HK1164158A - Methods and compositions for treating p. acnes - Google Patents

Description

Methods and compositions for treating propionibacterium acnes

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application serial No. 61/120,221, filed on 5.12.2008, which is incorporated herein by reference.

Technical Field

The present disclosure relates generally to antigenic compositions for immunization against propionibacterium acnes (p.acnes). Methods for producing vaccines for preventing propionibacterium acnes-related diseases and conditions, including rosacea in humans and animals, vaccines against propionibacterium acnes in humans and animals, and methods of producing vaccines against propionibacterium acnes are disclosed.

Background

Gram-positive anaerobic corynebacterium acnes (Propionibacterium acnes), a member of the human microflora, is found mainly in the sebaceous glands of the skin. However, it can also be isolated from the conjunctiva, the external auditory canal, the oral cavity, the upper respiratory tract and in some individuals also from the intestine. Propionibacterium acnes has a size of 10²To 10^5-6cm^-2The estimated skin density of. Propionibacterium acnes isRecognized opportunistic pathogens are particularly associated with medical implants such as central nervous system shunts, silicone implants and prosthetic hip joints. It also causes infections and endophthalmitis in and around the eye and is also involved in periodontal and dental infections. Dental exploration and treatment results in the transmission of propionibacterium acnes in the blood, a recognized cause of endocarditis associated with lesions or prosthetic valves. Propionibacterium acnes also plays a role in inflammatory acne because antibacterial treatment against propionibacterium acnes results in improvement, whereas resistance of propionibacterium acnes to antibiotics is associated with recurrence. The common form of acne, acne vulgaris, sometimes affects up to 80% of the population in life, making it the most common skin infection. There is also a strong correlation between severe forms of acne and joint pain, bone inflammation (osteitis) and arthritis. In patients with this disorder, known as the SAPHO (synovitis, acne, impetigo, hypertrophy of bone and osteitis) syndrome, propionibacterium acnes isolates have been obtained from biopsies of bone as well as synovial fluid and tissue.

Two distinct phenotypes of propionibacterium acnes, type I and type II, have been identified based on serological agglutination assays and cell wall sugar analysis. Recently, sequence analysis based on recA has shown that Propionibacterium acnes type I and II represent different groups of phylogeny (McDowell et al, 2005).

Propionibacterium acnes produce a co-hemolytic reaction with both sheep and human erythrocytes (Choudhury, 1978) similar to the Christie-Atkins-Munch-Petersen (CAMP) reaction first demonstrated in 1944 (Christie et al, 1944). The CAMP reaction describes the synergistic hemolysis of sheep red blood cells by a CAMP factor from Streptococcus agalactiae (Streptococcus agalactiae) and a toxin from Staphylococcus aureus (sphingomyelinase C), demonstrating the non-enzymatic affinity of the CAMP factor for ceramides (Bernheimer et al, 1979). Examination of sphingomyelinase treated immune erythrocytes has shown that discrete membrane pores are formed by the recombinant streptococcus agalactiae CAMP factor (Lang and Palmer, 2003). In addition to the extensive studies of CAMP factors in Streptococcus agalactiae (Bernheimer et al, 1979; Brown et al, 1974; Jurgens et al, 1985, 1987; Ruhlmann et al, 1988; Skala et al, 1980), it is known that many other gram-positive and gram-negative bacteria produce positive CAMP reactions, including Pasteurella haemolytica (Pasteurella haemolytica) (Fraser, 1962), Aeromonas (Aeromonas) (Figura and Guglielmetti, 1987), several species of the genus Vibrio (Vibrio) (Kohler, 1988) and group G streptococci (Soedermanto and Lammler, 1996). Some of these species may utilize, in addition to the use of staphylococcus aureus toxin, phospholipase C from Clostridium perfringens (Clostridium perfringens) (alpha-toxin) or phospholipase D from Corynebacterium pseudotuberculosis (Frey et al, 1989) as a cofactor for hemolysis. The CAMP factor genes of Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) and Streptococcus uberis (Streptococcus uberis) have been identified, cloned and expressed in E.coli (Frey et al, 1989; Jiang et al, 1996).

The exact role of CAMP molecules in bacterial virulence is still unknown. It is likely that the co-hemolytic reaction represents a laboratory phenotype, or side-phenomenon, that facilitates CAMP factor detection, but this may not be directly related to the role of the molecule in colonization and pathogenesis. CAMP factors from streptococcus agalactiae bind to the Fc region of IgG and IgM molecules, similarly to IgG binding to staphylococcus aureus protein a (Jurgens et al, 1987), and partial amino acid sequence similarity between streptococcus agalactiae CAMP factor proteins and staphylococcus aureus protein a has been demonstrated (Ruhlmann et al, 1988).

Disclosure of Invention

The present disclosure provides compositions and methods for treating or preventing propionibacterium acnes infection. In one embodiment, the methods and compositions include an asmase inhibitor, including, for example, a small molecule inhibitor or an anti-asmase antibody. In another embodiment, the compositions and methods comprise a vaccine comprising a propionibacterium acnes CAMP factor. In yet another embodiment, the methods and compositions comprise anti-propionibacterium acnes CAMP factor antibodies. In yet another embodiment, the methods and compositions comprise a combination of an anti-CAMP factor vaccine or antibody and an asmase inhibitor or antibody.

The present disclosure also provides immunogenic compositions comprising a substantially pure polypeptide comprising a sequence as indicated in table 1, immunogenic fragments thereof, and combinations of any of the foregoing. In one embodiment, the CAMP factor, lipase, or sialidase polypeptide or fragment thereof is used in an immunogenic composition. In yet another embodiment, the polypeptide comprising SEQ ID NO: 2. 3,7, 9 or 11 or an immunogenic fragment thereof for use in the preparation of an immunogenic composition. In yet another embodiment, the polypeptide comprises a sequence encoding SEQ ID NO: 2. 3,7, 9 or 11 or an antigenic fragment thereof in a vector administered to a subject. In one embodiment, the vector comprises an attenuated bacterial vector or an attenuated viral vector. In yet another embodiment, the antigen is expressed by a plant or plant cell.

The present disclosure also provides compositions comprising at least one recombinant attenuated bacterial or viral vector and an inhibitor of ASM enzyme activity, wherein the at least one recombinant attenuated bacterial or viral vector comprises at least one polynucleotide encoding one or more propionibacterium acnes polypeptides selected from the group consisting of CAMP factor, lipase, or sialidase, whereby the polypeptides are expressed in the at least one recombinant attenuated vector.

The present disclosure also provides a method of inducing protective immunity in a subject comprising administering to the subject the above composition and contacting the subject with an asmase inhibitor.

The present disclosure also provides an immunoprotective composition comprising at least one attenuated vector or plant preparation expressing an antigen for inducing an immunoprotective response against propionibacterium acnes, said antigen comprising a propionibacterium acnes extracellular or immunogenic protein or immunogenic fragment thereof linked to a transcriptional promoter and a termination signal. In one embodiment, the propionibacterium acnes protein or fragment thereof is selected from the group consisting of CAMP factor, lipase, sialidase, and any combination thereof.

The present disclosure provides compositions comprising ASM enzyme inhibitors for treating propionibacterium acnes infection. In yet another embodiment, a CAMP antigen or vaccine can be used in combination with an asmase inhibitor. In yet another embodiment, an antigenic composition comprising disrupted non-infectious propionibacterium acnes cells and further comprising an ASM enzyme inhibitor is used.

The present disclosure provides methods of treating propionibacterium acnes comprising administering to a subject a vaccine comprising a CAMP factor and a composition comprising an ASM enzyme inhibitor.

Drawings

Figures 1A-B show otitis and thickness following injection of propionibacterium acnes and staphylococcus epidermidis (s. epidermidis). When ICR mice were injected subcutaneously with 25. mu.l Propionibacterium acnes (10)⁸CFU) otitis (a) was observed. Injection of 25 μ l PBS into the other ear of the same mouse did not cause visible inflammation. Propionibacterium acnes (10)⁵To 10⁸CFU) and Staphylococcus epidermidis (10)⁸) Injected subcutaneously into the skin of mice. Ear thickness was measured daily using a peach thickness meter for 3 days (B). The ears of two mice per group were measured.

FIGS. 2A-F show that Propionibacterium acnes causes a granulomatous response and colonizes the root of the hair follicle. H&E staining showed injection of Propionibacterium acnes into the mouse ear (10)⁸CFU) increased ear thickness and caused a granulomatous response (short arrow) for 1 day (B). PBS injection served as control (a). Granuloma reaction zones were stained with Accustain gram stain (gram positive bacterial staining kit) (Sigma, st. louis, MO). Propionibacterium acnes (circles; purple staining) was surrounded by a densely packed granulomatous infiltrate 1 day after injection (C). No propionibacterium acnes (D) accumulated in the hair follicles 1 day after injection. However, 2 days after injection, propionibacterium acnes (circles and arrows) migrated to the hair follicle and colonized the root of the hair follicle (F). Follicular histology from mice injected with PBS for 2 days was used as control (E). A scale: 100 μm.

Figures 3A-C show the implantation of a tissue chamber and phagocytic cells within the fluid of the tissue chamber. Tissue compartments (1.5 and 3mm inner and outer diameter, respectively, length 1 cm; internal volume 80. mu.l) were implanted subcutaneously into the abdominal skin of ICR mice 7 days before bacterial injection (A). The tissue chamber consists of a closed teflon cylinder with 12 regularly spaced 0.1mm holes. A scale: 1 cm. H & E staining showed that the mouse tissue enclosed the tissue compartment 7 days after implantation (B). A scale: 1.0 mm. Tissue chamber fluid is withdrawn by percutaneous aspiration. After centrifugation, the infiltrated cells (phagocytes) were stained with the nuclear dye Hoechst 33258 (C). Arrows indicate phagocytes within the tissue compartment. A scale: 5 μm.

FIGS. 4A-B show the determination of Macrophage Inflammatory Protein (MIP) -2 concentration and the growth of Propionibacterium acnes in the tissue chamber fluid. 7 days after implantation in the tissue chamber, Propionibacterium acnes and Staphylococcus epidermidis (20. mu.l; 10)⁷CFU) or PBS (20 μ l). Tissue chamber fluid sampling was performed 3 days after the bacterial injection. Measurement of MIP-2 in fluid supernatant by use of Quantikine M mouse MIP-2 kit (R)&D systems, Minneapolis, MN) (a). Propionibacterium acnes growth in vivo was detected by spreading the tissue chamber fluid on MHB agar plates to quantify CFU (B).

FIGS. 5A-D show the quantitative analysis of Propionibacterium acnes proteome changes using isotope-encoded protein tags (ICPL): identification of CAMP factor and lipase. Propionibacterium acnes grow under aerobic and anaerobic conditions. Lysates from aerobically and anaerobically grown Propionibacterium acnes (1mg) were individually labeled with ICPL Label C¹²-N-nicotinic acid oxy-succinimide (Nic-NHS) and C¹³-Nic-NHS marker. All lysine side chains of the protein in the lysate were selectively modified. In the process of mixing C¹²-Nic-NHS labelled sample with C¹³After mixing the Nic-NHS labeled samples, the mixture was subjected to protein identification and quantification using an LTQ mass spectrometer (Thermo Electron Corp. Waltham, Mass.). The labeling of the two labels causes a mass difference of 6Da at each labeled site in the mass spectrum. Over 300 propionibacterium acnes proteins were identified. 23 proteins in anaerobic or aerobic conditionsDown or up or down (table 1). Secreted virulence factors (lipase and CAMP factor) with double charge and 3Da mass difference (a and B) are shown. Under anoxic conditions, both virulence factors in propionibacterium acnes are more highly expressed. Two peptides (SYSEKHLGVAFR (SEQ ID NO: 1) and DLLKAAFDLR (SEQ ID NO: 2)) were sequenced and designated as internal peptides for lipase (C) and CAMP factor (D), respectively.

Figures 6A-G show that removal of sialic acid by sialidase increases the susceptibility of human sebaceous gland cells to propionibacterium acnes. Sialic acid on the cell surface of immortalized human sebaceous gland cells (SZ95) was detected by reaction with biotinylated Maackia Amurensis (AA) lectin I (10. mu.g/ml) and streptavidin-FITC conjugate. FITC-fluorescence intensity was counted by flow cytometry to reflect sialic acid levels (A). Sebaceous gland cells were pretreated with PBS (vehicle), sialidase (10. mu.g/ml) (green, A) or GFP (10. mu.g/ml) (B) at pH6 for 2 hours. The decrease in FITC-fluorescence intensity in sialidase-treated sebaceous gland cells indicates that pure sialidase is the effector enzyme. Sebaceous gland cells were pretreated with Propionibacterium acnes (10. mu.g/ml, 2 hours) after sialidase (10. mu.g/ml)⁷CFU/10⁶Cells) were co-cultured for 24 hours. Cell death induced by propionibacterium acnes in vehicle, sialidase or GFP-treated sebaceous gland cells was counted by trypan blue staining (C). After washing off the suspended Propionibacterium acnes, the number of Propionibacterium acnes attached to sebaceous gland cells was counted by plating trypsin-affected sebaceous gland cells on MHB agar plates to quantify CFU/cell (D). Adhesion of propionibacterium acnes into vehicle (E), sialidase (F, arrow) or gfp (g) treated sebaceous gland cells was visualized by staining with Accustain gram kit.

Figures 7A-C show that sialidase is immunogenic when mice are immunized with either an e.coli vector based vaccine or recombinant protein/freund's (non) complete adjuvant. An irradiated E.coli vector-based vaccine (E.coli BL21(DE3) T7/lacO sialidase) was constructed by inserting the PCR product of sialidase into the pEcoli-Nterm6xHN vector (Clontech). Post-vaccination by vaccination 6Wechsler western blot analysis detection of E.coli vectors (10)⁹CFU) production of antibodies in immunized mice (a). Mice immunized with the empty vector of E.coli (lacZ) served as negative controls. ICR mice were also immunized with recombinant sialidase-6 xNH fusion protein or GFP using Freund/(non) complete adjuvant. For the first injection of subcutaneous vaccination, with complete Freund's adjuvant emulsion of 200 u g sialidase-6 xNH fusion protein or GFP inoculated mice. A second injection was performed two weeks after the injection. Mice were injected intramuscularly with the same amount of antigen as was fully slurried with incomplete freund's adjuvant. Anti-sialidase antibodies were detected by western blot (B) and antigen microarray (C) one week after the second vaccination. 0.35 μ g of purified sialidase-6 xNH fusion protein and the indicated IgG were spotted twice onto the antigen microarray. Data represent three independent experiments with similar results. Sialidase antibodies were elicited when mice were immunized with both an E.coli vector-based vaccine and recombinant protein/Freund's adjuvant.

Figure 8 shows protective immunity of sialidase-based vaccines against propionibacterium acnes-induced ear thickness. ICR mice were immunized with recombinant sialidase-6 xNH fusion protein or GFP using Freund's (non) complete adjuvant. After confirmation of antibody production by western blot, Propionibacterium acnes (10)⁷CFU, 25 μ l) were injected subcutaneously into the ear of sialidase and GFP immunized mice. PBS (25. mu.l) was injected to serve as a control. Ear thickness was measured for 9 days post injection and calculated as% of ear thickness of PBS injected ears.

FIG. 9 shows an in vitro anti-sialidase antiserum. Propionibacterium acnes were preincubated with anti-sialidase antiserum for 2 hours. Immortalized human sebaceous gland cells (SZ-95) were co-cultured with antiserum-treated Propionibacterium acnes for 18 hours. After incubation, cell death of sebaceous gland cells induced by cytotoxicity of propionibacterium acnes was determined using pNPP. Immortalized human sebaceous gland cell line SZ95 cultured in 96-well plates up to 2X 10⁵Cell/well Density, cultured in Sebomed basal Medium (Biochrom, B) supplemented with 5ng/ml human recombinant epidermal growth factor (Sigma, St. Louis, MO), 10% (v/v) heat-inactivated fetal bovine serum (Mediatech Inc., Herndon, Va.)erlin, Germany), 5% (v/v) CO in air₂At 37 ℃. Propionibacterium acnes were cultured as described above and washed with PBS by centrifugation. Propionibacterium acnes were suspended in Sebomed basal medium containing 25% (v/v) anti-sialidase or anti-GFP (control) antiserum and incubated at 37 ℃ for 2 hours. Sebaceous gland cells were washed twice with PBS and then incubated with PBS containing 2X 10⁶A100. mu.l neutralization reaction mixture of CFU Propionibacterium acnes and 2.5. mu.l antiserum was incubated for 18 hours. As a control, an equal amount of PBS was added instead of propionibacterium acnes. As background, Triton-X was added to a final concentration of 0.1% (v/v) to kill sebaceous gland cells. After incubation, the neutralization mixture was tested for cytotoxicity using disodium p-nitrophenylphosphate (pNPP). Sebaceous gland cells were washed 3 times with PBS and incubated with 100. mu.l of 2.5% (w/v) pNPP in ACPI for 1 hour at 37 ℃. After the incubation, 10. mu.l of 1N NaOH was added to stop the reaction and the absorbance at 405nm was measured. The cytotoxicity of the neutralization mixture was calculated as (propionibacterium acnes-propionibacterium acnes addition group) ÷ (propionibacterium acnes-free group-background group) × 100.

FIGS. 10A-F show protective immunity of an inactivated Propionibacterium acnes vaccine. ICR mice heat killed Propionibacterium acnes (10)⁸CFU) and boosted twice at three week intervals. After 10 weeks of immunization (one week for the second booster immunization), live Propionibacterium acnes (10)⁷CFU, 25 μ l) or PBS (25 μ l) were injected subcutaneously into the ears of killed propionibacterium acnes immunized and PBS vaccinated mice. Ear thickness was calculated as% ear thickness of PBS-injected ears (a). The ears of the killed propionibacterium acnes immunized (B, D) and PBS inoculated (C, E) mice were shown to be red after 24(B, C) and 72 hours (D, E) of live propionibacterium acnes injection. Measurement of MIP-2 in the fluid supernatant was performed by sandwich ELISA. In killed mice immunized with Propionibacterium acnes, the mice were immunized with Propionibacterium acnes (20. mu.l; 10)⁷CFU) injection induced elevation of MIP-2 was considerably inhibited (F).

FIG. 11 shows characterization of Propionibacterium acnes CAMP factor. (A) Recombinant propionibacterium acnes CAMP factor is expressed in e.Coli transformed with the pEcoli-Nterm6XHN vector containing a cDNA insert encoding the CAMP factor was either incubated without (lane 1) or with (lane 2) IPTG, disrupted and separated by SDS-PAGE (10% acrylamide). Purified CAMP factor is shown in the right panel. (B) The expression and purity of the CAMP factor was confirmed by NanoLTQ MS/MS mass spectrometry. The internal peptide of the sequenced CAMP factor is presented (AVLLTANPASTAK; SEQ ID NO: 3)). (C) The co-hemolytic activity of the recombinant CAMP factor was examined on sheep blood agar plates. Staphylococcus aureus strain 113 (2X 10)⁵CFU/10. mu.l) were streaked onto agar plates. Mu.l of recombinant CAMP factor (250. mu.g/ml) or GFP (250. mu.g/ml) as a control protein was spotted next to the S.aureus streaks. (D) Immunogenicity of CAMP factor in ICR mice was assessed by Western blot. Mice were inoculated intranasally with UV-killed e.coli overexpressing CAMP factor or GFP. Mice were bled 14 days after vaccination. anti-CAMP factor (1: 2000 dilution; lanes 1 and 2) or anti-GFP antiserum (lanes 3 and 4) was reacted with recombinant CAMP factor (0.2. mu.g; lanes 1 and 3) or GFP (lanes 2 and 4). Immunoreactivity was detected using goat anti-mouse IgG (H + L) -HRP conjugate. (E) The titers of CAMP factor antibodies were determined by ELISA. Mice (n-10) were bled at 14 and 21 days after vaccination with CAMP factor or GFP vaccine. Antiserum (diluted 1: 10000) was reacted with CAMP factor immobilized on microtiter ELISA plates. With goat anti-mouse IgG (H + L) -HRP conjugate and OptEIA^TMThe kit detects the collected antibodies. The optical density of each well was measured at 450 nm. The horizontal line represents the average of 10 independent determinations. (F) CAMP factor in propionibacterium acnes culture supernatants was detected by Western blot. Recombinant CAMP factor (0.2. mu.g; lane 1) as a positive control, 10-fold concentrated Propionibacterium acnes culture supernatant (70. mu.g total protein; lane 2) and 10-fold concentrated RCM (70. mu.g total protein; lane 3) as a negative control were separated by SDS-PAGE (10% acrylamide), transferred to a polyvinylidene fluoride membrane and reacted with mouse anti-CAMP factor antiserum (1: 1000 dilution, left panel) or anti-GFP antiserum (right panel). The 6 × HN tag of the recombinant CAMP factor was removed by enterokinase prior to loading into SDS-PAGE. (G) In human keratinocyte cell line (HaCaT) or murine macrophage cell lineThe cytotoxicity of recombinant CAMP factors was examined (RAW 264.7). Cell (1X 10)⁵Per well) were incubated with the indicated concentrations of recombinant CAMP factor or GFP at 37 ℃ for 18 hours. After incubation, cell viability was determined and cytotoxicity calculated as described in methods. Data are expressed as mean ± SE (n ═ 6, p < 0.005 by Student's t-test compared to GFP control^**And p < 0.0005^***). (H) Intradermal injection of CAMP factor induced an inflammatory response in the ear of ICR mice. The left ear was injected intradermally with recombinant CAMP factor (10. mu.g/20. mu.l) or GFP (10. mu.g/20. mu.l) in PBS. The right ear received an equal amount of PBS (20. mu.l). Ear thickness was measured with a microcard after 24 hours of injection and changes were recorded as% of ear thickness of PBS-injected ears. Data are expressed as mean ± SE (n ═ 4, p < 0.005 by Student's t-test^**)。

FIGS. 12A-C show that bacterial CAMP factors and host ASM enzymes are involved in the in vitro pathogenicity of Propionibacterium acnes. (A) After co-culture with propionibacterium acnes, cell culture supernatants were examined for CAMP factor and ASM enzymes by Western blot. HaCaT (lanes 1 and 3) or RAW264.7 (lanes 2 and 4) (5X 10)⁵Perpore) and Propionibacterium acnes (5X 10)⁶CFU/well; MOI 1: 10) (lanes 1 and 2) or no co-culture with Propionibacterium acnes (lanes 3 and 4) in serum-free medium at 37 ℃ for 14 hours. A concentrate of cell culture supernatant (10. mu.g total protein) was subjected to Western blotting. The CAMP factor and ASM enzyme were detected with mouse anti-CAMP factor antiserum and goat anti-ASM enzyme IgG, respectively. (B) Propionibacterium acnes-mediated cell death was neutralized in vitro by anti-CMAP factor antiserum. HaCaT or RAW264.7 cells (1X 10)⁵Perpore) and Propionibacterium acnes (1X 10)⁶CFU/well; MOI 1: 10) were co-cultured for 14 hours in the presence of mouse anti-CAMP factor or anti-GFP antiserum (2.5% v/v). (C) Addition of ASM enzyme inhibitors reduced propionibacterium acnes-mediated cell death in vitro. HaCaT or RAW264.7 cells (1X 10)⁵Per well) with or without Propionibacterium acnes (1X 10)⁶CFU/well, MOI 1: 10) were incubated in medium containing the selective ASM enzyme inhibitor desipramine (10 μ M) or an equivalent amount of PBS (vehicle) at 37 ℃ for 14 hours. After incubation, cell viability was determined and cytotoxicity calculated as described in materials and methods. Data are expressed as mean ± SE (n ═ 10, p < 0.05 by Student's t-test^*And p < 0.0005^***)。

FIGS. 13A-D show that host ASM enzymes may be involved in Propionibacterium acnes pathogenicity in vivo. (A) The amount of soluble ASM enzyme in the mouse ear increased 24 hours after bacterial challenge. ICR mice were injected intradermally into the ear with Propionibacterium acnes (1X 10) in PBS⁷CFU/20 μ l; left ear) or PBS (20 μ l; right ear) and cut after 24 hours. Ear tissue was obtained using an 8mm biopsy and homogenized in PBS. The supernatant (1. mu.g total protein) was subjected to Western blotting. ASM enzyme (upper panel) and GAPDH (lower panel) were detected with goat anti-ASM enzyme IgG followed by anti-GAPDH IgG (left panel). Normal goat or mouse IgG was used as a negative control for detection (right panel). (B) Stimulation of propionibacterium acnes in mice attracts CD11b + macrophages, which highly express ASM enzyme. Frozen sections of mouse ears obtained 24 hours after bacterial stimulation were stained with biotinylated anti-mouse CD11b IgG, a conventional macrophage marker, and TRITC streptavidin conjugate, followed by goat anti-asmase IgG and anti-goat IgG-TRITC conjugate. Nuclei were stained with DAPI (blue). Transmission electron microscopy (10000 × magnification) for presentation of colonized propionibacterium acnes and ruptured cell membranes in ears of mice injected with propionibacterium acnes or PBS, scale 200 μm. PA, propionibacterium acnes; CM, cell membrane; NC, cell nucleus. Scale bar 1 μm. (D) Systemic pretreatment of ICR mice with selective ASM enzyme inhibitors reduced propionibacterium acnes-induced inflammation. ICR mice were injected intraperitoneally 30 minutes prior to bacterial challenge with desipramine (20mg/kg mice) or an equivalent amount of PBS (vehicle). After pretreatment, live Propionibacterium acnes (1X 10) in PBS were each separately added⁷CFU/20 μ l) or equivalent amounts of PBS (control) were injected intradermally into the left or right ear. Ear thickness was measured using a microcard before and 24 hours after bacterial stimulation and changes were recorded as% of ear thickness of PBS-injected ears. Data are expressed as mean ± SE (n ═ 3, p < 0.005 by Student's t-test^**)。

FIG. 14 shows that the combination of CAMP factor vaccine and local injection of anti-ASM enzyme IgG synergistically inhibits Propionibacterium acnesBacillosis induced inflammation. ICR mice were inoculated with UV killed e.coli overexpressing CAMP factor or GFP at 3 week intervals. 2 weeks after the second booster vaccination, Propionibacterium acnes was injected intradermally into the ears of vaccinated mice in the same manner as described above. Within 30 minutes, the left ear (which had received propionibacterium acnes) was injected with goat anti-ASM enzyme IgG (4 μ g/20 μ l) or normal goat IgG in PBS (control), and the right ear with an equal volume of PBS (n ═ 8). Ear thickness was measured 24 hours after bacterial challenge and changes were recorded as% of ear thickness of PBS-injected ears. Data are expressed as mean. + -. SE (p < 0.05 by Student's t-test^*，p＜0.005^**，p＜0.0005^***)。

Figures 15A-C show the effect of CAMP factor-based vaccines on propionibacterium acnes-induced inflammation in mice. (A) Intradermal injection of CAMP factor induced an inflammatory response in the ear of ICR mice. The left ear was injected intradermally with recombinant CAMP factor (10. mu.g/20. mu.l) or GFP (10. mu.g/20. mu.l) in PBS. The right ear received the same amount of PBS (20. mu.l). Ear thickness was measured using a micro caliper 24 hours after injection and changes were recorded as% of ear thickness of PBS injected ears. Data are expressed as mean ± SE (n ═ 4, P < 0.005 by Student's t-test^**). (B) The titers of CAMP factor antibodies were determined by ELISA. Mice were bled 14 and 21 days after vaccination with CAMP factor (n-10). Antiserum (1: 10000 dilution) was reacted with CAMP factor immobilized on microtiter ELISA plates. With goat anti-mouse IgG (H + L) -HRP conjugate and OptEIA^TMThe kit detects the collected antibodies. The optical density at 450nm of each well was measured. The horizontal line represents the average of 10 independent determinations. (C) Immunization of ICR mice with CAMP factor alone provided therapeutic immunity against propionibacterium acnes-induced inflammation. Live Propionibacterium acnes (1X 10) in PBS⁷CFU/20 μ l) or equivalent amounts of PBS (control) were injected intradermally into the left or right ear, respectively, of naive mice. After 24 hours, mice were immunized intranasally with UV killed e.coli (arrow) overexpressing CAMP factor or GFP. Ear thickness was measured at the indicated time post bacterial challenge and changes were recorded as% PBS injected ear thickness. Data are expressed as mean ± SE (n is 10, by Student's t testTest P < 0.05^*，P＜0.005^**，P＜0.0005^***)。

FIGS. 16A-B show the in vitro co-cytotoxic properties of CAMP factor and bacterial SM enzyme. HaCaT (A) or RAW264.7 cells (B) were pretreated with SM enzyme from Staphylococcus aureus (350mU/ml) or an equivalent amount of vehicle for 15 minutes, washed three times to remove the enzyme, and then incubated with recombinant CAMP factor (25. mu.g/ml) or GFP for 18 hours at 37 ℃. After incubation, cell viability was determined and cytotoxicity calculated. Data are expressed as mean ± SE (n ═ 6, p < 0.0005 by Student's t-test^***)。

FIGS. 17A-B show that Propionibacterium acnes CAMP factor exerts virulence activity. The ears of ICR mice were injected intradermally with recombinant GFP (left ear) and CAMP factor (right ear). (A) Redness of the ear (arrows) inducing inflammation was seen 24 hours after injection. (B) Ear swelling was observed in H & E stained frozen tissue sections of GFP (I, iii) or CAMP factor (ii, iv) injected ears. The magnified images [4 × (i, iii) and 20 × (ii, iv) ] show the deposition of ruptured erythrocytes (short arrows). And the scale (a) is 1 cm. Scale [ b (I, iii) ] -2 mm. Scale [ b (ii, iv) ] -0.5 mm.

FIGS. 18A-D show transient expression of CAMP factor and GUS in radish leaves. (a) Radish (radish sativus L.) leaves were infiltrated with 35S: (right) Agrobacterium tumefaciens (LBA4404 strain) of GUS construct. Leaves infiltrated with non-transformed LBA4404 cells (left) served as negative controls. The dotted circle indicates the site of injection of infiltrating A.tumefaciens. Blue stained areas indicate GUS expression. The dynamic pattern of GUS expression in radish leaves 1 to 5 days after infiltration was analyzed by (b) histochemistry and (c) GUS activity assay. (by Student's t-test^*P < 0.05 and^**p is less than 0.005). (d) CAMP factor expression was detected by Western blot analysis. Crushed Agrobacterium tumefaciens (20. mu.g) infiltrated with 35S:: CAMP factor-His (CAMP factor-His), 35S:: SCAP-MBP-His (SCAP-MBP-His) or recombinant GUS (rGUS) were separated on 10% (w/v) SDS-PAGE and blotted on nitrocellulose membranes. anti-CA production from mice immunized with UV-irradiated E.coli BL21(DE3) overexpressing CAMP factorMP serum detection membrane. Arrows indicate the presence of CAMP factor at a molecular weight of 29 kDa. Scale 6 mm.

Figure 19 shows that mice immunized with leaves impregnated with CAMP factor produce CAMP factor-specific antibodies. Purified CAMP factor (65 μ g) was separated on 10% (w/v) SDS-PAGE, blotted on nitrocellulose membrane and immunoreactive with serum obtained from mice immunized with either GUS (left) or CAMP factor (right) encapsulated in leaves. A single band with 29kDa indicated purified CAMP factor reactive with serum from CAMP factor immunized mice, confirming the immunogenicity of the CAMP factor.

Figures 20A-C show that passive immunization of mice with neutralizing antibodies to CAMP factor reduces propionibacterium acnes-induced inflammation. (A) Propionibacterium acnes (1X 10) treated with 5% (v/v) anti-GUS (open circles) or anti-CAMP factor (filled circles) serum as described in "materials and methods⁷CFU) were inoculated in the right ear of ICR mice to cause an increase in ear thickness. As a control, an equal volume of PBS was injected into the left ear of the same mouse. Ear thickness was measured using a micro caliper at the indicated time after bacterial injection. Ear thickness of propionibacterium acnes injected ears was calculated as% PBS injected control. Error bars represent mean + -SE of 4 mice (by Student's t-test)^**P is less than 0.005). (B) Propionibacterium acnes (10) treated with injection of anti-GUS serum (i) or anti-CAMP factor (ii) serum⁷CFU) redness of the ear was visible after 3 days (arrow). Scale 1 cm. (C) H in the ears of Propionibacterium acnes treated with PBS (i, iv) alone or with serum against GUS (ii, v) or CAMP factor (iii, vi)&Otitis was observed in frozen tissue sections stained with E. Granulomatous reactions (short arrows) were visible at 4 × (i, ii, iii; scale 2mm) and 20 × (iv, v, vi; scale 0.5mm) magnification.

Figures 21A-C show that passive neutralization of propionibacterium acnes CAMP factor reduces pro-inflammatory MIP-2 cytokine production and bacterial colonization, without altering the survival of propionibacterium acnes in other parts of the body. (A) Measurement of proinflammatory MIP-2 cytokines was performed by sandwich ELISA using Quantikine M mouse MIP-2 kit. Compared to neutralization with anti-GUS serum (open column),passive neutralization with anti-CAMP factor serum (solid bars) clearly inhibited Propionibacterium acnes-induced MIP-2 increase. (B) Mice were injected with Propionibacterium acnes (1X 10) in the left ear in the presence of anti-GUS serum (open bars) or anti-CAMP factor serum (solid bars)⁷CFU). (C) Injection of live Propionibacterium acnes alone into the right ear (1X 10)⁷CFU). Bacterial Colonization (CFU) was quantified on agar plates as described in "materials and methods". Error bars represent mean. + -. SE of 4 mice (M.), (M.)^*P < 0.05 by Student's t-test).

Figure 22 shows that vaccination with CAMP factor confers a protective effect on propionibacterium acnes-induced ear swelling. 7 weeks after inoculation with GUS- (open bars) and CAMP factor (solid bars), aliquots (1X 10) of 25. mu.l of live Propionibacterium acnes suspended overnight in PBS⁷CFU) was injected intradermally into the right ear to stimulate mice. As a control, 25. mu.l of PBS was injected into the left ear of the same mouse. The increase in ear thickness was measured with a microcard after bacterial stimulation. The increase in ear thickness of the propionibacterium acnes-stimulated ears was calculated as% PBS injection control.

Detailed Description

The exemplary descriptions provided herein are exemplary and explanatory only and are not limiting of the invention as claimed. Furthermore, the invention is not limited to the particular embodiments described, as such may, of course, vary. Furthermore, the terminology used to describe particular embodiments is not intended to be limiting.

With respect to ranges of values, unless otherwise expressly indicated herein, each intervening value, to at least one tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is encompassed by the invention. Moreover, any other stated intervening value is encompassed by the invention. Further, the invention also includes ranges excluding either or both of the upper and lower limits of the ranges, unless expressly excluded from the stated ranges.

Unless defined otherwise, all 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 belongs. One of ordinary skill in the art will also recognize that any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In addition, all publications mentioned herein are incorporated herein by reference.

It must be noted that, as used in the appended claims herein, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a plurality of such polypeptides and reference to "a bacterium" includes one or more bacteria and equivalents thereof known to those skilled in the art, and so forth.

Likewise, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprising," "include," "including," and "included" are interchangeable and are not intended to be limiting.

It will be further understood that when the term "comprises" is used to describe various embodiments, those skilled in the art will understand that in some particular instances embodiments may alternatively be described using the language "consisting essentially of or" consisting of.

Propionibacterium acnes are involved in a wide variety of human microbial diseases, including acne vulgaris, endocarditis, endophthalmitis, osteomyelitis, joint, nervous system and cranial neurosurgical infections, and contamination of implanted biomaterials. More than fifty million people in the united states have acne vulgaris. In addition, acne vulgaris is the most common skin disorder that sometimes affects 85-100% of people in their lives. Systemic antibiotic treatment for acne lesions non-specifically kills most skin bacteria, which affects the homeostasis of skin colonizing colonies. Prior to the present disclosure, no vaccine was available against acne vulgaris and propionibacterium acnes-induced disease. The present disclosure provides anti-propionibacterium acnes vaccines to inhibit propionibacterium acnes-induced skin inflammation.

The proliferation of propionibacterium acnes begins in the micropowder spikes, which are precursors to acne lesions characterized by hyperkeratosis, keratotic plug formation, and increased sebum secretion from the sebaceous glands. Microcomedones provide an anoxic, sebum-rich microenvironment in the hair follicle, which promotes the overgrowth of Propionibacterium acnes. The initial event in acne inflammation is the destruction of the follicular epithelium by this propionibacterium acnes overgrowth, allowing the bacteria to come into contact with the host immune system in the comedo, triggering granulomatous inflammation (typical inflammatory acne). Propionibacterium acnes stimulate the production of proinflammatory cytokines including interleukin-1 beta, interleukin-8, interleukin-12, and tumor necrosis factor-alpha through toll-like receptor 2.

Acne vulgaris is one of the most common skin diseases that can cause severe inflammatory lesions highly associated with propionibacterium acnes infection. Staphylococcus epidermidis and propionibacterium acnes have been recognized as the major skin bacteria responsible for the formation of acne vulgaris. In addition, these bacteria have the ability to synthesize lipases, which degrade the sebum triglycerides into free fatty acids that trigger an inflammatory response. Treatment of acne should be initiated as early as possible in order to minimize the risk of scarring and adverse psychological effects. Many antibiotics have been used for acne treatment, but these are generally non-specific, short in duration and often applied when acne lesions have occurred (e.g. in late stages of acne). As described herein, the development of anti-acne vaccines can prevent the progression of early stage acne and improve the specificity of treatment, as described herein.

Acne vulgaris is a multifactorial disease associated with a variety of microbial infections, hormonal regulation and immune responses. The inflammatory phase of acne vulgaris is often of the greatest concern to the patient. Inflammatory lesions can lead to scarring and adverse psychological effects. A vaccine that selectively inhibits propionibacterium acnes-induced inflammation would minimize the risk of altering body hormones and parasitic skin microbial homeostasis.

Hemolysis is a virulence factor employed by numerous bacterial pathogens to degrade, invade host cells, and resist host immune attack. This is achieved by a variety of mechanisms that target the cell membrane: enzymatic, pore forming or surfactant. Propionibacterium acnes synergistically enhance hemolysis when grown on sheep blood agar plates in close proximity to β -hemolytic microorganisms, such as Staphylococcus aureus and Clostridium perfringens, similar to typical Christie, Atkins, Munch-Peterson (CAMP). CAMP reactions are induced by the pore-forming toxin CAMP factor co-hemolysin in combination with sphingomyelinase (SM enzyme) from other bacterial partners. CAMP factor itself has only weak hemolytic activity on erythrocytes, but pretreatment of cells with SM enzyme enhances its activity. The SM enzyme initially hydrolyzes sphingomyelin on the cell membrane of erythrocytes to form ceramides, which makes the cells susceptible to the hemolytic activity of CAMP factor. The entire genomic sequence of propionibacterium acnes includes many genes whose products are involved in degrading host molecules, and 5 genes encoding homologs of the CAMP factor of streptococcus agalactiae (s. This comprehensive analysis of Propionibacterium acnes by proteome technology using isotopically encoded protein tags coupled with NanoLC-MS analysis showed that bacteria cultured under anaerobic conditions produced one of the CAMP factor homologs that showed 42% nucleotide sequence identity to the Streptococcus agalactiae CAMP factor at higher concentrations than bacteria cultured under aerobic conditions (accession number: gi/50842175, incorporated herein by reference). These data indicate the physiological significance of CAMP factor for propionibacterium acnes.

CAMP factors from streptococcus agalactiae bind to the Fc region of IgG and IgM molecules, similar to the binding of IgG to staphylococcus aureus protein a (Jurgens et al, 1987), and partial amino acid sequence similarity between streptococcus agalactiae CAMP factor proteins and staphylococcus aureus protein a has been demonstrated (Ruhlmann et al, 1988). Evidence suggests that there are differences between propionibacterium acnes types IA, IB and II in protein expression with sequence similarity to CAMP co-hemolysin.

The present disclosure demonstrates that propionibacterium acnes secretes CAMP factors, which are known to act synergistically with bacterial sphingomyelinase (SM enzyme) to lyse erythrocytes. Furthermore, the present disclosure demonstrates that recombinant propionibacterium acnes CAMP factor alone induces cell death in human keratinocyte (HaCaT) and murine macrophage (RAW2647) cell lines in a dose-dependent manner. For example, intradermal injection of CAMP factor into the mouse ear induced significant ear swelling. In addition, the host acid SM enzyme (ASM enzyme) is released/secreted from HaCaT and RAW264.7 cells when the cells are co-cultured with propionibacterium acnes. Propionibacterium acnes-induced cytotoxicity in both cell lines was significantly neutralized by inclusion of selective ASM enzyme inhibitors, anti-ASM enzyme antibodies, or anti-CAMP factor antisera. Intradermal injection of live Propionibacterium acnes into the mouse ear attracts numerous macrophages that strongly express ASM enzyme, which results in increased soluble ASM enzyme in the ear after Propionibacterium acnes stimulation. Most notably, vaccination of ICR mice with CAMP factor vaccines resulted in protective immunity against propionibacterium acnes-induced inflammation and development of skin lesions. In addition, vaccination with CAMP factor in combination with local injection of anti-ASM enzyme IgG synergistically reduced propionibacterium acnes-induced ear swelling. These data indicate that propionibacterium acnes benefit from host enzymes, which broaden their pathogenicity; propionibacterium acnes can use host ASM enzymes to enhance the toxicity of its CAMP factor, which contributes to its evasion of host immune defenses, degradation of host tissues and transmission of pathogens.

The complete genomic sequence of propionibacterium acnes is known and is incorporated herein by reference in its entirety. The present disclosure identifies virulence factors and provides a panel of vaccines that are capable of providing protective immunity against propionibacterium acnes. Other virulence factors and antigenic compositions will be apparent and are included within the disclosure. In particular, antigens upregulated under hypoxic conditions as identified in table 1 are targets for the development of vaccines based on the technology herein. The present disclosure establishes the proteome of propionibacterium acnes by comparing the differential expression of propionibacterium acnes proteins under hypoxic and aerobic conditions (see examples and table 1). This analysis revealed many genes that were up-regulated. The present disclosure further provides additional information on three secreted virulence factors (CAMP factor, lipase and sialidase) associated with propionibacterium acnes-induced host cell injury and inflammation.

The anti-propionibacterium acnes vaccines provided by the present disclosure benefit subjects with polymicrobial propionibacterium acnes-associated diseases, including acne vulgaris, endocarditis, endophthalmitis, osteomyelitis, joint, nervous system, and cranial neurosurgical infections, and contamination of implanted biomaterials.

In addition to serving as vaccine targets, three secreted virulence factors (CAMP factor, lipase and sialidase) as well as killed propionibacterium acnes serve as candidates for developing anti-propionibacterium acnes drugs. For example, based on the present disclosure, methods of identifying therapeutic agents for treating microbial infections can include methods ranging from proteomic protein identification to vaccine evaluation. The present disclosure provides a platform for the study of functional proteomics and vaccine/drug generation using identified secreted virulence factors.

For example, the present disclosure provides anti-propionibacterium acnes vaccines that target secreted CAMP factors, lipases, and sialidases, as well as antigens from killed propionibacterium acnes. The vaccines of the present disclosure are demonstrated both in vitro and in vivo. The vaccine reduces inflammation caused by propionibacterium acnes.

The present disclosure further demonstrates that targeting secreted virulence factors is an effective strategy to reduce propionibacterium acnes-induced inflammation.

The vaccines of the present disclosure can be used alone or in combination with systemic antibiotic therapy.

"Polynucleotide" generally refers to any polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA), which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions. Polynucleotides also include hybrid molecules comprising DNA and RNA that are single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. Polynucleotides also include a plurality of DNAs or RNAs comprising one or more modified bases and a plurality of DNAs or RNAs with backbones modified for stability reasons or for other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides, as typically found in nature, as well as viruses and cells characteristic of DNA and RNA in chemical form. Oligonucleotides are relatively short polynucleotides. Examples of polynucleotides useful in generating antigens for inducing an immune response in the methods of the present disclosure include those in table 1, including fragments thereof encoding antigenic epitopes.

The sequences associated with the aforementioned accession numbers are incorporated by reference herein in their entirety.

"polypeptide" refers to any polypeptide comprising two or more amino acids joined by peptide bonds or modified peptide bonds. "polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and longer chains, commonly referred to as proteins. The polypeptide may comprise amino acids other than those normally encoded by codons.

Polypeptides include amino acid sequences that are modified either by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in the literature and are known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Such modifications may occur to the same or varying degrees at several sites in a given polypeptide. A given polypeptide may also contain many types of modifications. Polypeptides may be branched due to ubiquitination, and they may be circular with or without branching. Circular, branched, and branched circular polypeptides may be derived from post-translational natural processing or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination. Examples of methods and compositions for use in the present disclosure include those set forth in SEQ ID NO: 2. 3,7 and 9 and the propionibacterium acnes polypeptide set forth in SEQ ID NO: 11, an antigenic fragment thereof, and a polypeptide that hybridizes to SEQ ID NO: 2. 3,7, 9 and 11, are antigenic fragments that are 80%, 85%, 90%, 95%, 98% or 99% identical. Such polypeptides and fragments thereof are useful for immunization and antibody production. Antigens comprising the polypeptides of the present disclosure are used to generate an immune response in a subject.

Through the interaction of the antigen with cells of the immune system, an immune response is generated against the antigen. The resulting immune response can be roughly distinguished into two terminal types, either a humoral or a cell-mediated immune response (traditionally characterized by antibody and cellular effector protective mechanisms, respectively). These types of responses have been termed Th 1-type responses (cell-mediated responses), and Th 2-type immune responses (humoral responses). Terminal Th 1-type immune responses can be characterized as the generation of antigen-specific, haplotype-restricted CTLs, and natural killer cell responses. In mice Th 1-type responses are often characterized by the production of antibodies of the IgG2a subtype, whereas in humans these antibodies correspond to antibodies of the IgG1 type. Th 2-type immune responses are characterized by the production of a wide range of immunoglobulin isotypes, including IgG1, IgA, and IgM in mice.

The driving force behind the development of these two types of immune responses is the cytokine, a number of identified protein messengers, which are used to help the cells of the immune system and to direct the final immune response either to a Th1 response or to a Th2 response. Thus high levels of Th 1-type cytokines tend to contribute to the induction of cell-mediated immune responses against a given antigen, whereas high levels of Th 2-type cytokines tend to contribute to the induction of humoral immune responses against an antigen. It is important to remember that the difference between Th1 and Th 2-type immune responses is not absolute. In fact, the individual will provide an immune response described as predominantly Th1 or predominantly Th 2. Traditionally, Th 1-type responses have been associated with the production of INF-gamma and IL-2 cytokines by T lymphocytes. Other cytokines often directly associated with induction of a Th 1-type immune response are not produced by T cells, such as IL-12. In contrast, Th 2-type responses are associated with IL-4, IL-5, IL-6, IL-10, and tumor necrosis factor-beta (TNF-beta).

The present disclosure provides propionibacterium acnes antigens that are immunoprotective. Such antigens can be delivered to the host in a variety of ways to stimulate a protective immune response against propionibacterium acnes. The antigen may be delivered by an attenuated vector, resulting in presentation by MHC class I (e.g., such vectors include listeria monocytogenes (l. monocytogenes), escherichia coli, propionibacterium acnes avirulent, or another attenuated bacterial vector such as Mycobacterium bovis (Mycobacterium bovis) BCG, shigella flexneri). The term "attenuated" when used with respect to a bacterium means that the bacterium has lost some or all of its ability to proliferate and/or cause disease or other adverse reactions when the bacterium infects an organism. For example, an "attenuated" bacterium may be incapable of replicating at all, or limited to one or a few rounds of replication, when present in an organism in which wild-type or other pathogenic forms of the attenuated bacterium can replicate. Alternatively or additionally, an "attenuated" bacterium may have one or more mutations in one or more genes involved in bacterial pathogenicity. Many genes, loci, or operons are known, where mutations will result in attenuated bacteria. Examples of attenuated bacteria for use as live vaccines include salmonella typhi (s.typhi) carrying a mutation in its galE or htrA gene and vibrio cholerae (v.cholerae) carrying a mutation in its ctxA gene.

Microorganisms that may be used to express Propionibacterium acnes for use in the immunoprotective composition include, but are not limited to, Campylobacter sp, Yersinia sp, Helicobacter sp, Pectinops sp, Klebsiella sp, Lactobacillus sp, Streptococcus Gracillus sp, Enterobacter sp, Salmonella sp, Shigella sp, Aeromonas sp, Vibrio sp, Clostridium sp, Escherichia coli, Lenticus sp, and Lenticus sp, as well as, for example, the strains described in U.S. Pat. Nos. 3,361, and 3,361, in "New Generation Vaccines Second Edition", editor Levine et al, Marcel Dekker, Inc. 437 + page 446 (1997), Butterton et al, in "New Generation Vaccines Second Edition", editor Levine et al, Marcel Dekker, Inc. 379 + page 385 (1997) and Fennelly et al, in "New Generation Vaccines Second Edition", editor Levine et al, Marcel Dekker, Inc. 363 + page 377 (1997)). For example, Campylobacter jejuni (Campylobacter jejuni), Campylobacter coli (Campylobacter coli), Listeria monocytogenes (Listeria monocytogenes), Yersinia enterocolitica (Yersinia enterocolitica), Yersinia pestis (Yersinia pestis), Yersinia pseudotuberculosis (Yersinia pseudotuberculosis), Escherichia coli, Shigella flexneri, Shigella sonnei (Shigella dysenteriae), Shigella Shigella (Shigella boydii), Salmonella typhi (Shigella boydii), Helicobacter pylori (Helicobacter pylori), Helicobacter feline stomach Helicobacter (Helicobacter), Helicobacter pylori (Salmonella typhimurium), Salmonella typhi (Salmonella typhimurium), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella cholera typhi (Salmonella choleraesuis), Salmonella choleraesuis, Salmonella cholera viridis (Salmonella), Salmonella cholera viridis, Salmonella cholera, Salmonella typhi, Salmonella cholera, and the genus Salmonella cholera, Salmonella enteritidis (Salmonella enteritidis), Klebsiella pneumoniae (Klebsiella pneumoniae), Enterobacter cloacae (Enterobacter cloacae), and Enterococcus faecalis (Enterococcus faecalis) may be used. Coli including, but not limited to, enterotoxic, enterohemorrhagic, enteroinvasive, enteropathogenic, or other strains may be used in the present disclosure.

Alternatively, or in addition to the above, non-bacterial attenuated vectors, such as replication-defective viral vectors, may be used in the methods and compositions of the present disclosure. Such viral vectors for use in the disclosed methods and compositions include, but are not limited to, vaccinia, avipox, adenovirus, AAV, vaccinia virus NYVAC, modified vaccinia strain ankara (mva), Semliki forest virus, venezuelan equine encephalitis virus, and herpes virus. Naked DNA vectors may be used in addition to antigenic proteins alone or in combination with adjuvants. Naked DNA can be taken up and expressed by cells of the vaccinated subject, resulting in the induction of an immune response against the expressed polypeptide.

Examples of suitable viral vectors include herpes simplex viral vectors, vaccinia or alphavirus vectors and retroviruses, including lentiviruses, adenoviruses and adeno-associated viruses. In one embodiment, these vectors are replication-defective viral vectors. Techniques for gene transfer using these viruses are known to those skilled in the art. Retroviral vectors, for example, can be used to stably integrate a polynucleotide of the present disclosure into a host genome, although such recombination is not desirable. In contrast, replication-defective adenovirus vectors remain episomal and thus allow for transient expression.

In one embodiment, the adenovirus used as a live vector is a replication-defective human or simian adenovirus. Typically, these viruses contain an E1 deletion and can be grown in cell lines transformed with the E1 gene. Suitable simian adenoviruses are, for example, viruses isolated from chimpanzees. Examples of viruses suitable for use in the present disclosure include C68 (also known as Pan 9) (U.S. patent No. 6,083,716, incorporated herein by reference) and Pan 5, 6, and Pan7(WO 03/046124, incorporated herein by reference). Thus, these vectors can be manipulated to insert heterologous polynucleotides encoding antigens such that the products are expressed. The use forms and manufacture of such recombinant adenoviral vectors are described in detail in WO 03/046142, which is incorporated herein by reference.

The present disclosure also contemplates the use of plant systems to express the antigens of the present disclosure. Plant tissues or purified polypeptides may be used for immunization. For example, agroinfiltration is a method by which gene transient expression or protein production is achieved in plants. In the method, an agrobacterium suspension is delivered to a plant leaf, where the agrobacterium transfers a desired coding sequence (e.g., a CAMP polynucleotide) to a plant cell. The agrobacterium strain is transformed with a polynucleotide encoding a sequence of interest. Subsequently, the strain is grown in culture and then the agrobacterium is injected into the plant tissue (e.g., through stomata into the space within the leaf). Once inside the leaf, the polynucleotide is then transiently expressed. Many plants can be transformed by this method, but the two most commonly used are Nicotiana benthamiana (Nicotiana benthamiana) and Nicotiana tabacum (Nicotiana tabacum).

In addition, the present disclosure contemplates immunization using a combination of antigens and/or carriers. The present disclosure contemplates either homologous or heterologous prime-boost vaccination strategies. Heterologous strategies may include priming with one vector, such as listeria monocytogenes expressing one or more proteins, and boosting with another vector, such as an adenovirus expressing one or more of the same proteins, or vice versa. Boosting may also include immunization with one or more proteins of propionibacterium acnes or fragments thereof in an adjuvant. The specific examples provided herein demonstrate the delivery of antigens to animal hosts using a variety of vaccination strategies and the resulting immune protection against propionibacterium acnes stimulation.

The present disclosure includes several types of vaccines. One set of vaccines includes an attenuated bacterial vector that expresses one or more propionibacterium acnes antigens. Other vaccines of the present disclosure include a propionibacterium acnes antigen (e.g., a polypeptide or fusion polypeptide) in a suitable adjuvant. Another group of vaccines of the present disclosure includes viral vectors (e.g., adenoviruses) that express one or more propionibacterium acnes antigens.

Each vaccine is administered to a mammalian host, e.g., transdermally, subcutaneously, intramuscularly, intranasally, by inhalation, or even orally. The vaccine may be administered as part of a homologous or heterologous prime-boost strategy. Most importantly, the vaccine protects the mammalian host against infection by propionibacterium acnes.

As used herein, "vaccine" refers to a composition of matter comprising a molecule that induces an immune response when administered to a subject. Vaccines can comprise polynucleotide molecules, polypeptide molecules, and saccharide molecules, as well as derivatives and combinations of each, such as glycoproteins, lipoproteins, saccharide-protein conjugates, fusions of two or more polypeptides or polynucleotides, and the like. The vaccine may further comprise a diluent, adjuvant, carrier, or combination thereof, as will be readily understood by those skilled in the art.

The vaccine may comprise separate components. As used herein, "separate components" refers to the situation where the vaccine actually comprises two separate vaccines that are administered separately to a subject. In this sense, a vaccine comprising individual components may be regarded as a kit or package comprising the individual vaccine components. For example, in the context of the present disclosure, a package may comprise a first immunogenic composition comprising an attenuated bacterial vector and a second immunogenic composition comprising an attenuated viral vector comprising the same or different propionibacterium acnes antigen (e.g., CAMP factor, lipase, or sialidase).

An immunogenic composition/vaccine "induces" an immune response when the antigen or antigens present in the vaccine elicit an immune response against the antigen or antigens in the vaccinated subject. As demonstrated by activation of the immune system, the vaccinated subject will develop an immune response, including the production of vaccine antigen-specific T cells, vaccine antigen-specific B cells, vaccine antigen-specific antibodies, and cytokines. The immune response elicited can be measured by several methods, including ELISPOT, ELISA, chromium release assays, intracellular cytokine staining, FACS analysis, and MHC tetramer staining (to identify peptide-specific cells). The skilled person can also use these methods to measure primary or secondary immune responses.

An "antigen" is a substance that is capable of generating an immune response in a subject that is exposed to the antigen. The antigen is typically a polypeptide and is the focus of the host immune response. An "epitope" or "antigenic determinant" is the portion of an antigen to which T cells and antibodies specifically bind. The antigen may comprise multiple epitopes. Antigens of the present disclosure include propionibacterium acnes extracellular or immunogenic polypeptides (e.g., lipases, CAMP factors, and those described in table 1). In particular embodiments, the propionibacterium acnes polypeptide comprises: comprises the amino acid sequence of SEQ ID NO: 2. 3,7 or 9, and the antigenic fragment of a polypeptide of the sequence set forth in SEQ ID NO: 11, or an antigenic fragment of the human ASM enzyme of SEQ ID NO: 2. 3,7, 9 and 11, or a fragment thereof having at least 80% -99% identity thereto.

In one embodiment, the vaccine comprises an antigenic fragment of a propionibacterium acnes CAMP factor. An antigenic fragment typically comprises at least 6 amino acids (e.g., 6-10, 10-12, 12-20, 30-50 or more amino acids). Typically, an antigenic fragment is a fragment of a protein that is present on the surface of the protein and comprises a soluble domain. In one embodiment, the antigenic fragment may be part of a fusion protein comprising one or more non-contiguous sequences of the protein (e.g., a non-contiguous sequence of a propionibacterium acnes CAMP factor). In one embodiment, the vaccine comprises a polypeptide comprising SEQ ID NO: 7. In one embodiment, the vaccine comprises a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 7, a polypeptide having a sequence of at least 80%, 90%, 95%, 98% or 99% identity. In yet another embodiment, the vaccine comprises a polypeptide having the sequence as set forth in SEQ ID NO: 7 or at least 6 amino acids thereof and capable of producing a fragment of an antibody that specifically binds to a factor of propionibacterium acnes CAMP (e.g., a polypeptide consisting of SEQ ID NO: 7).

The vaccine may comprise killed and destroyed propionibacterium acnes. As described more fully below, killing and destruction results in the release of multiple antigens that may not be secreted unless cultured in an anoxic environment. Thus, in one embodiment, the present disclosure contemplates culturing propionibacterium acnes under hypoxic conditions, followed by killing and/or destroying the bacteria and preparing a vaccine from the destroyed propionibacterium acnes preparation. The disrupted Propionibacterium acnes preparation can be further purified to enrich for the antigen or antigens in the immunogenic composition. In another embodiment, the hypoxia cultured propionibacterium acnes is killed by gamma irradiation or other methods known to those skilled in the art and whole bacterial cells are used in the immunogenic formulation.

The primary immunisation vaccines used in some embodiments of the present disclosure comprise a propionibacterium acnes CAMP factor, lipase or sialidase antigen. The primary vaccine comprises an antigenic epitope of a propionibacterium acnes antigen, a full-length antigen, a vector comprising a polynucleotide encoding the antigen, and the like. In one embodiment, the primary vaccine comprises a polynucleotide encoding an antigen under the control of a foreign promoter within a bacterium, plant cell, or virus. The polynucleotide of the priming vaccine is present in a suitable delivery vector, such as a plasmid or other vector such as a bacterial, plant cell or viral vector. The polynucleotide may be under the control of a suitable promoter, for example a promoter derived from the HCMV IE gene. The priming vaccine is administered in an amount effective to elicit an immune response against the Propionibacterium acnes antigen. As used herein, "priming" of an immune response occurs when an antigen is presented to a T cell or B cell. As a result, the primed cells react again to the same antigen as memory cells in a second subsequent immune response. Thus, priming generates an initial immune response and establishes immunological memory. The skilled person realizes that the primary immune response represents an adaptive immune response when initially exposed to an antigen in a particular situation, such as in a pathogen or in a vaccine. However, it should also be appreciated that the present disclosure is not limited to the use of a priming vaccine in a naive individual. More generally, priming may also occur in individuals who have been exposed to the antigen but who have not received a priming vaccine.

The priming immunogenic (vaccine) composition may be administered once prior to the administration of the boosting immunogenic (vaccine) composition. In another embodiment, the priming vaccine may be administered several times.

A booster vaccine for use in the methods of the present disclosure may comprise at least one propionibacterium acnes antigen (e.g., a CAMP factor, lipase, or sialidase antigen polypeptide, or fragment thereof) that corresponds to the antigen of the primary vaccine. In addition, a booster vaccine may comprise (in addition to a priming antigen) a different antigen or a vector comprising the antigen or a coding region thereof. In one embodiment, the booster vaccine comprises a propionibacterium acnes polypeptide antigen to enhance the immunogenicity of the subject to propionibacterium acnes. For example, in one embodiment, the booster vaccine comprises a propionibacterium acnes antigen expressed in a viral vector. The propionibacterium acnes antigen can be selected from the group of upregulated antigens listed in table 1, including, but not limited to, CAMP factor, lipase, sialidase antigens, fragments, or combinations thereof.

The booster vaccine is administered in an amount effective to "boost" the priming immune response to the propionibacterium acnes antigen. As used herein, "boosting" an immune response means inducing a secondary immune response in a subject that has been primed by initial exposure to an antigen (i.e., has been exposed to an antigen). The secondary immune response is characterized by the activation and proliferation of specific T and B cells. Thus, boosting a specific response enhances a priming immune response by inducing immune cell proliferation and differentiation upon subsequent exposure to the antigen. A booster vaccine may achieve one or more of the following effects: induction of CD4+ T cells, induction of antibodies against propionibacterium acnes (e.g., antibodies against antigens in a vaccine), boosting the activity of CD8+ T cells primed by the primary vaccine, and induction of additional CD8+ T cells not originally identified in the immune response initially elicited. Booster vaccines can also induce CD4+ T cells and induce antibodies against propionibacterium acnes (e.g., anti-CAMP factor antibodies).

The presence of an immune response to a first dose of the immunoprotective composition prior to administration of a subsequent dose can be determined by known methods (e.g., by obtaining serum from the individual before and after the initial immunization, and demonstrating a change in the immune status of the individual, e.g., an immunoprecipitation assay, or ELISA, or bactericidal assay, or western blot, or flow cytometry assay, etc.). The presence of an immune response to a first dose can also be assessed by waiting a period of time after the first immunization, the period of time being sufficient for an immune response and/or priming to occur based on prior trials. Booster doses of the immunoprotective composition can be administered as needed.

Certain vaccine adjuvants are particularly useful for stimulating either a Th1 or Th 2-type cytokine response. Traditionally, the best indication of Th 1: Th2 balance of immune response after vaccination or infection involves direct measurement in vitro of Th1 or Th2 cytokines produced by T lymphocytes after restimulation with antigen, and/or measurement of the IgG 1: IgG2a ratio of antigen-specific antibody response. Thus, a Th 1-type adjuvant is one that stimulates isolated populations of T cells to produce high levels of Th 1-type cytokines when restimulated with antigen in vitro, and induces antigen-specific immunoglobulin responses associated with Th 1-type isotypes.

The present disclosure also relates to antibodies for use in the prevention and/or treatment of propionibacterium acnes infection. In a first embodiment, antibodies are raised against the propionibacterium acnes antigens of the present disclosure. Such antibodies are produced by administering as a vaccine an antigenic composition comprising an antigenic polypeptide (e.g., a propionibacterium acnes CAMP factor), a vector expressing the antigenic polypeptide, or a purified preparation of a propionibacterium acnes antigen.

Antibodies according to the present disclosure (e.g., anti-CAMP factor or anti-ASM enzyme antibodies) will be administered in one or more doses, and the amount required will depend on the stage of disease for which treatment is being administered, as well as other factors. To produce such antibodies, a subject will be administered an antigenic composition according to the present disclosure (e.g., an antigenic fragment of a CAMP factor or ASM enzyme) to induce production of the above-described antibodies. The antibody may be a monoclonal antibody. Once obtained, such novel antibodies can be produced by conventional techniques and used for therapy. In general, Monoclonal Antibodies against an epitope can be prepared by using techniques for producing antibody molecules from continuous culture cell lines, and methods for preparing Antibodies are well known to those skilled in the art (see, e.g., Coligan (1991) Current Protocols in immunology, Wiley/Greene, NY; Harlow and Lane (1989) Antibodies: A laboratory Manual, Cold Spring Harbor Press, NY; and Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd ed) Academic Press, N.Y.). In addition, such antibodies can be humanized using techniques known in the art. In addition, the antibody may include antibody fragments known in the art.

Passive immunity is the induction of immunity obtained by the transfer of antibodies from another individual (a Keller and Stiehm, 2000). Passive immunization has many advantages. (a) Unlike active immunization (vaccines), the biological effects of passive immunization are immediate and valuable, as in cases where symptoms have already developed. Thus, the use of a means for passively neutralizing the Propionibacterium acnes CAMP factor may benefit patients who have already developed acne. (b) There is no cell-mediated immunity and no direct bactericidal effect, which would have a low impact on the prejudicial symbiosis of microorganisms. (c) No side effects derived from the adjuvant were caused. (d) The dosage administered may be adjusted according to the severity of the disease. (e) Can be easily combined with other treatments. In addition, unlike active immunity, which requires time to induce protective immunity and depends on the ability of the host to mount an immune response, it is theorized that passive antibodies can confer protection regardless of the immune status of the host (casadeval, 2002). The present disclosure demonstrates that passive immunization targeting secreted CAMP factors, rather than bacterial surface proteins, can neutralize propionibacterium acnes virulence without directly killing the bacteria, reduce the risk of producing drug-resistant propionibacterium acnes, and alter propionibacterium acnes symbiosis.

As demonstrated herein, synergy can also be seen in the treatment of propionibacterium acnes infection by immunization with antigenic CAMP factor peptides or polypeptides and also contacting the subject with antibodies against asmase or asmase inhibitors. For example, the present disclosure contemplates the use of (a) a vaccine comprising a propionibacterium acnes CAMP factor peptide or polypeptide, (b) an anti-ASM enzyme antibody, (c) an ASM enzyme inhibitor, and (d) any combination of the foregoing to treat or prevent propionibacterium acnes infection. On the other hand, as used herein, a CAMP factor peptide or polypeptide does not necessarily refer to the origin of the peptide or polypeptide, but rather refers to (a) a sequence having some degree of identity to a wild-type CAMP factor; and (b) the ability to develop antibodies against such sequences to recognize.

For therapeutic purposes, the antibodies are formulated for convenient administration by injection, together with conventional pharmaceutical or pharmaceutically acceptable vehicles. Vehicles include deionized water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, Hank's solution, and the like. Other additives include additives to provide isotonicity, buffers, preservatives, and the like. The antibody may be administered parenterally, typically intravenously or intramuscularly, bolus-dose, intermittently or continuously.

Also provided are methods for ameliorating propionibacterium acnes in a subject by administering one or more propionibacterium acnes antigens (e.g., propionibacterium acnes CAMP factor proteins, polypeptides, peptides) or a carrier comprising propionibacterium acnes antigens, alone or in combination with an anti-SM enzyme (e.g., anti-ASM enzyme) antibody or inhibitor thereof, in a pharmaceutically acceptable carrier to the subject. In addition, methods of ameliorating propionibacterium acnes in a subject by administering to the subject an antibody that binds a propionibacterium acnes antigen or ASM enzyme in a pharmaceutically acceptable carrier are also provided.

The attenuated vaccine may be administered directly to the mammal. The immunogenic compositions and vaccines obtained using the disclosed methods can be formulated into pharmaceutical compositions for administration in any suitable manner. One route of administration is oral. Other routes of administration include intrarectal, intrathecal, buccal (e.g., sublingual) inhalation, intranasal, and transdermal, etc. (see, e.g., U.S. patent No. 6,126,938). While more than one route may be used to administer a particular composition, one particular route may often provide a more direct and more effective response than another route.

The immunoprotective compositions to be administered are provided in pharmaceutically acceptable solutions, such as aqueous solutions, often saline or buffered solutions, or they are provided in powder form. There are many suitable formulations of the pharmaceutical compositions of the present disclosure. See, e.g., Lieberman, Pharmaceutical document Forms, Marcel Dekker, Vol.1-3 (1998); remington's Pharmaceutical Science, 17 th edition, Mack publishing company, Easton, Pa, (1985) and similar publications. The composition may also include an adjuvant. Examples of known suitable adjuvants include aluminium, aluminium phosphate, aluminium hydroxide and MF59 (4.3% w/v squalene, 0.5% w/v Tween80, 0.5% w/v Span 85) -which are the only adjuvants currently licensed for use in humans. For experimental animals Freund's adjuvant, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1 ' -2 ' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria in a 2% squalene/Tween 80 emulsion, namely monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS), or Bacillus Calmette-Guerin (BCG). The effectiveness of an adjuvant can be determined by measuring the amount of antibody directed against the immunogenic antigen.

The concentration of the immunogenic antigens of the present disclosure in the pharmaceutical formulation can vary widely, for example, from less than about 0.1%, typically at or at least about 2% up to 20% to 50% or higher by weight, and will be selected primarily by fluid volume, viscosity, and the like, depending on the particular mode of administration selected.

Formulations suitable for oral administration may include (a) a liquid solution, e.g., an effective amount of the recombinant bacterium or polypeptide suspended in a diluent, e.g., buffered water, saline, or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as a lyophilized powder, liquid, solid, granules or gelatin; (c) suspensions in appropriate liquids; and (d) a suitable emulsion. Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, wetting agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms may comprise the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like, in addition to comprising the active ingredient in a carrier known in the art. It will be appreciated that attenuated vaccines must be protected from digestion when administered orally. This is typically achieved by complexing the vaccine with components to impart acid resistance and enzymatic hydrolysis, or by packaging the vaccine in a suitable resistant carrier such as liposomes or enteric-coated capsules. Methods for protecting attenuated bacteria or antigens from digestion are well known in the art. The pharmaceutical compositions may be encapsulated, for example, in liposomes, or in dosage forms that provide slow release of the active ingredient.

The vaccines can be formulated as aerosol formulations for administration by inhalation (e.g., they can be "nebulized") alone or in combination with other suitable ingredients. The aerosol formulation may be placed in a propellant that is acceptably pressurized, such as dichlorodifluoromethane, propane, nitrogen, and the like.

The dose administered to a subject in accordance with the context of the present disclosure should be sufficient to elicit a beneficial therapeutic and/or prophylactic response in the subject over time. The dosage can be determined by the effectiveness of the particular vaccine employed and the condition of the subject, as well as the body weight or vascular surface area of the subject to be treated. The size of the dose can also be determined by the presence, nature and extent of any adverse side effects associated with administration of a particular vaccine to a particular subject.

In determining the effective amount of vaccine to be administered in the treatment or prevention of infection or other disorder, the physician evaluates vaccine toxicity, disease progression, and anti-vaccine antibody production as needed.

Administering the composition to a subject at risk of infection by propionibacterium acnes, or administering the composition prevents or at least partially prevents the development of infection and its complications. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose" or "therapeutically effective amount". Amounts effective for therapeutic use will depend, for example, on the antigen composition, the mode of administration, the weight and general health of the subject, and the judgment of the prescribing physician. Depending on the dose and frequency required and the tolerance of the subject and the route of use, one or more doses of the antigenic composition may be administered. In addition, booster doses may be administered in the same or different dosage forms. For example, the methods contemplate administering a first composition comprising a propionibacterium acnes antigen in an attenuated bacterial vector and a second composition comprising a propionibacterium acnes antigen in an attenuated non-bacterial vector. The second composition may be administered simultaneously with or subsequent to the administration of the first immunogenic composition.

In particular embodiments, a therapeutically effective dose of the immunoprotective composition is administered to a subject. The amount of live attenuated bacteria or non-bacteria expressing Propionibacterium acnes or other antigens present in the initial immunization will typically range from 5X 10 per subject⁵To 5X 10¹¹Individual organism, and more typically from about 5X 10 per subject⁸To 5X 10⁹And (4) an organism.

Typically, the immunoprotective composition is administered to an individual who is naturally immune to propionibacterium acnes. Typically, 2-4 doses of the disclosed immunological composition are sufficient, however additional doses are required to achieve a high level of immunity. Additional booster doses may be given every 1-5 years to maintain high levels of immunity, as needed.

In general, administration to any individual should begin prior to the first disease symptom or prior to the first sign of possible or actual exposure to propionibacterium acnes.

The vaccines of the present disclosure can be packaged in containers, dispensing devices, and kits for administering the vaccines to mammals. For example, a packaging or dispensing device containing one or more unit dosage forms is provided. Typically, instructions for administering the compound will be provided in the package along with appropriate instructions on a label that the compound is appropriate for treating the indicated condition. For example, the label may describe the active compound within the package for use in treating a particular infectious disease or for use in the prevention or treatment of other diseases or conditions mediated by or potentially susceptible to an immune response in a mammal.

The following specific examples are meant to be illustrative and non-limiting. Those skilled in the art will recognize that numerous modifications and substitutions can be made to the compositions and methods below. Such modifications and substitutions do not depart from the disclosure and are intended to be included herein.

Examples

The present disclosure shows that intra-aural injection of Propionibacterium acnes (ATCC 6919; 10) into ICR mice⁸CFU) induced an increase in ear thickness (fig. 1) and granulomatous response (fig. 2A, B). One day after injection, propionibacterium acnes was surrounded by thick granulomatous infiltrates (fig. 2C). Two days after injection, propionibacterium acnes migrated to the hair follicle and accumulated in the sebaceous gland (fig. 2D, E). Although Staphylococcus epidermidis (ATCC 12228; 10) was injected⁸CFU) has less swelling, but the swelling can quickly subside within 4 days. Accordingly, the present disclosure provides models for measuring and testing agents that affect propionibacterium acnes. For example, the mouse ear models of the present disclosure can be used to test the cytotoxicity of propionibacterium acnes virulence factors. In addition, the model was used for evaluationAnd the anti-inflammatory effect of the Propionibacterium acnes vaccine.

Propionibacterium acnes, when grown on sheep blood agar plates in close proximity to β -hemolytic microorganisms such as Staphylococcus aureus and Clostridium perfringens, synergistically enhance hemolysis similar to that of typical Christie, Atkins, Munch-Peterson (CAMP). The CAMP response is induced by a combination of CAMP factor co-hemolysins, a pore-forming toxin, and sphingomyelinase (SM enzyme) from other bacterial partners. CAMP factor itself has weak hemolytic activity on erythrocytes, but pretreatment of cells with SM enzyme enhances its activity. The entire genomic sequence of propionibacterium acnes includes many genes whose products are involved in degrading host molecules, and 5 genes encoding homologs of the CAMP factor of streptococcus agalactiae have been found in the genomic information. This analysis of Propionibacterium acnes by proteomic techniques using isotopically encoded protein tags coupled with NanoLC-MS analysis showed that bacteria cultured under anaerobic conditions produced one of the CAMP factor homologs that showed 42% nucleotide sequence identity to the Streptococcus agalactiae CAMP factor at higher concentrations than bacteria cultured under aerobic conditions (accession number: gi/50842175, incorporated herein by reference). These data indicate the physiological significance of CAMP factor for propionibacterium acnes.

SM enzymes have been widely isolated and characterized from bacteria, yeast and various tissues and biological fluids of mammals. Despite the low identity of bacterial and mammalian SM enzymes, the amino acid sequences share many conserved residues, suggesting a common catalytic mechanism. The present disclosure demonstrates that propionibacterium acnes benefits from the host SM enzyme, which amplifies propionibacterium acnes CAMP factor-mediated pathogenicity. The present disclosure shows the involvement of host SM enzymes in CAMP factor-mediated pathogenicity of propionibacterium acnes in vitro and in vivo, as well as the synergistic potential of vaccine treatments targeting CAMP factor and local injection of IgG against host SM enzymes in propionibacterium acnes-related inflammatory acne vulgaris.

The tissue chamber model was used to detect proinflammatory cytokines and bacterial growth. The tissue compartment model was first described and characterized extensively in guinea pigs and then adapted for use in mice. This model accurately simulates bacterial infection in vivo. Since the bacteria are inoculated directly into the room, there is no adhesion and invasion step across the epithelium, and the lowest infectious dose of propionibacterium acnes required for sustained infection reflects virulence. Host responses are exclusively mediated by phagocytes and include antimicrobial peptides, cytokines, chemokines, leukocyte infiltration, and apoptosis. The tissue chamber model was used to evaluate the effectiveness of the vaccine against propionibacterium acnes. The tissue chamber (fig. 3) was implanted subcutaneously into the abdominal skin of ICR mice for 7 days.

After implantation into the tissue compartment, Propionibacterium acnes, Staphylococcus epidermidis (20. mu.l; 10)⁷CFU) or PBS (50 μ l) were injected into the tissue chamber. Tissue compartment fluid was collected 3 days after bacterial injection to detect Macrophage Inflammatory Protein (MIP) -2. MIP-2 levels were significantly increased 3-fold and 2-fold in mice injected with Propionibacterium acnes and Staphylococcus epidermidis, respectively, compared to mice injected with PBS (FIG. 4A). In vivo survival (colonies) of propionibacterium acnes was detected after spreading the tissue chamber fluid onto MHB agar plates (fig. 4B).

In humans, propionibacterium acnes thrive in an anoxic environment in which sebum accumulates in clogged hair follicles. It has been demonstrated that the level of lipase production is significantly increased by propionibacterium acnes in hypoxic conditions. In an effort to identify virulence factors highly expressed under hypoxic conditions, comprehensive quantitative proteomic analysis was performed on propionibacterium acnes in the presence or absence of oxygen. The identified virulence factors are selected as propionibacterium acnes vaccine candidates. Analysis of individual proteins for O-presence using a gel-free isotope-encoded protein labeling (ICPL) -based method₂Growing Propionibacterium acnes with O-free₂Abundance changes between growing propionibacterium acnes. 342 Propionibacterium acnes proteins were identified and sequenced by LTQ MS/MS. 152 of 342 proteins were successfully labeled with ICPL. 23 proteins were identified as up-or down-regulated under hypoxic or aerobic conditions (table 1). Two secreted virulence factors (CAMP factor and lipase) were highly expressed under hypoxic conditions (fig. 5A andB) in that respect Two internal peptides of lipase and CAMP factor are given (SYSEKHLGVAFR (SEQ ID NO: 1) and DLLKAAFDLR (SEQ ID NO: 2)), respectively (FIGS. 5C and D). Although it is well known that lipases are involved in the pathogenesis of acne lesions, the role of CAMP factors in acne development is completely unknown. Recently, it has been reported that streptococcus agalactiae CAMP factor acts as a pore-forming toxin. Thus, CAMP factor was selected as a target for the development of a propionibacterium acnes vaccine.

Sialidase treatment increases the susceptibility of human sebaceous gland cells to propionibacterium acnes. A number of sialidases have been disclosed in the Propionibacterium acnes genome. Three sialidases were selected from the Propionibacterium acnes genome for cloning, including a cell wall-anchored sialidase (accession number gi | 50843035; (SEQ ID NOS: 12 and 13, polynucleotides and polypeptides, respectively), a secreted sialidase B (accession number gi | 50842171; SEQ ID NOS: 14 and 15, polynucleotides and polypeptides, respectively) and a sialidase-like protein (accession number gi | 50843043; SEQ ID NOS: 16 and 17, polynucleotides and polypeptides, respectively). cell wall-anchored sialidase (accession number gi |50843035) exhibited the strongest enzymatic activity in removing sialic acid from the surface of human SZ95 sebaceous gland cells (EC 3.2.1.18) (accession number)Q02834) And the cell wall surface anchored family protein of Streptococcus pneumoniae serotype 2 (accession number)Q04M99) Identity (-30%), but the immunogenicity of the propionibacterium acnes sialidase was not studied.

The gene encoding sialidase was amplified by PCR from template DNA prepared from Propionibacterium acnes. Specific primers were designed including sense and antisense primers. The PCR product was inserted into the pEcoli-Nterm6xHN plasmid and expressed in E.coli [ E.coli BL21(DE3) ]. After IPTG induction, the overexpressed sialidase-6 xNH fusion protein from E.coli was detected in Coomassie blue stained SDS-PAGE gels at a molecular weight of approximately 53.1 kDa. Sialidase-6 xNH fusion proteins were purified using a talen resin column. Sialidase expression was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and NanoLC-MS/MS sequencing. The purified sialidase-6 xNH fusion protein was subjected to in-gel digestion with trypsin prior to NanoLC-MS/MS analysis. Nineteen internal peptides from sialidase were completely sequenced by NanoLC-MS/MS analysis of an HCTultra PTM system ion trap mass spectrometer. The MS/MS spectra of the sequenced peptides matched well to those of the propionibacterium acnes sialidase (accession number gi | 50843035). The internal peptide of the sialidase is shown (VVELSDGTLMLNSR; amino acid residues 316-329 of SEQ ID NO: 13). These results indicate that sialidases are expressed in E.coli BL21(DE3) and indicate that mass spectrometry provides an advantageous way to demonstrate that no antibodies are available for protein expression in western blots.

To determine enzyme activity, purified sialidase-6 xNH fusion protein (10. mu.g/ml) was added to human SZ95 sebaceous gland cell culture medium for 2 hours to remove sialic acid on the surface of the sebaceous gland cells. The amount of surface sialic acid was determined by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA) using a reaction of biotinylated locust lectin I and streptavidin-FITC conjugate. Fluorescence of MAA-labeled sialic acid decreased dramatically by 63% in sialidase-treated sebaceous gland cells (fig. 6A), while fluorescence did not change in GFP-treated sebaceous gland cells (fig. 6B). The data indicate that the purified sialidase retained enzymatic activity. Sebaceous gland cells (10. mu.g/ml) after 2 hours of treatment with sialidase (10. mu.g/ml)⁶Individual cell) exposure to live propionibacterium acnes (10)⁷CUF) overnight. Live Propionibacterium acnes induced approximately 15-20% cell death in untreated or GFP-treated sebaceous gland cells. However, in sialidase-treated sebaceous gland cells, propionibacterium acnes-induced cell death was significantly increased by 35% (fig. 6C), indicating that sialidase treatment increased the susceptibility of sebaceous gland cells to propionibacterium acnes. It has been demonstrated that the general populationIncubation of luminal epithelial cells with sialidase significantly increased the adhesion of Pseudomonas aeruginosa (Pseudomonas aeruginosa). Adhesion of propionibacterium acnes into sialidase-treated sebaceous gland cells was examined. The results show that pretreatment with sialidase (10. mu.g/ml, 2 hours), rather than GFP, considerably increased the adhesion of Propionibacterium acnes into sebaceous gland cells (FIG. 6D). Gram staining with Accustain showed that the number of propionibacterium acnes interacting with sebaceous gland cells increased once sialic acid on the surface of the sebaceous gland cells was removed by sialidase (fig. 6E-G). Thus, inhibition of sialidase activity or neutralization of sialidase by antibodies, siRNA, small molecule inhibitors, binding sites, antisense molecules, and the like can be used to provide protection against propionibacterium acnes infection. Antisense molecules can be generated based on the polynucleotide sequences provided herein.

Intact E.coli particles have been used as vectors for intranasal and intradermal vaccination. To engineer an escherichia coli vector-based vaccine targeting anthrax spore coat-associated protein (SCAP), the gene encoding SCAP was constructed in pET15b vector (EMD Biosciences, Inc.). After IPTG induction, escherichia coli carrying the empty expression vector or the SCAP expression plasmid was killed by ultraviolet irradiation. For immunization, the uv-irradiated e.coli vector-based vaccine, not mixed with exogenous adjuvants, was then administered directly into the nasal cavity of mice. Sera collected from each group of (n-4) mice 3 weeks after immunization were pooled. The production of anti-SCAP IgG was detected by antigen array. Antigen microarrays were generated by spotting recombinant SCAPs, SCAPs targeting Maltose Binding Protein (MBP) and mouse IgG (positive control). Sera from mice immunized with the empty expression vector and E.coli vectors with SCAP overexpression were hybridized on the array. Negative controls gave only background signal, positive controls (IgG) gave dilution-dependent signal reduction. Experiments showed that anti-SCAP antibodies were generated after immunization with E.coli vector-based vaccine (E.coli BL21(DE3) T7/lacO SCAP) compared to control sera. More importantly, anti-SCAP antibodies can be produced without booster immunizations.

The gene encoding the SCAP was also constructed into pCAL-n-FLAG vector (FIG. 7C) and then transformed into E.coli BL21 strain, which served as antigen carrier. For safety reasons, the transformed E.coli was destroyed by gamma irradiation. ICR mice were immunized by intranasal administration of irradiated e.coli vectors encoding SCAP. Antibody production was measured by western blot detection of reactivity with sera obtained from mice 1 month after immunization. The mice were able to produce detectable amounts of anti-SCAP antibodies without any booster immunization. Pretreatment of the skin surface of ICR mice with a non-ionic surfactant (tetradecyl-beta-D-maltoside; TDM) (Antatrace inc., Maumee, OH) at a concentration of 0.125% (in sterile water) for 15 minutes slightly breaches the stratum corneum barrier, but greatly enhanced the intradermal immunization of the e.coli based tetanus toxin C fragment vaccine (e.coli BL21nir/B tetC). The ICR mice skin surface was pretreated with TDM (0.125%) for 15 minutes, followed by washing and then intradermal application of the irradiated e.coli vector SCAP, and these mice could generate antibody responses against SCAP. Antibody production was measured by western blot detection of reactivity with sera obtained from mice 1 month after immunization. The mice were able to produce detectable amounts of anti-SCAP antibodies without any booster immunization. These results indicate that SCAP is an immunogenic anthrax protein when mice are non-invasively immunized with a vaccine based on an e. The results show that TDM disrupts the stratum corneum barrier of the skin, which clearly makes skin immunization possible. The present disclosure thus provides immunization protocols with CAMP factor and sialidase immunization.

Use of UV-irradiated E.coli vector-based vaccine [ E.coli BL21(DE3) T7/lacO sialidase]The immunogenicity of the propionibacterium acnes sialidase (accession gi |50843035) was tested. UV dose (4,500J/m) was administered²) All E.coli expressing both (sialidase-vector) and not expressing the sialidase gene (LacZ-empty vector) as demonstrated by the inability to form colonies on LB agar plates were irradiated. The amount of sialidase in the E.coli vector did not change after UV irradiation. ICR mice were immunized intranasally with UV-irradiated E.coli BL21(DE3) T7/lacO sialidase and in the first noseThe immunization was boosted 3 weeks after inoculation. Antibody (IgG) production in mouse sera was detected by western blot 3 and 6 weeks after immunization. When the sialidase-6 xNH fusion protein transfected membrane reacted with mouse serum collected 6 weeks after immunization, a strong band was seen at approximately 53.1 kDa. No production of antibodies reactive with sialidase was found in LacZ-empty vector immunized mice. ICR mice were also immunized with sialidase-6 xNH fusion protein or GFP using Freund/(non) complete adjuvant. Antibody production was detected by western blot analysis 3 weeks after immunization. When the sialidase-6 xNH fusion protein reacts with serum from sialidase-immunized mice, a strong band is seen at approximately 53.1 kDa. Antibody production was also confirmed by antigen microarray. Antigen microarrays were generated by printing with sialidase-6 xNH fusion protein and mouse IgG (positive control). After hybridization with mouse serum, the fluorescence signal shown in the antigen microarray indicates the production of sialidase antibodies. Sialidase antibodies were detected in sera collected from mice 3 weeks after immunization. No production of sialidase-reactive antibodies was observed in GFP-immunized mice. Data from both antigen microarrays and western blots confirmed that sialidases are immunogenic proteins.

Protective immunization of Propionibacterium acnes induced inflammation with a sialidase-based vaccine live Propionibacterium acnes (10)⁷CFU) stimulated ICR mice immunized with recombinant proteins (sialidase or GFP) using freund/(non-) complete adjuvant (fig. 8). 3 weeks after vaccination, mice were injected subcutaneously with 25. mu.l of Propionibacterium acnes (10. mu.l) into one ear⁷CFU) and 25 μ Ι PBS injected into the other ear as control. Injection of propionibacterium acnes induced thickening and reddening of the ear. Ear thickness was measured daily for 9 days. After 1 day of propionibacterium acnes stimulation, the ear thickness of GFP-immunized mice increased rapidly by more than 2-fold. When mice were immunized with sialidase, the increase in ear thickness was significantly reduced by more than 50% (fig. 8). Ear redness declined in GFP-immunized mice 7 days after propionibacterium acnes stimulation, while recovery of ear redness occurred in sialidase-immunized mice 3 days after propionibacterium acnes stimulation. These results indicate that sialidase-immunized mice inhibit acnePropionibacterium acnes-induced ear inflammation. Propionibacterium acnes-induced production of proinflammatory cytokines was also measured post-vaccination. A tissue compartment model (fig. 3) was used to detect the levels of proinflammatory cytokines in vivo. Propionibacterium acnes (10)⁷CFU) 7 days prior to injection, the tissue chamber was implanted subcutaneously into the abdominal skin of ICR mice. The data indicate that the tissue compartment fluid contains a variety of immune cells, including macrophages (CD11 b)⁺) Neutrophil (Gr-1)⁺) NK cells (CD49 b)⁺) And T cells (CD 3)⁺) Indicating the influx of immune cells into the tissue compartment. Tissue compartment fluid containing pro-inflammatory cytokines was withdrawn by percutaneous aspiration 3 days after propionibacterium acnes injection. MIP-2 cytokine levels in the immunized mice were measured by ELISA. In GFP-immunized mice, a significant increase in MIP-2 levels was observed 3 days after Propionibacterium acnes injection. Importantly, the increase in MIP-2 cytokine induced by Propionibacterium acnes was reduced by 61% in sialidase-immunized mice. These results demonstrate that sialidase-based vaccines are effective in reducing ear thickness and proinflammatory cytokine production in mice.

Pretreatment of propionibacterium acnes with serum from sialidase-immunized mice significantly reduced the cytotoxicity of propionibacterium acnes on human sebaceous gland cells (fig. 9). Culturing sebaceous gland cells with propionibacterium acnes treated with anti-GFP serum caused approximately 30% cell death, while cell death of sebaceous gland cells was reduced by almost 5% when the cells were co-cultured with propionibacterium acnes treated with anti-sialidase serum. The results indicate that sialidase-immunized mice stimulated production of antibodies that effectively neutralized the cytotoxicity of propionibacterium acnes on human sebaceous gland cells.

CAMP factors are up-regulated in propionibacterium acnes under anoxic or aerobic conditions. Thus, whether CAMP factor produces toxic effects on skin cells was examined. Keratinocytes are known to be one of the main targets of propionibacterium acnes during acne lesions. Thus, whether CAMP factor has a deleterious effect on keratinocytes in the ear of ICR mice was tested. By using the same protocol as for cloning and purifying sialidaseObtaining the purified recombinant CAMP factor. The PCR product of the CAMP factor was inserted into pEcoli-Nterm6xHN plasmid and cultured in E.coli [ E.coli BL21(DE3 CAMP factor)]Is expressed in (1). Following IPTG induction, expression of the CAMP factor-6 xNH fusion protein was detected at the approximately 36kDa molecular weight position in Coomassie blue stained SDA-PAGE gels (FIG. 11, lanes 1 and 2). The CAMP factor-6 xNH fusion protein was purified using a TALON resin column and confirmed by NanoLC-MS/MS sequencing. The 16 internal peptides from the CAMP factor were completely sequenced by NanoLC-MS/MS analysis by an HCTultra PTM system ion trap mass spectrometer. The MS/MS spectra of the sequenced peptides matched well with those of the propionibacterium acnes CAMP factor (accession number gi | 50842175). The internal peptide of the CAMP factor (AVLLTANPASTAK (SEQ ID NO: 3); amino acids 147-159 of SEQ ID NO: 11) is shown (FIG. 11B). Purified CAMP factor and GFP (1. mu.g/. mu.l) were injected subcutaneously into the ear of ICR mice. Apoptotic cells were examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay 1 day after injection. Injection of recombinant CAMP factor did not induce detectable ear thickening. No apoptotic cells were detected from mice injected with GFP. Apoptotic cells were detected only in mice injected with CAMP factor, indicating that CAMP factor is a toxic protein. During the TUNEL assay, tissue sections of CAMP factor-injected ears were double stained with the differentiated keratinocyte marker K10. Apoptotic cell localization in keratinocytes indicates that CAMP factor is detrimental to skin keratinocytes. To test the immunogenicity of CAMP factor, UV-irradiated E.coli vector-based vaccines [ E.coli BL21(DE3) T7/lacO CAMP factor]Mice were immunized. UV dose (4,500J/m) was administered²) All E.coli expressing both (CAMP factor-vector) and not expressing CAMP factor gene (LacZ-empty vector) were irradiated. ICR mice were immunized intranasally with UV-irradiated E.coli BL21(DE3) T7/lacO CAMP factor. anti-CAMP factor antibodies (IgG) can be detected in the mouse serum 3 weeks after immunization. The results show that if mice are irradiated with E.coli vector-based vaccine [ E.coli BL21(DE3 CAMP factor)]Immunization of mice can result in the production of anti-CAMP factor antibodies without the need for booster immunizations. No interactions with LacZ-empty vector immunized mice were foundAntibody production by CAMP factor response. anti-CAMP factor antibodies may also be produced when mice are immunized with recombinant protein/(non-) complete adjuvant. ICR mice were immunized with CAMP factor-6 xNH fusion protein or GFP using a protocol as described above, using freund/(non-) complete adjuvant. anti-CAMP factor antibodies were detectable in CAMP factor immunized mice but not in GFP immunized mice. These results indicate that CAMP factor is immunogenic when mice are immunized with either an e.coli vector based vaccine or a recombinant protein/(non) complete adjuvant. However, it is noteworthy that if mice were immunized with whole organism propionibacterium acnes, the mice could not produce anti-CAMP factor antibodies.

Heat killed Propionibacterium acnes was used as an inactivated anti-Propionibacterium acnes vaccine. After 30 minutes inactivation of propionibacterium acnes at 65 ℃, the inactivated propionibacterium acnes were spread on LB agar plates. Failure to form colonies indicated complete inactivation of propionibacterium acnes. For immunization, heat killed Propionibacterium acnes (10) were divided into three portions⁸A CFU; 50 μ l) was inoculated intranasally into ICR mice (first inoculation, first boost at week three and second boost at week 6). Mice inoculated with 50 μ l PBS were used as control. Antibody production was detected by western blot after 10 weeks of immunization. For the detection of protective immunity, live Propionibacterium acnes (10) was used⁷CFU) the ears of ICR mice immunized with heat killed propionibacterium acnes were stimulated subcutaneously. Ear thickness was measured for three days. Stimulation of PBS-inoculated mice by propionibacterium acnes induced a 1.8-fold increase in ear thickness. Importantly, the propionibacterium acnes-induced increase in ear thickness was reduced by 40% in killed propionibacterium acnes-immunized mice. Similarly, propionibacterium acnes-induced ear redness was significantly inhibited in killed propionibacterium acnes-immunized mice, indicating that mice immunized with killed propionibacterium acnes developed protective immunity against propionibacterium acnes infection. Following immunization, changes in the levels of proinflammatory cytokines were determined. The tissue compartment was implanted subcutaneously into the abdominal skin of ICR mice for 7 days, followed by Propionibacterium acnes(10⁷CFU) into the implanted tissue chamber. MIP-2 cytokine levels in the tissue chamber fluid were measured by ELISA 3 days after injection of propionibacterium acnes. In mice vaccinated with PBS, a significant increase in MIP-2 levels was observed 3 days after injection with propionibacterium acnes. The increase in MIP-2 cytokines was reduced by more than 50% when mice were immunized with killed Propionibacterium acnes.

The next procedure examined whether antibodies against sialidase and CAMP factor could be raised in killed propionibacterium acnes immunized mice. 50 μ g of purified recombinant sialidase, CAMP factor and Propionibacterium acnes lysate were subjected to 12.5% SDS-PAGE gel electrophoresis and transferred to PDVF membrane. The membranes were incubated overnight with mouse serum obtained from killed mice immunized with propionibacterium acnes. Many proteins with molecular weights greater than 50kDa reacted positively with sera obtained from killed mice immunized with Propionibacterium acnes. However, neither sialidase nor CAMP factor reacted with serum, suggesting that neither sialidase nor CAMP factor is immunogenic if mice were immunized with the whole organism propionibacterium acnes.

In summary, the data indicate that sialidases and CAMP factors are immunogenic when mice are immunized with e.coli vector based vaccines or recombinant proteins/(non) complete freund's adjuvant. Mice immunized with killed propionibacterium acnes developed antibodies against several proteins (> 50kDa) instead of sialidase and CAMP factor. Mice immunized with a sialidase-based vaccine or killed propionibacterium acnes produced significant protection against stimulation by live propionibacterium acnes.

Propionibacterium acnes (ATCC 6919) was cultured in Brucella broth agar supplemented with 5% (v/v) defibrinated sheep blood, vitamin K and hemin under anoxic or aerobic conditions using Gas-Pak (BDbiosciences, San Jose, Calif.) at 37 ℃. Bacteria isolated from a single colony were inoculated in Reinforced Clostridial Medium (RCM) (Oxford, Hampshire, uk) and cultured at 37 ℃ until the logarithmic growth phase (OD600 ═ 0.7-2.0). Staphylococcus aureus 113(ATCC 35556) was cultured on Tryptone Soya Broth (TSB) agar plates. Bacteria isolated from a single colony were inoculated in TSB and cultured overnight at 37 ℃. Bacterial pellets were harvested by centrifugation at 5000g for 10 minutes.

The product encoding the putative CAMP factor (accession number: gi/50842175) protein (amino acid residues 29-267) was generated by Polymerase Chain Reaction (PCR) using gene-specific primers designed based on the complete genomic sequence of Propionibacterium acnes. The forward PCR primer (5'-TAAGGCCTCTGTCGACGTCGAGCCGACGACGACCATCTCG-3'; SEQID NO: 4) included 16 nucleotides containing a SalI site that matched the end of an In-Fusion Ready pEcoli-Nterm6XHN vector (Clontech Laboratories, Inc., Mountain View, Calif.) and 26 nucleotides encoding the N-terminus of the CAMP factor. The reverse PCR primer (5'-CAGAATTCGCAAGCTTGGCAGCCTTCTTGACATCGGGGGAG-3'; SEQ ID NO: 5) included 16 nucleotides containing a HindIII site that matched the ends of the vector and 23 nucleotides encoding the C-terminus of the protein. PCR was performed using Propionibacterium acnes genomic DNA as a template. The amplified DNA product was inserted into the restriction enzyme site of an In-Fusion Ready pEcoli-Nterm expression plasmid and transformed into competent cells [ E.coli, BL21(DE3), Invitrogen, Carlsbad, CA]Subsequently, they were selected on Luria-Bertani (LB) plates containing ampicillin (50. mu.g/ml) and incubated overnight at 37 ℃. For expression of Green Fluorescent Protein (GFP) as a control, the pEcoli-Nterm-GFP vector (Clontech Laboratories) provided in the kit was used as a positive control for transformation. Aliquots of overnight cultures were diluted 1: 20 with LB medium and incubated at 37 ℃ until OD was reached₆₀₀0.7. Isopropyl-. beta. -D-thiogalactoside (IPTG) (1mM) was added to the culture and cultured for 4 hours to induce protein synthesis. The bacteria were harvested by centrifugation and disrupted by sonication on ice for 5 minutes and lysed by centrifugation at 3000g for 30 minutes. The pellet was washed with PBS and dissolved in 50mM sodium phosphate buffer containing 6M guanidine hydrochloride and 300mM NaCl. The expressed protein with the 6 × HN tag was purified under denaturing conditions using talen expression purification kit (Clontech Laboratories). Will be purifiedProtein against H₂O dialyzed, lyophilized, dissolved in ethylene glycol (1mg/1.2ml) and refolded overnight at 4 ℃ in 10ml 250mM Tris-HCl buffer, pH 8.4, containing 5mM cysteine, 0.5mM cystine and 1.5M urea. The refolded protein was dialyzed against PBS and concentrated. Protein expression was detected by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

Trypsin in-gel digestion and protein identification by NanoLC-LTQ Mass Spectrometry (MS) were performed. The automated NanoLC-LTQ MS/MS mechanism consisted of an Eksigent Nano 2D LC system, a switching valve, a C18 trap column (Agilent, Santa Clara, Calif.) and a capillary reversing column (10cm long, 75 μm inner diameter) filled with 5 μm, C18AQUASIL resin with a complete sprayer head (Picofrit, 15 μm tip, New Objective, Woburn, Mass.). Use from buffer A (H) in 100 minutes₂O plus 0.1% formic acid) to 50% buffer a plus 50% buffer B (acetonitrile plus 0.1% formic acid) reverse LC was performed directly coupled to an LTQ ion trap mass spectrometer (ThermoElectron, Waltham, MA). The instrument is operated in a data dependent mode. Collection by dynamic exclusion of more than 2X 10⁵Data for the 4 strongest ions of intensity and set the collision energy to 35%. By passing3.2(Thermo Scientific, San Jose, Calif.) Large-Scale MS/MS spectra were extracted using default values. Deconvolution and deisotoping of the charge state are not performed. Self Sorcerer with SEQUEST (v.27, rev.11) as a protein identification query program was used^TM2 System analysis of all MS/MS spectra. Sequence was set to query the target-decoy ipi.mouse.v3.14 database containing forward and reverse protein sequences (68627 in each direction) using trypsin as the digestive enzyme and allowing up to 5 missed breaks. False positive rates were roughly determined by doubling the direct ratio of the number of bait hits to the total number in the name. SEQUEST is queried with a fragment ion mass tolerance of 0.5Da and a parent ion tolerance of 1.0 Da.

The co-hemolytic reaction of the recombinant CAMP factor was detected on sheep blood agar. Staphylococcus aureus 113(ATCC 35556) (10. mu.l, 2X 10) as source of SM enzyme⁷CFU/ml in PBS) were streaked onto agar plates. Mu.l of recombinant CAMP factor (250. mu.g/ml) or GFP (250. mu.g/ml) as a control protein were spotted next to the S.aureus streaks and grown aerobically for 18 hours at 37 ℃.

Vaccination and anti-CAMP factor antibody titers 8 week old female ICR mice were used throughout the experiments. ICR mice were housed according to institutional guidelines. To pass UV (3500J/m)²) Radiation-inactivated Escherichia coli over-expressing CAMP factor or GFP [ BL21(DE3)]Mice were inoculated intranasally. Irradiated e.coli was unable to grow on LB agar plates (data not shown). Mu.l of E.coli suspension (1X 10)⁹CFU) were inoculated in the nasal cavity. At 2 and 3 weeks post-vaccination sera were collected individually for the detection of anti-CAMP factor antibody titers.

To determine the antibody titer against CAMP factor by enzyme-linked immunosorbent assay (ELISA), purified CAMP factor (5. mu.g/ml) was coated with coating buffer (0.015M Na)₂CO₂，0.35M NaHCO₂And 0.05% NaN₃) Diluted and coated with 96-well ELISA plates (Corning, Lowell, MA) overnight at 4 ℃. The plates were washed with PBS containing 0.1% (w/v) Tween-20 and blocked with PBS containing 1% (w/v) Bovine Serum Albumin (BSA) and 0.1% (w/v) Tween-20 for 2 hours at room temperature. Antiserum (1: 10000 dilution) from mice inoculated with E.coli overexpressing CAMP factor or GFP was added to the wells and incubated for 2 hours. Goat anti-mouse IgG (H + L) IgG-horseradish peroxidase (HRP) conjugate (Promega, Madison, Wis.) (1: 5000 dilution) was added, incubated for 2 hours, and then washed. Using OptEIA^TMThe HRP activity was determined using the kit (BD Biosciences, San Diego, Calif.). The Optical Density (OD) per well was measured at 450 nm.

Cell culture, cytotoxicity assay and neutralization assay the human keratinocyte line HaCaT or the murine macrophage line RAW264.7, respectively, were supplemented with 10% heat inactivationDulbecco's Modified Eagle Medium (DMEM) or Roswell Park Mechanical Institute (RPMI)1640 Medium of Fetal Bovine Serum (FBS) at 37 ℃ and 5% (v/v) CO₂Culturing under the condition. For determination of CAMP factor cytotoxicity, cells (1X 10)⁵Per well) were incubated with recombinant CAMP factor or GFP in 1% FBS-medium for 18 hours in 96-well microtiter plates. After incubation, cell viability was determined using the acid phosphatase (ACP) assay. Cells were washed three times with PBS and assayed with 100. mu.l of ACP assay buffer [1M sodium acetate buffer, pH5.5, containing 0.1% (w/v) triton-X]10mM p-nitrophenyl phosphate (pNPP) in (1) was incubated at 37 ℃ for 1 hour. The reaction was then stopped by adding 10. mu.l of 1N NaOH and the OD measured at 405 nm. Cytotoxicity was calculated as the percentage of cell death caused by triton-X (0.1%, v/v).

To detect secretion and/or release of CAMP factor and acid SM enzyme (ASM enzyme), HaCaT or RAW264.7 cells (5X 10)⁵) In serum-free Medium in 24-well plates [ 5X 10 ]⁶CFU/well; multiplicity of infection (MOI) 1: 10]Or not co-cultured with Propionibacterium acnes at 37 ℃ for 14 hours. The supernatant was centrifuged and filtered with a 0.22 μm pore size filter to remove cell debris and bacteria, then concentrated 10-fold using a 10kDa cut-off ultrafiltration membrane (Amicon inc., Beverly, MA). The concentrated supernatant (10 μ g) was subjected to 10% SDS-PAGE for Western blot analysis using mouse anti-CAMP factor antiserum and goat anti-ASM enzyme IgG (Santa Cruz biotechnology, inc., Santa Cruz, CA).

For neutralization assays, cells (1X 10)⁵Perwell) with Propionibacterium acnes (1X 10) in the presence of anti-CAMP factor or anti-GFP antiserum (2.5%, v/v)⁶CFU/well; MOI 1: 10) for 14 hours, in which complement has been inactivated by heating at 56 ℃ for 30 minutes. To examine the involvement of host ASM enzymes in the pathogenicity of propionibacterium acnes, cells were co-cultured with propionibacterium acnes in the presence or absence of the cell permeable selective ASM enzyme inhibitor desipramine (10 μ M) (Sigma, st. After incubation, cell viability was determined and cytotoxicity calculated as described above.

To examine the involvement of the CAMP factor IN the inflammation of Propionibacterium acnes IN vivo, recombinant CAMP factor (10. mu.g/20. mu.l) IN PBS or GFP (10. mu.g/20. mu.l) was injected intradermally into the ear of mice IN the area of Imprinting Control (ICR) (Harlan, Indianapolis, IN). The contralateral ear received the same amount of PBS (20. mu.l). Ear thickness was measured with a microcard ruler (Mitutoyo, Kanagawa, japan) 24 hours after injection and the increase in ear thickness induced by CAMP factor was reported as% of that of PBS-injected ears.

Detection of the enzyme ASM in ICR mice ear to intradermal injection of live Propionibacterium acnes (1X 10) in PBS into ICR mice ear⁷CFU/20. mu.l). The contralateral ear received an equal amount of PBS (20. mu.l). After 24 hours of bacterial stimulation, the ears were excised and perforated with an 8mm biopsy and homogenized in 200 μ l PBS using a tissue grinder. The supernatant (1 μ g total protein) was subjected to Western blotting using goat anti-ASM enzyme IgG (0.2 μ g/ml) (Santa Cruz Biotechnology, Inc.) followed by monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IgG (2 μ g/ml) (Fitzerald Inc, Concord, MA). Normal goat or mouse IgG was used as a negative control for the detection.

Injection of live propionibacterium acnes or PBS into the ear dermis of ICR mice was performed as described above. After 24 hours of bacterial challenge, ears were excised and mounted in Karnovsky's fixative (4% paraformaldehyde, 2.5% glutaraldehyde, 5mM CaCl)₂pH 7.4 in 0.1M sodium dimethylarsinate buffer) at 4 ℃ overnight, followed by 1% OsO in 0.1M sodium dimethylarsinate buffer, pH 7.4₄Medium fixation, bulk staining with 4% uranium acetate in 50% ethanol, followed by dehydration using a series gradient of ethanol, followed by dehydration with propylene oxide and infiltration with epoxy resin (Scipoxy812, Energy Beam Sciences, Agawam, MA). After polymerization at 65 ℃ overnight, thin sections were cut and stained with uranium acetate (4% uranium acetate in 50% ethanol) followed by staining with bismuth nitrate of the basic formula. The sections were examined using a Zeiss EM10C electron microscope (Oberkochen, Germany) at an accelerating voltage of 60 kV.

As described above, ICR mice were injected intradermally into the ear with live propionibacterium acnes or PBS. After 24 hours of bacterial stimulation, the ears were excised, fixed in Optimal Cutting Temperature (OCT) compounds (Sakura Finetek, Torrance, CA) and frozen at-80 ℃. Sections (7 μm) were fixed in 10% formamide in PBS. After blocking with PBS containing 5% BSA and anti-mouse Cluster of Differentiation (CD)16/CD32IgG (5. mu.g/ml) (BD Biosciences Pharmingen, spark, MD) for 30 minutes, sections were incubated with biotinylated anti-mouse CD11bIgG (5. mu.g/ml) (BD Biosciences Pharmingen), macrophage markers, and then goat anti-ASM enzyme IgG (5. mu.g/ml). Tetramethylrhodamine isothiocyanate (TRITC) -streptavidin conjugate (5. mu.g/ml) (ZYMED, Carlsbad, CA) and Fluorescein Isothiocyanate (FITC) -labeled anti-goat IgG (5. mu.g/ml) (Santa Cruz Biotechnology, Inc.) were added to the sections, incubated for 30 minutes at room temperature, followed by 4' -6-diamidino-2-phenylindole (DAPI) staining (Sigma). Images were obtained using an Olympus BX41 fluorescence microscope (Olympus, Center Valley, PA).

To examine the involvement of host ASM enzymes in propionibacterium acnes pathogenicity, ICR mice were injected intraperitoneally with either a selective ASM enzyme inhibitor in PBS (20mg/kg mice) or an equivalent amount of PBS as a control. After 30 minutes of desipramine treatment, ICR mice were injected intradermally with live propionibacterium acnes or PB as described above. Ear thickness was measured 24 hours after injection and the increase in the mouse ear induced by propionibacterium acnes was recorded as% of the ear thickness of the PBS-injected ear.

The combined effect of CAMP factor vaccine and local injection of anti-ASM enzyme IgG on Propionibacterium acnes-induced inflammation ICR mice were inoculated with inactivated E.coli overexpressing CAMP factor or GFP at 3 week intervals as described above. Two weeks after the second boost, the inoculated mice were injected intradermally into the ear with live propionibacterium acnes in the same manner as described above. Within 30 minutes, the left ear (receiving Propionibacterium acnes) was injected with goat anti-ASM enzyme IgG (4. mu.g/20. mu.l) or normal goat IgG (control) in PBS, and the right ear (receiving PBS) was injected with an equal amount of PBS. Ear thickness was measured 24 hours after injection and the increase in the mouse ear induced by propionibacterium acnes was recorded as% of the ear thickness of the PBS-injected ear.

To evaluate the expression of CAMP factor, e.coli transformed with an expression plasmid containing an insert encoding CAMP factor was incubated with or without IPTG. A major band of the expected size was detected in the insoluble fraction from IPTG-induced cells (the deduced molecular weight of the CAMP factor-6 × NH fusion protein was 324 kDa). The CAMP factor was purified using a talen resin column. The expression of CAMP factor was confirmed by in-gel tryptic digestion followed by NanoLC-LTQ MS/MS mass spectrometry sequencing. The 9 internal peptides derived from the CAMP factor were completely sequenced by NanoLC-LTQMS/MS mass spectrometry, matching well those from the Propionibacterium acnes CAMP factor (accession number: gi/50842175). The internal peptide of the CAMP factor was identified (AVLLTANPASTAK (SEQ ID NO: 3); amino acid residues 147-158), confirming the expression of the recombinant CAMP factor.

The biological activity of the recombinant CAMP factor was examined using a conventional CAMP assay, demonstrating that a co-hemolytic CAMP reaction occurs when spotted next to staphylococcus aureus expressing SM enzyme on sheep blood agar plates. The data indicate that recombinant CAMP factors are biologically active.

To examine the potential of CAMP factor as a vaccine target, ICR mice were immunized with CAMP factor or GFP control protein using an e. IgG against CAMP factor was detected by Western blot 14 days after immunization. No immunoreactivity against CAMP factor was detected in GFP-immunized mice. ELISA analysis showed a significant increase in antibody titer 14 days after immunization, increasing to 21 days after immunization. The anti-CAMP factor IgG titers in the antisera from CAMP factor immunized mice were > 100000 and those from GFP immunized mice < 100 after 21 days of immunization (antisera dilution curves not shown). These data indicate that CAMP factor is highly immunogenic.

To identify secreted CAMP factors, the propionibacterium acnes culture supernatant from the logarithmic growth phase was concentrated and Western blotted using mouse anti-CAMP factor antiserum. In propionibacterium acnes culture supernatant, a single band was detected at the position corresponding to the recombinant CAMP factor that had been treated with enterokinase to remove the 6x NH tag (fig. 11F, left panel, lane 1) (fig. 11F, left panel, lane 2), but not at the concentrated RCM used for the propionibacterium acnes culture (fig. 11F, left panel, lane 3). No bands were detected using the anti-GFP control antisera (fig. 11F, right panel). These data indicate that CAMP factor is a secreted protein.

To investigate the in vitro cytotoxicity of CAMP factor, human keratinocytes HaCaT or the murine macrophage line RAW264.7 were treated with recombinant CAMP factor or GFP control protein. Treatment with CAMP factor resulted in dose-dependent cytotoxicity in both HaCaT and RAW264.7 cells (fig. 11G). To examine the involvement of CAMP factor in propionibacterium acnes-induced inflammation, mice were injected intradermally with recombinant CAMP factor or GFP. A significant increase in ear thickness was observed in the CAMP factor-injected ears 24 hours after injection, but no increase was induced by GFP injection (fig. 11H). These data indicate that CAMP factor is involved in the propionibacterium acnes-induced inflammatory response.

To examine whether the ASM enzyme is released from the host cells when they were co-cultured with propionibacterium acnes, HaCaT or RAW264.7 cells were cultured with and without propionibacterium acnes for 14 hours. After incubation, cell culture supernatants were Western blotted, probed with mouse anti-CAMP factor antiserum and goat anti-ASM enzyme IgG. CAMP factor and ASM enzyme were detected in the cell culture supernatant only when the cells were co-cultured with propionibacterium acnes (fig. 12A, lanes 1 and 2). The homology between human and mouse ASM enzymes (mature proteins) is greater than 90% and their molecular weights are nearly identical. None of these ASM enzymes were detected in the cell culture without propionibacterium acnes (fig. 12A, lanes 3 and 4), and no ASM enzymes were detected in the propionibacterium acnes culture supernatant. These data indicate that ASM enzymes are released and/or secreted from host cells co-cultured with propionibacterium acnes.

To examine the neutralizing effect of CAMP factor on propionibacterium acnes-induced cytotoxicity, HaCaT and RAW264.7 cells were co-cultured with propionibacterium acnes in the presence of anti-CAMP factor or anti-GFP antiserum (fig. 12B). Propionibacterium acnes induced 29.3% and 44.0% death of HaCaT cells, respectively, in the presence of anti-GFP control antisera. Addition of anti-CAMP factor antiserum reduced propionibacterium acnes-induced cell death by 18.2% and 2.1%, respectively. To examine the involvement of host ASM enzymes in propionibacterium acnes pathogenicity, cells were co-cultured with propionibacterium acnes in the presence of desipramine, a selective ASM enzyme inhibitor, or an equivalent amount of PBS (vehicle) (fig. 12C). Propionibacterium acnes induced 43.4% and 45.4% of HaCaT and RAW264.7 cell death, respectively. The addition of desipramine significantly reduced the propionibacterium acnes-induced cell death by 21.9% and 30.6%, respectively. These data indicate that CAMP factor and ASM enzyme are involved in propionibacterium acnes-induced cytotoxicity.

To examine the host ASM enzyme participating in vivo propionibacterium acnes pathogenicity, propionibacterium acnes or PBS was injected intradermally into the ear of ICR mice, and the ear was excised 24 hours after bacterial stimulation and used in the following experiments. First, mouse ears were homogenized, and the supernatant of the homogenate was subjected to Western blotting using anti-ASM enzyme IgG (fig. 13A). A single band was detected at the expected molecular weight of the ASM enzyme (. about.60 kDa). Injection of propionibacterium acnes increased the amount of ASM enzyme in the ear compared to injection of PBS. To examine the localization of ASM enzyme in the mouse ear, the cryosections were co-stained with anti-mouse CD11B IgG, a conventional macrophage marker, followed by goat anti-ASM enzyme IgG (fig. 13B). This dual fluorescent staining showed that macrophages penetrated into the propionibacterium acnes application site 24 hours after bacterial stimulation; no CD11b + macrophages were observed in the PBS-injected control ears. ASM enzyme is highly expressed in infiltrated CD11b + macrophages. Transmission electron microscopy showed that p. acnes colonized and/or phagocytosed in macrophage-like cells and in the extracellular space 24 hours after bacterial stimulation (fig. 13C); no bacteria were observed in the PBS-injected ear. In addition, ruptured cell membranes were observed in the propionibacterium acnes injected ears, whereas the cell membranes in the PBS injected ears appeared intact. These data indicate that propionibacterium acnes stimulation induces macrophage infiltration that is highly expressing ASM enzyme. Furthermore, when mice were systemically pretreated with desipramine 30 minutes prior to bacterial stimulation, the propionibacterium acnes-induced ear swelling was significantly alleviated (fig. 13D), indicating that the host ASM enzyme is involved in the development of propionibacterium acnes-induced inflammation and skin lesions.

The effect of a combination of CAMP factor vaccination and local injection of anti-ASM enzyme IgG on propionibacterium acnes induced inflammation ICR mice were vaccinated with CAMP factor or GFP as a control. Propionibacterium acnes or PBS was injected intradermally into the ears of the vaccinated mice. After 30 minutes of stimulation, the left ear (receiving propionibacterium acnes) was injected topically with goat anti-ASM enzyme IgG or normal goat control IgG, while the right ear was injected with an equal volume of PBS. After 24 hours of bacterial stimulation, the combination of GFP vaccination with local injection of anti-ASM enzyme IgG and the combination of CAMP factor vaccination with local injection of normal IgG reduced the propionibacterium acnes-induced ear swelling to 202.8% and 193.5%, respectively, compared to the combination of GFP vaccination and normal IgG injection (224.5%). In contrast, the combination of CAMP factor vaccination with local injection of anti-ASM enzyme IgG synergistically reduced propionibacterium acnes-induced ear swelling to 153.7% (fig. 14). These data indicate that inhibition of both bacterial CAMP factor and host ASM enzyme synergistically reduces propionibacterium acnes-induced inflammation and skin damage.

To determine the antibody titer of CAMP factor by enzyme-linked immunosorbent assay (ELISA), purified CAMP factor (5. mu.g/ml) was coated with coating buffer (0.015M Na)₂CO₂，0.35M NaHCO₂And 0.05% NaN₃) Diluted and coated with 96-well ELISA plates (Corning, Lowell, MA) overnight at 4 ℃. Plates were washed with PBS containing 0.1% (w/v) Tween-20 and blocked with PBS containing 1% (w/v) BSA and 0.1% (w/v) Tween-20 for 2 hours at room temperature. Antiserum obtained from mice inoculated with E.coli overexpressing CAMP factor or GFP (1: 110000 diluted) were added to the wells and incubated for 2 hours. Goat anti-mouse IgG (H + L) IgG-horseradish peroxidase (HRP) conjugate (Promega, Madison, Wis.) (1: 5000 dilution) was added, incubated for 2 hours, and then washed. Using OptEIA^TMThe HRP activity was determined using the kit (BD Biosciences, San Diego, Calif.). The OD per well was measured at 450 nm.

Therapeutic Effect of CAMP factor vaccination on Propionibacterium acnes-induced inflammation live Propionibacterium acnes (1X 10) in PBS⁷CFU/20 μ l) injected intradermally into the middle portion of the left ear. Mu.l of PBS was injected into the right ear of the same mouse as a control. To examine the in vivo therapeutic effect of vaccination, ICR mice were inoculated with e.coli overexpressing CAMP factor or GFP 24 hours after bacterial stimulation as described above. Ear thickness was measured using a micro caliper for 98 days and the increase in ear thickness induced by propionibacterium acnes was recorded as% of the ear thickness of PBS-injected ears.

Human keratinocyte cell line HaCaT or murine macrophage cell line RAW264.7 were cultured in DMEM or RPMI1640 medium supplemented with 10% heat-inactivated FBS at 37 ℃ in air at 5% (v/v) CO2, respectively. To determine the co-cytotoxic activity of the SM enzyme and the CAMP factor, cells (1X 10)⁵Per well) in 96 well plates with 10mM MgCl₂In serum-free medium (5) SM enzyme from Staphylococcus aureus (350mU/ml, Sigma) or an equivalent amount of PBS (vehicle control) for 15 minutes. After pretreatment, cells were washed with PBS and then incubated with CAMP factor (25 μ g/ml) in 1% serum medium or GFP as a control for 18 hours. As a positive control for 100% cytotoxicity, triton-X was added to achieve a final concentration of 0.1% (v/v) for cell lysis. After incubation, cell viability was determined as described in the experimental procedures and cytotoxicity was calculated as the percentage of cell death caused by triton-X.

Hemolytic capacity is thought to be a virulence factor for many microbial pathogens to degrade tissue, invade host cells, disseminate themselves, and escape host immune attack. Microbial hemolysins generally have the ability to lyse red blood cells in vitro, but many are also toxic to other cell types. Propionibacterium acnes secreted CAMP factor as exotoxin (fig. 11). Although the hemolytic effects of CAMP factor have been demonstrated on erythrocytes and artificial plasma membranes, little attention has been paid to the cytotoxicity of CAMP factor against other cell types. Thus, the cytotoxic activity of CAMP factor on host cells, and the physiological significance of its pathogenicity in propionibacterium acnes associated with inflammatory acne vulgaris, were examined.

Human keratinocytes are one of the main targets of propionibacterium acnes. In addition, intradermal injection of live propionibacterium acnes into the ears of mice induced infiltration of numerous CD11B + macrophages (fig. 13B and C). Tissue chamber model data in combination with a dermal-based cell capture system was used to simulate the in vivo microenvironment of acne lesions, and injection of live propionibacterium acnes into the intradermally implanted tissue chamber draws Gr-1+ neutrophils and CD11b + macrophages into the chamber. Thus, the interaction between murine macrophages and propionibacterium acnes was studied in our model of propionibacterium acnes-induced inflammation in mice. Thus, the human keratinocyte line HaCaT and the murine macrophage line RAW264.7 were used to determine the in vitro cytotoxic activity of CAMP factor from propionibacterium acnes. The data indicate that CAMP factor is an important virulence factor for propionibacterium acnes degrading host cells (fig. 17G).

Evidence from a number of in vitro experiments suggests that in the absence of the SM enzyme, the CAMP factor co-hemolysin itself has only weak hemolytic activity. The CAMP factor has no significant homology to any other pore-forming toxins. Only the full-length recombinant CAMP factor was found to have co-hemolytic activity on sheep blood agar plates, but the structure-function relationship is still unclear. Lang and co-authors state that GBS CAMP factor binds to a Glycosylphosphatidylinositol (GPI) -anchored protein on the cell membrane of erythrocytes, which acts as a cell surface receptor for this toxin, and that interactions support its ability to form oligomer pores on sheep erythrocyte membranes. The amount of GPI-anchored proteins was increased by reducing sphingolipid levels on the cell membrane. Since GPI-anchored proteins are ubiquitous in mammalian cells, the same mechanism may be involved in the cytotoxic response of CAMP factors to keratinocytes and macrophages. Indeed, removal of sphingomyelin from cell membranes by enzymatic pretreatment with bacterial SM increased the susceptibility of cells to CAMP factors (fig. 17).

Several different forms of mammalian SM enzymes have been identified, including endosomal/lysosomal ASM enzymes (lytic enzymes), which are ubiquitous in mammalian tissues, and plasma membrane-associated or cytoplasmic neutral SM enzymes, which are mostly localized in the central nervous system. These enzymes catalyze the hydrolytic cleavage of sphingomyelin to ceramide on cell membranes by the same catalytic mechanism as bacterial SM enzymes. In turn, the released ceramide may serve as a cellular signal for a variety of activities, including apoptosis, differentiation, and proliferation. The activity of SM enzymes is regulated by a wide range of extracellular signals: growth factors, cytokines, neurotransmitters, hormones and stresses, such as ultraviolet light and reactive oxygen species. Ubiquitously expressed ASM enzymes play an important role in innate immune responses against infectious pathogens. Therefore, focus has been on the interaction of host ASM enzymes and bacterial CAMP factors associated with the pathogenicity of propionibacterium acnes. ASM enzymes were released and/or secreted from the host cells when the cells were co-cultured with propionibacterium acnes (fig. 18A). The cytotoxicity of propionibacterium acnes was neutralized in vitro in the presence of mouse anti-CAMP factor antiserum (fig. 18B). Furthermore, the addition of the specific ASM enzyme inhibitor desipramine to the co-cultured cells and propionibacterium acnes significantly reduced cytotoxicity. Data from in vitro experiments indicate that CAMP factor is a potential virulence factor for propionibacterium acnes and that host ASM enzymes are involved in the virulence of propionibacterium acnes.

Only a few studies have shown that CAMP factors are potentially virulent in vivo to pathogens. Rabbits and mice are lethal when high doses of partially purified CAMP factor from GBS are injected intravenously. Mice that have been infected with a sublethal dose of GBS develop fatal sepsis after receiving repeated injections of purified CAMP factor. The present disclosure demonstrates that intradermal injection of recombinant CAMP factor into the mouse ear induces ear swelling, suggesting that CAMP factor is involved in propionibacterium acnes-induced inflammation and skin damage in vivo.

To examine whether host ASM enzymes are involved in propionibacterium acnes pathogenicity and propionibacterium acnes-induced inflammation, propionibacterium acnes was injected intradermally into the ICR mouse ears according to the rat ear model described previously. The amount of soluble ASM enzyme in the ear increased after injection of propionibacterium acnes (fig. 13A). The characteristics of granulomatous inflammation in the mouse ear model (fig. 13B and C) were similar to those of inflammatory acne in human hair follicles; numerous propionibacterium acnes are observed in the infiltrating macrophage phagocytic body in the inflammatory acne lesions of the hair follicle. Propionibacterium acnes is resistant to killing by phagocytic cells and is able to survive within macrophages. It was demonstrated that GBS β -hemolysin/cytolysin, a pore-forming exotoxin, promotes the destruction of the immune defenses of phagocytic hosts. During the intracellular life cycle of listeria monocytogenes, the pore-forming toxin called listeria lyst O is primarily responsible for mediating the disruption of the phagosome membrane to allow its escape from the phagosome into the host cytoplasm, i.e., the appropriate environment for its replication. Lysosomal ASM enzymes are known to promote phagocytic killing of bacteria in the early stages of phagocytosis and require proper fusion of the late phagosome with the lysosome, which is critical for efficient transfer of lysosomal antibacterial hydrolases into the phagosome. Taken together, phagocytosed propionibacterium acnes within macrophages utilize host lysosomal ASM enzymes to enhance toxicity of CAMP factors to escape from the phagosomes, an interaction involved in the resistance of propionibacterium acnes to phagocytosis. Indeed, we observed a number of macrophages in the propionibacterium acnes-stimulated ear, among which many cells were destroyed by the colonizing propionibacterium acnes (fig. 19D). Infection with salmonella or escherichia coli triggers an early surge in the extracellular secretion and/or release of ASM enzyme activity from macrophages. Propionibacterium acnes ingeniously utilizes ASM enzymes released and/or secreted from macrophages to invade cells or to escape from cells in order to spread from cell to cell.

An effective vaccine for propionibacterium acnes-related inflammation as an alternative acne treatment consists of killed whole organisms propionibacterium acnes and propionibacterium acnes cell wall anchored sialidases. Thus, the possibility of CAMP factor as a target for acne vaccine development was examined. The propionibacterium acnes CAMP factor was highly immunogenic when vaccination was performed by an e.coli based vaccine delivery system (fig. 11D and E). Vaccination with Propionibacterium acnes CAMP factor elicited protective immunity against Propionibacterium acnes-induced inflammation. In addition, local injection of anti-ASM enzyme IgG into the mouse ear reduced propionibacterium acnes-induced inflammation. The combination of CAMP factor vaccination with local injection of anti-ASM enzyme IgG synergistically reduced propionibacterium acnes-induced ear swelling (figure 14). The data suggest that CAMP factor and host ASM enzymes interact synergistically in vivo.

Propionibacterium acnes utilizes host ASM enzymes to expand its CAMP factor-mediated pathogenicity lysosomal ASM enzymes in macrophages play an important role in innate immune responses against infectious pathogens; enzymes promote macrophage-mediated killing of pathogens and fusion of lysosomes with phagosomes. However, propionibacterium acnes can enhance its CAMP factor-mediated pathogenicity using ASM enzymes released and/or secreted from the host cell. The synergy promotes their escape from the host's immune defenses, degradation of host tissues and transmission of pathogens from cell to cell. Recent studies provide sufficient evidence that propionibacterium acnes is involved not only in acne vulgaris, but also in a number of diseases including endocarditis, endophthalmitis, osteomyelitis, joint, nervous system, cranial neurosurgical infections, and implanted biomaterial contamination. Therapies targeting propionibacterium acnes CAMP factor and host ASM enzymes would have the potential to be widely applied to these propionibacterium acnes-related diseases to inhibit pathogen expansion.

Japanese radish sprouts (Kaiware-daikon) (radish) were obtained from commercial suppliers (ICREST International, JCP, Carson, Calif.). A9 cm long Japanese radish bud with two leaves was used and grown under a 23 watt fluorescent bulb (Philips, Portland, OR) at room temperature and sprayed with water daily.

Binary vector pBI121(Jefferson, 1987) carrying reporter gene GUS driven by cauliflower mosaic virus 35S promoter was used for gene construction. Cloning the CAMP factor cDNA open reading frame in pEcoli-Nterm6XHN vector, by using a forward primer (5'-CCTTCTAGAGGAGATATACCATGGGTCATAATCAT-3'; SEQ ID NO: 18) and a reverse primer (5'-TCCCCCGGGTTAATTAATTAAGCGGCCGCC-3' (SEQ ID NO: 19) using a forward primer (5'-AGATCTAGAATGTCTGGTTCTCATCATCATCATC-3'; SEQ ID NO: 20) and a reverse primer (5'-GCCCCCGGGTTAGCCTTCGATCCCGAGGTT-3' (SEQ ID NO: 21) amplification of SCAP cDNA cloned into pIVEX-MBP vector (Liu et al, 2008.) primers were designed to add restriction sites at the end of the PCR product specifically, restriction sites XbaI and SmaI were encoded in the forward and reverse primers respectively, the PCR product was treated with XbaI and SmaI, the polylinker site of the pBI121 vector was then cloned to generate 35S: CAMP factors-His and 35S: the SCAP-MBP-His construct.

All constructs were transformed into Agrobacterium tumefaciens strain LBA4404 using a liquid nitrogen freeze-thaw method (An et al, 1988). A single colony of LBA4404 cells was inoculated in 5ml of YEP medium [10mg/ml bacto-trypton (DIFCO, Detroit, MI), 10mg/ml yeast extract (DIFCO, Detroit, MI), and 5mg/ml NaCl (Sigma, St. Louis, MO; pH 7.5)]Shaking was carried out overnight at 250rpm at 28 ℃. Subsequently, 2ml of the liquid culture was inoculated into 50ml of fresh YEP and incubated at 28 ℃ with shaking at 250rpm until OD₆₀₀Up to 0.8. The bacteria were centrifuged at 3000 Xg for 5 minutes at 4 ℃ and the pellet was resuspended in 1ml of 20mM calcium chloride. The bacteria (0.2ml) were transferred to a 1.5ml centrifuge tube and 1. mu.g of the gene construct was added. The mixture was frozen in liquid nitrogen for 5 minutes and then thawed in a water bath at 37 ℃ for 5 minutes. To the mixture was added 1ml of YEP medium and incubated at 28 ℃ for 2 to 4 hours with shaking at 150 rpm. The bacteria were centrifuged at 3000 Xg for 5 minutes and then resuspended in 0.1ml of YEP. Transformants were selected by plating the bacteria in YEP-agar medium (YEP medium containing 1.5% agar) containing antibiotics (50g/ml kanamycin and 50g/ml streptomycin) and culturing at 28 ℃ for 2 to 3 days.

Gene constructionIn vivo to the leaf of Agrobacterium infiltration and protein extraction A single colony of Agrobacterium tumefaciens transformants was included in 50g/ml kanamycin and 50g/ml streptomycin 2ml YEP medium, at 28 ℃ with shaking at 250rpm until OD₆₀₀Up to about 0.5. Thereafter, the bacteria were harvested by centrifugation at 1,300 Xg for 5 minutes and resuspended in 2ml of sterile ddH₂And (4) in O. All bacterial suspensions were kept at room temperature for 30 minutes until agroinfiltration. Non-transformed Agrobacterium served as a negative control and was cultured under the same conditions as the transformants were cultured but without kanamycin addition to the medium. For injection infiltration, the center sub-epidermis of potted seedling leaves (i.e., 25mm of culprit center)²Zone) was cut with a sterile scalpel (No. 15, Feather SafetyRazor co., Osaka, japan) and 0.1ml of agrobacterium bacterial suspension (5 × 10)⁷CFU) was injected into the wound site, which was placed between the finger and a 1ml syringe (BD, Bioscience, San Diego, CA). Infiltration was confirmed by visual monitoring of the spread of the bacterial suspension to the leaf margins (Schob et al, 1997). The agrobacterium infiltrated leaves were grown for 5 days before GUS assay and immunization was performed. Using a catalyst consisting of 0.1M NaPO₄(pH 7.0)、0.5mM K₃Fe(CN)₆、0.5mM K₄Fe(CN)₆Histochemical GUS assay solution consisting of 0.1% (v/v) Triton X-100 and 0.05% (w/v) 5-bromo-4-chloro-3-indolyl-. beta. -D-glucuronic acid, cyclohexylammonium salt (Sigma, St. Louis, Mo.) stained leaves infiltrated with Agrobacterium. The leaves were immersed in the staining solution and incubated overnight at 37 ℃ in the dark. After incubation, the leaves were removed from the staining solution and immersed in a stop solution containing 42.5% (v/v) ethanol, 10% (v/v) formaldehyde and 5% (v/v) acetic acid (Jefferson, 1987). Quantitative determination of GUS activity was achieved by fluorimetry. The whole leaf was treated with 200. mu.l of 1 XCCLR [100mM K-phosphate (pH 7.8), 1mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100 and 7 mM. beta. -mercaptoethanol]And (6) grinding. The mixture was centrifuged at 10000 Xg for 5 minutes at 4 ℃ and 200. mu.l of the supernatant was taken and added to a new microtube on ice, followed by mixing with 1mM 4-methylumbelliferone-D-glucuronide buffer for 1 hour at 37 ℃ (Jefferson, 1987). Fluorescence spectrophotometer (Molecular) by SpectraMAX GeminiEM using fluorescence spectrometer methodDevices, Sunnyvale, Calif.) in OD₃₆₅Excitation and OD of₄₅₅The emission of (a) measures the enzymatic reaction. To study the dynamic expression of antigen in radish leaves, leaves were removed at 0, 1,3 and 5 days to quantify GUS expression.

Purification of CAMP factor and SCAP from leaf tissue was performed on Ni-NTA agarose columns (Qiagen, Valencia, CA) by performing affinity chromatography with certain modifications. The column was washed with water and buffer A (8M guanidine, 100mM NaH)₂PO₄10mM Tris-HCl, pH 8.0). Leaf material (1g) was triturated in 15ml of ice-cold extraction buffer A under liquid nitrogen using a mortar and pestle. Guanidine-solubilized protein was centrifuged at 12000 Xg for 20 minutes to remove debris and insoluble material, and the supernatant was gently stirred with 1.6ml of Ni-NTA agarose resin at room temperature for 1 hour. The mixture was loaded onto a column that had previously been equilibrated with buffer a. Briefly, buffer B (8M Urea, 100mM NaH) was used₂PO₄10mM Tris-HCl, pH 6.8). Finally, buffer C (8M Urea, 100mM NaH) was used₂PO₄10mM Tris-HCl, pH6.3), buffer D (8M Urea, 100mM NaH)₂PO₄10mM Tris-HCl, pH 5.9) and buffer E (8M Urea, 100mM NaH)₂PO₄10mM Tris-HCl, pH 4.5).

Intranasal immunization with female ICR mice (3 to 6 weeks old; Harlan, Indianapolis, IN) maintained the potential to induce mucosal immune responses (Mantis, 2005). Mice were housed according to institutional guidelines. Central region (25 mm) of 5 radish leaves expressing GUS or CAMP factor alone²) Cut with a sterile scalpel. To avoid gene incorporation for Agrobacterium transgenesis, leaves were mixed and grown at 700 lddH₂O and then passed through a UV cross-linker (Spectronics, Westbury, NY) at 7000J/m²Sterilizing for the next 30 minutes. They were inactivated as evidenced by the inability of the sterilized agrobacterium to form colonies on YEP agar plates (data not shown). Then, whole leaves containing only CAMP factor or GUS (as a negative control) were inoculated intranasally to the nasal cavity of ICR mice without mixing with adjuvantInternal (25 l/mouse). Three boosts were performed at the same dose at 1, 2 and 4 weeks after the first immunization.

To detect antigen expression, 15g of recombinant GUS and 15g of whole leaves expressing only the CAMP factor or SCAP were separated using 10% SDS-PAGE. The bands were transferred to nitrocellulose membranes by electrophoresis (Gil et al, 2006). The membranes were probed with anti-CAMP factor serum obtained from mice immunized with UV-irradiated escherichia coli BL21(DE3) (Liu et al, 2008) overexpressing the propionibacterium acnes CAMP factor. To confirm antibody production in the immunized mice, purified CAMP factor (65g) was loaded on 10% SDS-PAGE and transferred to nitrocellulose membrane. Blotting membranes were immunoreactive with serum (1: 500 dilution) obtained from mice immunized with whole leaves containing CAMP factor for 4 weeks. Antibodies [ immunoglobulin G (IgG) ] were detected with anti-mouse horseradish peroxidase-conjugated IgG (1: 5000 dilution, Promega, Madison, Wis.). Peroxidase activity was visualized using a western lighting chemiluminescence kit (PerkinElmer, Boston, MA).

anti-CAMP factor serum against Propionibacterium acnes induced inflammation passive immunity by heating at 56 ℃ for 30 minutes to inactivate the complement in the serum. Propionibacterium acnes were pretreated with 5% (v/v) inactivated anti-GUS serum or anti-CAMP factor serum in culture medium at 37 ℃ for 2 hours. anti-GUS serum (3.63 + -1.47X 10)⁸CFU) and anti-CAMP factor serum (3.3 + -1.2X 10)⁸CFU) incubation for 2 hours each did not significantly affect the growth of propionibacterium acnes. ICR mice were injected intradermally with 25. mu.l of anti-GUS or anti-CAMP neutralized Propionibacterium acnes (1X 10) suspended overnight in PBS⁷CFU). As a control, 25. mu.l of PBS was injected into the right ear of the same mouse. The increase in ear thickness was measured using a micro caliper (Mitutoyo, japan) after bacterial injection and the increase in ear thickness of propionibacterium acnes-injected ears was calculated as% PBS injection control. For histological observation, the ear was cut 3 days after injection, cross-sectioned, and incubated with H&E stained and viewed under a Zeiss Axioskop2 plus microscope. To count bacterial colonies, bacteria-injected ears were placed on a vibrating homogenizer (mini-loader, Biospec Products, Bartlesville, OK) on 0.5ml of 2.0mm zirconia beads (BiospecPr)oducts, Bartlesville, OK) for 1 minute. The number of bacteria in the homogenate was quantified by serially diluting the bacteria and plating them on RCM plates. After centrifugation at 1,300 Xg, MIP-2 in the supernatant was measured by an ELISA kit according to the manufacturer's instructions (BD Biosciences, San Diego, Calif.).

To investigate whether passive administration of neutralizing antiserum affected the survival of other sites of Propionibacterium acnes, ICR mice were intradermally injected with 25 μ l aliquots of either anti-GUS serum or anti-CAMP factor serum-neutralized Propionibacterium acnes (1X 10)⁷CFU). The right ear of the same mouse was injected with only the same amount of live Propionibacterium acnes (1X 10)⁷CFU) overnight. The number of bacteria was calculated by counting colonies on RCM plates.

Compared with the active immunization of CAMP factor targeted vaccine, the passive neutralization CAMP factor has approximately equal potential in inhibiting propionibacterium acnes-induced otitis. Therapeutic antibodies against CAMP factor described herein may be extended for the treatment of a variety of propionibacterium acnes-associated human diseases, including implant infections, pulmonary sarcoidosis, osteomyelitis, and endocarditis (nakatsui et al, 2008 c; Nishiwaki et al, 2004; Zouboulis, 2004). With a view to use in humans, further studies will include the generation of therapeutic monoclonal antibodies against the propionibacterium acnes CAMP factor. The topical administration of human monoclonal antibodies against CAMP factor on the skin of patients with severe acne will eliminate propionibacterium acnes locally without interfering with the colonization of propionibacterium acnes and other commensals in other parts of our body.

The following sequences are incorporated herein by reference. GenBank accession NC-006085 (full-length genome of Propionibacterium acnes, whose sequence is incorporated herein by reference; the polypeptide sequence of Propionibacterium acnes CAMP factor is identified as SEQ ID NO: 7 and the corresponding encoding polynucleotide is identified as SEQ ID NO: 6; and the polypeptide of lipase is identified as SEQ ID NO: 9 and the corresponding encoding polynucleotide is identified as SEQ ID NO: 8. the human ASM enzyme is identified as SEQ ID NO: 11; and the corresponding encoding polynucleotide sequence is identified as SEQ ID NO: 10. homologs and variants of the ASM enzyme are known. for example, sphingomyelin phosphodiesterase 1, acid lysosomal isoform 2 precursor [ human (Homo sapiens gig) ] |56117842| ref | NP-001007594.1 [56117842], sphingomyelin phosphodiesterase 1, acid lysosomal isoform 1 precursor [ Homo sapiens ] gi |56117840| NP-000534.3 |56117840 ]; sphingomyelin phosphodiesterase 1, acidic lysosome [ mus musculus (mususculus) ] gi |6755582| ref | NP _035551.1| [6755582 ]; sphingomyelin phosphodiesterase 1, acidic lysosome [ mus musculus ] gi |21961231| gb | AAH34515.1| 21961231; sphingomyelin phosphodiesterase 1, acid lysosome [ mus musculus ] gi |15030106| gb | AAH11304.1| 15030106, sequences accompanying accession numbers are incorporated herein by reference. The propionibacterium acnes sialidase sequence is set forth in SEQ ID NO: 13 and the corresponding polynucleotides are provided in SEQ ID NO: 12 is provided. Propionibacterium acnes sialidase B is shown in SEQ ID NO: 15 and the corresponding polynucleotides are provided in SEQ ID NO: 14 is provided. Sialidase-like polypeptide and coding sequence are set forth in SEQ ID Nos: 17 and 16.

Claims (26)

1. An immunogenic composition comprising a substantially purified polypeptide comprising a sequence as indicated in table 1, an immunogenic fragment thereof, and any combination of the foregoing.

2. The immunogenic composition of claim 1, comprising a CAMP factor, lipase or sialidase polypeptide or fragment thereof.

3. The immunogenic composition of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. 3,7, 9 and 11 or immunogenic fragments thereof.

4. The immunogenic composition of claim 1, 2 or 3 wherein the polypeptide is expressed in a vector.

5. The immunogenic composition of claim 4, wherein the vector comprises an attenuated bacterial vector or an attenuated viral vector.

6. The immunogenic composition of claim 5, wherein the vector comprises an E.coli or adenovirus.

7. The immunogenic composition of claim 1, wherein the composition comprises at least one attenuated bacterial vector expressing or comprising at least one polypeptide selected from the group consisting of a CAMP factor, a lipase, or a sialidase.

8. A composition comprising at least one recombinant attenuated bacterial or viral vector comprising at least one polynucleotide encoding one or more Propionibacterium acnes (Propionibacterium acnes) polypeptides selected from CAMP factors, lipases, or sialidases, and an inhibitor of ASM enzyme activity, such that the polypeptides are expressed in the at least one recombinant attenuated vector.

9. The composition of claim 1, wherein the inhibitor of ASM enzyme activity comprises an antibody.

10. The composition of claim 1, wherein the ASM enzyme inhibitor comprises a small molecule inhibitor.

11. A method of inducing protective immunity in a subject, comprising administering to the subject the composition of any one of claims 1-3 or 8 and contacting the subject with an asmase inhibitor.

12. The method of claim 10 or 11, further comprising boosting the subject comprising administering an immunogenic composition comprising the same components or different components comprising the same antigenic polypeptide.

13. An immunoprotective composition comprising at least one attenuated vector expressing an antigen for inducing an immunoprotective response against propionibacterium acnes, said antigen comprising an extracellular or immunogenic protein of propionibacterium acnes or an immunogenic fragment thereof linked to a transcriptional promoter and a termination signal.

14. The immunoprotective composition of claim 13, wherein the propionibacterium acnes protein or fragment thereof is selected from the group consisting of CAMP factor, lipase, sialidase, and any combination thereof.

15. The immunoprotective composition of claim 10 or 11, wherein the composition comprises a strain selected from Campylobacter jejuni (Campylobacter jejuni), Campylobacter coli (Campylobacter coli), Listeria monocytogenes (Listeria monocytogenes), Yersinia enterocolitica (Yersinia enterocolitica), Yersinia pestis (Yersinia pestis), Yersinia pseudotuberculosis (Yersinia pseudotuberculosis), Escherichia coli (Escherichia coli), Shigella flexneri (Shigella flexneri), Propionibacterium acnes (Propionibacterium acnes), Shigella dysenteriae (Shigella dysseniae), Shigella dysenteriae (Shigella dysenteriae), Shigella boydii (Shigella Vibrio), Vibrio pylori (Vibrio pylori), Helicobacter felis (Vibrio felis), Salmonella typhi (Vibrio bacterioides), Vibrio fragilis (Vibrio bacterioides), Vibrio fragilis (Vibrio typhi), Vibrio typhi (Vibrio lactis), Vibrio lactis (Vibrio lactis), Vibrio lactis (Vibrio lactis), Vibrio lacti, Attenuated vectors of Salmonella (Salmonella gallinarum), Salmonella pullorum (Salmonella pulmorum), Salmonella choleraesuis (Salmonella choleraesuis), Salmonella enteritidis (Salmonella enteritidis), Streptococcus gordonii (Streptococcus gordonii), Lactobacillus sp (Lactobacillus sp.), Klebsiella pneumoniae (Klebsiella pneumoniae), Enterobacter cloacae (Enterobacter cloacae), and Enterococcus faecalis (Enterococcus faecalis).

16. The immunoprotective composition of claim 13, further comprising a pharmaceutical diluent.

17. The immunoprotective composition of claim 13, wherein the composition further provides protective immunity against klebsiella pneumoniae (k. pneumoconia), staphylococcus aureus (s. aureus), and/or streptococcus pyogenes (s. pyogenes) infection.

18. A method of protecting a susceptible host against infection by propionibacterium acnes comprising administering to the host an amount of the immunoprotective composition of claim 1 or claim 13 sufficient to elicit an immunoprotective response in the host and administering an ASM enzyme inhibitor.

19. The method of claim 18, wherein the composition is in a pharmaceutically acceptable carrier.

20. A recombinant attenuated bacterial or viral vector comprising a polynucleotide encoding at least one antigenic polypeptide selected from the group consisting of a CAMP factor, lipase or sialidase from propionibacterium acnes.

21. A method of providing protective immunity to a subject comprising administering to the subject the recombinant attenuated vector of claim 18.

22. A composition for treating propionibacterium acnes infection comprising an ASM enzyme inhibitor.

23. The composition of claim 22, further comprising a CAMP antigen or vaccine.

24. An antigenic composition comprising disrupted non-infectious propionibacterium acnes cells and further comprising an ASM enzyme inhibitor.

25. A method of treating propionibacterium acnes comprising administering to a subject a vaccine comprising a CAMP factor and a composition comprising an ASM enzyme inhibitor.

26. The method of claim 25, wherein the vaccine and the composition are administered simultaneously.