Propionibacterium acnes hyaluronidase targeting vaccines for prevention and treatment of acne vulgaris

Abstract

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

Description

Cross-references to related applications

[0001] Pursuant to 35 U.S.SC §119(e), this application includes the priority claim of U.S. Provisional Patent Application No. 63 / 436,332, filed December 30, 2022, which is incorporated herein by reference in its entirety. References to sequence lists

[0002] This application contains a sequence list submitted in computer-readable form with the name “SequenceListing_065472_000903WOPT.xml”, which is 35,558 bytes in size and was created on December 29, 2023. The information contained in this computer-readable form is incorporated herein by reference in its entirety. Statement regarding federally funded research or development

[0003] This invention was made with government support from the National Institutes of Health (NIH) under grant numbers AI141401 and AI138053. The U.S. government holds certain rights to this invention. Technical Field

[0004] This invention relates to the treatment and prevention of acne. Background Technology

[0005] All publications herein are incorporated by reference to the same extent that each individual publication or patent application is expressly and individually indicated as being incorporated by reference. The following description includes information that may aid in understanding the invention. This is not an admission that any information provided herein is prior art or related to the currently claimed invention, or an admission that any publication expressly or implicitly referenced is prior art.

[0006] Acne vulgaris affects four out of five individuals at some point in their lives. Susceptibility to acne depends on both host and environmental factors. The contribution of the dermatosymbiotic bacterium *Propionibacterium acnes* (C. acnes) is controversial, as both healthy and acne-susceptible subjects are robustly colonized by *C. acnes*. More detailed characterization of *C. acnes* isolated directly from acne lesions has demonstrated the importance of *C. acnes* genetic elements as major determinants of acne, as acne development and severity are clearly strain- and phylotype-dependent. Therefore, *C. acnes* strains have been classified based on their association with health or acne. Subsequent metagenomic studies have revealed gene sets significantly present in acne-associated or health-associated strains of *C. acnes*, opening new potential frontiers for understanding acne pathogenesis. However, the pathogenesis of acne remains poorly understood due to the lack of robust animal models and the poor survival of *C. acnes* in rodents.

[0007] Therefore, there remains a need for a better understanding of Propionibacterium acnes, as well as unmet needs for the prevention and treatment of Propionibacterium acnes. Summary of the Invention

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

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

[0010] In various embodiments, fragments of HylA may be linked to or fused to an adjuvant.

[0011] In various embodiments, the fragment of HylA may comprise one or more peptides selected from the following. (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATLLTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATLLTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATLLTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0012] In various embodiments, the adjuvant may be tetanus protein, pertussis toxoid, diphtheria toxoid, cytokine, or fragments thereof.

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

[0014] Various embodiments provide polypeptides comprising one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLOKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

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

[0016] In various embodiments, the adjuvant may be tetanus protein, pertussis toxoid, diphtheria toxoid, cytokine, or fragments thereof.

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

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

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

[0020] In various embodiments, the connector may be G, polyserine, polyglycine, glycine-serine, GGGGS (SEQ ID NO:5), GGGGGS (SEQ ID NO:6), leucine zipper, r aliphatic or helical peptide.

[0021] Various embodiments provide compositions comprising the polypeptides and adjuvants of the present invention.

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

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

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

[0025] Various embodiments provide methods for reducing the likelihood of acne, the methods comprising: administering to a subject in need the polypeptide of the present invention, or the composition of the present invention, or a composition comprising the mRNA molecule of the present 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 various features of embodiments of the invention. Attached Figure Description

[0027] The figures mentioned illustrate exemplary embodiments. The embodiments and figures disclosed herein are intended to be illustrative rather than restrictive.

[0028] Figure 1 (Subplots a-j) show that the HylA enzyme is a major virulence factor in the pathogenesis of acne. A, a pie chart, shows the phylogenetics of *Propionibacterium acnes* in health-associated and acne-associated forms and their association with the hylA or hylB genes. Bf, using 2 × 10⁻⁶ pie charts...7 CD1 mice (n=10) were infected intradermally with CFU WT (HL043PA1 or HL110PA3) or isogenetic mutants (ΔhylA or ΔhylB) of Propionibacterium acnes (id), followed by topical application of sebum daily. Bacterial load (b), disease score (c), and cytokines (df) were measured 2 days post-infection. Gi, CD1 mice (n=10) were infected as above with HL043PA1, ΔhylA, or ΔhylA plus recombinant I HylA protein (10 μg). Disease score (g) and histocytokines (h, i) were measured 2 days post-infection. Bi, Data from two independent experiments, each data point representing one mouse. Bars represent medians. j, Histocytokines 2 days post-infection. Data in j were analyzed by one-way ANOVA and Tukey post-hoc test. Data in b, c, and eh were analyzed by one-way ANOVA and Tukey post-hoc test. The data in d and i were analyzed using a nonparametric Kruskal-Wallis one-way ANOVA.

[0029] Figure 2 (Sub-figures a-e) show the HA degradation and structural characteristics of HylA and HylB enzymes. A, b, HPLC chromatograms of HMW HA (2 mg / mL) digested with rHylA or rHylB (1 μg) for 24 hours. Purified HA oligosaccharides were used at known concentrations (see [link to documentation]). Figure 10 and Figure 11 Quantification of the digested HA peaks (HA-2-, 4-, and 6-) was performed. Larger HA fragments, highlighted with green circles, are visible only in HA digested with recombinant HylA (rHylA). Results represent at least two independent experiments. C, Comparison of HylA and HylB crystal structures. HylA and HylB are shown in magenta and orange cartoon diagrams, respectively. Structural domains and junctions are labeled. D, Comparison of active site rift components and electrostatic potential of solvent exclusion surfaces. Electrostatic surface views of HylA and HylB crystal structures are shown. The electrostatic potential of the solvent exclusion surface of the Hylase crystal structure was calculated using APBS in PyMol. Active site rifts are shown by dashed ellipses. Red and blue correspond to -5 kTe, respectively. -1 and 5kTe -1 The potential E was calculated based on the similarity and differences of residues at the active site clefts of HylA and HylB. Residues were highlighted at different structural components of the clefts, and their functional roles were labeled. The HA-6 ligand was derived from the crystal structure of Streptococcus pneumoniae Hyl(SpHyl) (PDB: 1LOH) and modeled in the HylA active site cleft.

[0030] Figure 3(Subfigures a-f) show comparisons of HylA and HylB with bacterial and animal Hyl. A, The crystal structure of HylA overlaps with Hyl from *Streptomyces coelicolor* (ScHyl). B, The crystal structure of HylA is compared with Hyl from *Streptococcus pneumoniae* (SpnHyl) and *Streptococcus agalactiae* (SaHyl). HylA, ScHyl, SpnHyl, and SaHyl are represented by cartoons in magenta, salmon red, green, and cyan, respectively. The PDB IDs of ScHyl, SpnHyl, and SaHyl are 2X03, 2BRW, and 1F1S, respectively. The dashed circles in subfigures a and b show regions of the β-domain of HylA, whose topology is similar to or different from that of its homologs. Structural domains and linkers are labeled. C, The conformation of the active site cleft is shown. The relative positions (i.e., the spacing) of the LI and / or LII rings from the α-domain and the LIV and / or LV rings from the β-domain define the open / closed conformation of the Hyl cleft. This is indicated by black arrows. Clefts of Hyl enzymes from different bacteria, including 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 shows the residues involved in substrate acid (Asx) neutralization and the catalytic tetrad (Tyr-His-Arg-Glu). The corresponding residues from HylA, HylB, ScHyl, SpnHyl, and SaHyl are shown with magenta, orange, salmon red, green, and cyan bars, respectively. The HA-6 ligand is derived 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 a representative of animal hyaluronidase. F, Structural elements HylA and HylB defining the catalytic cleft are shown in a cartoon representation. HylA and HylB are shown in magenta and orange, respectively. The HA-6 ligand is derived from the SpnHyl crystal structure (PDB: 1LOH) and is shown with a yellow stick.

[0031] Figure 4(Subfigures a-h) show the enzyme activity of the HylA mutant with a single amino acid substitution. A, Position of the amino acid residue mutated to the corresponding HylB residue on the HylA crystal. B, h, HPLC chromatograms of HMW HA after co-incubation with WT or mutant HylA (1 μg) for 24 hours: undigested (b), rHylB (c), rHylA (d), or rHylA with a single amino acid substitution (e, h). Quantification of the HA digested peak was performed using purified HA oligosaccharides at known concentrations (see [reference]). Figure 10 and Figure 11 An asterisk (*) indicates a nonspecific peak present in the water control (see [reference]). Figure 7 The data represent two independent experiments.

[0032] Figure 5 (Subfigures a-f) illustrate the pro-inflammatory properties and TLR2 dependence of Hyl degradation products. A, HaCaT cells were stimulated with HA digested from the supernatant of HylA+HL043PA1 or HylB+HL110PA3. IL-6 in the culture supernatant was measured. Bd, HaCaT cells were stimulated with WT or isogenetic ΔhylA HL043PA1 (2×10⁻⁶). 7 CFU) such as WT, TLR2 infection in Shangdi ID - / - and TLR4 - / - Mice. Disease score (b) and skin cytokines (c, d) 24 hours post-infection. E, WT or TLR2 after stimulation with HA digested with rHylA or rHylB. - / - IL-6 in BMDM culture supernatant. F, IL-6 in HaCaT cell culture supernatant stimulated with HA digested with WT or mutant rHylA. Data in a, e, f, are presented as mean ± SD, and each data point is a technical replicate. Bd, bars represent the median, and each data point represents one individual mouse (for TLR4 infected with HL043PA1). - / - TLR2 - / - Or WT mice, n=5-11; and for TLR4 infected with isogenetic ΔhylA. - / - TLR2 - / - Or WT mice, n=4-5). Data a, e, and f represent two independent experiments. The p-values ​​for a and f were calculated using one-way ANOVA and Tukey's post-hoc test. The p-values ​​for b and d were calculated using a nonparametric Mann-Whitney T-test.

[0033] Figure 6(Subfigures a-d) illustrate how vaccination against HylA improves acne lesions and reduces inflammation. Mice (n=10) were immunized intraperitoneally (ip) with alum containing C-terminal tetanus protein (TT) or multiple HylA epitopes linked to TT (TT-mHylA), followed by HylA... + HL043PA1 Propionibacterium acnes strain ID was used for challenge. 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 were from three (ad) independent experiments, with each data point representing one mouse. Data in ac were analyzed by a two-tailed unpaired nonparametric Mann-Whitney Student's t-test, and data in d were analyzed by a nonparametric Kruskal-Wallis one-way ANOVA.

[0034] Figure 7 (Subplots a-h) show the validation of ΔhylA and ΔhylB enzyme activities. A, Enzyme activity against HMW HA substrate was tested on WT and Δhyl culture supernatants. Arrows indicate regions of HA clearance from incubation with WT strains that are absent on plates containing Δhyl. Be, HMW HA substrate (2 mg / mL) was digested for 24 hours with supernatant (10 μL) from WT HL110PA3 or HL043PA1 (b, d) or from isogenetic ΔhylB or ΔhylA (c, e) and analyzed by HPLC. F, Quantification of HA digestion peaks using purified HA oligosaccharides (HA-2, HA-4, and HA-6) at known concentrations. G, h, Controls, undigested HA (g) and water (h). An asterisk (*) represents a nonspecific peak present in the water control. Green circles (f) show larger oligomers present only in HMW HA digested with HL043PA1 supernatant. The results represent at least two independent experiments.

[0035] Figure 8 (Subplots a-c) show the HA disaccharide from HylB digest by LC-MS analysis in negative ionization mode. A, Extracted ion chromatogram of the disaccharide after elution time (12-12 min). b, Mass (m / z) of the corresponding disaccharide in negative mode (MH). C, Extracted ion chromatogram of the HA-tetrasaccharide.

[0036] Figure 9 (Subfigures a-h) show that HylA enzyme is a major virulence factor in the pathogenesis of acne. A, using WT (HL043PA1 or HL110PA3) or isogenetic mutants (ΔhylA or ΔhylB) of Propionibacterium acnes (2 × 10⁻⁶) 7CD1 mice were infected with HL043P1 id (n=10). Representative images of skin lesions 2 days post-infection. B, c, CD1 mice were infected as above with HL043PA1, ΔhylA, or ΔhylA plus recombinant I HylA protein (10 μg). CFU (b) and IL-1β (c) from skin lesions 2 days post-infection. (d) IL-10 levels (n=6 for HL043PA3; n=7 for ΔHylB). Eg, CD1 mice were infected with HL043P1 id plus topical application of sebum, or topical application of sebum only or no treatment (n=3 for sebum and PBS; n=5 for HL043PA1 + sebum), and disease score I and cytokines (fh) were subsequently measured on day 2 (48 hr). Bars represent medians, and data are from one or two independent experiments. Data were analyzed using the nonparametric two-tailed Mann-Whitney U test (d) or by one-way ANOVA and Tukey post-hoc test (eh).

[0037] Figure 10 (Subfigures a-g) illustrate the kinetics of recombinant Hyl digestion of HA. Ad, HPLC chromatogram of HMW HA (2 mg / mL) digested with rHylB (1 μg) for 0-60 min. e, HPLC chromatogram of HMW HA (2 mg / mL) digested with 1 μg rHylA enzyme for 60 min. f, Water control. G, Quantification of the HA peak using purified HA oligosaccharides at known concentrations. Elliptical circles I indicate larger oligomers present only in HA digested with rHylA. The results represent at least two independent experiments.

[0038] Figure 11 (Subfigures a-f) illustrate the kinetics of HA digestion using supernatants from Propionibacterium acnes strains expressing HylA or HylB. Ad, using supernatants from HylA... + HL043PA1 or HylB + HMWHA substrate (2 mg / mL) was digested with HL110PA3 supernatant (10 μL) for 1 hr (a, c) or 24 hr (b, d) and then analyzed by HPLC. E, Quantification of the digested HA peaks (DP2, DP4, and DP6) using purified HA oligosaccharides of known concentrations. F, Water control. Oval circles (b) show the results when only HMWHA was used. + Large oligomers were present in HA digested from the supernatant of HL043PA1. The results represent at least two independent experiments.

[0039] Figure 12Levels of Hyl-A and Hyl-B proteins in healthy and acne-associated Propionibacterium acnes strains. Two acne-associated strains (HL043PA1 and HL043PA2) and two healthy-associated strains (HL110PA3 and HL110PA4) were grown for 4 days, and Hyl expression in the supernatant was analyzed by SDS-PAGE, using rHylA and rHylB proteins as positive controls. Results represent at least two independent experiments. Yellow arrows indicate expressed HylA and HylB.

[0040] Figure 13 The enzyme activities of HylA and HylB mutants are shown in the figure. The figure illustrates the enzyme activity profiles of single amino acid substitutions (point mutations) achieved in HylA and HylB enzymes.

[0041] Figure 14 (Sub-figures A-B) show proposed HylA residues that influence / alter the mechanisms and phenotypes of HA degradation. Proposed HylA / B structural elements involved in domain movement (Mello et al., 2002). Structural elements are shown in cartoon representation and labeled with L (loop) and H (helix) followed by numbers. HylA and HylB are shown in magenta and orange, respectively. The hexasaccharide (HA-6) substrate is derived from the SpHyl crystal structure (PDB: 1LOH) and is shown with yellow bars. Key residues from the loop LIV involved in cleft opening / closing movement are shown with bars. The point mutation S452G in one of these residues was observed to significantly affect the phenotype. (B) Differences between these residues in HylA and HylB are also highlighted in the sequence alignment. HylA sequence (SEQ ID NO:7); HylA sequence (SEQ ID NO:8)

[0042] Figure 15 (Subfigures a-e) show the HA degradation products from incubation of HA with rHylA or the rHylA monoamino acid mutant. Ab, HPLC chromatograms of digestion of HMW HA (2 mg / mL) for 24 hours with WT (a) or mutant rHylA (b) (1 μg). c, Quantification of the HA digestion peak using purified HA oligosaccharides at known concentrations. D, e, Undigested HA (d) and water I as negative controls. Oval circles (ab) indicate larger oligomers. Results are from one experiment. Asterisks (*) represent nonspecific peaks present in the water control.

[0043] Figure 16(Sub-figures a-e) a, b, HaCaT cells stimulated with HA pre-digested with rHylA or rHylB. IL-6 (a) and IL-8 (b) in the culture supernatant. Cd, C57Bl / 6 mouse BMDM stimulated with HA digested with rHylA or rHylB. IL-6 (c) and TNF-α (d) in the culture supernatant. E, f, Infection with HL043PA1 or isogenetic ΔhylAi.d. (2 × 10⁻⁶ cells) 7 CFU)C57Bl / 6WT, TLR2 - / - Or TLR4 - / - Mice. Bacterial load I and TNF-α (f) after 24 hours. Bars represent mean ± SD. Data represent at least two experiments. Bars represent median (d, e). Each data point represents one mouse (for WT and TLR4 mice infected with HL043PA1). - / - or TLR2 - / - Mice, n = 5–11; and for WT and TLR4 mice infected with ΔhylA - / - or TLR2 - / - Mice (n=4-5). Data were analyzed using one-way ANOVA, Tukey post-hoc test (ad), and nonparametric Mann-Whitney T-test (e, f).

[0044] Figure 17 rHylA vaccination protects mice from acne. A, Schematic diagram of rHylA immunization followed by challenge with Propionibacterium acnes. B, Serum anti-HylA and anti-HylB antibody titers after the third rHylA immunization. Cf, CD1 mice (n=10) immunized with alum (reagent control (Mock)) or alum-rHylA (rHylA) challenged with HL043PA1 id. Disease score I, bacterial load (d), IL-6I, and IL-1β (f). g, Serum anti-HylA and anti-HylB titers from CD1 mice (n=5) immunized with rHylB. Hj, Mice vaccinated with reagent control, rHylA, or rHylB, and then challenged with HL110PA3 id. Disease score (h), bacterial load (i), and IL-1β (j) on day 2 post-infection. Bars represent medians. Data were drawn from either two independent experiments (bf) or from a single experiment (gj), with each data point representing one mouse. Data in cf were analyzed using a two-tailed, unpaired, nonparametric Mann-Whitney Student's t-test. Data in b and hj were analyzed using one-way ANOVA and Tukey's post-hoc test.

[0045] Figure 18 (subplots a-d) illustrates the design of a HylA multi-epitope vaccine. A, b, Linear B-cell epitopes in the HylA protein were predicted using Bepipred LinearEpitope Prediction 2.0 (IEDB analysis resource, tools.iedb.org / bcell / ). The graph shows immunogenic peptides with scores higher than 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, Four predicted epitopes (underlined) are physically linked to the C-terminus of the tetanus toxoid protein (italic). A linker amino acid, glycine (G), is placed between each peptide and the C-terminus of the tetanus toxoid.

[0046] Figure 19 The effect of TT-mHylA vaccination on disease induced by HL043PA1 and HL110PA3 infection. Ab, as extended data. Figure 12 As shown, mice were vaccinated with TT or TT-mHylA and challenged with HL043PA1 7 days post-vaccination. TNF-α (a) and IL-6 (b) 48 hours post-infection. Cf, mice were vaccinated with TT or TT-mHylA as above and challenged with HL110PA3 7 days post-vaccination. Disease score (c), IL-1β (d), IL-6, I, and TNF-α (f) 48 hours post-infection. Bars represent medians. Data were from three independent experiments (ab) or one experiment (cf), with each data point representing one mouse. p-values ​​in af were calculated using a two-tailed unpaired nonparametric Mann-Whitney Student's t-test.

[0047] Figure 20(Subfigures a-i) show the enzyme activity of the HylA mutant with a single amino acid substitution. A, Positions of amino acid residues (shown with magenta bars) mutated to the corresponding HylB residues on the HylA (PDB: 8FYG [www.rcsb.org / structure / unreleased / 8FYG]) crystal structure. HA-6 ligands were derived from the SpnHyl crystal structure (PDB: 1LOH). Bf, HPLC chromatograms of HMW-HA after co-incubation with WT or mutant HylA (0.35 μg) for 24 hr: HA alone (b), rHylA (c), or rHylA with a single amino acid substitution (df). g, HPLC chromatogram of HMW-HA after co-incubation with WT rHylB (0.35 μg) for 24 hr. h, Quantification of the peaks from HA digestion using purified HA oligosaccharides at known concentrations. I, Water alone as a blank control. The asterisk (*) in bi represents a nonspecific peak also present in the water control. The data represent two independent experiments.

[0048] Figure 21 (Subfigures a-h) illustrate the pro-inflammatory properties and TLR2 dependence of Hyl degradation products. Ac, HaCaT cells were stimulated for 24 hours with HA pre-digested with rHylA or rHylB, and then IL-6 (a), IL-8 (b), and TNF-α (c) in the culture supernatant were measured. D, HaCaT cells were stimulated for 24 hours with HA pre-digested with supernatant from HL043PA1, HL110PA3, or corresponding isogenetic mutants, and then IL-6 in the culture supernatant was measured. Eg, HaCaT cells were stimulated with HA pre-digested with WT or isogenetic ΔhylA HL043PA1 (2 × 10⁻⁶). 7 CFU strains, such as those infected with WT and TLR2, are found in Shangdi ID. - / - and TLR4 - / - Mice. Disease score I and skin cytokines (f, g) 24 hours post-infection. H, stimulated with HA digested with rHylA or rHylB, WT or TLR2 - / - IL-6 in BMDM culture supernatant. Data in a (n=5 for HA+rHylA; n=6 for other conditions), b (n=3 for medium; n=6 for other conditions), c (n=6), d (n=10), and h (n=4) are expressed as mean ± SD, and each data point represents one well. Data represent two independent experiments. Eg., bars represent the median, and each data point represents one individual mouse (for TLR4 infected with HL043PA1). - / - n=5, for TLR2 - / - n=8 or n=11 for WT mice, and for TLR4 mice infected with isogenetic ΔhylA- / - n=4, for TLR2 - / - (n=5 or n=6 for WT mice). The p-values ​​for a and b were calculated using a one-way Welch ANOVA, the p-values ​​for c and d were calculated using a nonparametric Kruskal-Wallis one-way ANOVA, and the p-value for eh was calculated using a nonparametric two-tailed Mann-Whitney U test.

[0049] Figure 22 (Subfigures a-f) show the selective neutralization of HylA, improving acne lesions and reducing inflammation. A, b, CD1 mice (n=10) immunized with alum (reagent control) or alum-rHylA (HylA) were challenged with HL043PA1 via intraperitoneal (ip) immunization. Disease score (a) and IL-1b in skin homogenate on day 2 post-challenge. Ce, Mice (n=15) were immunized with alum-added to the C-terminus of tetanus protein (TT) or multiple HylA epitopes linked to TT (mEHylA) via intraperitoneal (ip) immunization, followed by challenge with HL043PA1 Propionibacterium acnes strain via intraperitoneal (ip) immunization. Disease score (c), bacterial load (d), and IL-1b I on day 2 post-challenge. F, Serum (1:100,000 dilution) anti-HylA or anti-HylB IgG antibody titers after the third immunization with mEHylA vaccine. Bars represent medians. Data were drawn from two (a, b, f) or three (ce) independent experiments, with each data point representing one mouse. Data in ae were analyzed using a nonparametric two-tailed Mann-Whitney U test, and data in f were analyzed using a nonparametric Kruskal-Wallis one-way ANOVA test.

[0050] Figure 23 (Subfigures a-c) show that the HylB digest, analyzed by HPLC and LC-MS, reveals only HA-disaccharide. A, HPLC of the HylB digest (50 μg HA + 1 μg HylB for 24 hr) shows two peaks, 12.12 and 10.48, corresponding to the isomer of the HA-disaccharide. B, LC-MS shows that the peak from (a) is the HA-disaccharide at m / z. c, HPLC shows no evidence of HA-tetrasaccharide from the HylB digest.

[0051] Figure 24(Subfigures a-e) show the functional regions of Hyl from Propionibacterium acnes, as well as the crystal structures of HylA and HylB. Ac shows the crystal structures of HylA and HylB (wild-type and mutant). D shows the overlap 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 shows the overlap of the three crystal structures of HylA and HylB. E shows an electrostatic surface view of HylA (by APBS Electrostatics, PyMOL Molecular Graphics System, Version 2.4). The proposed functional portion of the Hyl enzyme from *Propionibacterium acnes* is shown in the preparation by LLC. All these regions, including the positively charged cleft region, aromatic / hydrophobic patch, active site, and negatively charged patch, are located around the substrate-binding cleft. It also depicts the substrate inlet and product release outlet of the cleft region. These structural regions in homologous enzymes (including ScHyl (PDB: 2XO3), SpnHyl (PDB: 2BRW), and SaHyl (PDB: 1F1S)) have been shown to be involved in substrate attraction, binding, localization, translocation, and product release.

[0052] Figure 25 (Subplots a-b) show the degradation products of HA after incubation with the mutant rHylA protein. Ab, HPLC chromatograms of HMW HA (2 mg / mL plus 0.35 μg recombinant protein) digested for 24 hours with mutant N442D rHylA (a) or E346G (b). The asterisks (*) in a and b represent non-specific peaks also present in the water control. The green circle (a) shows larger oligomers. Data represent two independent experiments.

[0053] Figure 26 Domain motions in the HylA-wt, HylB-wt, and HylA mutants are shown. Normalized amplitudes of four types of domain motions in each model are shown. Crack opening / closing motion (Evec1), domain twisting motion (Evec2), substrate inlet opening / closing motion (Evec3), and product outlet opening / closing motion (Evec4) are derived from molecular dynamics simulations performed using GROMACS version 2022.4.

[0054] Figure 27 (Subfigures a-k) illustrate the effect of mEHylA vaccination on disease induced by HL043PA1 or HL110PA3 infection. Ab, as Figure 17 As shown, mice (n=15) were vaccinated with reagent control (alum-TT) or alum plus mEHylA (mEHylA) and challenged with HL043PA1 14 days after the last vaccination. TNF-α (a) and IL-6 (b) on day 2 post-infection. Cf, mice (n=5) were vaccinated with reagent control (alum-TT) or alum plus mEHylA (mEHylA) as above and challenged with HL110PA3 14 days after the last vaccination. Disease score (c), IL-1β (d), IL-6I, and TNF-α (f) on day 2 post-infection. Gk, CD1 mice (n=5) were vaccinated with reagent control (alum-TT) or alum plus mEHylA (mEHylA) as above, and subsequently, total CD3+ T cells were isolated from the spleen and adoptively transferred to untreated mice 10 days after the last vaccination. Recipient mice were challenged with HL043PA1 20 h later. CFU(g), disease score(h), and cytokines(ik) were measured on day 2 post-infection. Bars represent medians. Data were from three independent experiments (ab) or from one experiment (ck), with each data point representing one mouse. p-values ​​in af were calculated using a nonparametric two-tailed Mann-Whitney U test, and data in gk were calculated using one-way ANOVA.

[0055] Figure 28 (Subplots a-f) show the HylA enzyme neutralization assay. Ae, HPLC chromatograms after co-incubation of HMW-HA (2 mg / mL) with rHylA (0.3 μg) and serum (10 μL) for 20 hr: HA standard (a), HA alone (b), water as a blank run (c), reagent control serum (alum-TT) + rHylA + HA (d), and anti-mEHylA serum + rHylA + HAI. The asterisk (*) in ae represents a nonspecific peak, also present in the water control. Combined sera (n=5) were used for the assay, and the reagent control serum was performed in duplicate, and the anti-mEHylA serum was performed in triplicate. F) Antibody isotype titers in serum isolated from mice vaccinated with reagent control (alum-TT) or alum plus mEHylA (mEHylA) on day 14 after the last vaccination (serum 1:100,000 dilution). Statistical analysis in f was performed using one-way ANOVA and Tukey post-hoc tests. Green circles represent larger oligomers. Detailed Implementation

[0056] All references cited herein are incorporated herein by reference in their entirety as if fully explained. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 3rd Edition, Revised Edition, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 7th Edition, J. Wiley & Sons (New York, NY 2013); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) provide general guidance to those skilled in the art regarding many of the terms used in this application.

[0057] Those skilled in the art will recognize that many methods and materials are similar to or equivalent to those described herein, and that can be used in the practice of this invention. In fact, this invention is by no means limited to the methods and materials described. For the purposes of this invention, the following terms are defined as follows.

[0058] As used herein, unless otherwise specified, when used in conjunction with a referenced numerical indication, the term "about" means up to 5% of the referenced numerical indication plus or minus that referenced numerical indication. For example, the expression "about 50%" covers a range of 45% to 55%. In various embodiments, if specifically specified in the claims, when used in conjunction with a referenced numerical indication, the term "about" may mean up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication plus or minus that referenced numerical indication.

[0059] Sequence identity as a percentage (%) relative to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage sequence identity, without considering any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various known ways, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for sequence alignment can be determined, including algorithms required to achieve maximum alignment covering the full length of the compared sequences.

[0060] As used herein in the context of linked peptides, polypeptides, or proteins, "linked" means "directly or indirectly connected to". Indirect linkages can be mediated by polypeptide linkers, such as polyglycine or glycine-serine polypeptides, for example, (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 from 1-10, 10-20, 20-30, 30-40, or 50-100. Other such linkers are known in the art and are considered to be covered by this terminology.

[0061] Linear epitope scores can be determined using a semi-empirical method that utilizes the physicochemical properties of amino acid residues and their frequency of occurrence in experimentally known fragment epitopes to predict antigenic determinants on proteins. This method is based on a single parameter that predicts antigenic scores based on antigenic propensity, as described by Kolaskar and Tongaonkar (1990). (ASKolaskar and Prasad C. Tongaonkar. FEBS. 276(12). 172-174 (1990)).

[0062] In this paper, we demonstrate that HylA hydrolyzes HA into large HA fragments that drive robust TLR2-dependent inflammatory pathology. In contrast, HylB specifically degrades hyaluronic acid (HA) into HA disaccharides, resulting in a reduction in acne immunopathology. Structural and phylogenetic studies indicate that this enzyme evolved from a common hyaluronidase that acquired differentiated enzymatic properties. We demonstrate that selective inhibition of HylA through vaccination alleviates acne pathology, thus pointing to a virulence-based approach to acne treatment. We recently addressed the problem of *Propionibacterium acnes* growth in mice by applying human synthetic sebum to mouse skin infected with a virus that allows for the persistence of *Propionibacterium acnes*. We further demonstrated in a model a significant and uniformly enhanced pathogenicity of acne-associated strains compared to healthy-associated strains. Using this model, we set out to address the question of what genetic elements drive disease- or health-associated differentiation of *Propionibacterium acnes*.

[0063] Among the candidate factors revealed by comparative genomics studies comparing healthy-associated strains with acne-associated strains, we are particularly interested in the matrix-degrading enzyme hyaluronidase (Hyl), which produces the HA fragment in mammals and mediates inflammation via TLR2 / 4 signaling, a major pro-inflammatory pathway in acne pathogenesis. Two variants of the Hyl enzyme, HylA and HylB, expressed by *Propionibacterium acnes*, appear to be uniquely expressed in acne-associated and healthy-associated strains, respectively. We therefore seek to understand their relationship and their contribution to the association between *Propionibacterium acnes* health and acne.

[0064] Our study supports Hyl as a key virulence factor explaining the health and acne phenotype differentiation of *Propionibacterium acnes* strains. This is supported by the strong association between HylA and HylB and clinical disease or health, their TLR2 dependence in immunopathological mechanisms (consistent with acne vulgaris), and the contributions of the two Hyl variants to immunopathology and health. While our data support the prominence of HylA virulence, several other *Propionibacterium acnes* virulence factors have been reported. These include genes involved in the upregulation of toxic porphyrin biosynthesis with vitamin B12 supplementation, and cAMP factors that recruit cytotoxic host sphingomyelinases.

[0065] Based on our phylogenetic analysis, HylA is the only pro-inflammatory hyl expressed in detail by either a human commensal or pathogen. Since *Propionibacterium acnes* is both a human commensal and a soil bacterium, its clustering in the environmental microbiome within the phylogenetic tree is meaningful, perhaps representing a transition from soil to commensal. The association of HylA and HylB with hyls from soil-derived organisms *Streptomyces* and *Arthrobacter* could be consistent with this proposed transition from a multifunctional lysin to a more restrictive and processive enzyme. Since inflammatory environments are generally detrimental to pathogens, it was expected that *Propionibacterium acnes* hyls would evolve from the pro-inflammatory HylA to HylB. Despite this expectation, in our acne model, expression of either HylA or HylB did not appear to alter *Propionibacterium acnes* survival, thus not exerting selective pressure on survival. This is consistent with the discovery of abundant *Propionibacterium acnes* strains expressing either hyl. Although HylA and HylB differ by 26% in their genomic sequences, we demonstrated that a single amino acid substitution can significantly alter the enzyme's phenotype, suggesting that the major pathogenic potential of *Propionibacterium acnes* may be encoded and modified by only small changes that occurred during enzyme evolution. These single amino acid substitutions were observed to be conserved across different *Propionibacterium acnes* strains.

[0066] Reports indicate that, based on the discovery of different sequences upstream and downstream of hylA and hylB, *Propionibacterium acnes*, associated with both health and acne, acquired HylB and HylA respectively through different insertion events in the indel 14 genome region. The exact mechanism of these events is unclear, but the structural and sequence associations of HylA and HylB with other bacterial Hyl groups suggest they most likely originated within the *Cutibacterium* species. Current data best aligns with this interpretation until further genetic data becomes available.

[0067] Understanding the structural differences between HylA and HylB allows us to design selective therapeutics that target pro-inflammatory enzymes. Reports show that only 4%–17% of people, and only after early adulthood, develop neutralizing antibodies against Propionibacterium acnes Hyl. Therefore, a vaccine approach would have significant benefits. Propionibacterium acnes and hyaluronidase contribute to healthy or acne-prone skin.

[0068] To assess the potential importance of HylA or HylB enzymes in clinical acne, we investigated all hylA and hylB genes in NCBI and analyzed their associations with healthy and acne-associated Propionibacterium acnes strains (Table 1). Propionibacterium acnes strains were classified into phylogenetics, which exhibited different predispositions to association with acne. Phylogenetics IA-1, IA-2, IB-1, IB-2, and IC were associated with acne, while phylogenetics IB-3 and II were strongly associated with health. Figure 1 a). Different classifications based on ribotype (RT) demonstrate a strong association between RT2 / 6 and health, and between RT4 / 5 and acne (Table 1). Figure 1 As shown in figure a, the hylA gene is found almost exclusively in acne-associated phylogenetics, while hylB is found in health-associated phylogenetics, supporting their potential to contribute to either acne or health. Table 1 shows the phylogenetics of Propionibacterium acnes, the number and percentage of strains in each phylogenetic, the presence of the Hyl (A or B) gene, and its association with acne or healthy skin.

[0069] To directly investigate the roles of hylA and hylB in acne, we generated in-frame allele exchanges of hylB and hylA in prototype healthy (HL110PA3, germline II, RT6) and acne-prone (H043PA1, germline IA-2, RT5) strains, respectively. Based on our previous research, these two strains represent the least acne-prone and most acne-prone strains, respectively, in the RT2 / 6 and RT4 / 5 healthy and acne-prone groups tested in our acne mouse model. We validated the Hyl gene deletion by sequencing and the loss of Hyl activity by HA plate assays and HPLC. Figure 7 ah and Figure 8 We applied both WT and mutant strains to our mouse acne model and investigated disease scores and tissue cytokines after 2 days. Figure 9 a). Independent of bacterial load ( Figure 1 b) Compared with the parental acne-associated strain, the hylA deletion mutant induced a significant decrease in disease scores and pro-inflammatory cytokines. Figure 1 Conversely, compared to the healthy parent strain, the hylB deletion mutant showed a modest increase in disease score and pro-inflammatory cytokines. Figure 1This aligns with the explanation that HylB possesses anti-inflammatory properties. Notably, when comparing the immunopathology of acne induced by proto-acne-associated and health-associated Propionibacterium acnes strains, the pathological differences between acne and healthy strains were eliminated or even moderately reversed in the absence of the Hyl enzyme, pointing to the significant contribution of the Hyl variant to the differences between healthy and acne strains.

[0070] We provided confirmation of the pro-inflammatory phenotype of HylA by supplementing ΔhylA with WT HylA recombinant protein (rHylA). Figure 1 gi; Figure 9 (b, c). Overall, our findings suggest that the two Hyl variants play a major role in promoting or regulating the immunopathology of acne with HylA. HylA and HylB enzymes have different HA degradation modes and efficiencies.

[0071] We then inquired how HylA / B develop such different inflammatory properties. Reports have shown that mammalian and hyalurolytic streptococcal enzymes produce HA fragments longer than 4 meters, which contribute to the induction of pro-inflammatory cytokines. Recently, we demonstrated that pathogenic bacterial pathogens (Group B Streptococcus, Streptococcus pneumoniae, and Staphylococcus aureus) produce Hyl, which rigorously degrades pro-inflammatory HA into non-inflammatory or anti-inflammatory disaccharides (HA-2). Therefore, depending on the HA degradation products, the enzymatic activities of HylA and HylB can lead to different inflammatory outcomes.

[0072] Therefore, we incubated the supernatant from HL110PA3 or H043PA1, or recombinant HylA or HylB, with high molecular weight (HM) HA for 1 h or 24 h. Figure 10 ae and Figure 11 The HylB enzyme activity is rapid, and from the start to the end of the reaction, it mainly produces HA-2 (ad). Figure 2 a). The tetrasaccharide HA (HA-4) was briefly observed at 5 minutes. Figure 10 b), but thereafter, as the degradation reaction continued, only HA-2 was detected.

[0073] In contrast, HylA produces different oligosaccharides throughout the reaction, including HA-4 and the hexasaccharide HA (HA-6), as well as HA-2 ( Figure 2b). Notably, HA-4 and H-6, along with higher MW HA, persisted at the end of 24 hours. We performed thorough digestion with supernatant containing HylA for up to 6 days and demonstrated that digestion remained incomplete. These differences in degradation rate and pattern are consistent with findings from another group of strains producing 3 HylA strains (stereotype IA) and 2 HylB strains (stereotypes IB and II). They illustrate fundamental differences in degradation mechanisms—the progressive / gradual exolytic degradation mechanism of HylB and a combination of this mechanism and the "non-progressive random biteendolytic" degradation mechanism of HylA. Although lower amounts of HylA were secreted into the supernatant compared to HylB (… Figure 12 However, increasing enzyme concentration did not overcome the difference between the endokinesis mechanism and the progressive mechanism. Figure 10 ce and Figure 11 a,b). Functional differentiation of HylA and HylB promotes different HA degradation mechanisms.

[0074] Unlike human Hyl, enzymes reported to date secreted by symbiotic or pathogenic bacteria strictly degrade HA to HA-2. In the Hyl phylogenetic tree, individual bacterial clusters stand out due to their ability to generate fragments larger than the disaccharide. These clusters include environmental bacteria, such as *Streptomyces*, which degrades HA into large fragments. *Dermatobacteria*, being both a human symbiotic and environmental bacterium, also clusters with *Streptomyces*, thus raising the question of whether pro-inflammatory HylA originates from *Streptomyces* and anti-inflammatory HylB originates from bacterial pathogens (such as *Streptococcus*). HylA and HylB are 90% homologous in nucleotide sequence and 74% identical in amino acid sequence. Further studies of HylA / B homologous enzymes in related species suggest that these two enzymes most likely originated within the *Dermatobacteria* lineage. The structures of HylA and HylB reveal a high degree of structural similarity between them and with homologous glycosaminoglycan-degrading lyases from other bacteria.

[0075] To understand the structural basis for the difference in hyaluronic acid lyase activity between HylA and HylB, we resolved the X-ray crystal structures of the HylAY285F mutant and wild-type (WT) HylB to [missing information]. and

[0076] HylAY285F is the catalytically deficient form of the enzyme, and its structure is referred to below as "HylA". Suitable enzymes share 74% identity with each other, exhibiting high structural similarity, with rmsd at 751 residues. Overlap (comparison of two HylA molecules in a crystallographically asymmetric unit with HylB). As typical hyaluronic acid lyases, HylA and HylB consist of an N-terminal domain primarily composed of α-helices, a C-terminal domain primarily containing β-chains, and a catalytic site located primarily within a large cleft in the N-domain. Figure 2 c, d). The catalytic sites are closely overlapping and contain many conserved elements within the hyaluronic acid lyase, including two conserved tryptophans (HylA / BTrp161 / 157 and Trp162 / 158), several positively charged residues, and three residues of the catalytic triad (Asn226 / 222, His276 / 272, and Tyr285 / 281). Figure 2 e). We further resolved the structure of the catalytically deficient Y281F mutant of HylB to a resolution of [resolution missing]. (Table 2); This structure resolves in a different space group than wild-type HylB (P1 vs. two molecules in the crystallographically asymmetric unit, P41212 and one molecule for wild-type). Although crystallized through different crystal stacking interactions, HylB Y281F is conformationally almost identical to wild-type HylB (rmsd). Wild-type comparison of two Y281F molecules) Figure 24 ae). We were unable to produce crystals of HylA or HylB complexed with the HA fragment; in fact, the three structures reported here exhibit open catalytic rifts that may be incompatible with substrate binding ( Figure 3 ).

[0077] Table 2.

[0078] Crystal structures revealed a high structural similarity between *Propionibacterium acnes* HylA and HylB and glycosaminoglycan lyases from Gram-positive bacteria, including hyaluronic acid lyases from *Streptococcus agalactiae* and *Streptococcus pneumoniae*, xanthan gum lyases from *Bacillus sp.* strain GL1 and *Paenibacillus nanensis*, and chondroitin AC lyases from *Streptomyces aurantium* and *Arthrobacter aurescens*. Figure 3 Each of these enzymes is homologous to HylA and HylB, with a sequence identity of 23%–37% with HylA, and is also structurally homologous, with a root mean square deviation from HylA ranging from [missing value]. Furthermore, the conserved geometry of the catalytic residues in these enzymes is maintained in HylA and HylB. Figure 3 d, e).

[0079] To gain further insight into the functional differentiation of HylA and HylB, we identified four residue sites in the catalytic cleft that differ between HylA and HylB. Two of these residue pairs (HylA Arg397 / HylBVal393 and HylA Ser116 / HylB Glu112) are located deep within the cleft, close to the β-D-glucuronic acid moiety at the putative non-reducing end of the bound HA, and contribute to the HylA binding cleft exhibiting a more positively charged surface than that of HylB. Figure 2 d). The other two pairs (HylA Asp345 / HylB Asn341 and HylA Glu346 / HylB Gly342) are located closer to the predicted position of the previous β-D-glucuronic acid moiety.

[0080] Next, we 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 was more favored at that position. In this assay, hyaluronidase cleavage of HMW-HA was recorded by monitoring UV absorbance at 232 nm, which increased with the formation of unsaturated carbon-carbon bonds in the β-D-glucuronic acid moiety at the cleavage site.

[0081] The assay showed that HylB degraded HA at approximately twice the rate of HylA, while the control mutations catalyzing the tryptophan residues in the triplet (HylA / B Y285F / Y281F) severely impaired the HA degradation activity of both enzymes. Figure 13 For residue pairs, only positions 346 / 342 of HylA / HylB showed a clear preference, with both HylA and HylB exhibiting higher enzyme rates than glycine in the case of glutamate. However, inconsistently, the wild-type sequence containing glutamate at this position is not HylB, but rather the less active HylA of the two variants; therefore, this residue is unlikely to explain part of the difference in cleavage rates between HylA and HylB. Mutating the Ser 452 residue of HylA to glycine of HylB altered the enzymatic phenotype of HylA.

[0082] We further generated point mutations at multiple residues in HylA, transforming them into similar residues in HylB, to reproduce the phenotype of HylB with the size of the HA product. HylA S452G is located in the loop of the C-terminal domain, between chain β10 and β11 ( Figure 3 d、 Figure 20 a) Mutations in the preceding residue of the similar loop in *Streptococcus pneumoniae* Hyl have been shown to alter enzyme activity. As noted above, HylA S116E and E346G are located in the catalytic cleft near the non-reducing end of the HA substrate. HylA S284G is adjacent to the catalytically active Y285, and N442D is located at the exposed position within the C-terminal domain. Figure 20 ).

[0083] HylAS284G, S116E, and E346G had no significant effect on the size phenotype of HylA products, although E346G showed reduced overall enzyme activity, and N442D resulted in almost complete loss of activity. Figure 20 -i, and Figure 13 and Figure 25 However, HylA S452G successfully altered the enzymatic phenotype of HylA; S452G exhibited increased enzyme rate and a reduced amount of larger oligomers as products, reminiscent of HylB (…). Figure 20 However, the resulting product was not predominantly HA-2 as expected from a strictly HylB-like phenotype, but rather a mixture containing a higher HA-4 to HA-2 ratio compared to WT HylA. Interestingly, this amino acid residue, S452 in HylA and G448 in HylB, is conserved in Propionibacterium acnes strains, suggesting that similar hydrolysis processes may be conserved.

[0084] Earlier domain movements in SpHyl, SaHyl, and ScHyl enzymes have been implicated in substrate processing through molecular simulations. To understand whether substrate processing via HylA and HylB follows a similar mechanism, we performed molecular simulations of wild-type HylA and HylB, as well as the HylA mutants S452G and E346G, as described by Josh et al. Our molecular simulation results are consistent with the observations of Josh et al.; in short, PCA analysis of the simulated trajectories shows that HylB-WT is far more dynamic than HylA-WT, and the S452G mutation in HylA shows increased (amplitude) domain movements, similar to HylB-WT; cleft opening / closing movements (eigenvector 1) increased by approximately 20%, while other domain movements increased by 10%–40%. Figure 26 These observations are consistent with the hypothesis that complex structural dynamics are one of the key mechanisms by which HylA and HylB process substrates.

[0085] Furthermore, the overall three-dimensional structures of HylA and HylB are almost identical. Figure 2 c); the fold consists of an N-terminal α-domain and a C-terminal β-domain linked by a 12-residue linker. The substrate-binding cleft in both enzymes contains a highly conserved catalytic site. Figure 2(d and Table 3) and decorated with charged residues. Most differences were observed near the substrate-binding region (Table 4). Comparison of the substrate-binding domain with other Hyl from Streptomyces and Streptococcus species shows that the positively charged plaques in HylA / B are located close to the non-reducing end of the substrate, and the negatively charged plaques are located at the reducing end, participating in substrate binding and product release, respectively. A few aromatic residues form aromatic / hydrophobic plaques that are involved in substrate localization for catalysis. Figure 2 e and Table 3). Table 3. Crack components of active sites at the residue level Table 4. Major structural differences between HylA and HylB, and key residues around the crack. A comparison of the HylA / B structure with the Hyl structures of other bacteria further supports the differentiation of HylA / B.

[0086] We were unable to obtain HylA / B crystals complexed with the HA fragment. To further understand the functional differentiation of HylA / B, we compared the structural features of HylA / B with those of other bacterial Hyl components from Streptococcus and Streptomyces species. Figure 3 HylA / B was compared with chondroitinase. Based on previous research, it has been proposed that HylA / B evolved from a pre-existing chondroitinase. As expected, as HylA / B evolved to recognize and process different substrates, including HA, it showed overall structural similarity to chondroitin AC lyase from Arthroacae (ArthroAC; PDB: 1RW9) and HylA / B from other bacteria (Table 5). This observation suggests that HylA / B originated from a common enzyme and functionally differentiated from it. Table 5 Comparison of hyaluronidase crystal structures and sequences from different bacterial species

[0087] Although the end-product profile of HylB (producing only HA-2) is similar to that of Hyl from *Streptococcus pneumoniae* (SpHyl), *Streptococcus agalactiae* (SaHyl), and *Streptococcus spp.* (ScHyl), the end-product from HylA contains both larger fragments and disaccharides. Mechanistically, chondroitinase and Hylase degrade HA following two types of mechanisms. Based on the product profile, HylB utilizes a progressive / gradual exocclusive cleavage mechanism. We show that HylA follows a two-step process, initially involving non-progressive random occlusive endocleavage followed by exocclusive function. The observation that the cleft of HylA is more open than that of HylB (Tables 6 and 7) is related to the above inferences regarding the differences in their HA degradation mechanisms. Therefore, taken together, the data suggest that HylA and HylB functionally differentiate through the shift from endocleavage to exocclusive processing, thereby enabling efficient degradation of HWA-HA, accompanied by different biological effects on the host. Table 6: Comparison of the cleft conformations among bacteria Hyl. Table 7: Shortest distances around active site fissures, describing the degree of openness of active site fissures. Proposed HylA / B degradation mechanism and its relationship with amino acid residue changes

[0088] To understand the structural basis of the differences in HA degradation associated with amino acid changes in HylA / B, based on previous research, we categorized the residues into three groups: (1) residues involved in basic catalysis [residues forming the active site], (2) residues involved in substrate binding / localization and product release [residues forming positively charged gaps, aromatics, and negatively charged plaques], and (3) residues involved in substrate entry and translocation / slip regulation [residues involved in domain movement and structural flexibility]. We evaluated the activity of several of these residues through point mutations. Figure 13 ).

[0089] Since residues from Group 1 are highly conserved in HylA / B and involved in basal catalysis, we selected residues surrounding them, including 116S / 112E and 284S / 280G (HylA / B numbers), and a few others. In Group 2, significant differences were observed in residues forming positively charged plaques (charged rifts), including 346E / 342G and 397R / 393V, and a few others. In addition to the differences mentioned above, interdomain motion in SpHyl, SaHyl, and ScHyl enzymes has been implicated in substrate processing. Based on this concept, we identified loops and helices associated with these functions. Figure 3 c and Figure 14a) and the main differences in HylA / B, including 346E / 342G, 394A / 390S, 395S / 391T, 442N / 438D and 452S / 448G ( Figure 14 (b) These differences critically alter the HA degradation mechanism because residues involved in domain movement and structural flexibility can modulate the translocation / slippage between substrate entry and subsequent catalytic cycles of progressive degradation of polymeric / oligomeric HA substrates. Interdomain movement in HylA may allow the enzyme to initially engage in endokinetic activity and gradually switch to exokinetic activity depending on the size of the available substrate. The differentiation of enzymatic mechanisms driven by the single amino acid serine 452 located outside the substrate-binding domain.

[0090] Based on the potential impact of specific HylA / B residues on HA degradation, we performed point mutations on several residues of HylA. Figure 4 ah and Figure 14 b).

[0091] Notably, the HylA mutant S452G significantly reversed the enzymatic phenotype to HylB. Figure 4 g). This mutant protein digests HMW-HA substrates to HA-2 (phenotypic mimicking WT HylB), with a small amount of residual HA-4. Other point mutations, particularly 442N, accelerate the digestion of HA to HA-2 and reduce the amount of undigested HA. Figure 4 h and Figure 15 ).

[0092] Structurally, the 452S and 442N residues are located in the ring LIV originating from the β domain. Figure 14 a) This positional change may reverse the phenotype by affecting substrate localization and enzyme activity. Joshi et al. have compared the role of interdomain movement in other Hyl homologs. Briefly, their analysis showed that the corresponding residue of 451N(HylA) (the residue preceding 452S(HylA)) in SpHylA(580N) was shown to be involved in substrate binding / translocation, and the loop containing this residue (βLIII in SpHyl and LIV in Hyl) controls substrate entry. In SpHyl, the mutation from 580N to glycine was also shown to alter its enzymatic activity by further opening the cleft (domain movement (i)). Therefore, replacing Ser with Gly at this position in HylA may confer greater flexibility in the LIV loop and increase domain movement. Notably, the exonucleolytic ScHyl (PDB: 2WCO) and ArthroAC (PDB: 1RW9) contain glycine at this position, suggesting that HylB may have acquired an efficient way of processing HA through interdomain movement.

[0093] These observations suggest that HylA can differentiate into more efficient HylB by regulating substrate entry, binding, and translocation / sliding through interdomain movement. Pro-inflammatory properties of Hyl degradation products

[0094] After defining the structure and enzymatic function of *Propionibacterium acnes* Hyl, we inquired whether the HA degradation products from HylA and HylB induced the inflammatory pathology observed in in vivo experiments. For these assays, we used rHyl( Figure 16 ad) or supernatant from WT / ΔhylA ( Figure 5 a) HA was digested for 24 hours, and cell-specific cytokine secretion responses were measured. We demonstrated that HylA, regardless of whether higher concentrations of rHylB or the supernatant were used, significantly increased the activity of HylA in keratinocytes ( Figure 5 a and Figure 16 a), b) and macrophage cell lines (extended data) Figure 16 c) and d) induced higher levels of acne-related cytokines than the control. In contrast, HylB degradation induced reduced cytokine levels (IL-8) or no change in other cytokine levels compared to the control, consistent with the previously defined anti-inflammatory properties of the progressively producing HA-2 Hyl enzyme. As reported, HA-2 lacks pro-inflammatory properties, and the degradation of HA into HA-2 eliminates the pro-inflammatory properties of the larger HA fragment. Furthermore, HA-2 competes with the larger HA fragment for further blocking TLR2 activation.

[0095] The TLR2 dependence of acne vulgaris is a recognized characteristic of this skin disease, and therefore we investigated the TLR2 dependence of inflammation induced by HylA / B. Consistent with the TLR2 dependence of HA (…), Figure 5 The pathological differences induced by HL043PA1 and ΔhylA in TLR2 - / - It was eliminated in mice, but in TLR4 - / - It was not found in mice.

[0096] Finally, we inquired how a single amino acid substitution that modifies or reverses hyl degradation affects inflammation. Figure 5 As shown in f and consistent with Hyl HA degradation products, a single amino acid substitution (S452G) that reverses or accelerates HA processing also leads to a reversal of the inflammatory phenotype. We also noted several other HylA single amino acid mutants that reversed inflammation, which was associated with higher HA-2 and lower amounts of undigested larger oligosaccharides compared to WT rHylA.

[0097] Overall, our findings are consistent with the varying sizes of degradation products resulting from Hyl generation in different inflammatory outcomes. The potential importance of Hyl in humans is further explored by linking Hyl mechanisms and acne through their TLR2 dependence. Targeting Hyl to treat acne

[0098] Above, we have demonstrated that HylA plays a major role in the immunopathology of acne in our mouse model. HylA is highly conserved and possesses consistent enzymatic activity proven across the germline of Propionibacterium acnes. Therefore, it is a promising target for therapeutic intervention. Alternatively, the significant homology between HylA and HylB presents a potential challenge to therapeutic selectivity.

[0099] First, we tested whether immunization against HylA conferred protection against acne. We injected mice three times at weekly intervals, then challenged them with HL043PA1. Figure 17 a). Vaccination with HylA in alum induced a robust antibody response against HylA. Figure 17 b) and significantly reduced the immunopathology associated with mouse acne disease ( Figure 6 a, b and Figure 17 cd). However, HylA ( Figure 17 b) or HylB Figure 17 e) Immune responses induced cross-reactive antibodies against each other, and a moderate deterioration in disease scores when mice were challenged with a healthy, relevant HL110PA3 strain. Figure 17 fh).

[0100] To avoid the potential induction of inflammation associated with cross-reactive antibodies, we designed a HylA-specific peptide vaccine using an antigenic procedure (IEDB analysis resource, tools.iedb.org / bcell / ) (Figures 18a-d) and a combination of crystal structures of HylA and HylB. This peptide binds several HylA-specific epitopes and shows no significant homology to human proteins. We physically linked the predicted peptide to tetanus toxoid (TT), expressed the fusion protein in *E. coli*, and then validated the construct by mass spectrometry. This vaccine selectively inhibits acne induced by HL043PA1 (…). Figure 22 There is very little evidence of cross-reactivity of HylB from HL110PA3 (ce), Figure 22 f and Figure 27 af). In adoptive T-cell transfer experiments, CD3 from vaccinated mice + T cells have been shown to be dispensable in terms of protection against acne. Figure 27Next, we tested the effect of serum after mEHylA vaccination on rHylA enzyme activity. Serum after vaccination significantly reduced HA degradation by HylA enzyme (gk). Figure 28 ae). Notably, mEhylA generated the major IgG1 anti-HylA antibody ( Figure 28 f).

[0101] Therefore, various embodiments of the present invention are based at least in part on these findings. polypeptide

[0102] Various embodiments of the present invention provide polypeptides comprising a fragment of HylA. In various embodiments, the HylA fragment is about 12-29 amino acid residues. In various embodiments, the HylA fragment is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the HylA fragment length is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues. In various embodiments, the HylA fragment has an amino acid sequence of any one of SEQ ID NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0103] Various embodiments of the present invention provide polypeptides comprising a fragment of HylA linked to or fused to an adjuvant. In various embodiments, the adjuvant is a polypeptide adjuvant. In various embodiments, the HylA fragment is about 12-29 amino acid residues. In various embodiments, the HylA fragment is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the HylA fragment length is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues. In various embodiments, the HylA fragment has an amino acid sequence of any one of SEQ ID NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0104] In various embodiments, the fragment of HylA comprises one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0105] In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0106] In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0107] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 2 or 1 amino acid additions, substitutions, or deletions.

[0108] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0109] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 relative to its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference peptide.

[0110] In various embodiments, the adjuvant is tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0111] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker may be a peptide linker.

[0112] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0113] In various embodiments, the connector is G, polyserine, polyglycine, GGGGS (SEQ ID NO:5) or GGGGGS (SEQ ID NO:6), leucine zipper or aliphatic.

[0114] Various embodiments of the present invention provide polypeptides comprising one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0115] In various embodiments, the EPDAFASPDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EPDAFASPDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0116] In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0117] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 2 or 1 amino acid additions, substitutions, or deletions.

[0118] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0119] In various embodiments, the variant shares at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference polypeptide. In various embodiments, the variant shares at least 95%, 96%, 97%, 98%, or 99% sequence identity with its 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 relative to its reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference polypeptide.

[0120] In various embodiments, the one or more peptides are linked to or fused to an adjuvant. In various embodiments, the adjuvant is a peptide adjuvant.

[0121] In various embodiments, the adjuvant is tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0122] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker may be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the N-terminus or C-terminus of the one or more peptides and the adjuvant.

[0123] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0124] In various embodiments, the connector is G, polyserine, polyglycine, glycine-serine, GGGGS (SEQ ID NO:5) or GGGGGS (SEQ ID NO:6), leucine zipper, r aliphatic, or helical peptide.

[0125] Various embodiments provide compositions comprising peptides and adjuvants.

[0126] In various embodiments, the polypeptide is a fragment of HylA. In various embodiments, the HylA fragment has about 12-29 amino acid residues. In various embodiments, the HylA fragment has about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the length of the HylA fragment is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues. In various embodiments, the HylA fragment has the amino acid sequence of any one of SEQ ID NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0127] In various embodiments, the polypeptide comprises one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), wherein the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), 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)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0128] In various embodiments, the EMPDAASPDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EMPDAASPDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0129] In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0130] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 2 or 1 amino acid additions, substitutions, or deletions.

[0131] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0132] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 relative to its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference peptide.

[0133] In various embodiments, the adjuvant is alum, hydroxyphosphate sulfate, CpG1018, monophospholipid A, oil-in-water emulsion, CpG, or QS-21 saponin.

[0134] In various embodiments, the polypeptide further comprises 1-10 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1-5 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1-3 amino acid residues at the N-terminus, C-terminus, or both.

[0135] In various embodiments, the polypeptide comprises L-amino acids or D-amino acids and / or equivalent non-natural amino acids.

[0136] In various embodiments, the polypeptide comprises α-amino acids or β-amino acids.

[0137] In various embodiments, the polypeptide contains non-hydrolyzable bonds.

[0138] Various embodiments provide mRNA encoding the polypeptides of the present invention. Embodiments also provide mRNA compositions for inducing an immune response. The mRNA composition (e.g., an immune composition, a vaccine) functions by introducing a segment of mRNA encoding the polypeptide. Using this mRNA, cells can produce the polypeptide. As part of a normal immune response, the immune system recognizes the polypeptide as foreign and produces antibodies.

[0139] In short, in vitro transcribed mRNA can be produced from a linear DNA template using T7, T3, or Sp6 phage RNA polymerases. The resulting product should optimally contain an open reading frame encoding the protein of interest, a flanking UTR, a 5' cap, and a poly(A) tail. Therefore, the mRNA is engineered to resemble fully processed mature mRNA molecules because they are naturally present in the cytoplasm of eukaryotic cells.

[0140] In vitro and in vivo transfection reagents have been developed that promote cellular uptake of mRNA and protect it from degradation. Once the mRNA is transported to the cytosol, cellular translation mechanisms produce proteins that undergo post-translational modifications to become properly folded, fully functional proteins or peptides. This characteristic of mRNA pharmacology is particularly advantageous for protein replacement therapies and vaccines that require the delivery of cytosol proteins or transmembrane proteins to the correct cellular compartments for proper presentation or function.

[0141] Various embodiments of the present invention provide pharmaceutical compositions comprising any of the immunogenic peptides of the present invention as described herein, or mRNA encoding the immunogenic peptides of the present invention as described herein. In various embodiments, the compositions are in nanoparticle form.

[0142] The pharmaceutical compositions according to the invention may also comprise any pharmaceutically acceptable carrier. As used herein, “pharmaceuticalally acceptable carrier” means a pharmaceutically acceptable material, composition, or medium that participates in carrying or transporting the compound of interest from one tissue, organ, or site of the body to another tissue, organ, or site of the body. For example, a 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 “pharmaceuticalally acceptable” because it must be compatible with the other components of the formulation. It must also be suitable for contact with any tissue or organ it may come into contact with, meaning it must not pose a risk of toxicity, irritation, allergic reactions, immunogenicity, or any other complications that outweigh its therapeutic benefit.

[0143] Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate its preparation. Liquid carriers include syrups, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, kaolin, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. Carriers may also include sustained-release materials (e.g., glyceryl monostearate or glyceryl distearate) alone or with the wax.

[0144] The pharmaceutical compositions according to the invention can be delivered in a therapeutically effective amount. A precise therapeutically effective amount is the amount of composition that produces the most effective results in terms of therapeutic efficacy in a given subject. This amount will vary depending on a variety of 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, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the art of clinical and pharmacology will be able to determine the therapeutically effective amount through routine experiments, such as by monitoring the subject's response to administration of the compound and adjusting the dose accordingly. For further guidance, see Remington: The Science and Practice of Pharmacy (Gennaro, ed., 20th edition, Williams & Wilkins PA, USA) (2000).

[0145] Typical effective doses can be determined by in vitro reactions or responses in animal models, as directed by those skilled in the art. Such doses can typically be reduced in concentration or amount by up to about an order of magnitude without loss of the associated biological activity. Therefore, the actual dose will depend on the physician's judgment, the patient's condition, and the effectiveness of the treatment, based on, for example, the in vitro reactivity of the relevant primary cultured cell or tissue culture sample, or the responses observed in appropriate animal models as previously described.

[0146] In various embodiments, the pharmaceutical compositions according to the invention can be formulated for delivery via any route of administration. "Route of administration" can refer to any route of administration known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal, or parenteral administration.

[0147] Transdermal application can be achieved by using topical creams or ointments or transdermal patches.

[0148] "Parenteral" refers to routes of administration typically associated with injection, including intraorbital, infusion, intra-arterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrasheath, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal routes. Through a parenteral route, the composition may be in the form of a solution or suspension for infusion or injection, or as a lyophilized powder. Through a parenteral route, the composition may also be in the form of microspheres or nanospheres, lipid vesicles, or polymer vesicles that allow for controlled release.

[0149] The pharmaceutical composition can be delivered via the enteric route in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles, tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, or emulsions that allow controlled release.

[0150] Pharmaceutical compositions based on compounds according to the invention can be formulated for the treatment of skin and mucous membranes via a local route and are available in the form of ointments, creams, milks, powders, dip pads, solutions, gels, sprays, lotions, or suspensions. They can also be in the form of hydrogels and polymer patches or microspheres or nanospheres or lipid vesicles or polymer vesicles that allow for controlled release. Depending on the clinical indication, these local route compositions can be in anhydrous or aqueous forms.

[0151] Furthermore, the peptides of the present invention can be conjugated to nanoadjuvants. For example, the peptides of the present invention can be conjugated to polymersomes (polymer vesicles that are self-assembled from different arrays 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 conjugated 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. Reagent test kit

[0152] This invention also relates to a kit for treating acne or reducing the likelihood of acne, or for inhibiting Propionibacterium acnes hyaluronidase. The kit is useful for practicing the inventive methods of treating acne or reducing the likelihood of acne, or inhibiting Propionibacterium acnes hyaluronidase. The kit is a collection of materials or components comprising at least one of the inventive peptides or compositions. Therefore, in some embodiments, the kit comprises a composition containing any one or more of the immunogenic peptides of this invention as described above.

[0153] The exact properties of the components configured in the inventive kit depend on its intended purpose. In various embodiments, the kit is specifically configured for the purpose of treating mammalian subjects. In various embodiments, the kit is specifically configured for the purpose of treating human subjects.

[0154] Instructions for use may be included in the kit. These instructions typically include a tangible description of the techniques to be used when employing the kit's components to achieve the desired outcome (e.g., treating acne or reducing the likelihood of acne, or inhibiting Propionibacterium acnes hyaluronidase). Optionally, the kit may also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring instruments, dressing materials, or other useful instruments readily identifiable by those skilled in the art.

[0155] Materials or components assembled in the kit can be provided to the operator in any convenient and suitable manner that preserves their operability and usability. For example, components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room temperature, refrigerated, or frozen temperatures. Components are typically contained in suitable packaging materials. As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the kit (e.g., inventive compositions, etc.). The packaging material is constructed by well-known methods and preferably provides a sterile, uncontaminated environment. As used herein, the term "packaging" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., capable of holding a single kit component. Thus, for example, packaging can be a glass vial for containing a suitable amount of an inventive composition containing any one or more of the inventive immunogenic peptides as described herein. Packaging materials typically have external labels indicating the contents and / or purpose of the kit and / or its components. method

[0156] Various embodiments provide methods for treating acne, the methods comprising: administering to a subject in need a polypeptide or a composition of the invention as described herein.

[0157] Various implementations provide methods for reducing the likelihood of acne, the methods comprising: administering to a subject in need a polypeptide of the present invention as described herein or a composition of the present invention as described herein.

[0158] Various embodiments provide methods for inducing an immune response in a subject in need, the methods comprising administering to the subject in need a polypeptide or a composition of the invention as described herein. In various embodiments, the immune response is a protective immune response.

[0159] Various embodiments provide methods for treating acne, the methods comprising: administering to a subject in need a composition comprising an mRNA molecule encoding an mRNA molecule encoding a polypeptide of the present invention as described herein. Various embodiments also provide methods for reducing the likelihood of acne, the methods comprising: administering to a subject in need a composition comprising an mRNA molecule encoding an mRNA molecule encoding a polypeptide of the present invention as described herein.

[0160] Various embodiments provide methods for inducing an immune response in a subject in need, the methods comprising administering to the subject a composition comprising an mRNA molecule encoding a polypeptide of the present invention. In various embodiments, the immune response is a protective immune response.

[0161] Therefore, in various embodiments, the polypeptide comprises a fragment of HylA. In various embodiments, the fragment of HylA has about 12-29 amino acid residues. In various embodiments, the fragment of HylA has about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the fragment of HylA has 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 NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0162] Therefore, in various embodiments, the polypeptide comprises a fragment of HylA linked to or fused to an adjuvant. In various embodiments, the adjuvant is a polypeptide adjuvant. In various embodiments, the HylA fragment is about 12-29 amino acid residues. In various embodiments, the HylA fragment is about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the HylA fragment is about 10-14, 15-20, 21-25, 26-30, or 31-35 amino acid residues. In various embodiments, the HylA fragment has an amino acid sequence of any one of SEQ ID NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0163] In various embodiments, the fragment of HylA comprises one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), 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)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0164] In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0165] In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0166] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 2 or 1 amino acid additions, substitutions, or deletions.

[0167] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0168] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.5, 0.6, 0.7, 0.8, or 0.9 relative to its reference peptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference peptide.

[0169] In various embodiments, the adjuvant is tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0170] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker may be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the N-terminus or C-terminus of the one or more peptides and the adjuvant.

[0171] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0172] In various embodiments, the connector is G, polyserine, polyglycine, glycine-serine, GGGGS (SEQ ID NO:5) or GGGGGS (SEQ ID NO:6), leucine zipper, r aliphatic, or helical peptide.

[0173] Therefore, in other embodiments, the polypeptide used in these methods comprises one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0174] In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EMPDASAPSDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0175] In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0176] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 2 or 1 amino acid additions, substitutions, or deletions.

[0177] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0178] In various embodiments, the variant shares at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference polypeptide. In various embodiments, the variant shares at least 95%, 96%, 97%, 98%, or 99% sequence identity with its 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 relative to its reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference polypeptide.

[0179] In various embodiments, the one or more peptides are linked to or fused to an adjuvant. In various embodiments, the adjuvant is a peptide adjuvant.

[0180] In various embodiments, the adjuvant is tetanus protein, pertussis toxoid, diphtheria toxoid, or a fragment thereof.

[0181] In various embodiments, the polypeptide further comprises a linker between the one or more peptides and the adjuvant. The linker may be a peptide linker. In various embodiments, the polypeptide further comprises a linker between the N-terminus or C-terminus of the one or more peptides and the adjuvant.

[0182] In various embodiments, the polypeptide comprises at least two peptides and further comprises a linker between each of the at least two peptides.

[0183] In various embodiments, the connector is G, polyserine, polyglycine, glycine-serine, GGGGS (SEQ ID NO:5) or GGGGGS (SEQ ID NO:6), leucine zipper, r aliphatic, or helical peptide.

[0184] Therefore, in other embodiments, the composition used in these methods comprises a peptide and an adjuvant.

[0185] In various embodiments, the polypeptide is a fragment of HylA. In various embodiments, the HylA fragment has about 12-29 amino acid residues. In various embodiments, the HylA fragment has about 10-31 amino acid residues, 9-32 amino acid residues, or 8-33 amino acid residues. In various embodiments, the length of the HylA fragment is about 5-9, 10-14, 15-20, 21-25, 26-30, 31-35, or 36-40 amino acid residues. In various embodiments, the HylA fragment has the amino acid sequence of any one of SEQ ID NO:9-SEQ ID NO:29 and SEQ ID NO:38.

[0186] In various embodiments, the polypeptide comprises one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO:1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO:1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO:1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2) having at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO:2), having an addition, substitution, or deletion of up to 17 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (e)ENSSDRISVSRS(SEQ ID NO:3), (f) A variant of ENSSDRISVSRS (SEQ ID NO:3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO:3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO:4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

[0187] In various embodiments, the EMPDAFSDPDIW (SEQ ID NO:1) variant has up to 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the EMPDAFSDPDIW (SEQ ID NO:1) variant has up to 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0188] In various embodiments, the VATILTDLASSSSRTTLLSANLQKEESS (SEQ ID NO:2) variant has 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. In various embodiments, the VATILTDLASSSSRTTVLLSANLOKEESS (SEQ ID NO:2) variant has up to 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0189] In various embodiments, the ENSSDRISVSRS (SEQ ID NO:3) variant has up to 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0190] In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid additions, substitutions, or deletions. In various embodiments, the ALPKPTKPSLRASSYPLGLP (SEQ ID NO:4) variant has up to 4, 3, 2, or 1 amino acid additions, substitutions, or deletions.

[0191] In various embodiments, the variant has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with its reference polypeptide. In various embodiments, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with its 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 relative to its reference polypeptide. In various embodiments, the variant has a linear epitope score of at least 0.9 relative to its reference polypeptide.

[0192] In various embodiments, the adjuvant is alum, hydroxyphosphate sulfate, CpG1018, monophospholipid A, oil-in-water emulsion, CpG, or QS-21 saponin.

[0193] In various embodiments, the polypeptide further comprises 1-10 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1-5 amino acid residues at the N-terminus, C-terminus, or both. In various embodiments, the peptide further comprises 1-3 amino acid residues at the N-terminus, C-terminus, or both.

[0194] In various embodiments, the polypeptide comprises L-amino acids or D-amino acids and / or equivalent non-natural amino acids.

[0195] In various embodiments, the polypeptide comprises α-amino acids or β-amino acids.

[0196] In various embodiments, the polypeptide contains non-hydrolyzable bonds. Example

[0197] The following embodiments are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. References to specific materials are merely for illustrative purposes and are not intended to limit the invention. Equivalent means or reactants can be developed by those skilled in the art without inventiveness and without departing from the scope of the invention. Example 1 Propionibacterium acnes bacterial culture

[0198] This study used two acne-associated strains (HL043PA1 and HL043PA2) and two health-associated strains (HL110PA3 and HL110PA4). Clinical Propionibacterium acnes strains from cryopreserved storage were plated on blood agar plates using BDBBL. TM GasPak TM The system was anaerobically cultured at 37°C for 96 h. Single colonies of *Propionibacterium acnes* were anaerobically grown in 10 mL of Brain Heart Infusion (BHI) broth (catalog number #53286, Sigma-Aldrich, USA) for 3–4 days (OD = 0.15–0.3), followed by washing the bacterial pellet once with BHI medium at 2300 × g for 5 min. The pellet was resuspended in BHI medium to the desired OD value. 600nm (0.5) Used in in vitro and in vivo studies. Bacterial culture supernatants were collected and used for analysis of HA degradation activity in chicken combs (catalog number #H5388, Sigma-Aldrich, USA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and human keratinocyte HaCaT cell (ATCC) stimulation. Construction of ΔhylA and ΔhylB Propionibacterium acnes strains

[0199] The homologous recombination cloning strategy used was previously described (Sorensen et al., 2010) with slight modifications. Briefly, 1 kb (500 bp) upstream and downstream of the hyaluronidase gene were amplified by PCR, purified by gel electrophoresis, ligated together, and cloned into pGEM-T-easy (catalog #A137A, Promega, USA). An erythromycin resistance cassette from pDCerm was amplified by PCR and ligated between the upstream and downstream regions before transformation into *E. coli* (DH5α) (catalog #18265017, ThermoFisher Scientific). Plasmid DNA was purified from the ampicillin (100 μg / mL) resistant clone and verified by PCR. The correct plasmid was transformed into dam-negative *E. coli* (catalog #C2925I, New England Biolabs) and purified. Competent *Propionibacterium acnes* cells were prepared as previously described (Cheong et al., 2008). Briefly, *Propionibacterium acnes* was anaerobically grown in BHI medium at 37°C until OD. 600nm The concentration was 0.5-0.6. The cell pellet was washed twice in EP buffer (272 mM sucrose, 7 mM sodium phosphate, and 1 mM magnesium chloride). Plasmid DNA was mixed with freshly prepared electrocompetent *Propionibacterium acnes* cells and electroporated. Immediately after electroporation, 1 mL of BHI was added. The cell pellet was resuspended in 100 μL of BHI medium, plated on BHI agar plates, and incubated anaerobically overnight at 37°C. The next day, bacteria were removed with a swab, 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 mutant was validated by PCR, and the lack of activity was confirmed on agar plates containing hyaluronic acid. Hyaluronidase Plate Assay

[0200] Hyaluronic acid lyase (HA) from rooster combs was used as a substrate to measure the hyaluronic acid lysin activity of HylA or HylB in Propionibacterium acnes culture supernatants. 20 μL or 40 μL of supernatant from a single Propionibacterium acnes colony (anaerobic growth for 4 days followed by harvesting at 2600 × g for 10 min) was spotted onto BHI agar plates containing 1% bovine serum albumin (BSA) fraction V (catalog number #10735078001, Sigma-Aldrich, USA) and HA (400 μg / mL). The plates were incubated overnight at 37°C, and HA degradation was detected by washing the plates with 2N acetic acid for 3–5 min. Cell culture

[0201] Bone marrow-derived macrophages (BMDMs) were isolated from the femur and tibia of 12-week-old C57BL / 6 mice (Jackson laboratories) and suspended in complete RPMI 1640 medium (Gibco, ThermoFisher Scientific, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 10 ng / mL M-CSF (catalog number #PeproTech, Inc., USA), and 1% penicillin-streptomycin antibiotic (catalog number #P4333, Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in 92 mm non-adhesive culture dishes (ThermoFisher Scientific, USA) at 37°C and 5% CO2, with the medium replaced every two days with fresh medium containing an equivalent concentration of M-CSF. After seven days of culture, cells were harvested and stimulated with HA (40 μg) digested with bacterial supernatant or rHylA or rHylB enzymes.

[0202] HaCaT cells (ATCC) were cultured in complete DMEM medium (catalog number 10-013-CV, Corning Incorporated, USA) with 10% heat-inactivated FBS at 37°C and 5% CO2. Before cell stimulation with digested HA (40 μg), HaCaT cells were cultured at 10... 5 Cells were seeded at a concentration of 100 cells / mL in 96 wells. Cells were incubated in 96-well tissue culture plates (catalog number #353072, Corning Incorporated, USA) and incubated at 37°C with 5% CO2 for 6 hours, then washed with DMEM medium and stimulated. HA digestion for HPLC analysis and cell culture stimulation

[0203] HA (2 mg / mL) from chicken comb was digested with 1 μg of purified recombinant protein (rHylA, rHylB, or mutant protein) at a concentration of 0.35 μL / mL or 1 μL / mL, or with supernatant from Propionibacterium acnes culture (10 μL / mL). This digestion was performed at 37°C at different time points (0, 5 min, 15 min, 1 hr, and 24 hr) in a reaction buffer containing 100 mM Na acetate, 10 mM CaCl2, and 0.5 mM DTT (pH 5.5), and the reaction was stopped by inactivating the enzyme at 80°C for 10 min. The enzyme was then stored at -20°C until further use. A 50 kDa solution was used. The Ultra-15 centrifugal filter (catalog number #UFC905024, Millipore Sigma, USA) concentrates the supernatant from bacterial cultures used for HA digestion by 20×.

[0204] For BMDM and HaCaT cell assays, 10 saturations were performed using an equivalent of 40 μg of HA digestate. 5 Cells were plated for 8 hours and 16 hours. Cells were cultured in 200 μL of complete RPMI medium supplemented with 10% FBS and 1× penicillin-streptomycin antibiotic solution in 96-well tissue culture plates. After incubation at 37°C and 5% CO2 for 8 or 16 hours (or 24 hours), cells were centrifuged at 400×g and the culture supernatant was 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).

[0205] HA digestion products were analyzed by strong anion exchange high-performance liquid chromatography (HPLC) using an Ultimate 3000 HPLC system (Thermo Scientific, USA) equipped with an Ultimate 3000 variable wavelength detector. The chromatography was performed on a Pro Pack SAX-10 (4×250 mm) column attached to a Pack SAX-10G guard column (4×50 mm, Thermo-Dionex, USA) at 30 °C. 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 (linearity) are shown in the table below. Time (min) ％A ％B 2 100 0 25 75 25 27 40 60 32 100 0 45 100 0

[0206] Chromatograms were obtained at a UV absorbance of 232 nm. A known amount of the sample was dissolved in UP water and injected into HPLC. A standard mixture of 1 μg each of HA-DP2, HA-DP4, and HA-DP6 was injected, and the HA oligosaccharides in the sample were quantified by comparing the peak area with that of the standard mixture. Phylogenetic analysis

[0207] Phylogenetic analysis was performed using the FASTA amino acid sequences of HylA and HylB obtained from the NCBI or RCSB protein databases. Clustal alignment of the sequences was performed, and then a Neighbor-Joining tree was constructed using Geneious Prime. Expression and purification of recombinases

[0208] Propionibacterium acnes HylB (residues 37-801) and HylA (41-805) were cloned into pET His6 TEVLIC (catalog number #29653, Addgene) and pET His6 MBP TEVLIC (catalog number #29656, Addgene) cloning vectors, respectively, and propagated in E. coli Top10 cells (catalog number #C404010, ThermoFisher Scientific, USA). The recombinant plasmids were transformed into E. coli BL21(DE3)pLysS cells (catalog number #C606010, ThermoFisher Scientific, USA), and protein expression was induced by adding 0.1 mM IPTG (catalog number #16758, Sigma-Aldrich, USA) to the bacterial culture (OD = 0.6 nm). The cultures were then incubated at 18°C ​​for 16 hours. The bacteria were then precipitated at 10,000 rpm (or 17,700 × g) for 10 min, and the precipitate was resuspended in lysis buffer (50 mM Na₂HPO₄ (e.g., pH 8), 300 mM NaCl, 2 mM MgCl₂, 10 mM imidazole, 1% Triton X-100, 1 mg / mL egg white lysozyme, 1 mM PMSF, and 10 μg / mL DNases). The bacterial lysate was stored at -80°C for 24 hours, followed by freeze-thaw at 4°C and centrifugation at 10,000 rpm (or 17,700 × g) for 20 min. The supernatant was harvested and centrifuged with His60 Ni Superflow. TM The resin (catalog number #635660, Takara Bio USA, Inc.) was incubated for 4 hours, followed by passage of the mixture through a gravity column. The resin was washed three times (90 mL total) with wash buffer (50 mM Na₂HPO₄ (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 Na₂HPO₄ (pH 7.4), 300 mM NaCl, 300 mM imidazole, and 0.1% Tween 80). 50 kDa Amicon was used. TM Centrifuge the filter and wash the eluted protein three times with PBS-T buffer containing 0.1% Tween-80. Confirm the purity of the purified protein by SDS-PAGE analysis. Use Pierce... TM High-volume endotoxin removal centrifuge column (catalog number #88274, ThermoFisher Scientific, USA) removes LPS contamination from purified proteins according to the manufacturer's instructions.

[0209] The mutant constructs hylA and hylB with a single amino acid substitution were cloned and expressed as described above. Protein concentrations were estimated using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA) and stored at -80°C until further use.

[0210] 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 mixture. Samples were lysed by sonication, centrifuged at 17,000 rpm (or 23,700 x g) for 40 min to remove cell debris, and loaded onto a HisTrap FF crude purification column (GE Healthcare). Proteins were eluted using Ni buffer containing 500 mM imidazole. After incubation with TEV protease and dialyzing overnight into Ni buffer, the sample was reloaded onto a HisTrap FF crude purification column to remove uncleaved products. The sample was then purified on a Superdex 200 Increase 10 / 300GL column (GE Healthcare) into a buffer containing 100 mM Na acetate pH 5, 10 mM CaCl2 and 0.5 mM TCEP, concentrated and frozen with liquid nitrogen. Molecular dynamics simulation:

[0211] Molecular dynamics simulations were performed using GROMACS package version 2022.4, as described by Joshi HV et al. Briefly, the crystal structures of HylA-Y285F and HylB-WT apo were modeled with missing residues, and the residue Phe285 was reverted to Tyr285. Two mutant HylA models (S452G and E346G) were then generated. Four models (HylB-wt, HylA-wt, and the HylA mutant) were used as starting models for the simulation studies. All four simulations performed in this study were 100 ns MD runs. Domain motion (eigenvectors) for each model were determined using PCA analysis from the Gromacs package (manual.gromacs.org / 2022.4 / manual-2022.4). The fracture opening / closing motion (Evec1) was determined to be Cα-Cα separation of Ser97 and Thr636 (HylA number); the domain twisting motion (Evec2) was determined to be Cα-Cα separation of Glu208 and Pro216; the substrate inlet opening / closing motion (Evec3) was determined to be Cα-Cα separation of Thr80 and Thr636; and the product outlet opening / closing motion (Evec4) was determined to be Cα-Cα separation of Thr80 and Thr636. Design of HylA Multiepitope Vaccine

[0212] Linear B-cell epitopes within the HylA protein were predicted using Bepipred Linear Epitope Prediction 2.0 (IEDB analysis resource, tools.iedb.org / bcell / ). Immunogenic peptides with scores higher than 0.5 were selected and compared with HylB. Four peptides without 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 the tetanus toxoid protein. Figure 15 The fusion gene (TT-mHylA) was then optimized for protein expression in *E. coli*, 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. Crystallization, data collection and structure determination

[0213] HylB crystals were grown using the hanging drop method by adding equal volumes of protein and pore solution (0.2 M Na2-phosphate pH 6.5, 9% PEG 8000, 5 mM TCEP) and suspending the protein on the pore solution at 18°C. Streak inoculation was used to improve crystal size and quality. Crystals were cryoprotected in a pore solution containing 12% PEG 8000 and 25% glycerol, and then rapidly frozen in liquid nitrogen. HylB Y281F crystals were grown using a pore solution of 0.1 M bistris pH 6.5, 0.4 M MgCl2, 16% PEG 3350, and 5 mM TCEP as described above, and cryoprotected in a pore solution containing 20% ​​PEG 3350 and 25% glycerol. HylA Y285F crystals were grown and cryoprotected as described above for HylB, except that 0.1 M Na2-phosphate pH 6.5 was used. Diffraction data were collected on a Rigaku MicroMax-007HF rotating anode X-ray generator equipped with an R-Axis IV++ detector.

[0214] Diffraction data were processed using XDS and scaled using Scala. Molecular substitutions for HylB were performed using PHASER, with the N-terminal domain of *Streptococcus agalactiae* hyaluronic acid lyase (PDB: 1F1S) and the C-terminal domain of *Arthrobacter chondroitin AC lyase* (PDB: 1RWA) used as search models. The structures of HylB were used as search models to resolve HylBY281F and HylA Y285F. Model building and refinement were performed using COOT and PHENIX.

[0215] After refinement, the Laplace plot statistics for HylB WT were 97.6% favored, 2.4% allowed, and 0% outliers; for HylB Y281F, these were 96.82%, 3.11%, and 0.07%; and for HylA Y285F, they were 96.5%, 3.37%, and 0.13%. Visualization was performed using PyMOL (The PyMOL Molecular Graphics System, version 2.4). Structure diagrams were prepared by LLC. All crystallography and structural visualization and analysis tools / applications mentioned above were used on the SBGrid consortium platform [www.sbgrid.org]. Root mean square deviation between GAG lyase crystal structures was performed using the Dali server. Crystal structures and related data were available from the RCSB protein database. The PDB code for HylA is 8FYG [www.rcsb.org / structure / unreleased / 8FYG], and for HylB it is 8FNX [www.rcsb.org / structure / unreleased / 8FNX] 8G0O [www.rcsb.org / structure / unreleased / 8G0O].

[0216] Pairwise structure comparisons were performed using the Dali server. Structure diagrams were generated using the PyMOL visualization tool. Hyaluronidase assay:

[0217] Using an Infinite M200 Pro UV spectrophotometer (Tecan), 0.0075 μg / mL–0.3 μg / mL HylA or HylB and 0.2 mg / mL HMW-HA were added to a 96-well UV-Star transparent microplate (Greiner Bio-One, #655801) with a reaction volume of 100 μL. The reaction was monitored at 232 nm for over 10 min using the 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. The reaction rate (absorbance units / sec) was obtained by calculating the slope over reaction times from 1 min to 9.5 min using Magellan software v.7.0. All reactions were performed in triplicate. Enzyme-substrate curves were generated by using GraphPadPrism (Ref-Manual) to fit the enzyme kinetic equation—Michaelis-Menten—through nonlinear regression. Y = VmaxX / (Km + X) Where Y is the enzyme rate in absorbance units per second, X is the HMW-HA concentration, Vmax is the maximum enzyme rate, and Km is the HA-HMW concentration required to achieve the half-maximum enzyme rate. HMW-HA is sodium hyaluronate derived from rooster comb, Sigma#H5388, with a MW of 1-4 million Daltons. This method is adapted from previous work. mouse acne model

[0218] All animal studies were approved in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of California, San Diego (UCSD). Outbred 6-week-old female CD1 mice (The Charles River Laboratory) were housed in UCSD's animal facilities and cared for according to federal, state, local, and NIH guidelines.

[0219] Purchased 6-week-old C57BL / 6, TLR2 infants from Jackson Laboratories. - / - (Strain #: 004650) and TLR4 - / - (Strain #: 004650) Mice. TLR2 mice were bred in a specific pathogen-free facility. - / - and TLR4 - / - Mice. All mice were provided with unrestricted sterile food and water, and animal experiments were conducted at approximately 8 weeks of age.

[0220] To model human acne disease, 8-week-old mice were infected with Propionibacterium acnes strain (2 × 10⁶ in 50 μL of BHI medium). 7 CFU), and then topical application of synthetic sebum daily as previously described. Infection was performed under anesthesia with vaporized isoflurane (Fluriso, Vet One). Synthetic sebum was prepared by mixing fatty acids (17% oleic acid; catalog #O1008, Millipore Sigma), triglycerides (45% triolein; catalog #ICN10312201, Fisher Scientific), wax monoesters (25% jojoba oil, Trader Joe), and squalene (13%; catalog #AC215351000, Fisher Scientific). Disease scores were assessed one or two days post-infection, and mice were euthanized by CO2. Skin lesions were aseptically excised and harvested in phosphate-buffered saline (PBS, pH 7.4). The skin lesions were then homogenized, and 25 μL was serially diluted (10-fold) in PBS to determine CFU on BHI agar plates. BHI agar plates were anaerobically incubated at 37°C for 3–4 days. In addition, the homogenized skin lesions were centrifuged at the maximum speed (13000 rpm) for 20 min, and the supernatant was collected and stored at -80°C for additional analysis. Disease score

[0221] Gross skin pathology was scored based on the following list: erythematous changes (none = 0, mild = 1, moderate = 2, and significant = 3); papules (flat = 0, small = 1, large = 2, and very large = 3), based on the modified scheme. mouse immunization

[0222] Eight-week-old CD1 mice were vaccinated intraperitoneally (IP) with 200 μL of alum, alum-rHylA, alum-rHylB, or alum-tetanus toxoid-multi-epitope HylA fusion protein (TT-mHylA(mEHylA)) on days 1, 7, and 14. The rHylA or rHylB enzyme was then mixed with 500 μg of... Alum adjuvant (catalog number #vac-alu-50, InvivoGen) was mixed and then gently shaken on ice for 1 hour to prepare alum-rHylA and alum-rHylB. The vaccine was administered at a dose of 70 μg for the first injection and 50 μg for the subsequent two injections. Serum samples were collected seven days after the last vaccination to assess antibody titers against rHylA or rHylB. To assess the protective effect of the vaccine against acne, two weeks after vaccination, Propionibacterium acnes strains (2 × 10⁻⁶) were used to analyze the antibody titers against rHylA or rHylB. 7 Mice were challenged with CFU (CFU / mL) id. As previously described, bacterial counts (CFU / mL), skin lesion size, and pro-inflammatory cytokines were determined on days 1 and 2 post-challenge. Adoptive transfer of T cells

[0223] Spleens collected on day 10 after the last vaccination were homogenized in sterile PBS (pH 7.4) and then subjected to RBC lysis (catalog number: 00-4300-54, eBioscience). TM ), and use MojoSort TM Mouse CD3 T cell isolation kit isolated CD3 cells using negative selection according to the manufacturer's instructions. + T cells (catalog number #480031, Biolegend, USA). 1×10 7 CD3 + T cells were injected retro-orbitally into untreated recipient mice. The injection was performed under isoflurane (Fluriso, Vet One) anesthesia. Twenty hours after cell transfer, the cells were injected with HL043PA1 (2 × 10⁻⁶ cells). 7 Mice were challenged with CFU on day 2, followed by CFU determination, disease scoring, and skin cytokines analysis as described above. Serum neutralization of HylA enzyme activity

[0224] HylA enzyme (0.3 μg) was incubated with 10 μL of serum (n = 5) from either the reagent control or mEHylA-vaccinated mice at 37°C for 20 min. HA (2 mg / mL) was added to the mixture, and the mixture was incubated at 37°C with continuous shaking for 20 hr. Subsequent HPLC analysis was performed as described above. Identification of cytokines in skin lesions

[0225] The levels of IL-1β, IL-6, and TNF-α cytokines in skin homogenates previously stored at -80°C were measured using a solid-phase sandwich ELISA kit for mouse cytokines (Biolegend, San Diego, CA, USA). Assays were performed with biological replicates following the manufacturer's instructions. Skin homogenates (50 μL) for IL-1β and IL-6 were diluted 1:1 with blocking buffer (1% BSA plus 1×PBS-Tween 20), and undiluted skin homogenates (100 μL) for TNF-α were used in the assay along with cytokine standards (supplied with the kit) at known concentrations in each ELISA plate. Plates were developed and read at optical density (OD) at 450 nm using a multimode microplate reader (PerkinElmer, Waltham, MA, USA), with wavelength correction set to 570 nm. A standard curve generated from the OD of the cytokine standards was used to determine cytokine levels in the samples. To determine the cytokine levels in HaCaT cell culture supernatant, a human IL-6 and IL-8 cytokine ELISA kit was purchased from Biolegend. The culture supernatant was diluted 1:1 and the assay was performed as described above. Determination of antibody titer

[0226] Serum antibody titers against rHylA and rHylB were measured using an indirect ELISA method. Briefly, 96-well high-binding microplates (catalog number #655081, Greiner Bio-one) were coated overnight at 4°C with 500 ng of rHylA or rHylB in carbonate-bicarbonate buffer (0.2 M, pH 9.6). Plates were washed three times with PBS-T and blocked for 1 hour at room temperature with continuous shaking. After washing, serum samples diluted in PBS-T (1:100, 1:1000, 1:10,000, 1:100,000) were applied to the wells with biological replicates, and plates were incubated at room temperature with continuous shaking for 2 hours. Antibody titers (IgM, IgG, IgG1, IgG2b, and IgG3) were measured for 1 hour at a 1:5000 dilution in blocking buffer with continuous shaking at room temperature. After stepwise washing (three times), the plates were developed with TMB substrate (catalog number #555214, BD Bioscience, CA, USA) at room temperature for 3 min, and the reaction was stopped by adding 1N H2SO4. Plates were read at 450 nm optical density (OD) using a multimode microplate reader (PerkinElmer, Waltham, MA, USA), with wavelength correction set to 570 nm. Statistical analysis and data reproducibility

[0227] All data were analyzed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, graphpad.com). Specific statistical analyses are indicated in the legend. In vitro experiments were performed independently 2–3 times using at least three techniques. Data are presented as mean ± standard deviation. In vitro data were analyzed using the nonparametric Mann–Whitney Student's t-test and one-way ANOVA. All in vivo mouse data are presented 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 test). Multiple group comparisons were performed using one-way ANOVA and Tuckerey's post-hoc test. In the absence of normality, data were analyzed using nonparametric Kruskal-Wallis one-way ANOVA.

[0228] Various embodiments of the present invention have been described in the specific embodiments above. Although these descriptions directly illustrate the above embodiments, it will be understood that modifications and / or alterations to the specific embodiments shown and described herein will be conceived by those skilled in the art. Any such modifications or alterations falling within the scope of this specification are also intended to be included therein. Unless otherwise stated, the inventors intend that the words and phrases in the specification and claims be given the ordinary and conventional meaning understood by one of ordinary skill in the art.

[0229] The foregoing description of various embodiments of the invention known to the applicant at the time of filing this application has been given and is intended for illustrative and descriptive purposes. This description is not intended to be exhaustive, nor to limit the invention to the precise forms disclosed, but rather, given the foregoing teachings, many modifications and variations are possible. The described embodiments are intended to explain the principles of the invention and its practical application, and to enable others skilled in the art to utilize the invention in various embodiments with various modifications suitable for the particular use contemplated. Therefore, the invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention.

[0230] While specific embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made based on the teachings herein without departing from the invention and its broader aspects. Therefore, the appended claims are intended to cover all such changes and modifications within their scope that fall within the true spirit and scope of the invention. As used herein, the term “comprising / including / containing” is used to refer to compositions, methods, and corresponding components useful to embodiments, but also includes elements not specified, whether useful or not. Those skilled in the art will understand that, in general, the terms used herein are intended to be “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” the term “comprising” should be interpreted as “including but not limited to,” etc.). Although the open-ended term “comprising” is used herein as a synonym for terms such as “comprising,” “containing,” or “having,” it is also used to describe and claim the invention, but alternative terms such as “consisting of” or “substantially consisting of” may be used to describe the invention or embodiments thereof.

[0231] Unless otherwise stated, the terms “a / an” and “the”, and similar references used in the context of describing a particular embodiment of this application (particularly in the context of the claims), may be interpreted as encompassing both the singular and the plural. The enumeration of ranges of values ​​herein is intended only as a convenient method of individually referring to each separate value falling within that range. Each individual value is incorporated into the specification as if individually enumerated herein. All methods described herein may be performed in any suitable order unless otherwise stated herein or clearly contradicted by the context. The use of any and all instances or exemplary language (e.g., “such as”) provided herein with respect to certain embodiments is intended only to better elucidate the application and does not constitute a limitation on the scope of the otherwise claimed application. The abbreviation “eg” originates from the Latin *exempli gratia* and is used herein to denote a non-limiting instance. Therefore, the abbreviation “eg” is synonymous with the term “for example.” No language in the specification should be construed as indicating any unclaimed element essential to the practice of this application.

[0232] "Optional" or "optionally" means that the situation described below may or may not occur, and therefore the description includes both the possibility that the situation occurs and the possibility that the situation does not occur.

[0233] The grouping of alternative elements or embodiments of this disclosure herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, this specification is hereby deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

Claims

1. An immunogenic polypeptide, said immunogenic polypeptide comprising: A fragment of HylA.

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

3. The immunogenic polypeptide according to claim 1 or claim 2, wherein, The HylA fragment contains one or more peptides selected from the following: (a)EMPDAFASPDPDIW (SEQ ID NO: 1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO: 1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO: 1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), wherein the variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2) has at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), 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)ENSSDRISVSRS (SEQ ID NO: 3), (f) A variant of ENSSDRISVSRS (SEQ ID NO: 3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO: 3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO: 4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO: 4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO: 4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

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

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

6. A polypeptide, said polypeptide comprising: Selected from one or more of the following peptides: (a)EMPDAFASPDPDIW (SEQ ID NO: 1), (b) A variant of EMPDAFASPDPDIW (SEQ ID NO: 1) having at least 50% sequence identity with EMPDAFASPDPDIW (SEQ ID NO: 1), having an addition, substitution, or deletion of up to 7 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (c) VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), (d) A variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), wherein the variant of VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2) has at least 50% sequence identity with VATILTDLASSSSRTTVLLSANLQKEESS (SEQ ID NO: 2), 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)ENSSDRISVSRS (SEQ ID NO: 3), (f) A variant of ENSSDRISVSRS (SEQ ID NO: 3) having at least 50% sequence identity with ENSSDRISVSRS (SEQ ID NO: 3), having an addition, substitution, or deletion of up to 6 amino acids, or having a linear epitope score of at least 0.4, or a combination thereof. (g)ALPKPTKPSLRASSYPLGLP(SEQ ID NO: 4), or (h) A variant of ALPKPTKPSLRASSYPLGLP (SEQ ID NO: 4) having at least 50% sequence identity with ALPKPTKPSLRASSYPLGLP (SEQ ID NO: 4), having up to 10 amino acid additions, substitutions or deletions, or having a linear epitope score of at least 0.4, or a combination thereof.

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

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

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

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

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

12. The polypeptide according to any one of claims 9-11, wherein, The connector is G, polyserine, polyglycine, glycine-serine, GGGGS (SEQ ID NO:5), GGGGGS (SEQ ID NO:6), leucine zipper, r aliphatic or helical peptide.

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

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

15. An mRNA molecule encoding a polypeptide according to any one of claims 6, 11 or 12.

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

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