JP-2022532130-A5 -

Dates

Publication Date

20230515

Application Date

20200507

Description

Figure 5 shows a quantitative decrease in viable colony-forming units (CFUs) in an assay after treatment with Gardnerella prophage endolysin. The upper histogram in Figure 5 shows the results for viability in CFU/ml, while the lower histogram shows the results for the percentage of dead CFUs.Figure 6 shows a quantitative decrease in viable colony-forming unit (CFU) assays comparing G. vaginalis strain Gv_9 (in the strict sense) incubated in imidazole-containing and imidazole-free media at various pH values with untreated cells. Cells with 5 × 10⁷ CFU/ml were incubated under anaerobic conditions at 37°C for 5 hours under the conditions shown below the graph, and the surviving CFU/ml was then measured quantitatively by plate culture. The results indicate that the survival of G. vaginalis Gv_9 is highly dependent on the absence of imidazole and low pH under the test conditions. Figure 7 shows a quantitative decrease in viable colony-forming units (CFUs) in a viable colony-forming unit (CFU) assay comparing cells from the narrowly defined G. vaginalis strain Gv_9 treated with eluate solutions containing recombinant endolysin H10B1 and 250 mM imidazole at various pH values, or with a control containing 250 mM imidazole at various pH values. Cells with 5 × 10⁷ CFUs/ml were incubated under anaerobic conditions at 37°C for 5 hours under the conditions shown below the graph, and the surviving CFUs/ml were then measured by quantitative plate culture. The vertical bars labeled with the imidazole control show the same data as in Figure 6. The results indicate that the enzymatic activity of H10B1, as an example of all endolysins in the present invention, is higher at lower pH values and shows the strongest activity at pH 5.5 and pH 5.0. Figures 8A – 8D show quantitative reductions in viable colony-forming unit (CFU) assays measuring the killing activity of the native and recombinant Gardnerella prophage endolysin described herein against four major Gardnerella species. In Figures 8A , 8B , 8C , and 8D , the killing activity of endolysin is measured by detecting viable CFUs in suspensions of G. vaginalis strain Gv_9, G. leopoldii strain Gv_11, G. piocii strain Gv_17, and G. swidosinskii strain Gv_23, respectively. Strains showing 5e7 CFU/ml in 90 μl were incubated with 10 μl of endolysin solution under anaerobic conditions at pH 5.0 for 5 hours (concentration adjusted to 0.2 mg/ml where possible; see Table 4). The logarithmic Y-axis shows the total number of viable cells. The dotted line indicates the limit of detection (LOD) achieved by plate culture of 2 μl of the reaction mixture (500 CFU/ml). The results show that the endolysins of the present invention have the ability to dissolve four major Gardnerella species. The results also indicate that some of the genetically modified endolysins of the present invention have higher wilting activity than the naturally occurring endolysins of the present invention. Figure 9 shows the phylogenetic relationship tree (amino acid level) of H-domains created using Clustal Omega (Sievers et al., 2011 Mol. Syst. Biol. 7, 539). Figure 10 shows the phylogenetic relationship tree (amino acid level) of region B, created using Clustal Omega (Sieffers et al., 2011 MoL Syst. Biol7, 539). We analyzed the dependence of activity on three potentially important parameters: pH, anaerobic/microaerophilic/aerobic conditions, and the presence/absence of imidazole. I selected three criteria for evaluation for the following reasons. - pH: The endolysin-killing activity of the present invention was successfully demonstrated using experiments conducted at a pH of approximately 7. However, the pH of a healthy vagina is approximately 3.5, while in BV vaginas, the pH rises to approximately 5.5 and even higher. Therefore, the pH dependence of endolysin activity was investigated. - Oxygen: In the literature, *Gardenerella* is described as anaerobic or microaerophilic. Therefore, we investigated whether more untreated cells survived under anaerobic, microaerophilic, or aerobic conditions over the incubation period of the experiment (usually 5 hours). - Imidazole: Endorcine of the present invention was purified by a one-step Ni-NTA column according to the method described in Reference Example 1, where the buffer used to elute endolysine from the Ni-NTA matrix contained imidazole. Therefore, without a further step of dialyzing the sample, the resulting eluate contains 250 mM imidazole. In this regard, the effect of imidazole on Gardnerella was investigated. First, the susceptibility of G. vaginalis Gv_9 to incubation in media with and without imidazole at different pH values was evaluated (see Figure 6). Cells at 5 × 10⁷ CFU/ml were incubated under anaerobic conditions at 37°C for 5 hours under the conditions shown below the graph. Next, viable CFU/ml was measured quantitatively by plate culture. As shown in Figure 6, at pH 6.0, median 1 × 10⁷ and 1 × 10⁶ cells survived incubation with and without imidazole, res