxy group, a t-butyloxycarbonyl group, a chloroacetyl group or a formyl group.Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form 0-acyl or 0-alkyl derivatives. Peptides containing naturally occurring amino acid derivatives of the 20 standard amino acids are also included as chemical derivatives. For example, proline can be substituted with 4-hydroxyproline. 5-hydroxylysine can be substituted for lysine. 3-methylhistidine can be used in place of histidine. Homoserine can be substituted for serine, and ornithine can be substituted for lysine. Derivatives also include peptides containing one or more additions or deletions, so long as the required activity is maintained. Other included modifications are terminal modifications such as amidation, amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation, e.g., with ammonia or methylamine. It will be further understood by those skilled in the art that peptidomimetic compounds can also be useful. Thus, "polypeptide" includes peptidomimetic compounds that exhibit endolysin activity. The term "peptidomimetic" refers to a compound that mimics the conformation and desirable characteristics of a particular polypeptide as a therapeutic agent.

[0049] Methods for the production of endolysins, or fragments, variants, fusions or derivatives thereof, for use according to the invention are well known in the art. Advantageously, the endolysins, or fragments, variants, fusions or derivatives thereof for use according to the invention are or comprise recombinant endolysins. Endolysins for use according to the invention can be produced by standard techniques of genetic engineering, including the use of recombinant vectors comprising a polynucleotide encoding an endolysin as described herein. A number of expression systems can be used, including bacterial plasmids and derivative vectors, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papillomaviruses such as SV40, vaccinia viruses, adenoviruses, foxpox viruses, pseudorabies viruses, retroviruses, cosmids or phagemid derivatives. The nucleotide sequence can be inserted into a recombinant expression vector by methods well known to those skilled in the art, for example as described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. The recombinant vector can contain nucleotide sequences that control the regulation, expression, transcription, and / or translation of the polynucleotide encoding the endolysin, these sequences being selected according to the host cell used. The recombinant vector can further contain nucleotide sequences, such as those encoding a His tag, to facilitate the purification process.Such recombinant vectors are then introduced into host cells according to the methods described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis basic methods in molecular biology, Davis et al., 2nd ed., McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL (supra) (e.g., calcium phosphate transfection, DEAE dextran transfection, transfection, microinjection, cationic lipid transfection, electroporation, transduction or infection). Host cells can be, for example, bacterial cells such as E. coli, fungal cells such as yeast, and Aspergillus, Streptomyces cells, insect cells, Chinese hamster ovary cells (CHO), C127 mouse cell line, Syrian hamster cell BHK cell line, human embryonic kidney 293 (HEK 293) cells. Preferably, the host cells are E. coli. Such host cells are then cultured under appropriate conditions to produce the endolysins described herein, which can then be further purified from the culture medium or host cell lysates by any standard purification method, including immobilized metal affinity chromatography (IMAC) (Block et al. 2008, Protein Expr. Purif. 27:244.254).

[0050] The endolysins used in the therapeutic uses of the present invention preferably exhibit a low or no observable resistance profile. Bacterial resistance to antibacterial agents can be measured by assays described herein and known to those skilled in the art.

[0051] The endolysin used in the therapeutic uses of the present invention may be provided as a composition, for example a pharmaceutical composition comprising an endolysin as described herein for use in the present invention.Thus, the present invention also provides a pharmaceutical composition comprising a Gardnerella-specific endolysin as described herein, preferably a recombinant Gardnerella-specific endolysin, and optionally a pharma- ceutically acceptable carrier and / or excipient, for use in the treatment of bacterial vaginosis as described herein.

[0052] The term "pharmaceutical composition" refers to a formulation in such a form that the biological activity of the active ingredient is clearly effective and does not contain additional ingredients that are toxic to the patient to whom the composition is administered. As used herein, "pharmaceutical composition" refers to a therapeutically effective formulation for use in the method 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 administration regimen. It is a predetermined amount of active agent calculated to produce a desired therapeutic effect in association with the required additives and diluents, i.e., carriers or administration vehicles. Moreover, it is intended to mean an amount sufficient to reduce, and most preferably prevent, clinically significant deficits in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement of a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. An appropriate dosage may include a predetermined amount of active composition calculated to produce a desired therapeutic effect in combination with the required diluents. In the methods for the manufacture and use of the compositions of the present invention, a therapeutically effective amount of active ingredient is provided. The therapeutically effective amount can be determined by an ordinary skilled medical or veterinary practitioner based on the characteristics of the patient, such as age, weight, sex, condition, co-morbidities, other diseases, etc., as is well known in the art. In one embodiment of the use of the present invention, the pharmaceutical composition is as described herein above comprising a recombinant Gardnerella-specific endolysin and is for use in the treatment of bacterial vaginosis as described herein above. Thus, the pharmaceutical composition may comprise an amount of endolysin, or a fragment, variant, fusion or derivative thereof, sufficient to at least partially inhibit the proliferation of cells of the genus Gardnerella in a patient infected or susceptible to infection with such cells as described herein above. Preferably, the pharmaceutical composition comprises an amount of endolysin, or a fragment, variant, fusion or derivative thereof, sufficient to kill cells of the genus Gardnerella in a patient treated as defined herein above.It will be understood by those skilled in the art that the endolysins used according to the invention will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (see, for example, Remington: The Science and Practice of Pharmacy, 19th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA). For example, the endolysins may be administered topically, i.e., locally in the vagina of a female subject, and / or in or on the glans penis, foreskin or urethral opening in a male subject. As used herein, the term "administration in or on the glans penis" also includes "administration in and on the glans penis". Accordingly, the term "administration in or on the glans penis, foreskin or urethral opening of a male subject" also includes "administration in or on the glans penis, foreskin and urethral opening of a male subject". In another embodiment, the endolysin may be co-administered with a compound or composition that adjusts the vaginal pH, hi some embodiments, the compound or composition adjusts the vaginal pH to between pH 4.0 and 6.0, preferably pH 5.0.

[0053] The term "pharmacologically acceptable" refers to a carrier composed of materials that are not biologically or otherwise undesirable.

[0054] The term "carrier" refers to any ingredient present in a pharmaceutical formulation other than an active agent, and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, etc.

[0055] A composition for use according to the 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 such an endolysin polypeptide or a fragment thereof.

[0056] Pharmaceutical compositions for use in the present invention may further comprise one or more additional pharma- ceutically acceptable ingredients, such as alum, stabilizers, antibacterial agents, buffers, colorants, flavorings, adjuvants, etc. Pharmaceutical compositions for use in the present invention are preferably imidazole-free.

[0057] The Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, for medical uses and methods of treatment provided herein can be in the form of pharmaceutical compositions and unit doses thereof together with conventionally used adjuvants, carriers, diluents or excipients, in the form of solids such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs or capsules filled therewith, all for oral use or in the form of suppositories for local (including vaginal) use. Furthermore, other means of administration are also contemplated in the context of the present invention. Such means may include, inter alia, parenteral administration. Also contemplated are pharmaceutical compositions for local and / or regional administration, for example in the form of (topical) gels, lotions or creams, or via pessaries, i.e. vaginal suppositories. Such pessaries / suppositories can be coated with the Gardnerella-specific endolysin, preferably recombinant Gardnerella-specific endolysin, as described herein. The pharmaceutical compositions and unit dosage forms thereof may contain the ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredients consistent with the intended daily dose range used. Compositions for use in the present invention may be liquid preparations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monolaurate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol.Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials and processing techniques are described in Part 5 of Remington's "The Science and Practice of Pharmacy", 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins.

[0058] The solid compositions for use in the present invention can be in the form of tablets or lozenges formulated in a conventional manner.The tablets can be coated according to methods well known in the art.Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable carriers known in the art.

[0059] The compositions for use in the present invention may also be formulated as suppositories, which may include suppository bases, including but not limited to cocoa butter or glycerides. The compositions of the present invention may also be formulated into transdermal formulations, including but not limited to, aqueous or non-aqueous vehicles, including but not limited to, creams, ointments, lotions, pastes, medicated plasters, patches, or membranes. The compositions for use in the present invention may also be formulated for parenteral administration, including but not limited to, by injection or continuous infusion. The injectable preparations may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents, including but not limited to, suspending agents, stabilizing agents, and dispersing agents. The compositions may also be provided in powder form for reconstitution with a suitable vehicle, including but not limited to, sterile pyrogen-free water.

[0060] The compositions for use in the present invention can also be formulated as a depot preparation, which can be administered by implantation or 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 sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[0061] Compounds for use in the present invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in Remington's "The Science and Practice of Pharmacy."

[0062] Also provided by the present invention is a method of treating bacterial vaginosis as described herein above in a patient who has previously failed antibiotic treatment and / or who is suffering from bacterial vaginosis and the infectious bacteria is resistant to antibiotic treatment, the method comprising administering to the patient a therapeutically effective amount of a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin as described herein, or a pharmaceutical composition comprising a Gardnerella-specific endolysin, preferably a recombinant Gardnerella-specific endolysin as described herein.

[0063] The embodiments and definitions set out herein above for the therapeutic use according to the present invention apply mutatis mutandis in the context of the method of treating BV.

[0064] In one embodiment of the use of the present invention, a host cell or a pharmaceutical composition comprising a host cell is used to deliver an endolysin, preferably to a host cell.

[0065] It will be appreciated that the endolysins and pharmaceutical compositions for use described herein may be administered to a subject in combination with one or more additional therapeutic agents. For example, the endolysins and pharmaceutical compositions described herein may be administered to a subject in combination with: (a) one or more conventional antibiotic treatments: such antibiotics may include clindamycin, metronidazole, or any other suitable antibiotic known to one of skill in the art; (b) one or more additional endolysins, or a nucleic acid molecule, vector, host cell or bacteriophage capable of expressing same; (c) a compound or composition that adjusts the vaginal pH, preferably to a pH between 4.0 and 6.0, more preferably to about pH 5.0; such pH adjusting compounds may include phosphates, lactic acid (e.g., natural acidifying substances secreted to establish an acidic environment) or other organic acids, such as carboxy-substituted polymers; (d) therapy to neutralize toxins released upon bacterial lysis of G. vaginalis cells in the vagina; suitable neutralizing therapies may include antibodies (see Babcock et al., 2006, Infect. Immun. 74:6339-6347) and toxin absorbents such as torebamer (see Barker et al., 2006, Aliment. Pharmacol. Ther. 24:1525-1534); (e) Probiotics.

[0066] The invention is further illustrated by reference to the following non-limiting figures and examples. [Brief description of the drawings]

[0067] The drawings show: [Figure 1]Reduction of biofilm CFU of a representative Gardnerella strain (G. vaginalis ATCC14018) by MDZ, CLI, and PM-477(H2B10). Figure 1 shows that G. vaginalis (ATCC14018) grown in biofilms can be eradicated by MDZ and PM-477(H2B10), but not by CLI treatment. Biofilms were grown for 40 h and then incubated with the indicated antimicrobial agents for an additional 24 h. For analysis, antimicrobial agents were washed off, biofilms were mechanically removed, and viable cells were counted (CFU / mL) by quantitative plating. LOD: limit of detection. Abbreviations: ctrl: control, medium only. LOD: limit of detection.

[0068] [Diagram 2] Resistance development by serial passaging. Figure 2 shows that six Gardnerella strains were passaged for up to 25 days in the presence of sub-MIC concentrations of MDZ or MDZ-OH as indicated in the legend. One representative Gardnerella strain was passaged for 25 days with PM-477 (H2B10) as indicated in the figure legend. MICs (μg / mL) were determined daily before the next round of passaging.

[0069] [Diagram 3] Lytic effect of MDZ and PM-477(H2B10) on wild-type and MDZ-passaged G. vaginalis (ATCC 14018T) cells in suspension. Figure 3 shows that wild-type cells naive to MDZ and a strain passaged for 25 rounds with sub-MIC concentrations of MDZ were treated with MDZ (A) and PM-477(H2B10) (B) for 1, 5 and 24 hours, respectively. Abbreviations: ctr: control, medium only; LOD: limit of detection.

[0070] [Figure 4-1]MDZ passaged strains grown as biofilms are resistant to MDZ and sensitive to PM-477(H2B10). Figure 4 shows that 72-h preformed biofilms were treated for 24 h with different concentrations of MDZ (A) and PM-477(H2B10) (B), as indicated on the x-axis, and viable cells were quantified. Abbreviations: ctr: control, medium only; LOD: limit of detection. MDZ-OH: hydroxymetronidazole. Numbers indicate rounds of passaging with sub-MIC concentrations of MDZ or MDZ-OH. [Figure 4-2] MDZ passaged strains grown as biofilms are resistant to MDZ and sensitive to PM-477(H2B10). Figure 4 shows that 72-h preformed biofilms were treated for 24 h with different concentrations of MDZ (A) and PM-477(H2B10) (B), as indicated on the x-axis, and viable cells were quantified. Abbreviations: ctr: control, medium only; LOD: limit of detection. MDZ-OH: hydroxymetronidazole. Numbers indicate rounds of passaging with sub-MIC concentrations of MDZ or MDZ-OH.

[0071] [Diagram 5] The lytic effect of H2B10B11 is maintained in MDZ-resistant Gardnerella isolates. Shown are MIC and MBC99.5 values ​​for H2B10B11 and MDZ determined for a panel of Gardnerella BV patient isolates (n=18). The MBC99.5 for H2B10B11 could not be analyzed for one strain, and growth of another strain was too low to determine the MIC for MDZ (n=17). The same strains were tested for both antimicrobial agents. Boxes (25th-75th percentiles) and whiskers (10th-90th percentiles) are shown, medians are shown as lines, and data points below and above the whiskers are shown as circles. The breakpoint for resistance for MDZ is shown as a line (32 μg / mL). Values ​​above the ULOQ are arbitrarily displayed as 16 μg / mL (H2B10B11) and 4096 μg / mL (MDZ). Abbreviations: LLOQ: lower limit of quantification. ULOQ: upper limit of quantification.

[0072] [Figure 6]Gardnerella BV patient isolates of various origins are susceptible to H2B10B11. Figure 6 shows the MIC and MBC99.5 values ​​of H2B10B11 determined for Gardnerella BV patient isolates received or isolated in-house as described in Table 1. The number of strains analyzed, n, is indicated on the right side of the graph. MICs for a subset of fast growing strains were determined after 48 hours. MICs for most strains were determined after 72 hours, as most clinical isolates grow poorly at pH 5.5. Boxes (25th-75th percentiles) and whiskers (10th-90th percentiles) are shown, with medians shown as lines and data points below and above the whiskers shown as circles. Values ​​above the ULOQ are displayed as 16 μg / mL. Abbreviations: LLOQ: lower limit of quantification. ULOQ: upper limit of quantification. The following examples further illustrate the invention.

[0073] overview Antibiotics are the mainstay of treatment for bacterial vaginosis (BV). However, the treatment failure rate in patients with recurrent BV is approximately 50%. Herein, we investigated potential mechanisms of treatment failure, including the tendency for resistance development and biofilm activity of metronidazole (MDZ), clindamycin (CLI) and PM-477 (H2B10), an engineered endolysin with specificity for bacteria of the Gardnerella genus. Minimum inhibitory concentration (MIC) determinations showed that 60% of a panel of 22 Gardnerella isolates from four different species were resistant to MDZ, whereas all strains were highly susceptible to CLI and endolysin PM-477 (H2B10). Similar results were obtained with a panel of 18 Gardnerella isolates from vaginal swabs of BV patients, 10 of which were resistant to MDZ (MIC > 32 μg / mL) but inhibited by endolysin H2B10B11 at 4–8 μg / mL (MIC 90 =4.4μg / mL).

[0074] Six strains that were initially susceptible to MDZ were passaged with MDZ or its more potent hydroxy metabolites. All of them developed complete resistance after 5–10 passages, resulting in MICs >512 μg / mL. In contrast, only a mild increase in MIC was observed with PM-477(H2B10). There was also no formation of cross-resistance, as MDZ-resistant Gardnerella strains remained highly susceptible to PM-477(H2B10) both in suspension and in preformed biofilms. Strains that were resistant to MDZ in suspension were also resistant to >2048 μg / mL of MDZ when grown as biofilms. All strains were susceptible to PM-477(H2B10) when grown as preformed biofilms, with minimum biofilm eradicating concentrations (MBECs) ranging from 1–4 μg / mL. Surprisingly, the MBEC of CLI was >512 μg / mL for seven of the nine tested Gardnerella strains, all of which were susceptible to CLI when grown in suspension. The observed challenges of MDZ and CLI due to resistance formation and ineffectiveness against biofilms may be one explanation for the frequent treatment failures in uncomplicated or recurrent BV, respectively. Thus, the high efficacy of PM-477(H2B10) in eliminating Gardnerella in in vitro biofilms, as well as its high resilience against resistance formation, make PM-477(H2B10) a promising alternative for the treatment of bacterial vaginosis, especially in patients with frequent recurrences. Furthermore, we investigated the MIC of H2B10B11 for a total of 104 Gardnerella isolates from different geographical origins as well as several types of strains, but found no significant differences in their susceptibility to H2B10B11.

[0075] Materials and Methods Bacterial isolates and culture conditions Gardnerella spp. isolates of different species (i.e. G. vaginalis sensu stricto, G. leopoldii, G. pyoti and G. swidszynskii, cf. Vaneechoutte et al., 2019, Int. J. Syst. Evol. Microbiol. 69, 679-687) were obtained from the Laboratory of Bacteriology, University of Ghent, Belgium. These isolates include strains purchased from culture collections and fresh isolates from BV patients obtained from the University Clinic Bruges. Gardnerella isolates were grown for 48 h on chocolate (Choc) agar plates (Becton Dickinson) under anaerobic conditions in an anaerobic chamber equipped with anaerobic atmosphere generating bags (Sigma Aldrich). All isolates were cultured in New York City broth III (NYCB) consisting of 10 mM HEPES (Sigma Aldrich), 15 g / L proteose peptone (Sigma Aldrich), 3.8 g / L yeast extract (Thermo Fisher Scientific), 86 mM sodium chloride (Carl Roth), 28 mM α-D-glucose (Sigma Aldrich) supplemented with 10% horse serum (HS) (Thermo Fisher Scientific). Table 1 lists all Gardnerella isolates tested. For purposes of enablement, at least one strain of each identified species of the genus Gardnerella, i.e., G. vaginalis sensu stricto, G. leopoldii, G. piochii, and G. swidszynskii (following the nomenclature of Vanechoutte et al., 2019, Int. J. Syst. Evol. Microbiol. 69, 679-687), is publicly available to one of skill in the art. These publicly available strains are identified in Table 1 below with "T" as the type strain (T) for the corresponding Gardnerella species.As illustrative examples, ATCC 14018 is a publicly available type strain of G. vaginalis sensu stricto, UGent 18.01 is a publicly available type strain of G. pioti, UGent 06.41 and UGent 09.48 are both publicly available type strains of G. leopoldii, and GS 9838-1 is a publicly available type strain of G. swidszynskii. As will be apparent to those skilled in the art, other bacterial strains from each species can also be used. Additionally, additional Gardnerella strains were obtained via isolation from vaginal swabs from BV patients received from several medical centers, namely Ghent (BE), Vilnius (LT), Vienna (AT) and Durban (ZA). All samples were obtained with informed consent and ethical approval by AZ St. Jan (Bruges, BE), the Lithuanian Bioethics Committee (LT), the Medical University in Vienna (AT) and the University of KwaZulu-Natal (Durban, ZA). Swabs were streaked onto Choc or Gardnerella selective agar plates (both Becton Dickinson). Single strains were isolated by picking individual colonies and restreaking onto Choc or Gardnerella selective agar plates (both Becton Dickinson), incubating the plates anaerobically at 37°C for 48 hours, and repeating the isolation and restreaking process at least two more times. Following this purification of individual strains, glycerol frozen stocks were prepared and strains were subjected to identification by MALDI-TOF MS (Bruker Biotyper). With this method, G. vaginalis could be distinguished from G. leopoldii and G. swidszynskii, however, without making it possible to distinguish between the latter two, and G. piochii could not be identified to species level.

[0076] Preparation of PM-477 (also referred to interchangeably as "H2B10") Endolysin PM-477 (H2B10, SEQ ID NO: 1) was engineered and produced as previously reported (Landlinger et al., (2021 Pathogens 10, 1-19) and WO 2020 / 225335 A1 brochure). Endolysin H2B10B11 differs from H2B10 in one amino acid substitution in one of the binding domains (see SEQ ID NO: 37). Briefly, PM-477 (H2B10) and H2B10B11 were recombinantly expressed in E. coli BL21(DE3). Protein purification was performed by affinity chromatography on a nickel-nitrilotriacetic acid (Ni-NTA) HISTrap column. Proteins were eluted with 50 mM MES (Carl Roth) pH 7, 150 mM NaCl (Carl Roth), 150 mM to 500 mM imidazole fractions (2-fold dilution). Compared to the preparation used in the study by Landlinger et al. (2021, Pathogens 10, 1-19), the N-terminal His tag was cleaved by digestion with 1:100 w / w 3C protease. The removed tag and protease were separated from PM-477 (H2B10) and H2B10B11 by anion exchange chromatography. Untagged proteins were concentrated (if necessary) and dialyzed against MES buffer (50 mM MES pH 5.5, 200 mM NaCl, 8 mM MgSO4 (Sigma Aldrich)).

[0077] Protein concentrations were determined by OD 260 / 280 nm or by using the Pierce™ BCA (Bicinchoninic Acid) Protein Assay Kit (Thermo Fisher Scientific). Purified aliquots of 1000 μL containing 0.7 mg / mL PM-477 (H2B10) or 1.3 mg / mL H2B10B11 were stored at −80° C. until use.

[0078] The amino acid sequence of PM-477 (H2B10) is shown in SEQ ID NO: 1. The amino acid sequence of H2B10B11 is shown in SEQ ID NO:37.

[0079] Culture-based assessment of bactericidal activity Bacterial suspension (OD 600 0.1, about 10 7 ~10 8 Bacterial suspensions were prepared by diluting 10 μL of endolysin (200 μg / mL) with 90 μL of bacterial suspension in NYCB + 10% HS, pH 5.5. Reactions were performed in triplicate by mixing 10 μL of endolysin (200 μg / mL) with 90 μL of bacterial suspension in wells of a 96-well plate. 10 μL of MES buffer without endolysin was used as a control. The 96-well reaction plate was incubated anaerobically at 37 °C for 5 h. Ten-fold dilution series (10 -1 ~10 -6 ) were prepared in NYCB + 10% HS and 2 μL of each dilution was spotted onto Choc agar plates. After 48 h of anaerobic incubation at 37 °C, colonies were counted, CFU / mL was calculated, and log 10 Decided to decrease.

[0080] MIC evaluation The minimum inhibitory concentration (MIC), a standard measure of activity of antimicrobial substances, was determined according to the Clinical and Laboratory Standards Institute protocol (2018) for antimicrobial susceptibility testing of anaerobic bacteria (Carpenter et al., 2018). 5 ~10 6 Bacterial suspensions of CFU / mL were treated with a two-fold dilution series of PM-477 (H2B10) or the antibiotic clindamycin (clindamycin hydrochloride, Sigma Aldrich) tested at a starting concentration of 64 μg / mL, or with a two-fold dilution series of the antibiotic metronidazole (MDZ, Gatt-Koller) and its hydroxy metabolite 1-(2-hydroxyethyl)-2-hydroxy-methyl-5-nitroimidazole (MDZ-OH, Sigma Aldrich) starting at 2048 or 512 μg / mL, respectively, or with a two-fold dilution series of tinidazole (TDZ, Sigma Aldrich) starting at 128 μg / mL. Controls for growth in the absence of antimicrobial agents were also included. OD 600The OD was recorded by a microplate reader (Tecan, Grodig, Austria) after 48 h of incubation at 37°C or, for some fast-growing strains, after 24 h of incubation. Absence of growth was indicated by an OD 600 The MBC was defined as ≦0.14. Because most of the clinical isolates summarized in Figures 5 and 6 grew slowly and poorly at pH 5.5, overnight cultures in NYCB (unadjusted pH of about 7 instead of adjusted to pH 5) + 10% HS were inoculated and used as input for MIC determination. MIC readings and MBC 99.5 Spotting was performed after 72 hours of incubation for H2B10B11. 99.5 MBC is the minimum concentration of an antimicrobial agent that results in bacterial death of all cells in a suspension up to a defined detection limit, resulting in the killing of at least 99.5% of the bacteria. 99.5 To determine the MICs, 2 μL of the antibacterial dilution series and bacterial suspensions in the MIC plates were spotted onto NYCB + 10% HS agar plates and incubated anaerobically for 2-3 days at 37 °C. To bridge the MIC results determined after 72 h of incubation with previously obtained results, MICs for a subset of the rapidly growing strains were determined after 48 h of incubation (Figure 6).

[0081] Serial passaging for resistance development profiling NYCB+10%HS 10 5 ~10 6 Bacterial suspensions of CFU / mL were prepared and treated with a two-fold dilution series of either endolysin PM-477(H2B10) produced as previously reported (Landlinger et al., 2021, Pathogens 10, 1-19), PM-477(H2B10) with modifications defined herein, metronidazole (MDZ, Gatt-Koller), or 1(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MDZ-OH, Sigma Aldrich). The final volume of the reaction was 100 μL and reactions were performed in triplicate in 384-well plates. OD 600was measured using a microplate reader (Tecan, Grodig, Austria) to obtain baseline values ​​at time 0. The reactions were incubated anaerobically for 24 h and then OD 600 Measure again and change MIC to OD 600 The lowest concentration at which growth was detected was determined as <0.14. Bacterial suspensions treated with the highest concentration of antimicrobial agent at which growth was detected ("sub-MIC") were 5 ~10 6 CFU / mL and treated anew with a dilution series of antimicrobial agents. This process was repeated for up to 25 rounds. At each round, the input CFU / mL was determined by quantitative plating.

[0082] Biofilm formation and MBEC measurements Gardnerella spp. (see figure for strain details) were resuspended in Brain Heart Infusion Broth supplemented with 2% (w / v) gelatin, 0.5% yeast extract (w / v), 0.1% starch (w / v) and 0.25% glucose (w / v) (sBHIG). Passaged strains were resuspended in unbuffered NYCB (sNYCB) supplemented with 1% glucose (w / v). OD 600 to 0.1 (approximately 10 7 ~10 8CFU / mL) and cell suspensions were diluted 1:10 in growth medium as input for biofilm formation. A total of 200 μL of each bacterial suspension was added to a 96-well flat-bottom plate (treated for tissue culture, Sigma-Aldrich). Biofilms were grown under anaerobic conditions at 37 °C for 40–72 h depending on the isolate. The supernatant was subsequently removed and biofilms were treated with 100 μL of antimicrobial dissolved in sBHIG (pH 5 for treatment with PM-477 (H2B10) and unbuffered for treatment with antibiotics) and incubated anaerobically at 37 °C for an additional 24 h. After treatment, biofilms were washed twice with 200 μL of 1× PBS and lysed by vigorous pipetting (40× up and down). Serial dilutions of lysed cells were spotted onto chocolate agar plates. Plates were incubated anaerobically for 2–3 days and the minimum biofilm eradicating concentration (MBEC) of the endolysin or antibiotic being used was calculated.

[0083] statistical analysis Where necessary, data were log-normalized before applying statistical tests (e.g., for CFU / mL values, as indicated in the figure legends). When only two groups were compared, unpaired two-tailed t-tests were used as indicated in the respective figure legends. Multiple groups were compared by two-tailed one-way ANOVA tests. GraphPad Prism8 software was used for statistical analysis. Differences between groups were considered statistically significant if p<0.05.

[0084] Example 1: 59% of Gardnerella strains and patient isolates tested are MDZ resistant but highly susceptible to PM-477 (H2B10) and H2B10B11 The minimum inhibitory concentrations (MICs) of MDZ, tinidazole (TDZ), CLI, and PM-477 (H2B10) were determined for 22 strains of Gardnerella according to the Clinical and Laboratory Standards Institute (CLSI) protocol for anaerobic bacteria (Carpenter et al., 2018). The resistance breakpoints defined by EUCAST for Gram-positive anaerobes are >4 μg / mL for both CLI and MDZ (EUCAST, 2021). However, topical antibiotic administration is recommended for BV therapy, which allows reaching concentrations of active ingredients in the mg / mL range. Therefore, in this example, we used resistance breakpoints of ≥8 μg / mL and ≥32 μg / mL for CLI and MDZ, respectively, as previously described for bacteria associated with BV (Petrina et al., 2017 Anaerobe 47, 115-119). The susceptibility of Gardnerella strains to the antimicrobial agents PM-477 (H2B10), MDZ, CLI and TDZ was evaluated. The MIC and MBC values ​​for each strain are shown in Table 1. 99.5 Values ​​(minimum concentration that reduces CFU by 99.5% within 24 hours of treatment) are shown. Abbreviations: nd, not determined; SJHB, St. Jan Hospital Bruges. [Table 1]

[0085] The minimum inhibitory concentrations (MICs) of MDZ, CLI and PM-477 (H2B10) for Gardnerella type strains and patient isolates are summarized below in Table 2. Resistance is defined as ≥ 32 μg / mL and ≥ 8 μg / mL for MDZ and CLI, respectively. MICs 90 is defined as the MIC value for 90% of the test strains. For PM-477 (H2B10), no resistance breakpoints have been defined and therefore no "% resistant" has been defined (nd). [Table 2]

[0086] For MDZ, MICs ranging from 8 to >256 μg / mL were observed, with MIC values ​​for 90% of the strains (MIC 90 ) exceeded 256 μg / mL. Thirteen of the 22 isolates tested (59%) had MICs for MDZ of 32 μg / mL or higher. MDZ resistance in different Gardnerella strains ranged from 25% for G. vaginalis to 100% for G. leopoldii and G. swidszynskii. MIC values ​​for MDZ and TDZ were very similar, and for the most part the same isolates were resistant or susceptible, indicating very similar mechanisms of activity and resistance development. Thus, without being bound by any theory, these findings are expected to hold true for all nitroimidazoles, including secnidazole, since the resistance status across strains is found to be comparable for MDZ and TDZ. In contrast, all Gardnerella strains of all four species had CLI (MIC 90 = 0.5 μg / mL, range < 0.06 to 2 μg / mL) and endolysin PM-477 (H2B10) (MIC 90 = 1 μg / mL, range < 0.03 to 1 μg / mL).

[0087] MICs and MBCs of H2B10B11 and MDZ 99.5 was determined for 18 autologous Gardnerella strains isolated from vaginal swabs of BV patients (Figure 5). The majority of the analyzed isolates had a median MIC of 64 μg / mL and a median MBC of 1536 μg / mL. 99.5 and were resistant to MDZ (resistance breakpoint 32 μg / mL). Importantly, the same isolates were highly susceptible to H2B10B11, with a median MIC of 2 μg / mL H2B10B11 and a median MBC of 1. 99.5 was 8 μg / mL. Note that the MIC value for H2B10B11 was determined after 72 hours of incubation, which is different from the MIC determined above for PM-477 (H2B10).

[0088] Example 2: Pronounced killing of Gardnerella cells in biofilms by MDZ, CLI and PM-477 (H2B10) Gardnerella -dominated biofilms covering human vaginal epithelial cells are now generally considered a hallmark of BV. Therefore, we tested how antibiotics and PM-477 (H2B10) penetrate and kill in vitro preformed 40-72 hour old biofilms of various Gardnerella strains. The MBEC values ​​of ancestral (non-passaged) Gardnerella isolates are shown in Table 3 below. Interestingly, Gardnerella strains were all highly susceptible to CLI in suspension (MICs ranging from 0.01 to 1 μg / mL) and became resistant to CLI when grown as biofilms (Table 3 and Figure 1), with MBECs up to ≥ 512 μg / mL. MDZ eliminated biofilms above the LOD with MBECs ranging from 8 to 128 μg / mL, whereas endolysin PM-477(H2B10) was able to kill all six Gardnerella strains grown as biofilms at MBECs lower than either antibiotic, i.e., <2 to 32 μg / mL. T ) biofilm CFU reduction is shown in Figure 1. [Table 3]

[0089] Example 3: Gardnerella species that are initially susceptible to MDZ rapidly become resistant with successive rounds of passage The first was against MDZ (G. vaginalis ATCC14018 TSix Gardnerella strains that were susceptible to MDZ (MIC 8 μg / mL) or at least to the more potent MDZ hydroxy metabolite (MDZ-OH, MIC between 2 and 16 μg / mL, data not shown) were serially passaged in MDZ or MDZ-OH concentrations below the MIC such that growth was impaired but not completely inhibited. Upon passaging, the MICs for both MDZ and MDZ-OH metabolites increased strongly, with all Gardnerella strains reaching a resistance breakpoint of ≥ 32 μg / mL within five passages (see Figure 2). After nine rounds of passaging, four of the six strains could no longer be inhibited, even by the highest concentration used (MIC > 512 μg / mL) (Figure 2). In parallel, G. vaginalis (ATCC 14018 T ) was passaged 25 times with PM-477(H2B10). The MIC of PM-477(H2B10) increased only slightly over the 25 passages, to 8 μg / mL (Figure 2).

[0090] Example 4: Subcultured isolates that have acquired MDZ resistance remain sensitive to PM-477 (H2B10) We next tested to what extent the resistance of Gardnerella cells to MDZ influenced their susceptibility to the endolysin PM-477 (H2B10). G. vaginalis (ATCC 14018), passaged for 25 days on MDZ, was exposed to different concentrations of MDZ and PM-477 (H2B10) for 1, 5, and 24 h, and then the bactericidal effect was assessed by quantitative plating on Choc agar plates. The susceptibility of Gardnerella strains before and after 8-9 passages with sub-MIC concentrations of MZD or MDZ-OH is shown in Table 4 below. All MICs were determined after 48 h of incubation. [Table 4]

[0091] For the ancestral strain, there is a clear dose / time dependence of viability upon exposure to MDZ (Figure 3A). In contrast, G. vaginalis strains passaged on MDZ were able to tolerate very high concentrations of MDZ up to 2 mg / mL for 1 and 5 hours without any loss of viability, and treatment with 2 mg / mL for 24 hours resulted in only a 2 log reduction compared to buffer-treated controls. When G. vaginalis (ATCC 14018) was exposed to PM-477(H2B10), the susceptibility was similar to the ancestral and passaged strains (Figure 3B). After 1 hour of treatment, 10 μg / mL PM-477(H2B10) reduced viability by 3.0 and 2.8 log units for the ancestral and passaged strains, respectively, and by ... 8 CFU / mL of the suspension, and the MIC values ​​of PM-477(H2B10) were very similar for the ancestral and MDZ-passaged strains (Table 4), indicating that acquired resistance to MDZ does not interfere with the mode of action of the endolysin.

[0092] All MDZ or MDZ-OH passaged Gardnerella strains were still able to form biofilms of similar thickness and CFU numbers as their respective unpassaged ancestors (data not shown). The MBEC of MDZ on Gardnerella strains passaged on MDZ or MDZ-OH was >2048 μg / mL for all six passaged strains (Figure 4A). Of these, one G. vaginalis strain (ATCC 14018) initially had an MIC below the resistance breakpoint. However, given that intravaginal MDZ (typically 0.75% cream, i.e., 7.5 mg / mL) can establish concentrations in the mg / mL range in vaginal fluid, strains with MICs above the EUCAST breakpoint were also passaged and tested (pre-passage MIC range 8-256 μg / mL, see Table 4).

[0093] In contrast, all strains were highly susceptible to PM-477(H2B10), with MB EC ranged from 1 to 8 μg / mL (Figure 4B, Table 3).

[0094] Example 5: Gardnerella BV patient isolates of various origins are susceptible to H2B10B11 MICs and MBCs of H2B10B11 against a panel of approximately 100 Gardnerella type strains and patient isolates from various geographic origins 99.5 were determined according to CLSI protocols. As most clinical isolates were poorly growing strains, the MIC reaction plates were incubated for 72 h. For a subset of fast growing strains and some additional strains, MIC determinations were performed after 48 h of incubation to align the results with previously evaluated MIC values ​​(Figure 6). In total, 104 MICs were determined for different strains (MICs for 15 strains were determined both after 48 and 72 h). MIC and MBC 99.5 Both values ​​were 1 concentration lower when the readout was performed after 48 h compared to 72 h. Strains summarized in "Total Gardnerella (72 h)" were subgrouped according to their geographic origin or their species (note that two isolates could not be identified to the species level, which is why they do not appear in the species subgroup). The number of strains in each group is shown on the right side of the graph. Importantly, there was no significant difference between the susceptibility of isolates from African and European BV patients to H2B10B11 (see Materials and Methods for source of isolates) (unpaired t-test for log2-transformed data, p=0.1221). Similarly, there was no significant difference between the MICs of G. vaginalis and G. leopoldii / Swidszynskii (unpaired t-test for log2-transformed data, p=0.5076).

[0095] [ka] [ka] [ka] [ka] [ka]

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Claims

1. A recombinant Gardnerella-specific endolysin comprising the amino acid sequence of SEQ ID NO:

37.

2. An endolysin described in claim 1, consisting of the amino acid sequence provided in SEQ ID NO:

37.

3. A pharmaceutical composition comprising the recombinant Gardnerella-specific endolysin described in claim 1 and, optionally, a pharmaceutically acceptable carrier and / or diluent.

4. A composition comprising an endolysin as described in claim 1 or a pharmaceutical composition as described in claim 3 for use in treating bacterial vaginosis in a patient.

5. A composition or pharmaceutical composition for use as described in claim 4, wherein the patient is suffering from bacterial vaginosis in which previous antibiotic treatment has failed and / or the infecting bacteria are resistant to antibiotic treatment, and the antibiotic treatment is treatment with metronidazole, tinidazole, secnidazole, clindamycin or any combination thereof.

6. 5. The composition or pharmaceutical composition for use according to claim 4, wherein the patient is suffering from recurrent bacterial vaginosis, preferably wherein the patient has had two or more episodes of BV within six months or three or more episodes of BV within twelve months.

7. 5. The composition or pharmaceutical composition for use according to claim 4, wherein the bacterial vaginosis is characterized by the presence of infectious bacteria of the species Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti, and Gardnerella swidszynskii, and / or any other species of the Gardnerella genus.

8. 5. The composition or pharmaceutical composition for use according to claim 4, wherein the endolysin has killing activity against Gardnerella vaginalis sensu stricto, Gardnerella leopoldii, Gardnerella pyoti, and / or Gardnerella swidszynskii.

9. 5. The composition or pharmaceutical composition for use according to claim 4, wherein the endolysin does not have killing activity against Lactobacillus crispatus, Lactobacillus gasseri, and / or Lactobacillus yensenii.

10. 5. A composition or pharmaceutical composition for use according to claim 4, which is administered locally into the vagina of a female subject and / or into or on the glans penis, foreskin or urethral opening of a male subject.

11. 5. The composition or pharmaceutical composition for use according to claim 4, wherein the endolysin is co-administered with a compound or composition that adjusts the vaginal pH to between 4.0 and 6.

0.

12. 5. The pharmaceutical composition for use according to claim 4, further comprising a compound or composition that adjusts the vaginal pH to 4.0 to 6.0.

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