Vaccines targeting C. acnes hyaluronidase for the prevention and treatment of acne vulgaris

Abstract

The present invention provides immunogenic peptides and compositions for the treatment of acne or reducing the likelihood of having acne in a subject in need thereof. TIFF2026502948000224.tif173134

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 436,332, filed December 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application contains a Sequence Listing, which has been submitted in computer-readable form under the name "SequenceListing_065472_000903WOPT.xml," having a size of 35,558 bytes, created on December 29, 2023. The information contained in this computer-readable form is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers AI141401 and AI138053 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] FIELD OF THE INVENTION The present invention relates to the treatment and prevention of acne. [Background technology]

[0005] background All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description contains information that may be useful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is any publication mentioned expressly or impliedly prior art.

[0006] Four out of five people will suffer from acne vulgaris at some point in their lives. Predisposition to acne depends on both host and environmental factors. Among these, the contribution of the skin commensal C. acnes (C. acnes) has been debated, given the robust colonization of healthy and acne-prone subjects with C. acnes. More careful characterization of C. acnes strains isolated directly from acne lesions has demonstrated the importance of C. acnes genetic elements as major acne determinants, as acne onset and severity are clearly dependent on the C. acnes lineage and phylotype. Accordingly, C. acnes strains have been classified based on their health- or acne-associated status. Subsequent metagenomics studies have revealed gene sets that are significantly present in acne- or health-associated C. acnes strains, thereby opening up new avenues for understanding acne pathogenesis. Acne pathogenesis remains poorly understood due in part to the lack of robust animal models and the poor survival of C. acnes in rodents.

[0007] Therefore, a better understanding of C. acnes remains necessary, and there is an unmet need for prevention and treatment of C. acnes. Summary of the Invention

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0009] Various aspects of the present invention provide immunogenic polypeptides comprising fragments of HylA.

[0010] In various embodiments, a fragment of HylA can be linked or fused to an adjuvant.

[0011] In various embodiments, the fragment of HylA is: (a) TIFF2026502948000002.tif4128; (b) TIFF2026502948000003.tif4128 variant, TIFF2026502948000004.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000005.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000006.tif4128; (d) TIFF2026502948000007.tif4128 variant, TIFF2026502948000008.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000009.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000010.tif4128; (f) TIFF2026502948000011.tif4128 variant, TIFF2026502948000012.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000013.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000014.tif4128; or (h) TIFF2026502948000015.tif4128 variant, TIFF2026502948000016.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000017.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The peptide may comprise one or more peptides selected from:

[0012] In various embodiments, the adjuvant can be a tetanus protein, pertussis toxoid, diphtheria toxoid, a cytokine, or a fragment thereof.

[0013] Various embodiments provide mRNA molecules that encode the immunogenic polypeptides of the invention described herein.

[0014] Various aspects include: (a) TIFF2026502948000018.tif4128; (b) TIFF2026502948000019.tif4128 variant, TIFF2026502948000020.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000021.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000022.tif4128; (d) TIFF2026502948000023.tif4128 variant, TIFF2026502948000024.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000025.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000026.tif4128; (f) TIFF2026502948000027.tif4128 variant, TIFF2026502948000028.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000029.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000030.tif4128; or (h) TIFF2026502948000031.tif4128 variant, TIFF2026502948000032.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000033.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The present invention provides a polypeptide comprising one or more peptides selected from:

[0015] In various embodiments, one or more peptides can be linked or fused to an adjuvant.

[0016] In various embodiments, the adjuvant can be a tetanus protein, pertussis toxoid, diphtheria toxoid, a cytokine, or a fragment thereof.

[0017] In various embodiments, the polypeptide may further comprise a linker between the one or more peptides and the adjuvant.

[0018] In various embodiments, the polypeptide may further comprise a linker between the one or more peptides and the C-terminus of the adjuvant.

[0019] In various embodiments, the polypeptide can comprise at least two peptides and can further comprise a linker between each of the at least two peptides.

[0020] In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, TIFF2026502948000034.tif4128, leucine zipper, r aliphatic, or helical peptides.

[0021] Various aspects provide compositions comprising a polypeptide of the invention and an adjuvant.

[0022] In various embodiments, the adjuvant can be alum, hydroxyphosphate sulfate, CpG1018, monophosphoryl lipid A, oil-in-water emulsion, CpG, or QS-21 saponin.

[0023] Various embodiments provide mRNA molecules that encode the polypeptides of the invention described herein.

[0024] Various embodiments provide methods of treating acne comprising administering to a subject in need thereof a composition comprising a polypeptide of the invention or a composition of the invention, or an mRNA molecule of the invention.

[0025] Various embodiments provide methods of reducing the likelihood of acne comprising administering to a subject in need thereof a composition comprising a polypeptide of the invention or a composition of the invention, or an mRNA molecule of the invention.

[0026] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of aspects of the invention. [Brief explanation of the drawings]

[0027] Exemplary embodiments are illustrated in the referenced drawings, in which: It is intended that the embodiments and figures disclosed herein be considered illustrative and not restrictive.

[0028] [Figure 1-1](Panels a–j) show that the HylA enzyme is a major virulence factor in acne pathogenesis. [a] Pie charts showing the association between health-associated and acne-associated C. acnes phylotypes and the hylA or hylB gene. [b–f] CD1 mice (n=10) were infected intradermally (id) with 2×107 CFU of wild-type (HL043PA1 or HL110PA3) or isogenic mutant (ΔhylA or ΔhylB) C. acnes and then topically administered sebum daily. Bacterial burden (b), disease score (c), and cytokines (d–f) at 2 days postinfection. [g–i] CD1 mice (n=10) were infected with either HL043PA1, ΔhylA, or ΔhylA + recombinant I HylA protein (10 μg) as described above. Disease score (g) and tissue cytokines (h, i) at 2 days postinfection. [b-i] Data are from two independent experiments, and each data point represents one mouse. Bars indicate median values. [j] Tissue cytokines at 2 days post-infection. Data in j were analyzed by one-way ANOVA with Tukey's post-hoc test. Data in b, c, and e-h were analyzed by one-way ANOVA with Tukey's post-hoc test. Data in d and i were analyzed by the nonparametric Kruskal-Wallis one-way ANOVA test. [Figure 1-2] See description of Figure 1-1. [Figure 2](Panels a–e) show HA degradation and structural features of the HylA and HylB enzymes. [a, b] HPLC profiles of HMW HA (2 mg / ml) digested with rHylA or rHylB (1 μg) for 24 hours. The digested HA peaks (HA-2, 4, and 6) were quantified using known concentrations of purified HA oligosaccharides (see Figures 10 and 11). The larger HA fragments, highlighted by green circles, are only visible in HA digested with recombinant HylA (rHylA). Results are representative of at least two independent experiments. [c] Comparison of the crystal structures of HylA and HylB. HylA and HylB are shown in schematic form in magenta and orange, respectively. Structural domains and linkers are labeled. [d] Comparison of the electrostatic potential of the active site cleft components and the solvent-excluded surface. Electrostatic surface views are shown for the crystal structures of HylA and HylB. The electrostatic potential of the solvent-excluded surface of the Hylase crystal structure was calculated using APBS in PyMol. The active site cleft is indicated by a dashed oval. Red and blue correspond to potentials of -5 kTe-1 and 5 kTe-1, respectively. [e] Similarities and differences in residues in the active site clefts of HylA and HylB. Residues are highlighted in different structural components of the cleft and labeled for their functional roles. The HA-6 ligand was taken from the crystal structure of Streptococcus pneumoniae Hyl (SpHyl) (PDB: 1LOH) and modeled in the HylA active site cleft. [Figure 3]Panels a–f show a comparison of HylA and HylB with bacterial and animal Hyls. [a] Superposition of the HylA crystal structure with Hyl from Streptomyces coelicolor (ScHyl). [b] Comparison of the HylA crystal structure with Hyl from Streptococcus pneumoniae (SpnHyl) and Hyl from Streptococcus agalactiae (SaHyl). HylA, ScHyl, SpnHyl, and SaHyl are shown in schematic representation in magenta, salmon red, green, and cyan, respectively. The PDB IDs for ScHyl, SpnHyl, and SaHyl are 2X03, 2BRW, and 1F1S, respectively. The dashed circles in panels a and b indicate regions of the HylA β domain whose topology is similar or different from that of homologs. Structural domains and linkers are labeled. [c] Conformations of the active site cleft are shown. The relative positions (i.e., the distance between) of the LI and / or LII loops from the α domain and the LIV and / or LV loops from the β domain define the open / closed conformations of the Hyl cleft, indicated by black arrows. The clefts of Hyl enzymes from different bacteria, including the crystal structures HylA, HylB, 2XO3, 2WCO, 2BRW, 1LOH, 1F1S, and 1LXM, are shown in magenta, orange, salmon red, slate blue, green, split pea green, cyan, and gray, respectively. [d] The catalytic tetrad (Tyr-His-Arg-Glu) and residues involved in neutralizing the substrate acid (Asx) are shown. Corresponding residues from HylA, HylB, ScHyl, SpnHyl, and SaHyl are shown as magenta, orange, salmon red, green, and cyan sticks, respectively. The HA-6 ligand was taken from the SpnHyl crystal structure (PDB: 1LOH). [e] Structure of human Hyl (hHyl1, human hyaluronidase 1) (PDB: 2PE4). The structural organization of hHyl1 is shown as representative of animal hyaluronidases.[f] The structural elements of HylA and HylB that define the catalytic cleft are shown in schematic representation. HylA and HylB are shown in magenta and orange, respectively. The HA-6 ligand is taken from the SpnHyl crystal structure (PDB: 1LOH) and is shown as a yellow stick. [Figure 4] (Panels a–h) show the enzymatic activity of HylA mutants with single amino acid substitutions. [a] Position of the amino acid residue on the HylA crystal that was mutated to the corresponding HylB residue. [b–h] HPLC profiles of HMW HA after 24 h of co-incubation with WT or mutant HylA (1 μg): undigested (b), rHylB (c), rHylA (d), or rHylA with single amino acid substitutions (e, h). Quantitation of the HA digestion peak was performed using known concentrations of purified HA oligosaccharides (see Figures 10 and 11). Asterisks (*) indicate nonspecific peaks present in the water control (see Figure 7). Data are representative of two independent experiments. [Figure 5](Panels a–f) demonstrate the pro-inflammatory properties and TLR2 dependence of Hyl degradation products. [a] HaCaT cells were stimulated with HA digested with either the supernatant from HylA+HL043PA1 or HylB+HL110PA3. IL-6 levels were measured in the culture supernatant. [b–d] WT, TLR2– / –, and TLR4– / – mice were infected i.d. with WT or isogenic ΔhylA HL043PA1 (2 × 107 CFU) as described above. Disease scores (b) and skin cytokines (c, d) were measured 24 hours post-infection. [e] IL-6 levels in the culture supernatant of WT or TLR2– / – BMDMs after stimulation with HA digested with rHylA or rHylB. [f] IL-6 levels in the culture supernatant of HaCaT cells after stimulation with HA digested with WT or mutant rHylA. Data in a, e, and f are shown as mean ± SD, and each data point is a technical replicate. [b-d] Bars indicate median values, and each data point represents one individual mouse (n = 5-11 for TLR4- / -, TLR2- / -, or WT mice infected with HL043PA1; n = 4-5 for TLR4- / -, TLR2- / -, or WT mice infected with isogenic ΔhylA). [a, e, f] Data are representative of two independent experiments. p values ​​in a and f were calculated by one-way ANOVA with Tukey's post-hoc test. p values ​​in b-d were calculated by the nonparametric Mann-Whitney T-test. [Figure 6](Panels a–d) show that vaccination against HylA improves acne lesions and reduces inflammation. [a–d] Mice (n = 10) were immunized intraperitoneally (ip) with alum and the C-terminus of the tetanus protein (TT) or multiple HylA epitopes linked to TT (TT-mHylA) and then challenged i.d. with the HylA+HL 043 PA1 C. acnes strain. Disease score (a), bacterial load (b), and IL-1b (c) at 48 hours. [d] Serum anti-HylA or anti-HylB antibody titers after the third immunization with TT-mHylA. Data are from three independent experiments (a–d), and each data point represents one mouse. Data in a–c were analyzed by a two-tailed, unpaired, nonparametric Mann-Whitney Student's t-test, and in d by a nonparametric Kruskal-Wallis one-way analysis of variance test. [Figure 7] (Panels a–h) show validation of the enzymatic activity of ΔhylA and ΔhylB. [a] Culture supernatants of WT and Δhyl strains were tested for enzymatic activity against HMW HA substrate. The arrow indicates the area of ​​HA clearance from incubation with the WT strain, which was not observed in plates with Δhyl. [b–e] HMW HA substrate (2 mg / ml) was digested with supernatants (10 μl) from WT HL110PA3 or HL043PA1 (b, d) or isogenic ΔhylB or ΔhylA (c, e) for 24 h and analyzed by HPLC. [f] HA digestion peaks were quantified using known concentrations of purified HA oligosaccharides (HA-2, HA-4, and HA-6). [g, h] Undigested HA control (g) and water control (h). Asterisks (*) indicate nonspecific peaks present in the water control. The green circle (f) indicates a larger oligomer present only in HMW HA digested with HL043PA1 supernatant. Results are representative of at least two independent experiments. [Figure 8](Panels a–c) show HA disaccharides from HylB digests analyzed by LC-MS in negative ionization mode. [a] Extracted ion chromatogram of the disaccharide elution time (12.12 min). [b] Mass (m / z) of the corresponding disaccharide in negative ionization mode (MH). [c] Extracted ion chromatogram of the HA-tetrasaccharide. [Figure 9-1] (Panels a–h) demonstrate that the HylA enzyme is a major virulence factor in acne pathogenesis. [a] CD1 mice (n = 10) were infected i.d. with WT (HL043PA1 or HL110PA3) or isogenic mutant (ΔhylA or ΔhylB) C. acnes (2 × 10 cfu). Representative images of skin lesions at 2 days postinfection. [b, c] CD1 mice (n = 10) were infected with HL043PA1, ΔhylA, or ΔhylA + recombinant I HylA protein (10 μg) as described above. CFU (b) and IL-1β (c) from skin lesions at 2 days postinfection. (d) IL-10 levels (n = 6 for HL110PA3 and n = 7 for ΔHylB). [e-g] CD1 mice (n = 3 for sebum and PBS, and n = 5 for HL043PA1 + sebum) were topically infected with HL043P1 id + sebum, topically administered sebum alone, or untreated, and then disease scores I and cytokines (f-h) were measured on day 2 (48 hours). Bars represent median values, and data are from one to two independent experiments. Data were analyzed by the nonparametric two-tailed Mann-Whitney U test (d) or one-way ANOVA with Tukey's post-hoc test (e-h). [Figure 9-2] See description of Figure 9-1. [Figure 10](Panels a–g) show the kinetics of HA digestion by recombinant HylB. [a–d] HPLC profiles of HMW HA (2 mg / ml) digested with rHylB (1 μg) for 0–60 min. [e] HPLC profile of HMW HA (2 mg / ml) digested with 1 μg of rHylA enzyme for 60 min. [f] Water control. [g] HA peaks were quantified using known concentrations of purified HA oligosaccharides. Oval circle I indicates larger oligomers present only in rHylA-digested HA. Results are representative of at least two independent experiments. [Figure 11] (Panels a–f) show the kinetics of HA digestion by supernatants from either HylA-expressing or HylB-expressing C. acnes strains. [a–d] HMW HA substrate (2 mg / ml) was digested with 10 μl of supernatant from HylA+HL043PA1 or HylB+HL110PA3 for 1 hour (a, c) or 24 hours (b, d), followed by HPLC analysis. [e] Digested HA peaks (DP2, DP4, and DP6) were quantified using known concentrations of purified HA oligosaccharides. [f] Water control. The oval circle (b) indicates larger oligomers present only in HA digested with HylA+HL043PA1 supernatant. Results are representative of at least two independent experiments. [Figure 12] Hyl-A and Hyl-B protein levels in healthy and acne-associated C. acnes strains. Two acne-associated strains (HL043PA1 and HL043PA2) and two healthy-associated strains (HL110PA3 and HL110PA4) were grown for 4 days, and supernatants were analyzed by SDS-PAGE for Hyl expression, using rHylA and rHylB proteins as positive controls. Results are representative of at least two independent experiments. Yellow arrows indicate expressed HylA and HylB. [Figure 13] Enzyme activity of HylA and HylB mutants. The enzymatic activity profiles for single amino acid substitutions (point mutations) made in the HylA and HylB enzymes are shown in the graph. [Figure 14](Panels A-B) show proposed HylA residues that affect / alter HA degradation mechanism and phenotype. Structural elements of HylA / B have been proposed to be involved in domain behavior (Mello et al., 2002). Structural elements are shown in schematic representation and labeled with L (loop) and H (helix) before the number. HylA and HylB are shown in magenta and orange, respectively. The hexasaccharide (HA-6) substrate is taken from the SpHyl crystal structure (PDB: 1LOH) and is shown as yellow sticks. Key residues from loop LIV, involved in cleft gating behavior, are shown as sticks. A point mutation in one of these residues, S452G, has been observed to have a significant effect on phenotype. (B) Differences in these residues between HylA and HylB are also highlighted in the sequence alignment. HylA sequence (SEQ ID NO:7); HylA sequence (SEQ ID NO:8). [Figure 15] (Panels a-e) show HA degradation products from HA incubation with rHylA or single amino acid mutants of rHylA. [a-b] HPLC profiles of HMW HA (2 mg / ml) digested with WT (a) or mutant rHylA (b) (1 μg) for 24 hours. [c] Quantification of HA digestion peaks using known concentrations of purified HA oligosaccharides. [d, e] Undigested HA (d) and water I served as negative controls. Oval circles (a-b) indicate larger oligomers. Results are from a single experiment. Asterisks (*) represent nonspecific peaks present in the water control. [Figure 16](Panels a–e) [a, b] HaCaT cells were stimulated with HA predigested with rHylA or rHylB. IL-6 (a) and IL-8 (b) were measured in the culture supernatant. [c–d] C57Bl / 6 mouse BMDM were stimulated with HA digested with rHylA or rHylB. IL-6 (c) and TNF-α (d) were measured in the culture supernatant. [e, f] C57Bl / 6 WT, TLR2- / -, or TLR4- / - mice were infected i.d. with HL043PA1 or isogenic ΔhylA (2 × 107 CFU). Bacterial load I and TNF-α (f) were measured 24 h later. Bars represent the mean ± SD. Data are representative of at least two experiments. Bars represent the median (d, e). Each data point represents one mouse (n = 5–11 for WT, TLR4- / -, or TLR2- / - mice infected with HL043PA1; n = 4–5 for WT, TLR4- / -, or TLR2- / - mice infected with ΔhylA). Data were analyzed by one-way ANOVA with Tukey's post-hoc test (a–d) and nonparametric Mann-Whitney T-test (e, f). [Figure 17]Vaccination with rHylA protects mice from acne. [a] Schematic of immunization with rHylA followed by C. acnes challenge. [b] Serum anti-HylA and anti-HylB antibody titers after the third immunization with rHylA. [c-f] CD1 mice (n=10) immunized with alum (mock) or alum-rHylA (rHylA) were challenged i.d. with HL043PA1. Disease score I, bacterial load (d), IL-6 I, and IL-1b (f). [g] Serum anti-HylA and anti-HylB titers from CD1 mice (n=5) immunized with rHylB. [h-j] Mice were vaccinated with mock, rHylA, or rHylB and then challenged i.d. with HL110PA3. Disease score (h), bacterial load (i), and IL-1β (j) at 2 days postinfection. Bars represent median values. Data are from two independent experiments (b–f) or one experiment (g–j), and each data point represents one mouse. Data from c–f were analyzed by two-tailed, unpaired, nonparametric Mann-Whitney Student's t-test. Data from b, h–j were analyzed by one-way ANOVA with Tukey's post-hoc test. [Figure 18A] (Panels a–d) show the design of the HylA multi-epitope vaccine. [a, b] Linear B cell epitopes in the HylA protein were predicted using Bepipred Linear Epitope Prediction 2.0 (IEDB analysis resources, tools.iedb.org / bcell / ). The chart shows immunogenic peptides with a score above 0.5 (a), and the table shows a list of predicted peptides (b). [c] Alignment of four selected predicted peptides (underlined) shows no sequence homology with HylB. [d] The four predicted epitopes (underlined) are physically linked to the C-terminus (italicized) of the tetanus toxoid protein. A linker amino acid, glycine (G), was placed between each peptide and the C-terminus of tetanus toxoid. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 19] Effect of TT-mHylA vaccination on disease caused by HL043PA1 and HL110PA3 infection. [a-b] As in Extended Data Fig. 12, mice were vaccinated with either TT or TT-mHylA and challenged with HL043PA1 7 days post-vaccination. TNF-α (a) and IL-6 (b) at 48 hours post-infection. [c-f] As above, mice were vaccinated with either TT or TT-mHylA and challenged with HL110PA3 7 days post-vaccination. Disease score (c), IL-1β (d), IL-6, and TNF-α (f) at 48 hours post-infection. Bars represent median values. Data are from three independent experiments (a-b) or one experiment (c-f), and each data point represents one mouse. p values ​​in a-f were calculated by two-tailed, unpaired, nonparametric Mann-Whitney Student's t-test. [Figure 20] (Panels a-i) show the enzymatic activity of HylA mutants with single amino acid substitutions. [a] Positions of amino acid residues (shown as magenta sticks) mutated to the corresponding HylB residues in the HylA (PDB: 8FYG [www.rcsb.org / structure / unreleased / 8FYG]) crystal structure. The HA-6 ligand was obtained from the SpnHyl crystal structure (PDB: 1LOH). [b-f] HPLC profiles of HMW-HA after 24 h of co-incubation with WT or mutant HylA (0.35 μg): HA alone (b), rHylA (c), or rHylA with single amino acid substitutions (d-f). [g] HPLC profiles of HMW-HA after 24 h of co-incubation with WT rHylB (0.35 μg). [h] Quantitation of the HA digestion peak was performed using known concentrations of purified HA oligosaccharides. [i] A water-only run as a blank control. The asterisks (*) in b–i represent nonspecific peaks, also present in the water control. Data are representative of two independent experiments. [Figure 21](Panels a–h) show the pro-inflammatory properties and TLR2 dependence of Hyl degradation products. [a–c] HaCaT cells were stimulated with HA predigested with either rHylA or rHylB for 24 h, followed by measurement of IL-6 (a), IL-8 (b), and TNF-α (c) in the culture supernatant. [d] HaCaT cells were stimulated with HA predigested with either HL043PA1, HL110PA3, or the corresponding isogenic mutants for 24 h, followed by measurement of IL-6 in the culture supernatant. [e–g] ​​WT, TLR2- / -, and TLR4- / - mice were infected i.d. with the WT strain or the isogenic ΔhylA HL043PA1 strain (2 × 10 CFU) as described above. Disease score I and skin cytokines (f, g) at 24 h postinfection. [h] IL-6 in WT or TLR2- / - BMDM culture supernatants after stimulation with HA digested with rHylA or rHylB. Data in a (n = 5 for HA + rHylA and 6 for other conditions), b (n = 3 for medium and 6 for other conditions), c (n = 6), d (n = 10), and h (n = 4) are expressed as mean ± SD. Each data point represents one well. Data are representative of two independent experiments. [e-g] Bars indicate median values, and each data point represents one individual mouse (n = 5 for TLR4- / - mice, n = 8 for TLR2- / - mice, or n = 11 for WT mice infected with HL043PA1; n = 4 for TLR4- / - mice, n = 5 for TLR2- / - mice, or n = 6 for WT mice infected with isogenic ΔhylA). The p values ​​in a and b were calculated using the one-way Welch analysis of variance test, the p values ​​in c and d were calculated using the nonparametric Kruskal-Wallis one-way analysis of variance test, and the p values ​​in e to h were calculated using the nonparametric two-tailed Mann-Whitney U test. [Figure 22](Panels a-f) show that selective neutralization of HylA improves acne lesions and attenuates inflammation. [a, b] CD1 mice (n=10) immunized with alum (mock) or alum-rHylA (HylA) were challenged i.d. with HL043PA1. Disease score (a) and IL-1b in skin homogenates 2 days postchallenge. [c-e] Mice (n=15) were immunized intraperitoneally (ip) with alum and either the C-terminus of tetanus protein (TT) or multiple HylA epitopes linked to TT (mEHylA) and then challenged i.d. with the HL043PA1 C. acnes strain. Disease score (c), bacterial load (d), and IL-1b I. 2 days postchallenge. [f] Serum (1:100,000 dilution) anti-HylA or anti-HylB IgG antibody titers after the third immunization with mEHylA vaccine. Bars represent median values. Data are from two (a, b, f) or three (c–e) independent experiments, and each data point represents one mouse. Data in a–e were analyzed by the nonparametric two-tailed Mann-Whitney U test, and data in f by the nonparametric one-way Kruskal-Wallis ANOVA test. [Figure 23] (Panels a-c) show that the HylB digest analyzed by HPLC and LC-MS shows only HA-disaccharide. [a] HPLC of the HylB digest (50 μg HA + 1 μg HylB, 24 h) shows two peaks, 12.12 and 10.48, which correspond to isomeric forms of the HA-disaccharide. [b] LC-MS shows that the peak from (a) is an HA-disaccharide by m / z. [c] HPLC shows no evidence of HA-tetrasaccharide from the HylB digest. [Figure 24](Panels a-e) show the crystal structures of HylA and HylB, as well as the functional region of Hyl from C. acnes. [a-c] Crystal structures of HylA and HylB (wild-type and mutant) are shown. [d] Superposition of three crystal structures: HylA (PDB: 8FYG [www.rcsb.org / structure / unreleased / 8FYG]), and HylB (wild-type (PDB: 8FNX [www.rcsb.org / structure / unreleased / 8FNX]), and mutant (PDB: 8G0O [www.rcsb.org / structure / unreleased / 8G0O]). [d] Superposition of three crystal structures of HylA and HylB are shown. [e] The putative functional portions of the C. acnes Hyl enzyme are shown in the electrostatic surface view of HylA (APBS Electrostatics, PyMOL Molecular Graphics System, version 2.4, created by Schrodinger, LLC.). These regions, including the positively charged cleft region, aromatic / hydrophobic patch, active center, and negative patch, are all located around the substrate-binding cleft. Additionally, the substrate entry and product release ports in the cleft region are also depicted. These structural regions in homologous enzymes, including ScHyl (PDB:2X03), SpnHyl (PDB:2BRW), and SaHyl (PDB:1F1S), have been shown to be involved in substrate attraction, binding, positioning, and translocation, as well as product release. [Figure 25] (Panels a-b) show HA degradation products from HA incubation with mutant rHylA proteins. [a-b] HPLC profiles of HMW HA (2 mg / ml + 0.35 μg recombinant protein) digested with mutant N442D rHylA (a) or E346G (b) for 24 hours. Asterisks (*) in a and b represent nonspecific peaks, also present in the water control. Green circles (a) indicate larger oligomers. Data are representative of two independent experiments. [Figure 26]Domain behaviors in HylA-wt, HylB-wt, and mutant HylA are shown. The normalized amplitudes of the four domain behaviors in each model are shown. Molecular dynamics simulations using GROMACS version 2022.4 were used to calculate cleft opening and closing (Evec1), domain twisting (Evec2), substrate entry and exit (Evec3), and product exit and entry (Evec4). [Figure 27-1] (Panels a-k) show the effect of mEHylA vaccination on disease caused by HL043PA1 or HL110PA3 infection. [a-b] As in Figure 17, mice (n = 15) were vaccinated with either mock (Alum-TT) or Alum + mEHylA (mEHylA) and challenged with HL043PA1 14 days after the last vaccination. TNF-α (a) and IL-6 (b) were measured 2 days after infection. [c-f] As above, mice (n = 5) were vaccinated with either mock (Alum-TT) or Alum + mEHylA (mEHylA) and challenged with HL110PA3 14 days after the last vaccination. Disease score (c), IL-1β (d), IL-6, and TNF-α (f) were measured 2 days after infection. [g-k] CD1 mice (n = 5) were vaccinated with either mock (Alum-TT) or Alum + mEHylA (mEHylA) as described above. Then, 10 days after the last vaccination, total CD3+ T cells were isolated from the spleen and adoptively transferred into naive recipient mice, which were then challenged with HL043PA1 20 hours later. CFU (g), disease score (h), and cytokines (i-k) were measured 2 days postinfection. Bars represent median values. Data are from three independent experiments (a-b) or one experiment (c-k), and each data point represents one mouse. p values ​​in a-f were calculated by the nonparametric two-tailed Mann-Whitney U test, and in g-k, by one-way analysis of variance. [Figure 27-2] See description of Figure 27-1. [Figure 28]Panels a–f show the HylA enzyme neutralization assay. [a–e] HPLC profiles of HMW-HA (2 mg / ml) after 20 h of co-incubation with rHylA (0.3 μg) and serum (10 μl): HA standard (a), HA alone (b), a water run as a blank (c), mock serum (Alum-TT) + rHylA + HA (d), and anti-mEHylA serum + rHylA + HA. The asterisks (*) in a–e represent nonspecific peaks, also present in the water control. Pooled serum (n = 5) was used for the assay; mock serum was performed in duplicate, and anti-mEHylA serum was performed in triplicate. [f] Antibody isotype titers in sera isolated from mice vaccinated with either mock (Alum-TT) or Alum + mEHylA (mEHylA) 14 days after the last vaccination (serum diluted 1:100,000). Statistical analysis in f was performed by one-way ANOVA with Tukey's post-hoc test. Green circles represent larger oligomers. DETAILED DESCRIPTION OF THE INVENTION

[0029] Description of the Invention All documents cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides those of skill in the art with a general guide to many of the terms used in this application.

[0030] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Moreover, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0031] As used herein, the term "about," when used in conjunction with a referenced numerical indication, means the referenced numerical indication plus or minus up to 5% of the referenced numerical indication, unless otherwise specified herein. For example, the expression "about 50%" encompasses a range of 45% to 55%. In various embodiments, when specifically defined in the claims, the term "about," when used in conjunction with a referenced numerical indication, can mean the referenced numerical indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication.

[0032] The sequence identity percentage (%) with respect to reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to maximize the sequence identity percentage, and not considering any conservative substitutions as part of sequence identity.The alignment for determining amino acid sequence identity percentage can be achieved by various known methods, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequence to be compared.

[0033] As used herein, "linked" in the context of linked peptides, polypeptides, or proteins refers to being "connected," either directly or indirectly. Indirect linkages can be mediated by polypeptide linkers such as polyglycine or glycine-serine polypeptides, e.g., (GGGGS)n (SEQ ID NO:5)n or (GGGGGS)n (SEQ ID NO:6)n, where n is an integer. In various embodiments, n is an integer between 1 and 10, 10 and 20, 20 and 30, 30 and 40, or 50 and 100. Other such linkers are known in the art and are considered to be encompassed by this term.

[0034] Linear epitope scores can be determined by semi-empirical methods that utilize the physicochemical properties of amino acid residues and their frequency of occurrence in experimentally known segmental epitopes to predict antigenic determinants on proteins. This method is based on a single parameter that predicts antigenicity scores based on antigenic propensity, as described in Kolaskar and Tongaonkar (1990). AS Kolaskar and Prasad C. Tongaonkar. FEBS. 276 (12). 172-174 (1990).

[0035] Herein, we show that HylA hydrolyzes HA into large HA fragments, which contribute to robust TLR2-dependent inflammatory pathology. In contrast, HylB degrades hyaluronic acid (HA) exclusively into HA disaccharides, resulting in reduced acne immunopathology. Structural and phylogenetic studies suggest that these enzymes evolved from a common hyaluronidase and acquired divergent enzymatic properties. We demonstrated that selective inhibition of HylA by vaccination alleviates acne pathology, thus demonstrating a virulence-based approach to acne treatment. We recently addressed the growth of C. acnes in mice by applying synthetic human sebum to infected mouse skin, which allowed C. acnes to persist. Furthermore, we demonstrated that acne-associated strains exhibited significantly and uniformly enhanced virulence compared with healthy-associated strains in our model. Using this model, we set out to address the question of what genetic factors drove the divergence of C. acnes into disease- or health-associated forms.

[0036] Among the candidate factors that emerged from the comparative genomic study of health-associated versus acne-associated strains, we were particularly interested in the matrix-degrading enzyme hyaluronidase (Hyl), which generates HA fragments that mediate inflammation through TLR2 / 4 signaling, a major pro-inflammatory pathway in acne pathogenesis in mammals. Two variants of the Hyl enzyme, HylA and HylB, are expressed by C. acnes and appear to be differentially expressed by acne-associated and health-associated strains, respectively. Therefore, we aimed to gain a deeper understanding of their relationship and their contribution to the association of C. acnes with health and acne.

[0037] Our studies support Hyl as a key virulence factor explaining the divergence of healthy and acne phenotypes in C. acnes strains. This is supported by the high association of HylA and HylB with clinical disease or healthy outcomes, their TLR2 dependency in immunopathological mechanisms consistent with acne vulgaris, and the contribution of two Hyl variants to immunopathology and healthy outcomes. While our data support the virulence prominence of HylA, several other C. acnes virulence factors have been reported. These include toxic porphyrin biosynthetic genes that are upregulated by vitamin B12 supplementation and CAMP factors that recruit cytotoxic host sphingomyelinases.

[0038] Based on our phylogenetic analysis, HylA is the only pro-inflammatory Hyl produced by human commensals or pathogens. Because C. acnes is both a human commensal and a soil bacterium, its clustering among environmental microorganisms in the phylogenetic tree makes sense, likely representing a transition from soil to commensals. The relatedness of HylA and HylB to Hyls from soil-derived organisms, Streptomyces and Arthrobacter, may be consistent with this proposed transition from multifunctional lyases to more restrictive and processive enzymes. Because an inflammatory environment is usually harmful to pathogens, we expect that C. acnes Hyl would have evolved from pro-inflammatory HylA to HylB. Although this is expected, expression of HylA or HylB does not appear to alter or exert selective pressure on the survival of C. acnes in our acne model. This is consistent with the discovery of a large number of C. acnes strains expressing either HylA or HylB. Although HylA and HylB differ by 26% in their genome sequences, we show that one single amino acid substitution can significantly alter the enzyme's phenotype, suggesting that the major pathogenic potential of C. acnes may be encoded and modified simply by small changes that occur during enzyme evolution. These single amino acid substitutions were observed to be conserved among different C. acnes strains.

[0039] Based on the discovery of distinct sequences upstream and downstream of hylA and hylB, health-associated C. acnes and acne-associated C. acnes have been reported to have acquired HylB and HylA, respectively, through distinct insertion events in the indel 14 genomic region. While it is unclear how these events occurred, the structural and sequence relatedness of HylA and HylB compared to Hyls in other bacteria suggests that they likely originated within the Cutibacterium species. Until further genetic data become available, current data are most consistent with that interpretation.

[0040] Understanding the structural differences between HylA and HylB allowed us to design selective therapeutics that target pro-inflammatory enzymes. One report showed that only 4-17% of humans develop neutralizing antibodies against C. acnes Hyl, and only after early adulthood. Therefore, a vaccine approach would be of great benefit.

[0041] C. acnes hyaluronidase contributes to healthy or acneic skin To assess the potential importance of the HylA or HylB enzymes in clinical acne, we surveyed all hylA and hylB genes in NCBI and profiled their association with health- and acne-associated C. acnes strains (Table 1). C. acnes strains are classified into phylotypes with different biases for association with acne. Phylotypes IA-1, IA-2, IB-1, IB-2, and IC are associated with acne, whereas phylotypes IB-3 and II are closely associated with health (Figure 1a). Another classification based on the presence of ribotypes (RTs) demonstrates a strong association between RT2 / 6 and health, and RT4 / 5 and acne disease (Table 1). As shown in Figure 1a, hylA genes are found almost exclusively in acne-associated phylotypes, and hylB genes are found almost exclusively in health-associated phylotypes, supporting their potential contribution to acne or health.

[0042] Table 1. C. acnes phylotypes, the number and percentage of strains belonging to each phylotype, the presence of the Hyl (A or B) gene, and their association with acne or healthy skin. TIFF2026502948000035.tif51143

[0043] To directly examine the role of hylA and hylB in acne, we generated in-frame allelic exchanges of hylB and hylA in a prototypic healthy strain (HL110PA3, phylotype II, RT6) and an acne strain (H043PA1, phylotype IA-2, RT5), respectively. Based on our previous studies, these two strains represented the least acne-causing and most acne-causing strains, respectively, among the panels of healthy and acne strains, RT2 / 6 and RT4 / 5, tested in our mouse acne model. We confirmed the deletion of the Hyl gene by sequencing and the loss of Hyl activity by HA plate assay and HPLC (Figures 7a–h and 8). We applied both the wild-type and mutant strains to our mouse acne model and examined disease scores and tissue cytokines after 2 days (Figure 9a). Regardless of bacterial load (Figure 1b), hylA deletion mutants induced dramatic decreases in disease scores and pro-inflammatory cytokines compared with the parent acne-associated strain (Figure 1c-f). Conversely, hylB deletion mutants showed modest increases in disease scores and pro-inflammatory cytokines compared with the parent health-associated strain (Figure 1c-f), consistent with the interpretation that HylB has an anti-inflammatory effect. Notably, when comparing the immunopathology of acne induced by prototypic acne-associated and health-associated C. acnes strains, the differences in pathology between acne and health-associated strains were suppressed or even slightly reversed in the absence of the Hyl enzyme, pointing to the important contribution of Hyl variants to the differences between health and acne strains.

[0044] We provided corroboration of the HylA pro-inflammatory phenotype by complementing WT HylA recombinant protein (rHylA) injection with ΔhylA (Fig. 1g-i; Fig. 9b, c). Overall, our findings suggest that the two Hyl variants play a major role in promoting or modulating HylA-mediated acne immunopathology.

[0045] HylA and HylB enzymes have different HA degradation patterns and efficiencies We next sought to understand how HylA / B evolved such distinct inflammatory properties. Reports suggest that enzymes from mammals and Streptomyces hyalurolyticus produce HA fragments longer than 4-mers, which contribute to the induction of proinflammatory cytokines. More recently, we demonstrated that pathogenic microorganisms (Group B Streptococcus, S. pneumoniae, and S. aureus) produce Hyls that specifically degrade proinflammatory HA into either non-inflammatory or anti-inflammatory disaccharides (HA-2). Therefore, the enzymatic activities of HylA and HylB may lead to distinct inflammatory outcomes depending on the HA degradation products.

[0046] Therefore, we incubated supernatants from HL110PA3 or H043PA1, or recombinant HylA or HylB, with high molecular weight (HM) HA for 1 or 24 hours (Fig. 10a-e and Fig. 11a-d). HylB enzyme activity is rapid, producing primarily HA-2 throughout the reaction (Fig. 2a). Tetrasaccharide HA (HA-4) is briefly observed at 5 minutes (Fig. 10b), after which only HA-2 is detected as the degradation reaction continues.

[0047] In contrast, HylA produced distinct oligosaccharides throughout the reaction, including HA-4 and a hexasaccharide (HA-6) along with HA-2 (Figure 2b). Notably, at the end of 24 h, HA-4, H-6, and higher molecular weight HA remained. We performed extensive digestion for up to 6 days using HylA-containing supernatants and demonstrated that digestion was still incomplete. These differences in degradation rates and patterns are consistent with findings from another group on three HylA (phylotype IA)-producing strains and two HylB (phylotypes IB and II)-producing strains. They suggest a fundamental difference in the degradation mechanisms: a processive / progressive exolytic degradation mechanism for HylB, combined with a "non-processive random-bite exolytic" degradation mechanism by HylA. Although less HylA was secreted into the supernatant than HylB (Fig. 12), increasing the enzyme concentration did not overcome the difference in the endolytic versus processive mechanism (Fig. 10c-e and Fig. 11a, b).

[0048] Divergent functions of HylA and HylB promote distinct mechanisms of hyaluronan degradation Unlike human Hyl, enzymes secreted by resident or pathogenic bacteria reported to date strictly degrade HA to HA-2. One cluster of bacteria stands out in the Hyl phylogenetic tree for its ability to generate fragments larger than disaccharides. This cluster includes environmental bacteria, such as Streptomyces, which degrade HA to larger fragments. Cutibacteria, both resident and environmental bacteria, also cluster with Streptomyces, raising the question of whether the pro-inflammatory HylA may have originated from Streptomyces, while the anti-inflammatory HylB may have originated from pathogenic microorganisms such as Streptococci. HylA and HylB share 90% nucleotide sequence identity and 74% amino acid sequence identity. Further investigation of HylA / B homologous enzymes across closely related species indicates that the two enzymes likely originated within the Cutibacterium lineage.

[0049] The structures of HylA and HylB reveal high structural similarity between them and with homologous glycosaminoglycan-degrading lyases from other bacteria To understand the structural basis for the difference in the hyaluronate lyase activity of HylA and HylB, we solved the X-ray crystal structures of the HylA Y285F mutant and wild-type (WT) HylB to 2.05 Å and 2.1 Å, respectively.

[0050] HylA Y285F is a catalytically deficient form of the enzyme, and this structure is hereafter referred to as "HylA." As befits enzymes with 74% identity between each other, the structures are highly similar, overlapping with an rmsd of 0.8 Å over 751 residues (both HylA molecules in the crystallographic asymmetric unit versus HylB). In keeping with hyaluronate lyases, HylA and HylB consist of a largely α-helical N-terminal domain, a C-terminal domain containing primarily β-strands, and a catalytic site located primarily in a large cleft within the N-domain (Fig. 2c, d). The catalytic site is closely overlapping and contains many elements conserved among hyaluronate lyases, including two conserved tryptophans (Trp161 / 157 and Trp162 / 158 in HylA / B), several positively charged residues, and three residues of the catalytic triad (Asn226 / 222, His276 / 272, and Tyr285 / 281) (Fig. 2e). We further solved the structure of the catalytically deficient Y281F mutant of HylB to 2.1 Å resolution (Table 2); this structure was solved in a different space group from wild-type HylB (P41212 and one molecule for wild-type, versus P1 due to two molecules in the crystallographic asymmetric unit). Although crystallized with different crystal packing interactions, HylB Y281F is nearly conformationally identical to wild-type HylB (rmsd 0.6 Å, wild-type vs. both Y281F molecules) (Fig. 24a-e). We were unable to crystallize either HylA or HylB complexed with HA fragments; indeed, the three structures reported here show an open catalytic cleft that appears incompatible with substrate binding (Fig. 3).

[0051] (Table 2) TIFF2026502948000036.tif226166

[0052] The crystal structures of C. acnes HylA and HylB showed that they share high structural similarity with glycosaminoglycan lyases from Gram-positive bacteria, including hyaluronate lyases from Streptococcus agalactiae and Streptococcus pneumoniae, xanthan lyases from Bacillus sp. strain GL1 and Paenibacillus nanensis, and chondroitin AC lyases from Streptomyces coelicolor and Arthrobacter aurescens (Figure 3a–c). Each of these enzymes is homologous to HylA and HylB, with sequence identities ranging from 23–37% to HylA, and they are also structurally homologous with HylA, with root-mean-square deviations ranging from 2.2–3.4 Å. Furthermore, the shape of the catalytic residues conserved in these enzymes is maintained in HylA and HylB (Fig. 3d, e).

[0053] To gain further insight into the functional divergence of HylA and HylB, we identified four residue positions within the catalytic cleft that differ between HylA and HylB. Two of these residue pairs (HylA Arg397 / HylB Val393 and HylA Ser116 / HylB Glu112) are located deep within the cleft, close to the β-D-glucuronic acid moiety at the nonreducing end of the putative bound HA, and contribute to cleft binding in HylA, which displays a more positively charged surface than that of HylB (Fig. 2d). The other two pairs (HylA Asp345 / HylB Asn341 and HylA Glu346 / HylB Gly342) are located closer to the predicted location of the preceding β-D-glucuronic acid moiety.

[0054] We then examined these residue pairs by mutating HylA residues to match their HylB counterparts, and vice versa, and measured their hyaluronidase activity to determine which amino acid in each pair is preferred at that position. In this assay, cleavage of HMW-HA by hyaluronidase is recorded by monitoring UV absorbance at 232 nm, which increases due to the formation of an unsaturated carbon-carbon bond in the β-D-glucuronic acid moiety at the cleavage site.

[0055] This assay demonstrates that HylB degrades HA approximately twice as quickly as HylA, whereas control mutations of tryptophan residues in the catalytic triad (HylA / B Y285F / Y281F) significantly reduced the HA degradation activity of both enzymes (Figure 13). For this residue pair, only positions 346 / 342 of HylA / HylB showed a clear preference, with both HylA and HylB exhibiting faster enzymatic rates for glutamate than glycine. Disproportionately, however, the wild-type sequence containing glutamate at this position is not that of HylB, but rather that of HylA, the less active of the two variants; therefore, this residue is unlikely to account for part of the difference in cleavage rates between HylA and HylB.

[0056] Mutation of HylA residue Ser 452 to glycine in HylB alters the enzymatic phenotype of HylA. To recapitulate the HylB phenotype with respect to HA product size, we further mutated several residues in HylA to analogous residues in HylB. HylA S452G is located in the loop between strands β10 and β11 in the C-terminal domain (Fig. 3d, Fig. 20a); mutation of the preceding residue in the analogous loop of S. pneumoniae Hyl has been shown to alter enzyme activity. HylA S116E and E346G reside in the catalytic cleft toward the non-reducing end of the HA substrate, as described above. HylA S284G is adjacent to the catalytic Y285, and N442D is located at an exposed site within the C-terminal domain (Fig. 20).

[0057] Although S284G, S116E, and E346G of HylA had no significant effect on the product size phenotype of HylA, E346G showed a decrease in overall enzymatic activity, and N442D almost completely lost activity (Figure 20i and Figures 13 and 25). However, S452G of HylA successfully altered the enzymatic phenotype of HylA; reminiscent of HylB, S452G showed an increased enzymatic rate and a decreased amount of larger oligomers as products (Figure 20). However, the resulting product size was not HA-2 dominated, as expected for a strictly HylB-like phenotype, but rather a mixture containing a higher ratio of HA-4 to HA-2 compared to WT HylA. Interestingly, the amino acid residues S452 in HylA and G448 in HylB are conserved among C. acnes strains, suggesting that a similar hydrolysis process may be conserved.

[0058] Molecular simulations have correlated the initial domain behaviors in SpHyl, SaHyl, and ScHyl enzymes with substrate processing. To understand whether substrate processing by HylA and HylB follows a similar mechanism, we performed molecular simulations of wild-type HylA and HylB and the HylA mutants S452G and E346G, as described by Josh et al. The results of our molecular simulation studies are consistent with Josh et al.'s observations. Briefly, PCA analysis of the simulated trajectories suggested that HylB-WT was much more dynamic than HylA-WT, and the S452G mutation in HylA exhibited an increase in domain behavior (amplitude) similar to that of HylB-WT. The cleft opening and closing behavior (eigenvector 1) increased by approximately 20%, while other domain behaviors increased by 10–40% (Figure 26). These observations are consistent with the hypothesis that complex structural dynamics is one of the key mechanisms for substrate processing by HylA and HylB.

[0059] Furthermore, the overall three-dimensional structures of HylA and HylB are nearly identical (Figure 2c); the fold consists of an N-terminal α-domain and a C-terminal β-domain connected by a 12-residue linker. The substrate-binding cleft in both enzymes contains a highly conserved catalytic site (Figure 2d and Table 3) and is decorated with charged residues. The majority of differences are observed near the substrate-binding region (Table 4). Comparing the substrate-binding domains with other Hyls from Streptomyces and Streptococcus species suggests that the positive patch in HylA / B is located toward the non-reducing end of the substrate, while the negative patch is located toward the reducing end, suggesting that these are involved in substrate binding and product release, respectively, with fewer aromatic residues forming an aromatic / hydrophobic patch implicated in substrate positioning for catalysis (Figure 2e and Table 3).

[0060] Table 3. Active site cleft components at the residue level TIFF2026502948000037.tif94161

[0061] Table 4. Major structural differences between HylA and HylB and key residues around the cleft. TIFF2026502948000038.tif201152

[0062] Comparison of the HylA / B structure with other bacterial Hyl structures further supports the divergence of HylA / B. We were unable to obtain crystals of HylA / B complexed with HA fragments. To gain further insight into the functional divergence of HylA / B, we compared the structural features of HylA / B with other bacterial Hyl structures (Figure 3a–d) and chondroitinases from Streptococcus and Streptomyces species. Based on previous studies, it has been proposed that Hyl evolved from existing chondroitinases. As expected, since Hyl enzymes evolved to recognize and process different substrates, including HA, HylA / B share overall structural similarity with chondroitin AC lyase from Arthrobacter aurescens (ArthroAC; PDB: 1RW9) and Hyl enzymes from other bacteria (Table 5). This observation suggests that HylA / B originated from a common enzyme and functionally diverged.

[0063] Table 5. Alignment of hyalase crystal structures and sequences from different bacterial species. TIFF2026502948000039.tif78166

[0064] The end-product profile of HylB (producing only HA-2) is similar to that of HA degradation by Streptococcus pneumoniae Hyl (SpHyl), Streptococcus agalactiae Hyl (SaHyl), and Streptomyces coelicolor Hyl (ScHyl), whereas the end-product from HylA contains both larger fragments and disaccharides. Mechanistically, HA degradation by chondroitinases and Hyl enzymes follows two distinct mechanisms. Based on the product profile, HylB utilizes a processive / processive exolytic cleavage mechanism. We suggest that HylA follows a two-step process that initially involves nonprocessive random-bite endolytic cleavage followed by the adoption of exolytic functionality. The observation that the cleft of HylA is more open than that of HylB (Tables 6 and 7) correlates with the above speculation of a different mechanism for their HA degradation. Thus, combined, these data suggest that HylA and HylB have functionally diverged by switching from endolytic to exolytic processing for efficient degradation of HWA-HA concomitantly with different biological effects on the host.

[0065] Table 6. Comparison of cleft conformations among bacterial Hyl TIFF2026502948000040.tif89161

[0066] Table 7. The closest distances around the active site cleft, indicating the degree of opening of the active site cleft. TIFF2026502948000041.tif121165

[0067] Proposed HylA / B degradation mechanism and its relationship to the location of amino acid residue changes To understand the structural basis for the differences in HA degradation associated with the amino acid changes in HylA / B, based on previous studies, we classified residues into three groups: (1) residues involved in basic catalysis [residues forming the active center], (2) residues involved in substrate binding / positioning and product release [residues forming the positive cleft, aromatic patch, and negative patch], and (3) residues involved in regulating substrate entry and translocation / sliding [residues involved in domain behavior and structural flexibility]. We assessed some of these residues for their activity by point mutation (Figure 13).

[0068] Because group 1 residues are highly conserved in HylA / B and involved in basic catalysis, we selected residues surrounding them, including 116S / 112E and 284S / 280G (HylA / B numbering), among few other residues. From group 2, significant differences were observed in residues forming the positive patch (charged cleft), including 346E / 342G and 397R / 393V, among few other residues. In addition to the above differences, interdomain behavior in SpHyl, SaHyl, and ScHyl enzymes has been implicated in substrate processing. Based on this idea, we identified key differences in HylA / B, including the loops and helices associated with these functions (Fig. 3c and Fig. S14a), as well as residues 346E / 342G, 394A / 390S, 395S / 391T, 442N / 438D, and 452S / 448G (Fig. S14b). These differences crucially alter the HA degradation mechanism, as residues involved in domain movement and structural flexibility can regulate substrate entry and translocation / sliding during subsequent catalytic cycles of processive degradation of polymeric / oligomeric HA substrates. Interdomain movements in HylA may allow the enzyme to initially engage in endolytic activity and gradually switch to exolytic activity depending on the size of the available substrate.

[0069] A single amino acid, serine 452, located outside the substrate-binding domain, diverges the enzyme mechanism Based on the potential influence of specific HylA / B residues on HA degradation, we performed point mutations on several of the residues in HylA (Fig. 4a-h and Fig. S14b).

[0070] Notably, the HylA mutation S452G significantly reversed the enzyme phenotype to HylB (Fig. 4g). The mutant protein digested the HMW-HA substrate to HA-2, with trace amounts of HA-4 remaining (phenocopying WT HylB). Other point mutations, particularly 442N, accelerated the digestion of HA to HA-2 and reduced the amount of undigested HA (Fig. 4h and Fig. S15).

[0071] Structurally, residues 452S and 442N are located in loop LIV from the β domain (Figure 14a). Changing this position reversed the phenotype, likely by affecting substrate localization and enzymatic activity. Joshi et al. compared the role of interdomain behavior in other Hyl homologs. Briefly, their analysis showed that in SpHylA, the corresponding residue (580N) of 451N (HylA) (the preceding residue of 452S (HylA)) is involved in substrate binding / translocation, and the loop containing this residue (βLIII in SpHyl and LIV in Hyl) controls substrate entry. Mutation of 580N to glycine in SpHyl also altered its enzymatic activity by further opening the cleft (domain behavior (i)). Therefore, replacing Ser with Gly at this position in HylA likely confers greater flexibility to loop LIV, resulting in greater domain behavior. Notably, the exolytics ScHyl (PDB: 2WCO) and ArthroAC (PDB: 1RW9) have a glycine at this position, suggesting that HylB may have acquired an efficient way to process HA through interdomain behavior.

[0072] These observations suggest that HylA may have diverged into the more efficient HylB by regulating substrate entry, binding, and translocation / sliding through interdomain behavior.

[0073] Pro-inflammatory properties of Hyl degradation products Having defined the structure and enzymatic function of C. acnes Hyl, we sought to determine whether HA degradation products from HylA and HylB could cause the inflammatory pathology noted in in vivo experiments. For these assays, we digested HA with rHyl (Fig. 16a–d) or supernatant from WT / ΔhylA (Fig. 5a) for 24 h and measured cell-specific cytokine secretion responses. We showed that HylA induced higher levels of acne-associated cytokines than controls in keratinocytes (Fig. 5a, Fig. 16a,b) and macrophage cell lines (Extended Data Fig. 16c,d), regardless of whether we used supernatant or higher concentrations of rHylB. In comparison, degradation by HylB reduced or completely altered other cytokine levels compared to controls (IL-8). This is consistent with the anti-inflammatory properties of the previously defined processive HA-2-producing Hyl enzyme. As reported, HA-2 lacks pro-inflammatory properties, and degradation of HA to HA-2 abrogates the pro-inflammatory properties of larger HA fragments. Furthermore, HA-2 competes with larger sized HA to further block TLR2 activation.

[0074] Because TLR2 dependency of acne vulgaris is a well-known feature of skin disease, we investigated the TLR2 dependency of inflammation induced by HylA / B. Consistent with the TLR2 dependency of HA (Fig. 5b-e), the differences in pathology induced by HL043PA1 and ΔhylA were due to the TLR2 dependency. - / - It disappeared in mice, but TLR4 - / - It did not disappear in mice.

[0075] Finally, we sought to determine how single amino acid substitutions that altered or reversed HylA degradation affected inflammation. As shown in Figure 5f, and consistent with the product of HylA degradation, a single amino acid substitution (S452G) that restored or accelerated HA processing also resulted in a reversal of the inflammatory phenotype. We also observed a reversal of inflammation by several other HylA single amino acid mutants, which correlated with the higher production of HA-2 and lower amounts of undigested larger oligos compared to WT rHylA.

[0076] Overall, our findings are consistent with the generation of distinct degradation product sizes by Hyl, which result in different inflammatory outcomes. The potential importance of Hyl in humans is furthered by linking the Hyl mechanism to acne through their TLR2 dependency.

[0077] Targeting Hyl to treat acne disease Above, we demonstrated in our mouse model that HylA plays a key role in the immunopathology of acne. HylA is highly conserved across C. acnes phylotypes, with consistent enzymatic activity demonstrated. Therefore, it is an excellent target for therapeutic intervention. Alternatively, the significant homology between HylA and HylB poses a potential challenge for therapeutic selectivity.

[0078] First, we tested whether immunization against HylA confers protection against acne. We injected mice three times at weekly intervals and then challenged them with HL043PA1 (Figure 17a). Vaccination with HylA in alum induced robust antibody responses to HylA (Figure 17b) and significantly reduced immunopathology associated with murine acne disease (Figures 6a, b and 17c-d). However, immunization with HylA (Figure 17b) or HylB (Figure 17e) induced cross-reactive antibodies and induced a modest worsening of disease scores when mice were challenged with the health-related HL110PA3 strain (Figures 17f-h).

[0079] To avoid the potential for inducing inflammation associated with cross-reactive antibodies, we designed a HylA-specific peptide vaccine using an antigenicity program (IEDB analysis resource, tools.iedb.org / bcell / ) (Fig. 18a-d) in combination with the crystal structures of HylA and HylB. This peptide combines several HylA-specific epitopes and has no significant homology to the human protein. We physically linked the predicted peptide to tetanus toxoid (TT), expressed the fusion protein in Escherichia coli (E. coli), and then validated the construct by mass spectrometry. This vaccine selectively inhibited acne caused by HL043PA1 (Fig. 22c-e), with minimal evidence of inflammation from cross-reactivity with HylB derived from HL110PA3 (Fig. 22f and Fig. 27a-f). In adoptive T cell transfer experiments, CD3 + T cells were shown to be dispensable for protection against acne disease (Fig. 27g-k). Next, we examined the effect of serum from mEHylA vaccination on the enzymatic activity of rHylA. Post-vaccination serum significantly reduced HA degradation by the HylA enzyme (Fig. 28a-e). Notably, mEhylA generated predominant IgG1 anti-HylA antibodies (Fig. 28f).

[0080] Accordingly, various aspects of the present invention are based, at least in part, on these findings.

[0081] Polypeptides Various embodiments of the present invention provide polypeptides comprising a fragment of HylA. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues in length. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0082] Various embodiments of the present invention provide a polypeptide comprising a fragment of HylA linked or fused to an adjuvant. In various embodiments, the adjuvant is a polypeptide adjuvant. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues in length. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0083] In various embodiments, the fragment of HylA is: (a) TIFF2026502948000042.tif4128; (b) TIFF2026502948000043.tif4128 variant, TIFF2026502948000044.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000045.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000046.tif4128; (d) TIFF2026502948000047.tif4128 variant, TIFF2026502948000048.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000049.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000050.tif4128; (f) TIFF2026502948000051.tif4128 variant, TIFF2026502948000052.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000053.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000054.tif4128; or (h) TIFF2026502948000055.tif4128 variant, TIFF2026502948000056.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000057.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The compound comprises one or more peptides selected from:

[0084] In various embodiments, TIFF2026502948000058.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000059.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0085] In various embodiments, TIFF2026502948000060.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000061.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0086] In various embodiments, TIFF2026502948000062.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000063.tif4128 Variants have up to two or one amino acid addition, substitution, or deletion.

[0087] In various embodiments, TIFF2026502948000064.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000065.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0088] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0089] In various embodiments, the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0090] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker can be a peptide linker.

[0091] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0092] In various embodiments, the linker is G, polyserine, polyglycine, TIFF2026502948000066.tif4128, leucine zipper, or aliphatic.

[0093] Various aspects of the present invention include: (a) TIFF2026502948000067.tif4128; (b) TIFF2026502948000068.tif4128 variant, TIFF2026502948000069.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000070.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000071.tif4128; (d) TIFF2026502948000072.tif4128 variant, TIFF2026502948000073.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000074.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000075.tif4128; (f) TIFF2026502948000076.tif4128 variant, TIFF2026502948000077.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000078.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000079.tif4128; or (h) TIFF2026502948000080.tif4128 variant, TIFF2026502948000081.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000082.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The present invention provides a polypeptide comprising one or more peptides selected from:

[0094] In various embodiments, TIFF2026502948000083.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000084.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0095] In various embodiments, TIFF2026502948000085.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000086.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0096] In various embodiments, TIFF2026502948000087.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000088.tif4128 Variants have up to two or one amino acid addition, substitution, or deletion.

[0097] In various embodiments, TIFF2026502948000089.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000090.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0098] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0099] In various embodiments, the one or more peptides are linked or fused to an adjuvant, hi various embodiments, the adjuvant is a polypeptide adjuvant.

[0100] In various embodiments, the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0101] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker can be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the N-terminus or C-terminus of the adjuvant.

[0102] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0103] In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, TIFF2026502948000091.tif4128, leucine zipper, r aliphatic, or helical peptide.

[0104] Various aspects provide a composition comprising a polypeptide and an adjuvant.

[0105] In various embodiments, the polypeptide is a fragment of HylA. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues in length. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0106] In various embodiments, the polypeptide is: (a) TIFF2026502948000092.tif4128; (b) TIFF2026502948000093.tif4128 variant, TIFF2026502948000094.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000095.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000096.tif4128; (d) TIFF2026502948000097.tif4128 variant, TIFF2026502948000098.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000099.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000100.tif4128; (f) TIFF2026502948000101.tif4128 variant, TIFF2026502948000102.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000103.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000104.tif4128; or (h) TIFF2026502948000105.tif4128 variant, TIFF2026502948000106.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000107.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The compound comprises one or more peptides selected from:

[0107] In various embodiments, TIFF2026502948000108.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000109.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0108] In various embodiments, TIFF2026502948000110.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000111.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0109] In various embodiments, TIFF2026502948000112.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000113.tif4128 Variants have up to two or one amino acid addition, substitution, or deletion.

[0110] In various embodiments, TIFF2026502948000114.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000115.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0111] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0112] In various embodiments, the adjuvant is alum, hydroxyphosphate sulfate, CpG1018, monophosphoryl lipid A, an oil-in-water emulsion, CpG, or QS-21 saponin.

[0113] In various embodiments, the polypeptide further comprises 1 to 10 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1 to 5 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1 to 3 amino acid residues at the N-terminus, C-terminus, or both.

[0114] In various embodiments, the polypeptides include L- or D-amino acids and / or equivalent unnatural amino acids.

[0115] In various embodiments, the polypeptide comprises α-amino acids or β-amino acids.

[0116] In various embodiments, the polypeptide comprises a non-hydrolyzable bond.

[0117] Various embodiments provide mRNA encoding a polypeptide of the invention. Embodiments also provide mRNA compositions for eliciting an immune response. The mRNA compositions (e.g., immune compositions, vaccines) function by introducing a portion of an mRNA that encodes a polypeptide. This mRNA enables cells to produce the polypeptide. As part of a normal immune response, the immune system recognizes the polypeptide as foreign and produces antibodies.

[0118] Briefly, in vitro transcribed mRNA can be generated from a linear DNA template using T7, T3, or Sp6 phage RNA polymerase. The resulting product should optimally contain an open reading frame encoding the protein of interest, flanking UTRs, a 5' cap, and a poly(A) tail. Thus, the mRNA is engineered to resemble a fully processed mature mRNA molecule as naturally present in the cytoplasm of eukaryotic cells.

[0119] In vitro and in vivo transfection reagents have been developed that facilitate cellular uptake of mRNA and protect it from degradation. Once mRNA enters the cytosol, the cellular translational machinery produces proteins that undergo post-translational modifications to produce properly folded, fully functional proteins or polypeptides. This feature of mRNA pharmacology is particularly advantageous for vaccines and protein replacement therapies, where cytosolic or transmembrane proteins must be delivered to the correct cellular compartment for proper presentation or function.

[0120] Various aspects of the present invention provide pharmaceutical compositions comprising any one of the immunogenic peptides of the present invention described herein or mRNA encoding the immunogenic peptides of the present invention described herein. In various aspects, the composition is in the form of nanoparticles.

[0121] The pharmaceutical composition according to the present invention may also contain any pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of the formulation. This further means that it must be suitable for use in contact with any tissue or organ with which it may come into contact, and must not be associated with the risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs the therapeutic benefit.

[0122] Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the composition or to facilitate the preparation of the composition.Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol and water.Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.Carriers can also include sustained-release materials such as glycerin monostearate or glyceryl distearate alone or with wax.

[0123] The pharmaceutical composition according to the present invention can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that will produce the most effective results in terms of therapeutic efficacy in a given subject. This amount can vary depending on various factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dosage, and type of administration), the nature of the pharmaceutically acceptable carrier(s) in the formulation, and the route of administration. Those skilled in the clinical and pharmacological fields will be able to determine a therapeutically effective amount through routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0124] Typical dosages of effective amounts can be determined by in vitro responses or animal model responses as indicated by those skilled in the art. Such dosages can usually be reduced in concentration or amount by up to about one order of magnitude without losing relevant biological activity. Therefore, the actual dosage depends on the physician's judgment, the patient's condition, and the effectiveness of the treatment method, based on, for example, the in vitro responses of relevant primary cultured cells or tissue culture samples, and the responses observed in appropriate animal models, as previously described.

[0125] In various embodiments, pharmaceutical compositions according to the present invention may be formulated for delivery via any route of administration, which may refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, transmucosal, transdermal, or parenteral.

[0126] "Transdermal" administration can be accomplished using topical creams or ointments or by means of a transdermal patch.

[0127] "Parenteral" generally refers to an administration route related to injection, including intraorbital, infusion, intraarterial, intraarticular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In parenteral routes, the composition can be in the form of a solution or suspension for infusion or injection, or in the form of a lyophilized powder. In parenteral routes, the composition can be in the form of microspheres or nanospheres, or lipid vesicles or polymer vesicles that allow controlled release.

[0128] For the enteral route, the pharmaceutical composition may be in the form of tablets, gel capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres allowing controlled release, or lipid or polymer vesicles.

[0129] For topical administration, pharmaceutical compositions based on the compounds according to the present invention can be formulated to treat the skin and mucous membranes and are in the form of ointments, creams, emulsions, salves, powders, impregnated pads, solutions, gels, sprays, lotions, or suspensions. They can also be in the form of microspheres or nanospheres, lipid or polymer vesicles, or polymer patches and hydrogels, which allow controlled release. These compositions for topical administration can be in either anhydrous or aqueous form, depending on the clinical indication.

[0130] Furthermore, the peptides of the present invention can be linked to nanoadjuvants.For example, the peptides of the present invention can be linked to polymersomes (polymer vesicles self-assembled from a diverse array of synthetic amphiphilic block copolymers containing hydrophilic and hydrophobic blocks), as described in Levine et al., Methods 46 (2008) 25-32.The peptides of the present invention can also be linked to nanoparticles, such as albumin, liposomes, polymers, gold nanoparticles, and iron oxide nanoparticles, such as those described in Mu et al., Nanoscale. 2015 November 21; 7(43): 18010-18014.These publications are incorporated herein by reference as if fully set forth herein.

[0131] kit The present invention also relates to a kit for treating or reducing the likelihood of acne, or inhibiting C. acnes hyaluronidase. The kit is useful for carrying out the method of the invention for treating or reducing the likelihood of acne, or inhibiting C. acnes hyaluronidase. The kit is a collection of materials or components, including at least one of the polypeptides or compositions of the invention. Thus, in some embodiments, the kit includes a composition comprising any one or more of the immunogenic peptides of the invention described above.

[0132] The exact nature of the components disposed in an inventive kit will depend on its intended purpose. In various embodiments, the kit is specifically configured for treating mammalian subjects. In various embodiments, the kit is specifically configured for treating human subjects.

[0133] The kit may include instructions for use. "Instructions for use" typically include a substantive description of the techniques to be employed when using the components of the kit to achieve a desired result, such as treating or reducing the likelihood of acne, or inhibiting C. acnes hyaluronidase. Optionally, the kit may also include other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring devices, bandages, or other useful tools that will be readily recognized by those skilled in the art.

[0134] The materials or components assembled in the kit can be stored and provided to the practitioner in any convenient and appropriate manner that maintains their operability and usability. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated, or frozen temperatures. The components are typically housed in suitable packaging. As used herein, the phrase "packaging" refers to one or more physical structures used to contain the kit contents, such as the inventive compositions. The packaging is preferably constructed by well-known methods to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material, such as glass, plastic, paper, or foil, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain an appropriate amount of the inventive composition, including any one or more of the immunogenic peptides of the invention described herein. The packaging typically has an exterior label indicating the contents and / or purpose of the kit and / or its components.

[0135] method Various aspects provide methods of treating acne comprising administering to a subject in need thereof a polypeptide of the invention as described herein or a composition of the invention as described herein.

[0136] Various aspects provide methods of reducing the likelihood of acne comprising administering to a subject in need thereof a polypeptide of the invention as described herein or a composition of the invention as described herein.

[0137] Various embodiments provide methods of eliciting an immune response in a subject in need thereof, comprising administering to the subject a polypeptide of the invention described herein or a composition of the invention described herein. In various embodiments, the immune response is a protective immune response.

[0138] Various embodiments provide methods of treating acne comprising administering to a subject in need thereof a composition comprising an mRNA molecule encoding a polypeptide of the invention as described herein.Various embodiments provide methods of reducing the likelihood of acne comprising administering to a subject in need thereof a composition comprising an mRNA molecule encoding a polypeptide of the invention as described herein.

[0139] Various embodiments provide methods of eliciting an immune response in a subject in need thereof, comprising administering to the subject a composition comprising an mRNA molecule encoding a polypeptide of the invention. In various embodiments, the immune response is a protective immune response.

[0140] As such, in various embodiments, the polypeptide comprises a fragment of HylA. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 10-14, 15-20, 21-25, 26-30, or 31-35 amino acid residues. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0141] Also as such, in various embodiments, the polypeptide comprises a fragment of HylA linked or fused to an adjuvant. In various embodiments, the adjuvant is a polypeptide adjuvant. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 10-14, 15-20, 21-25, 26-30, or 31-35 amino acid residues. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0142] In various embodiments, the fragment of HylA is: (a) TIFF2026502948000116.tif4128; (b) TIFF2026502948000117.tif4128 variant, TIFF2026502948000118.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000119.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000120.tif4128; (d) TIFF2026502948000121.tif4128 variant, TIFF2026502948000122.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000123.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000124.tif4128; (f) TIFF2026502948000125.tif4128 variant, TIFF2026502948000126.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000127.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000128.tif4128; or (h) TIFF2026502948000129.tif4128 variant, TIFF2026502948000130.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000131.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The compound comprises one or more peptides selected from:

[0143] In various embodiments, TIFF2026502948000132.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000133.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0144] In various embodiments, TIFF2026502948000134.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000135.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0145] In various embodiments, TIFF2026502948000136.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000137.tif4128 Variants have up to two or one amino acid addition, substitution, or deletion.

[0146] In various embodiments, TIFF2026502948000138.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000139.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0147] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0148] In various embodiments, the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0149] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker can be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the N-terminus or C-terminus of the adjuvant.

[0150] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0151] In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, TIFF2026502948000140.tif4128, leucine zipper, r aliphatic, or helical peptides.

[0152] As such, in other aspects, the polypeptide used in these methods is: (a) TIFF2026502948000141.tif4128; (b) TIFF2026502948000142.tif4128 variant, TIFF2026502948000143.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000144.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000145.tif4128; (d) TIFF2026502948000146.tif4128 variant, TIFF2026502948000147.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000148.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000149.tif4128; (f) TIFF2026502948000150.tif4128 variant, TIFF2026502948000151.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000152.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000153.tif4128; or (h) TIFF2026502948000154.tif4128 variant, TIFF2026502948000155.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000156.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The compound comprises one or more peptides selected from:

[0153] In various embodiments, TIFF2026502948000157.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000158.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0154] In various embodiments, TIFF2026502948000159.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000160.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0155] In various embodiments, TIFF2026502948000161.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000162.tif4128 Variants have up to two or one amino acid addition, substitution, or deletion.

[0156] In various embodiments, TIFF2026502948000163.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000164.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0157] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0158] In various embodiments, the one or more peptides are linked or fused to an adjuvant, hi various embodiments, the adjuvant is a polypeptide adjuvant.

[0159] In various embodiments, the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0160] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker can be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the N-terminus or C-terminus of the adjuvant.

[0161] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0162] In various embodiments, the linker is G, polyserine, polyglycine, glycine-serine, TIFF2026502948000165.tif4128, leucine zipper, r aliphatic, or helical peptide.

[0163] As such, in yet other embodiments, the compositions used in these methods comprise a polypeptide and an adjuvant.

[0164] In various embodiments, the polypeptide is a fragment of HylA. In various embodiments, the fragment of HylA is about 12-29 amino acid residues. In various embodiments, the fragment of HylA is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues in length. In various embodiments, the fragment of HylA has the amino acid sequence of any one of SEQ ID NOs: 9-29 and 38.

[0165] In various embodiments, the polypeptide is: (a) TIFF2026502948000166.tif4128; (b) TIFF2026502948000167.tif4128 variant, TIFF2026502948000168.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000169.tif4128, or has up to 7 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (c) TIFF2026502948000170.tif4128; (d) TIFF2026502948000171.tif4128 variant, TIFF2026502948000172.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000173.tif4128, or has up to 17 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (e) TIFF2026502948000174.tif4128; (f) TIFF2026502948000175.tif4128 variant, TIFF2026502948000176.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000177.tif4128, or has up to 6 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof; (g) TIFF2026502948000178.tif4128; or (h) TIFF2026502948000179.tif4128 variant, TIFF2026502948000180.tif4128 variant, the variant has at least 50% sequence identity to TIFF2026502948000181.tif4128, or has up to 10 amino acid additions, substitutions, or deletions, or has a linear epitope score of at least 0.4, or a combination thereof. The compound comprises one or more peptides selected from:

[0166] In various embodiments, TIFF2026502948000182.tif4128 variants have up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000183.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0167] In various embodiments, TIFF2026502948000184.tif4128 variants have up to 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000185.tif4128 variants have up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0168] In various embodiments, TIFF2026502948000186.tif4128 variants have up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000187.tif4128 variants have up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0169] In various embodiments, TIFF2026502948000188.tif4128 variants have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. TIFF2026502948000189.tif4128 variants have up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0170] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 to the reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 to the reference polypeptide.

[0171] In various embodiments, the adjuvant is alum, hydroxyphosphate sulfate, CpG1018, monophosphoryl lipid A, an oil-in-water emulsion, CpG, or QS-21 saponin.

[0172] In various embodiments, the polypeptide further comprises 1 to 10 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1 to 5 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1 to 3 amino acid residues at the N-terminus, C-terminus, or both.

[0173] In various embodiments, the polypeptides include L- or D-amino acids and / or equivalent unnatural amino acids.

[0174] In various embodiments, the polypeptide comprises α-amino acids or β-amino acids.

[0175] In various embodiments, the polypeptide comprises a non-hydrolyzable bond. [Example]

[0176] The following examples are provided to better illustrate the claimed invention and are not to be construed as limiting the scope of the invention. To the extent that specific elements are referred to, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art will be able to develop equivalent means or reactants without the use of inventive capacity and without departing from the scope of the invention.

[0177] Example 1 C. acnes bacterial culture Two acne-associated strains (HL043PA1 and HL043PA2) and two health-associated strains (HL110PA3 and HL110PA4) were used in this study. Clinical C. acnes strains from frozen stocks were anaerobically cultured on blood agar plates for 96 hours at 37°C using a BD BBL™ GasPak™ system. A single colony of C. acnes was grown anaerobically in 10 ml of brain heart infusion (BHI) broth (catalog number #53286, Sigma-Aldrich, USA) for 3-4 days (OD = 0.15-0.3), and the bacterial pellet was then washed once with BHI medium at 2300 × g for 5 minutes. The pellet was then cultured at the desired OD for in vitro and in vivo testing. 600nm The bacterial culture supernatant was collected and used for roostercomb HA (catalog number H5388, Sigma-Aldrich, USA) degradation activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and stimulation of human keratinocyte HaCaT cells (ATCC).

[0178] Construction of ΔhylA and ΔhylB C. acnes strains The homologous recombination cloning strategy employed was slightly modified from that previously described (Sorensen et al., 2010). Briefly, 1 kb (500 bp) upstream and downstream regions of the hyaluronidase gene were amplified by PCR, gel-purified, ligated together, and cloned into pGEM-T-easy (catalog #A137A, Promega, USA). The erythromycin resistance cassette from pDCerm was PCR-amplified and ligated between the upstream and downstream regions, followed by transformation into E. coli (DH5α) (catalog #18265017, ThermoFisher Scientific). Plasmid DNA from ampicillin (100 μg / mL)-resistant clones was purified and verified by PCR. Correct plasmids were transformed into dam-negative E. coli (catalog #C2925I, New England Biolabs) and purified. Competent C. acnes cells were prepared as previously described (Cheong et al., 2008). Briefly, C. acnes was cultured at an OD of 0.5–0.6. 600nm The cells were grown anaerobically in BHI medium at 37°C until viable. The cells were pelleted and washed twice in EP buffer (272 mM sucrose, 7 mM sodium phosphate, and 1 mM magnesium chloride). The plasmid DNA was mixed with freshly prepared electrocompetent C. acnes cells and electroporated. Immediately after electroporation, 1 mL of BHI was added. The cells were pelleted, resuspended in 100 μl of BHI medium, plated on BHI agar plates, and incubated anaerobically at 37°C overnight. The next day, bacteria were swabbed, placed in fresh BHI medium, plated on BHI plates containing erythromycin (10 μg / ml) (catalog number #E5389, Sigma-Aldrich), and incubated at 37°C until colonies appeared (5–7 days). The mutants were verified by PCR to confirm lack of activity on hyaluronan-containing agar plates.

[0179] Hyaluronidase plate assay The hyaluronate lyase activity of HylA or HylB in C. acnes culture supernatants was measured using roostercomb-derived hyaluronan as a substrate. 20 μL or 40 μL of supernatant from a single C. acnes colony grown anaerobically for 4 days and harvested at 2600 × g for 10 min was spotted onto BHI agar plates containing 1% bovine serum albumin (BSA) Fraction V (Cat. No. 10735078001, Sigma-Aldrich, USA) and hyaluronan (400 μg / mL). Plates were incubated overnight at 37°C, and hyaluronan degradation was detected by flushing the plates with 2N acetic acid for 3–5 min.

[0180] cell culture Bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibias of 12-week-old C57BL / 6 mice (Jackson Laboratories) and suspended in complete RPMI 1640 medium (Gibco, ThermoFisher Scientific, USA) with 10% heat-inactivated fetal bovine serum (FBS), 10 ng / ml M-CSF (PeproTech, Inc., USA), and 1% penicillin-streptomycin antibiotic (P4333, Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in 92-mm nonadherent dishes (ThermoFisher Scientific, USA) at 37°C under 5% CO2, and the medium was replaced with fresh medium containing an equal concentration of M-CSF every 2 days. Cells were then harvested after 7 days of culture and stimulated with bacterial supernatant or HA (40 μg) digested with either rHylA or rHylB enzymes.

[0181] HaCaT cells (ATCC) were cultured in complete DMEM medium (Cat. No. 10-013-CV, Corning Incorporated, USA) supplemented with 10% heat-inactivated FBS at 37°C in 5% CO. Prior to cell stimulation with digested HA (40 μg), HaCaT cells were plated at 10 in a 96-well Falcon® tissue culture plate (Cat. No. 353072, Corning Incorporated, USA). 5 The cells were seeded at a concentration of 1000 cells / ml and incubated at 37°C in 5% CO2 for 6 hours, then washed with DMEM medium and stimulated.

[0182] HA digestion for HPLC analysis and cell culture stimulation HA from chicken comb (2 mg / ml) was digested with either supernatant from C. acnes bacterial culture (10 μl / ml) or 1 μg of purified recombinant protein (rHylA, rHylB, or mutant protein) at a concentration of 0.35 or 1 μl / ml. Digestion was carried out at 37°C for various time points (0, 5, 15 min, 1 h, and 24 h) in a reaction buffer containing 100 mM Na acetate, 10 mM CaCl, and 0.5 mM DTT (pH = 5.5). The reaction was stopped by inactivating the enzyme at 80°C for 10 min and then stored at -20°C until further use. The supernatant from the bacterial culture used for HA digestion was concentrated 20-fold using a 50 kDa Amicon® Ultra-15 centrifugal filter (catalog number #UFC905024, Millipore Sigma, USA).

[0183] For BMDM and HaCaT cell assays, the equivalent of 40 μg of HA digest was used, with 10 5Cells were stimulated for 8 and 16 hours, respectively. Cells were plated in Falcon® 96-well tissue culture plates and cultured in 200 μl of complete RPMI medium supplemented with 10% FBS and 1x penicillin-streptomycin antibiotic solution. After incubation at 37°C under 5% CO2 for 8 or 16 hours (or 24 hours), the cells were centrifuged at 400 × g, and the culture supernatants were collected for analysis of pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, and IL-8, by solid-phase sandwich enzyme-linked immunosorbent assay (ELISA; Biolegend, CA, USA).

[0184] The HA digestion products were analyzed by strong anion-exchange high-performance liquid chromatography (HPLC), which was performed at 30°C using an Ultimate 3000 HPLC system (ThermoScientific, USA) equipped with an Ultimate 3000 variable wavelength detector connected to a Pro Pack SAX-10 (4 × 250 mm) column attached to a Pack SAX-10G guard column (4 × 50 mm, Thermo-Dionex, USA). Two different solvents were used: solvent A (HPLC-water, pH 3.5) and solvent B (2 M NaCl, pH 3.5) at a flow rate of 1 mL / min. The gradient conditions (linear) are listed in the table below. TIFF2026502948000190.tif34128

[0185] Chromatograms were acquired with UV absorbance set at 232 nm. Known amounts of samples were dissolved in UP water and injected into the HPLC. 1 μg each of a standard mixture of HA-DP2, HA-DP4, and HA-DP6 was injected, and the HA oligosaccharides in the samples were quantified by comparing the area under the peak with that of the standard mixture.

[0186] phylogenetic analysis FASTA amino acid sequences of HylA and HylB obtained from the NCBI or RCSB Protein Data Bank were used for phylogenetic analysis. Sequences were aligned using Clustal, and then a neighbor-joining phylogenetic tree was generated using Geneious Prime.

[0187] Expression and purification of recombinant enzymes C. acnes HylB (residues 37–801) and HylA (residues 41–805) were cloned into pET His6 TEV LIC (catalog no. #29653, Addgene) and pET His6 MBP TEV LIC (catalog no. #29656, Addgene) cloning vectors, respectively, and propagated in Escherichia coli Top10 cells (catalog no. #C404010, ThermoFisher Scientific, USA). The recombinant plasmids were transformed into E. coli BL21(DE3)pLysS cells (catalog no. #C606010, ThermoFisher Scientific, USA). Protein expression was induced by adding 0.1 mM IPTG (catalog no. #16758, Sigma-Aldrich, USA) to the bacterial culture (OD = 0.6 nm), and the culture was then incubated at 18°C ​​for 16 hours. The bacteria were then pelleted at 10,000 rpm (or 17,700 × g) for 10 minutes, and the pellet was resuspended in lysis buffer (50 mM NaHPO (e.g., pH 8), 300 mM NaCl, 2 mM MgCl, 10 mM imidazole, 1% Triton X-100, 1 mg / ml egg white lysozyme, 1 mM PMSF, and 10 μg / ml DNase). The bacterial lysate was stored at −80°C for 24 hours, then freeze-thawed at 4°C and centrifuged at 10,000 rpm (or 17,700 × g) for 20 minutes. The supernatant was collected and incubated with His60 Ni Superflow™ resin (catalog number ##635660, Takara Bio USA, Inc.) for 4 hours, and the mixture was then passed through a gravity chromatography column. The resin was washed three times (total 90 ml) with wash buffer (50 mM NaHPO (e.g., pH 7.4), 300 mM NaCl, and 25 mM imidazole), and the protein was eluted with 15 ml of elution buffer (10 mM NaHPO (pH 7.4), 300 mM NaCl, 300 mM imidazole, and 0.1% Tween 80). The eluted protein was collected at 50 kDa. The purified protein was washed three times with PBS-T buffer containing 0.1% Tween-80 using an Amicon™ centrifugal filter. The purity of the purified protein was confirmed by SDS-PAGE analysis. LPS contamination of the purified protein was removed using Pierce™ High Capacity Endotoxin Removal Spin Columns (Cat. No. 88274, ThermoFisher Scientific, USA) according to the manufacturer's instructions.

[0188] Mutant hylA and hylB constructs with single amino acid substitutions were cloned and expressed as described above. Protein concentrations were estimated with a NanoDrop 2000 Spectrophotometer (ThermoScientific, USA) and stored at -80°C until further use.

[0189] For protein crystallization studies, BL21 bacterial cells containing HylA and HylB proteins were harvested by centrifugation at 4000 rpm for 15 min and then resuspended in Ni buffer (30 mM HEPES, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 5 mM β-mercaptoethanol; pH 7.5) supplemented with Roche Complete EDTA-free protease inhibitor cocktail. The sample was lysed by sonication, centrifuged at 17,000 rpm (or 23,700 × g) for 40 min to remove cell debris, and applied to a HisTrap FF crude column (GE Healthcare). Protein was eluted using Ni buffer containing 500 mM imidazole. After incubation with TEV protease and overnight dialysis against Ni buffer, the sample was again applied to the HisTrap FF crude column to remove uncleaved products. The sample was then purified through a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) into a buffer containing 100 mM Na acetate (pH 5), 10 mM CaCl2, and 0.5 mM TCEP, concentrated, and frozen in liquid nitrogen.

[0190] Molecular dynamics simulation: Molecular dynamics simulations were performed using the GROMACS software package version 2022.4 and as described by Joshi HV et al. Briefly, the apo crystal structures of HylA-Y285F and HylB-WT were modeled with missing residues, and residue Phe285 was mutated back to Tyr285. Two mutant HylA (S452G and E346G) models were then generated. Four models (HylB-wt, HylA-wt, and HylA-mutant) were used as starting models for the simulation studies. 100 ns MD runs were performed for all four simulations performed in this study. Domain behavior (eigenvectors) for each model were determined using PCA analysis in the Gromacs package (manual.gromacs.org / 2022.4 / manual-2022.4). The cleft gating behavior (Evec1) was determined as the Cα-Cα separation between Ser97 and Thr636 (HylA numbering), the domain twisting behavior (Evec2) was determined as the Cα-Cα separation between Glu208 and Pro216, the substrate entry gating behavior (Evec3) was determined as the Cα-Cα separation between Thr80 and Thr636, and the product exit gating behavior (Evec4) was determined as the Cα-Cα separation between Thr80 and Thr636.

[0191] Design of a HylA multi-epitope vaccine Linear B cell epitopes within the HylA protein were predicted using Bepipred Linear Epitope Prediction 2.0 (IEDB analysis resources, tools.iedb.org / bcell / ). Immunogenic peptides with a score above 0.5 were selected and aligned with HylB. Four peptides with no homology to HylB were selected and physically linked to the C-terminus of the tetanus toxoid protein. A linker amino acid, glycine (G), was placed between each peptide and the C-terminus of tetanus toxin (Figure 15). The fusion gene (TT-mHylA) was then optimized for E. coli protein expression, cloned into pET28a(+) (GenScript), and transformed into E. coli BL21(DE3)pLysS cells. The HylA multi-epitope construct was then expressed and purified as described above, and sequence accuracy was assessed by mass spectrometry.

[0192] Crystallization, data collection, and structure determination Crystals of HylB were grown using the hanging drop method by adding equal amounts of protein and well solution (0.2 M Na phosphate monobasic pH 6.5, 9% PEG 8000, 5 mM TCEP) and suspending them over the well solution at 18 °C. The use of streak seeding improved the size and quality of the crystals. Crystals were cryoprotected in well solution containing 12% PEG 8000 and 25% glycerol and flash-frozen in liquid nitrogen. Crystals of HylB Y281F were grown as above using well solution (0.1 M Bis-Tris pH 6.5, 0.4 M MgCl2, 16% PEG 3350, and 5 mM TCEP) and cryoprotected in well solution containing 20% ​​PEG 3350 and 25% glycerol. Crystals of HylA Y285F were grown and cryoprotected as described above for HylB, except that 0.1 M Na dihydrogen phosphate pH 6.5 was used. Diffraction data were collected using a Rigaku MicroMax-007HF rotating anode X-ray generator with an R-Axis IV++ detector.

[0193] Diffraction data were processed using XDS and scaled using Scala. Molecular replacement for HylB was performed using PHASER, using the N-terminal domain of S. agalactiae hyaluronate lyase (PDB: 1F1S) and the C-terminal domain of A. aurescens chondroitin AC lyase (PDB: 1RWA) as search models. To analyze HylB Y281F and HylA Y285F, the structure of HylB was used as the search model. Model building and refinement were performed using COOT and PHENIX.

[0194] After refinement, the Ramachandran statistics for HylB WT were 97.6% favorable, 2.4% accepted, and 0% outliers, whereas for HylB Y281F they were 96.82%, 3.11%, and 0.07%, respectively, and for HylA Y285F they were 96.5%, 3.37%, and 0.13%, respectively. Structure diagrams were generated using the PyMOL visualization tool (The PyMOL Molecular Graphics System, version 2.4, Schrodinger, LLC.). All of the above crystallographic and structural visualization and analysis tools / applications were used on the SBGrid Consortium platform [www.sbgrid.org]. The root mean square deviation between the crystal structures of GAG lyases was calculated using the Dali Server. The crystal structures and related data are available from the RCSB Protein Data Bank. The PDB codes are (8FYG [www.rcsb.org / structure / unreleased / 8FYG]) for HylA and (8FNX [www.rcsb.org / structure / unreleased / 8FNX], 8G0O [www.rcsb.org / structure / unreleased / 8G0O]) for HylB.

[0195] Pairwise structural comparisons were performed using Dali Server, and structural diagrams were created using the PyMOL visualization tool.

[0196] Hyaluronidase Enzyme Assay: Using an Infinite M200 Pro UV spectrophotometer (Tecan), HylA or HylB at concentrations of 0.0075–0.3 μg / mL in assay buffer and HMW-HA at a concentration of 0.2 mg / mL were added to a 96-well UV-Star clear microplate (Greiner Bio-One, #655801) in a reaction volume of 100 μL. The reaction was monitored over 10 minutes at 232 nm using an Infinite M200 Pro UV spectrophotometer (Tecan). The reaction volume was 100 μL. The assay buffer contained 100 mM Na acetate pH 5.5, 10 mM CaCl2, and 0.5 mM TCEP. Reaction rates (absorbance units / second) were obtained using the slope calculated with Magellan software v.7.0 over reaction times of 1–9.5 minutes. All reactions were performed in triplicate. Enzyme-substrate curves were obtained using GraphPad Prism (Ref-Manual) using the equation for enzyme kinetics (Michaelis-Menten): Y = Vmax * X / (Km + X) The equation was generated by applying a nonlinear regression fit to the ΔΨ ( ). where Y is the enzyme velocity in absorbance units / second, X is the HMW-HA concentration, Vmax is the maximum enzyme velocity, and Km is the HA-HMW concentration required to achieve half-maximum enzyme velocity. HMW-HA was hyaluronic acid sodium salt from rooster comb (Sigma #H5388, MW 1-4 million Da). This method was adapted from a previous study.

[0197] Mouse acne model All animal studies were approved under the guidelines of the University of California, San Diego (UCSD) Institutional Animal Care and Use Committee. Outbred, 6-week-old female CD1 mice (The Charles River Laboratory) were housed in the UCSD animal facility under standard care in accordance with federal, state, local, and NIH guidelines.

[0198] 6-week-old C57BL / 6, TLR2 - / - (strain #:004650), and TLR4 - / - (Strain #:004650) mice were purchased from Jackson Laboratories. - / - Mice and TLR4 - / - Mice were housed in a specific pathogen-free facility. All mice were fed sterile food and water ad libitum and were subjected to animal experiments at approximately 8 weeks of age.

[0199] To model human acne disease, 8-week-old mice were inoculated with C. acnes strain (2 × 10 in 50 μl of BHI medium). 7Mice were infected intraperitoneally with 1000 mg of 1000 CFU (1000 mg of ... The skin lesions were then homogenized, and 25 μl of each sample was serially diluted (10-fold) in PBS and plated on BHI agar plates to measure CFU. The BHI agar plates were anaerobically incubated at 37°C for 3–4 days. The homogenized skin lesions were then centrifuged at maximum speed (13,000 rpm) for 20 minutes, and the supernatant was collected and stored at −80°C for further analysis.

[0200] Disease Scoring Gross skin pathology was scored based on the following tally: Based on the modified protocol, erythematous changes (none = 0, mild = 1, moderate = 2, and marked = 3); papules (flat = 0, small = 1, large = 2, and extra large = 3).

[0201] Immunization of mice Eight-week-old CD1 mice were intraperitoneally vaccinated with 200 μl of alum, alum-rHylA, alum-rHylB, or alum-tetanus toxoid-multiepitope HylA fusion protein (TT-mHylA(mEHylA)) on days 1, 7, and 14. Alum-rHylA and alum-rHylB were prepared by mixing rHylA or rHylB enzyme with 500 μg of Alhydrogel® alum adjuvant (catalog number #vac-alu-50, InvivoGen) and then gently rocking on ice for 1 hour. The vaccine was administered at 70 μg for the first injection and 50 μg for the two subsequent injections. Serum samples were collected 7 days after the final vaccination to assess antibody titers against rHylA or rHylB. To evaluate the protective effect of vaccination against acne, mice were challenged with clinical HL043PA1 or Hl110PA3 C. acnes strains (2 × 10 7 Bacterial counts (CFU / ml), skin lesion size, and pro-inflammatory cytokines were measured 1 and 2 days after challenge as previously described.

[0202] Adoptive transfer of T cells Spleens harvested 10 days after the final vaccination were homogenized in sterile PBS (pH 7.4), followed by red blood cell lysis (Cat. No.: 00-4300-54, eBioscience™) and CD3 T cells were isolated by negative selection using the MojoSort™ Mouse CD3 T Cell Isolation Kit. + T cells (Cat. No. 480031, Biolegend, USA) were isolated according to the manufacturer's instructions. 1 × 10 7 CD3 + T cells were injected retro-orbitally into naive recipient mice. Retro-orbital injection was performed under isoflurane (Fluriso, Vet One) anesthesia. 20 hours after cell transfer, mice were injected with HL043PA1 (2 × 10 7 CFU) was challenged id, and then CFU determinations, disease scores, and skin cytokines were performed on day 2 as described above.

[0203] Serum neutralization of HylA enzyme activity HylA enzyme (0.3 μg) was incubated with 10 μl of pooled serum (n=5) isolated from either mock- or mEHylA-vaccinated mice for 20 min at 37°C. HA (2 mg / ml) was added to the mixture and incubated at 37°C for 20 h under continuous rocking. Subsequent HPLC analysis was performed as described above.

[0204] Measurement of cytokines in skin lesions IL-1β, IL-6, and TNF-α cytokine levels in skin homogenates previously stored at -80°C were measured by solid-phase sandwich ELISA using a commercially available mouse cytokine ELISA kit (Biolegend, San Diego, CA, USA). Assays were performed in biological replicates according to the manufacturer's instructions. For IL-1β and IL-6, 50 μl of skin homogenate was diluted 1:1 with blocking buffer (1% BSA + 1X PBS-Tween 20). For TNF-α, 100 μl of undiluted skin homogenate was used in the assay along with known concentrations of cytokine standards (provided in the kit) in each ELISA plate. Plates were developed and read at an optical density (OD) of 450 nm with wavelength compensation set to 570 nm in a multimode microplate reader (PerkinElmer, Waltham, MA, USA). Cytokine concentrations in samples were determined using a standard curve constructed from the OD of cytokine standards. Human IL-6 and IL-8 cytokine ELISA kits were purchased from Biolegend to measure cytokine levels in HaCaT cell culture supernatants. Culture supernatants were diluted 1:1 and assayed as described above.

[0205] Measurement of antibody titers Serum antibody titers against rHylA and rHylB were measured by indirect ELISA. Briefly, 96-well high-binding microplates (catalog no. 655081, Greiner Bio-one) were coated overnight at 4°C with 500 ng of either rHylA or rHylB in carbonate-bicarbonate buffer (0.2 M, pH 9.6). The plates were washed three times with PBS-T and blocked with 1% BSA (dissolved in PBS-T buffer) for 1 hour at room temperature with continuous rocking. After washing, serum samples diluted in PBS-T (1:100, 1:1000, 1:10,000, 1:100,000) were applied to the wells in biological replicates, and the plates were incubated at room temperature with continuous rocking for 2 hours. Antibody titers (IgM, IgG, IgG1, IgG2b, and IgG3) were detected using goat anti-mouse HRP-conjugated antibodies (SouthernBiotech) at a 1:5000 dilution in blocking buffer for 1 h at room temperature with continuous rocking. After three washing steps, the plate was developed using TMB substrate (catalog no. #555214, BD Bioscience, CA, USA) for 3 min at room temperature, and the reaction was stopped by adding 1N H2SO4. Plates were read at an optical density (OD) of 450 nm with wavelength compensation set to 570 nm in a multimode microplate reader (PerkinElmer, Waltham, MA, USA).

[0206] Statistical analysis and data reproducibility All data were analyzed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, graphpad.com). Specific statistical analyses are noted in the figure legends. In vitro experiments were performed independently two to three times with at least three technical replicates. Data are presented as mean ± standard deviation. In vitro data were analyzed using the nonparametric Mann-Whitney Student's t-test and one-way analysis of variance. All in vivo mouse data were expressed as the median of two or more independent experiments. Two-group analyses were performed using the nonparametric Mann-Whitney unpaired Student's t-test (two-tailed). Comparisons of multiple groups were performed using one-way analysis of variance with Tuckey's post hoc test. In cases of lack of normality, data were analyzed using the nonparametric Kruskal-Wallis one-way analysis of variance.

[0207] Various aspects of the present invention have been described above in the detailed description. While these descriptions directly describe the above aspects, it is understood that those skilled in the art may consider modifications and / or variations to the specific aspects shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be encompassed by this description as well. Unless otherwise specified, it is the inventors' intention that the words and phrases in this specification and claims be given the ordinary and accustomed meaning to those skilled in the applicable technical field.

[0208] The foregoing description of various aspects of the invention known to applicant at the time this application was filed is presented and is intended for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described aspects serve to illustrate the principles of the invention and its practical application, and to enable those skilled in the art to utilize the invention in various aspects and with various modifications as may be suitable for the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular aspects disclosed for carrying out the invention.

[0209] While particular embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the present invention and its broader aspects; therefore, it is intended that the appended claims encompass within their scope all such changes and modifications as are within the true spirit and scope of the present invention. As used herein, the terms "comprising" or "comprise" are used in reference to compositions, methods, and components thereof that are useful in an embodiment, leaving room for the inclusion of elements not expressly specified, whether useful or not. In general, those skilled in the art will understand that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "include" should be interpreted as "including, but not limited to," etc.). The open-ended term "comprising," as synonymous with terms such as including, containing, or having, is used herein to describe and claim the invention, and the invention or embodiments thereof may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of."

[0210] Unless otherwise stated, the terms "a," "an," and "the," and similar references, when used in the context of describing particular embodiments of this application (particularly in the context of the claims), can be construed to cover both the singular and the plural. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of placing individual reference to each individual value within that range. Unless otherwise stated herein, the individual values ​​are incorporated herein as if they were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to particular embodiments herein is intended merely to better describe the application and does not pose a limitation on the scope of the application as otherwise claimed. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0211] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the description includes cases where the circumstance occurs and cases where it does not occur.

[0212] Groupings of alternative elements or aspects of the disclosure disclosed herein are not to be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification herein is deemed to include the modified group, and therefore satisfies the written description of all Markush groups used in the appended claims.

Claims

1. An immunogenic polypeptide comprising a fragment of HylA.

2. The immunogenic polypeptide of claim 1, wherein the HylA fragment is linked or fused to an adjuvant.

3. The fragment of HylA is: （a） ； （b） A variant, The variant is or having at least 50% sequence identity to, or having up to 7 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （c） ； （d） A variant, The variant is or having at least 50% sequence identity to, or having up to 17 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （e） ； （f） A variant, The variant is or having at least 50% sequence identity to, or having up to 6 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （g） ;or, （h） A variant, The variant is or has a linear epitope score of at least 0.4, or a combination thereof.

3. The immunogenic polypeptide of claim 1 or 2, comprising one or more peptides selected from:

4. 4. The immunogenic polypeptide of claim 2 or 3, wherein the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, cytokine, or fragment thereof.

5. An mRNA molecule encoding the immunogenic polypeptide of claim 1 or claim 3.

6. below: （a） ； （b） A variant, The variant is or having at least 50% sequence identity to, or having up to 7 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （c） ； （d） A variant, The variant is or having at least 50% sequence identity to, or having up to 17 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （e） ； （f） A variant, The variant is or having at least 50% sequence identity to, or having up to 6 amino acid additions, substitutions, or deletions, or having a linear epitope score of at least 0.4, or a combination thereof; （g） ;or, （h） A variant, The variant is or has a linear epitope score of at least 0.4, or a combination thereof. A polypeptide comprising one or more peptides selected from:

7. The polypeptide of claim 6 , wherein the one or more peptides are linked or fused to an adjuvant.

8. 8. The polypeptide of claim 7, wherein the adjuvant is a tetanus protein, pertussis toxoid, diphtheria toxoid, cytokine, or fragment thereof.

9. The polypeptide of any one of claims 7 to 8, further comprising a linker between the one or more peptides and the adjuvant.

10. The polypeptide of any one of claims 7 to 8, further comprising a linker between the one or more peptides and the C-terminus of the adjuvant.

11. The polypeptide of any one of claims 6 to 10, comprising at least two peptides and further comprising a linker between each of the at least two peptides.

12. The linker may be G, polyserine, polyglycine, glycine-serine, 12. The polypeptide of any one of claims 9 to 11, which is a leucine zipper, aliphatic, or helical peptide.

13. A composition comprising the polypeptide of claim 6 and an adjuvant.

14. 14. The composition of claim 13, wherein the adjuvant is alum, hydroxyphosphate sulfate, CpG1018, monophosphoryl lipid A, an oil-in-water emulsion, CpG, or QS-21 saponin.

15. 13. An mRNA molecule encoding the polypeptide of any one of claims 6, 11, or 12.

16. 16. A method of treating or reducing the likelihood of acne, comprising administering to a subject in need thereof a composition comprising a polypeptide according to any one of claims 1 to 4 or 6 to 12, or a composition according to claim 13 or 14, or an mRNA molecule according to claim 5 or 15.

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