Propionibacterium acnes prophylactic and therapeutic immune treatment

Abstract

To provide a method of generating a vaccine, and a vaccine, for use in the treatment or prevention of P. acnes-related infections.SOLUTION: Provided is a method including selecting at least one antigen comprising at least one P. acnes epitope, wherein the P. acnes epitope is an epitope that is directly accessible to human serum antibodies when presented by live P. acnes bacteria, specific binding of the epitope by the human immune system leads to a reduction in bacterial number, fitness, growth, pathogenicity or a combination thereof, binding of the epitope by the immune system is demonstrated by using a flow cytometry assay; and the reduction in bacterial number, fitness, growth, pathogenicity or a combination thereof is demonstrated by an opsonophagocytic killing assay (OPK) in the presence of antibodies specific for the at least one antigen comprising at least one P. acnes epitope.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention refers to vaccine antigens that can be used for the preventive and therapeutic treatment of P. acnes-associated pathologies. [Background technology]

[0002] 1. Background The Gram-positive bacterium Propionibacterium acnes (P. acnes; recently proposed to be renamed Cutibacterium acnes) is a skin commensal that resides primarily within the sebaceous glands, which provide a unique lipid-rich environment due to sebum secretion. Although aerotolerant, P. acnes prefers anaerobic growth conditions and utilizes sebum, cellular debris, and metabolic by-products from surrounding skin tissue as its primary source of energy and nutrients. Away from the skin, P. acnes has also been found in the conjunctiva, respiratory tract, genitourinary tract, and digestive tract of humans and other animals.

[0003] P. acnes is best known for its role in skin disorders such as acne vulgaris. Acne is a disease of the pilosebaceous unit of the skin that affects over 85% of young people, with over 20% of the population continuing to experience significant symptoms beyond the teenage years. Acne vulgaris presents in a range of severity: mild, moderate, and severe. Moderate and severe acne account for over one-third of all cases and require medical treatment. Acne can also appear after puberty as an adult-onset condition often associated with hormonal fluctuations that are more common in women. While its relationship with acne vulgaris is most firmly established, P. acnes is suspected to play a role in other severe forms of acne, such as acne conglobata, acne fulminans, and cystic acne. In addition to dermatological pathologies, P. acnes has also been found in corneal ulcers and is a common cause of chronic endophthalmitis after cataract surgery. A variety of other inflammatory diseases have been associated with P. acnes, including postoperative prosthetic implant- and device-associated infections (implant-associated infections), endocarditis, sarcoidosis, osteomyelitis, allergic alveolitis, pulmonary vasculitis, SAPHO syndrome (joint synovitis, acne, pustulosis, hyperostosis, and osteitis), and inflammation of the lumbar nerve roots leading to sciatica. More recent studies have suggested a potential pathogenic role for P. acnes in noninfectious diseases such as prostate cancer, where its ability to persist intracellularly can lead to altered gene expression.

[0004] Antibiotics have been used for decades as one of the most common treatments for both localized and systemic acne. However, antibiotics are not specifically effective against P. acnes and cannot be administered for long periods, especially because widespread use of antibiotics leads to the proliferation of antibiotic-resistant bacteria. Vitamin A derivatives (retinoids) represent the second most widely used treatment option because they suppress sebaceous gland activity, thereby indirectly reducing inflammatory lesions. However, oral retinoids are not effective in all patients and are not curative because recurrence after treatment cessation is frequent; furthermore, they have been associated with severe side effects, including elevated serum triglyceride levels, acute pancreatitis, hepatotoxicity, clinical depression, and severe birth defects in pregnant women. Other traditional acne treatments include oral contraceptives and topical antiseptics, such as benzoyl peroxide. Like retinoids, none of these treatments are long-term effective or curative, are not suitable for all patients, and long-term use is associated with unwanted side effects. Furthermore, drugs currently being tested in clinical trials are not significantly different from those discovered decades ago and have shown disappointing results (Non-Patent Document 1). Therefore, there is a need for more effective acne treatments with improved safety profiles. Furthermore, Preventive acne treatments, particularly for teenagers with a family history of acne, an important predisposing factor, would be extremely beneficial, yet currently lack one. Also, preventative treatment in the form of a vaccine specifically designed to induce higher levels of protective immunity against P. acnes would significantly reduce the risk of implant-associated infections and other conditions in which P. acnes plays a pathogenic role. Despite significant medical need and decades of research, progress in developing immune-based treatments for P. acnes-associated pathologies has been extremely slow and hindered by confusion in the field regarding disease pathogenesis and the role of the human immune system in disease.

[0005] The manner and extent to which P. acnes contributes to acne pathogenesis and other pathologies remains debated due to the multifactorial nature of the disease; the fact that P. acnes colonizes all individuals, as Koch disease claims, may not be applicable; its identification in different clinical samples has often been attributed to contamination during sample processing by laboratory personnel. A further complicating factor is the lack of a proven predictive animal model that accurately represents the complexity and organization of human tissue and recapitulates human host interactions with skin-colonizing microorganisms such as P. acnes. Animal skin differs from human skin histologically, biochemically, and immunologically; P. acnes does not colonize animal skin and does not induce acne lesions. Several different P. acnes animal models have been published by different research groups, but they have not been accepted by the broader acne research community because they lack key symptoms of the human disease (Non-Patent Document 2). For all these reasons, research efforts in the field have focused on evaluating the growth and behavior of P. acnes in various in vitro systems, examining acne-affected skin compartments by various histological methods, and recently much attention has been devoted to analyzing the microbial communities that colonize human skin, collectively known as the human skin microbiota, and their interactions with host cells leading to inflammation or infection, which remain the subject of intense research efforts.

[0006] Thus, the current state of the art in the treatment of acne is summarized in Table III of Non-Patent Document 3:

[0007] [Table 1]

[0008] In this guideline table, all available evidence for acne treatments was evaluated using a uniform system called the Strength of Recommendation Taxonomy (SORT), developed by the editors of the journal US Family Medicine and Primary Care. Evidence was graded using a three-point scale based on methodological quality (e.g., randomized trials, case-control, prospective / retrospective cohorts, case series, etc.) and the overall focus of the study (i.e., diagnosis, treatment / prevention / screening, or prognosis), as follows: I. Good quality patient-directed evidence (i.e., evidence measuring outcomes that are important to patients: morbidity, mortality, symptom improvement, cost reduction, and quality of life). II. Limited quality patient-oriented evidence. III. Other evidence, including consensus guidelines, opinions, case studies, or disease-oriented evidence (i.e., evidence measuring intermediate, physiologic, or surrogate endpoints that may or may not reflect improved patient outcomes).

[0009] Clinical recommendations were made with respect to the best available evidence tabulated in the guidelines. The strength of the recommendations was ranked as follows: A. Recommendations based on consistent, good-quality, patient-directed evidence. B. Recommendations based on inconsistent or limited patient-directed evidence. C. Recommendations based on consensus, opinion, case studies, or disease-directed evidence.

[0010] (While most treatments aim to kill or reduce P. acnes) It is noteworthy that the chin vaccination approach did not make it to the clinic.

[0011] Furthermore, recent review articles in this field, which contain current knowledge and perspectives on potential future treatments, either do not fully address vaccination (Non-Patent Document 4), are highly skeptical of vaccination as a viable approach (Non-Patent Document 1), or do not provide specific solutions regarding the selection and composition of vaccine antigens, but only provide an overview of the various P. acnes virulence factors involved in pathogenesis and suggest that more thorough and extensive research is needed to further define the appropriate role of these virulence factors in P. acnes pathogenesis (Non-Patent Document 2, Non-Patent Document 5). Notably, Zouboulis et al. concluded in a 2017 review that "the potential for vaccination against P. acnes has been investigated" (Non-Patent Document 1, p. 819), "however, relevant research was halted in 2011." Vaccines against P. acnes antigenic structures have not been clearly shown to be effective in humans with acne. Given the results obtained with such vaccination approaches, the authors questioned the "potential role of vaccination" in principle to combat acne, "particularly with regard to patient selection, the role of P. acnes in acne, and the efficacy of vaccination in disease with a no-virus background." [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Zouboulis et al., 2017 [Non-patent document 2] O'Neill and Gallo 2018 [Non-patent document 3] “Guidelines of care for the management of acne vulgaris”, Zaenglein et al., 2016 [Non-patent document 4] Lee et al., 2019 [Non-patent document 5] McLaughlin et al., 2019 [Non-patent document 6] Chattopadhyay et al. Prot. Sci. Vol. 28 (2019), pp. 1127-1134 [Non-Patent Document 7] Martin et al., 2014-NanoTemper Technologies GmbH-Application_Note_NT-PR-001_-_Thermal_Unfolding [Non-patent document 8] Krakowiak et al., J. Biol. Phys. 45 (2019), pp. 161-172 [Non-Patent Document 9] Xiao et al., Biochemistry 49 (2010), 5588-5599 [Non-Patent Document 10] Wingfield, Curr. Protoc. Protein Sci. Vol. 88 (2018), 6.14.1-6.14.3 Summary of the Invention [Problem to be solved by the invention]

[0013] 2. Objectives of the Invention It is an object of the present invention to overcome the shortcomings of the prior art teachings of P. acnes vaccines and to provide improved means and methods for preventing and combating P. acnes infections and P. acnes-related disorders.

[0014] A further object is to provide improved P. acnes vaccines and immunotherapy against P. acnes.

[0015] More particularly, another object of the present invention is to provide a wide range of different pathogens capable of causing disease. The objective is to provide a vaccine that is protective against strains, phylotypes, and variants of P. acnes, particularly at least two or more ribotypes of P. acnes, or at least two or more CC types of P. acnes, or at least two or more MLST phylotypes of P. acnes, or all three major phylotypes of P. acnes, namely types I, II, and III.

[0016] Preferably, the present invention can provide protection from P. acnes strains with various states and levels of antigen expression on the surface of P. acnes, particularly from strains (pathogenic variants and pathogenesis-associated variants) that express genes that mediate host invasion and infection, and whose expression products can induce antibodies with opsonizing and killing or neutralizing activity that can increase the efficiency of the host adaptive immune response against the bacteria.

[0017] A further object of the present invention is to select antigens and antigenic epitopes that are immunogenic, accessible to antibody binding on the surface of P. acnes, and that induce antibodies that have functional activity against P. acnes, such as opsonophagocytic impairment, neutralization of pathogenic potential (e.g., reducing the potential for adhesion to host tissue and cell invasion, reducing intracellular survival / persistence, reducing bacterial fitness through interference with iron acquisition and growth, reducing the potential for bacterial biofilm formation, or preventing the spread of infection by activating the adaptive immune response, etc.).

[0018] As a further preferred object, the present invention may provide improved antigens that have increased immunogenicity and induce cross-binding and / or cross-reactive antibodies, especially cross-type reactive antibodies.

[0019] It is a further object of the present invention to provide immunorelevant polypeptides and vaccines that, upon immunization, induce antibodies that greatly increase the ability of phagocytes against bacteria, preventing their growth and pathogenic behavior.

[0020] Furthermore, another preferred object is that the present invention may provide vaccine compositions and formulations with increased stability, purity, and amenability for vaccine manufacture and administration to a human host.

[0021] Furthermore, another preferred object of the present invention is to select an antigen that drives the production of antibodies in the human host during infection, which can specifically bind to P. acnes in its pathogenic state and instruct phagocytes to absorb and remove it, reducing its numbers at the site of infection and preventing the spread of infection to surrounding tissues.

[0022] Furthermore, another preferred object of the present invention is to induce an immune response against immunologically relevant antigens that are expressed and accessible on the surface of P. acnes biofilms formed by strains of different genetic backgrounds and that similarly direct the immune response to the bacterial biofilm to help prevent its formation and spread, and / or that are specifically bound by antibodies so that their degradation and removal by immune effectors will help enable control of P. acnes in patients. [Means for solving the problem]

[0023] 3. Summary of the Invention Thus, the present invention provides subject matter, including embodiments, as claimed and further described herein. The present invention provides substantial and clinically relevant strategies for immunologically targeting P. acnes and therapeutic outcomes associated with this microorganism. The present invention is intended to immunologically address the immune system of a patient (human) to enable effective prevention or treatment of P. acnes symptoms. By the products, particularly vaccines and therapeutic and preventive methods, according to the present invention, P. acnes symptoms are effectively controlled, ameliorated, or cured. In contrast to the current situation in the field of P. acnes symptom treatment (particularly for acne vulgaris: Zaenglein et al., 2016), where a live vaccination approach has not been suggested and the immunopathology of acne is shown as one of the gaps in knowledge of disease pathogenesis, the present invention provides an appropriate and relevant preventive treatment approach for such disorders. In addition, drugs currently being tested in clinical trials are not significantly different from those discovered decades ago and have shown disappointing results. Furthermore, vaccine strategies based on P. acnes antigens have been questioned regarding their feasibility (Zouboulis et al., 2017). Thus, for the first time, the present invention opens up a live vaccination approach for P. acnes symptoms.

[0024] Preferably, vaccines containing the innovative polypeptides of the present invention, which can be used as therapeutic or prophylactic treatments, are protective against a wide range of genetically diverse P. acnes strains that can colonize human hosts and become pathogenic in specific environments or conditions that favor the expression of virulence genes and traits. Preferably, the present invention identifies and characterizes epitopes that, when presented by viable P. acnes bacteria, are directly accessible to antibodies, and specific binding of the epitope by the immune system leads to a reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof. Preferably, the epitopes characterized in the process of the present invention are presented by P. acnes bacteria so as to be recognized as surface-accessible epitopes and are specifically bound by human serum immunoglobulins raised against P. acnes. Also preferably, the epitopes are specifically bound by animal serum antibodies raised against the protein itself or by P. acnes, as observed in the process of the present invention for experimental demonstration purposes.

[0025] According to certain embodiments of the invention, vaccine antigens characterized according to the invention induce antibodies that lead to specific binding of at least two P. acnes MLST phylotypes, and / or at least two different CC types of P. acnes, and / or at least two ribotypes of P. acnes and a substantial increase in opsonophagocytic injury, as determined by surface binding measurements using flow cytometry and bactericidal assays using immune sera raised against such vaccine antigens.

[0026] Preferably, the cross-reactivity, especially cross-type reactivity, of antibodies induced after immunization with a product of the invention can be determined by specifically binding to and inducing the opsonophagocytic cytotoxicity of at least two or three genetically distinct P. acnes strains. According to a particular embodiment of the invention, the cross-reactivity / cross-type reactivity is with at least one type I and at least one type II or type III strain. According to a further embodiment of the invention, the cross-reactivity / cross-type reactivity is with at least one type II and at least one type I or type III strain. According to a further embodiment of the invention, the cross-reactivity / cross-type reactivity is with at least one type III and at least one type I or type II strain. According to a further embodiment of the invention, the cross-reactivity / cross-type reactivity is with at least one type I, at least one type II, and at least one type III strain.

[0027] Preferably, the cross-reactivity / cross-type reactivity of antibodies induced by immunization with the vaccine of the present invention is with two or more P. aeruginosa strains selected from the group consisting of types IA1, IA2, IB, IC, II, and III, or types I-Ia, I-Ib, I-2, and types II and III, as defined according to the MLST typing scheme. Preferably, the cross-reactivity of antibodies induced by immunization with the vaccine of the present invention is directed against two or more different P. acnes strains, each of which is selected from types IA1, IA2, IB, IC, II, and III strains, or types I-Ia, I-Ib, I-2, II, and III, as defined according to the MLST typing scheme.

[0028] Preferably, the cross-reactivity / cross-type reactivity of antibodies induced by immunization with the vaccine of the present invention is against two or more P. acnes strains selected from a group of different ribotypes, determined, for example, according to 16S ribosomal sequence differences (Fitz-Gibbon et al., 2013; Tomida et al., 2013).

[0029] Preferably, the cross-reactivity / cross-type reactivity of antibodies induced by immunization with the vaccine of the present invention is against two or more phylotypes as determined based on the analysis of single loci (SLST) as described by Scholz et al., 2014, including over 140 SLS types A1-L10 documented in the online SLST database: http: / / medbac.dk / slst / pacnes (updated: September 14, 2019; ST number: 142).

[0030] Preferably, the cross-reactivity / cross-type reactivity of antibodies induced by immunization with the vaccine of the present invention is with the strains NCTC737, KPA171202 (DSMZ, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), SK137, HL005PA1, HL005PA4, HL013PA1, HL030PA1, HL043PA1, HL053PA1, HL053PA2, HL050PA1, HL050PA2, HL060PA1, HL110PA4 (BEI, Biodefense and Emerging Infections Research Resources Repository, Manassas, VA), P. acn31, PV66, and Asn12 (McDowell et al., 2012), Hung.#2 (Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged), and IAI 008, IAI031, IAI034, IAI035, IAI038, IAI040, IAI042, IAI045, IAI041 (Charite Berlin, Pro-Implant foundation), more preferably for at least two different phylogenetic types, more preferably for three, and even more preferably for four, five, or six phylogenetic types.

[0031] Preferably, the vaccines described herein are protective against P. acnes invasion, the progression of pathology induced by infection, or can prevent or reduce the progression of P. acnes-associated symptoms. Protection can be determined by at least two or more of the following tests: a) induction of antigen-specific antibodies that bind to the surface of P. acnes; and / or b) functional inhibition of the antibacterial activity of other cognate or antigen-specific antibodies against P. acnes; and / or c) Induction of opsonophagocytic killing activity against P. acnes d) Preventing, inhibiting, or reducing biofilm formation by P. acnes e) Prevention of P. acnes infections of implants or medical devices f) reducing inflammation or tissue damage caused by P. acnes infection g) reduced ability to adhere to human cells, extracellular matrix components, or tissues h) Reduced cell invasion and intracellular viability i) a reduced ability to evade or compete with the human immune response (e.g., by inhibiting the function or expression of virulence factors); j) Reduced ability to absorb nutrients and minerals required for niche adaptation and survival k) Reduction of tissue inflammation induced by P. acnes l) Increased rate of tissue healing and recovery after injury caused by P. acnes.

[0032] Preferably, the vaccine preparation is administered to a human repeatedly, preferably by at least two or three administrations, or by at least four, five, or more repeated administrations. Preferably, the vaccine can be used in one or more treatment cycles, each comprising at least two or three consecutive administrations at intervals of at least one or two weeks, for example, within a period of one year. Preferably, the treatment cycle can be repeated 1x, 2x, 3x, or 4x or more times, for example, within a period of five years or less.

[0033] According to certain embodiments, the vaccine is formulated for administration to a human subject, e.g., a pediatric, adolescent, or adult subject. Typically, the vaccine is provided in a formulation suitable for use in a treatment regimen that includes both prime and boost immunizations, and preferably, the formulation is suitable for prime and boost administration.

[0034] Preferably, the preparation consists of or comprises one or more antigens or epitopes as active ingredients, formulated with an adjuvant.

[0035] Preferably, the adjuvant is selected from the group consisting of inorganic salts, oil-in-water emulsions, liposomes, TLR agonists, monophosphoryl lipid A, saponins, phospholipids, or combinations thereof.

[0036] Preferably, the vaccine is formulated to be suitable for intradermal, subcutaneous (sc), parenteral, e.g., intramuscular (im), mucosal, transdermal, or topical administration. Preferably, various types of formulations can be used to treat the same human subject, e.g., starting with a systemic treatment or injection, followed by a chronic treatment by local or topical administration, e.g., by (repeated) application of a vaccine patch.

[0037] Preferably, the vaccine contains 0.1 μg to 5 mg, preferably 0.5 to 1000 μg, more preferably 1 to 500 μg, even more preferably 5 to 300 μg, especially 10 to 200 μg of each antigen per dose.

[0038] Preferably, the antigen is provided as antigen-encoding DNA or RNA. Thus, the present invention further provides human vaccines comprising DNA or RNA encoding the vaccine antigens described herein, preferably as mRNA vaccines having an mRNA molecule with the following structure: 5'UTR-signal peptide-encoded antigen or epitope-3'UTR. In such cases, the RNA / DNA vaccine dose can be in the range of 1 μg to 5 mg of DNA or RNA.

[0039] Preferably, the dose may be varied when administered repeatedly, for example starting with a higher treatment dose followed by a reduced treatment dose.

[0040] In contrast to current literature / state of the art practice, vaccines and formulations according to the present invention are versatile and can be used for a wide variety of P. acnes-associated indications. In surgery, specific strains believed to cause specific diseases have been investigated and identified. For example, acne is caused by IA1 strains (McLaughlin, 2019), progressive patchy melanosis is caused by type III strains (Barnard, 2016), prostate cancer is more strongly promoted by thiopeptide-producing type IB strains (Sayanjali et al., 2016), and high antibodies are thought to have a pathogenic rather than protective role, for example, in sarcoidosis (Schupp et al., 2015). Phylotype-independent virulence has been primarily described in the context of implant-associated infections caused by more biofilm-producing strains, independent of phylotype (Achermann et al., 2014; Kuehnast et al., 2018). However, the products according to the present invention can be used against a wide variety of P. acnes-associated infections.

[0041] According to certain embodiments, the present invention provides a human anti-P. acnes vaccine for use in treating human subjects at risk for or suffering from a P. acnes symptom, preferably an infection of the human body, including but not limited to acne vulgaris, keratitis, synovitis acne pustulosis ossificans osteitis (SAPHO) syndrome, endocarditis, medical implant biofilm infections, prosthetic joint infections, surgical wound infections, vascular graft infections, anaerobic arthritis, cardiovascular device-associated infections, e.g., prosthetic valve endocarditis; ophthalmic implant infections, breast implant disease, sciatica, conjunctivitis, shunt-related and / or spinal hardware central nervous system infections, shunt-associated central nervous system infections, It is provided in an amount effective to prevent, treat, or ameliorate diseases selected from the group consisting of sarcoidosis, endophthalmitis osteomyelitis, allergic alveolitis, chronic rheumatoid arthritis, infectious arthritis, chronic juvenile arthritis, chronic destructive oligoarthritis, degenerative disc disease, dental infection, ulcerative colitis hyperthermia, brain abscess, subdural empyema, peritonitis, periodontitis, endodontic infection, endophthalmitis, keratitis, chronic sinusitis, folliculitis, keratitis, corneal ulcer, endophthalmitis, prostatic inflammation, chronic prostatitis, primary biliary cirrhosis, hidradenitis suppurativa, acne inversa, pulmonary vasculitis, atherosclerosis, prostate cancer, progressive patchy amelanosis, and acne conglobata. The specific disease state to be treated by the medical use described herein is any hospital-acquired infection or inflammation-related disease when P. acnes plays a causative role.

[0042] This "broad spectrum" of applicability of the vaccine of the present invention also stands in contrast to the current research goal review and summary in Bruggemann et al. (2019): In this review paper, which provides an "overview of the current status of C. acnes research in the postgenomic era," the authors conclude that there is still a lack of understanding of the underlying disease pathogenesis mechanisms, and that to better understand the pathogenic mechanisms, it is important to better characterize the bacterial-host interaction; this is currently a challenge due to the lack of effective research tools. Finally, their take-home message is summarized as follows: "The interactions of C. acnes (and other skin microorganisms) with components of the human immune system and their consequences are currently being thoroughly investigated. These interactions are complex and involve various cell types [...]." Hoping to address future treatment options, the paper points to "novel therapeutic strategies" currently being developed: "In light of the threat of ineffective antibiotics and the severe side effects of excessive antibiotic treatment and other anti-acne treatments, alternative strategies are preferable. Probiotics for the skin are currently being developed [...]" Notably, there is no mention of a vaccine being one of these "promising future strategies," nor is there any hint in the paper of how such a P. acnes vaccine might work or how it could be designed.

[0043] Preferably, the vaccine is used to induce immunity to prevent and / or ameliorate P. acnes infection and / or alleviate at least one symptom of such infection and / or enhance the effectiveness of another dose of vaccine. The vaccine can be conveniently used to prevent, ameliorate, or treat P. acnes symptoms. When introduced into a subject, the vaccine induces the production of antibodies and / or cytokines, and / or can elicit an immune response that includes activation of cytotoxic T cells, B cells, antigen-presenting cells, helper T cells, dendritic cells, and / or other cellular responses.

[0044] Preferably, the subject is treated either prophylactically or therapeutically.

[0045] Preferably, the subject is a patient with a skin or other diseased organ or tissue condition in which strains derived from either type I, type II, or type III P. acnes, or a combination of at least two phylotypes of types I, II, and III, or at least two ribotypes of P. acnes, are implicated as pathogenic factors, or a healthy individual who may be susceptible to infection by any of the P. acnes strains.

[0046] Preferably, the present invention provides a method of treating a subject for any of the above-mentioned symptoms, or any of the more common P. acnes symptoms, in which an effective amount of a vaccine antigen is administered to the subject.

[0047] Preferably, the antigen is administered at least once in an effective amount ranging from 0.1 μg to 5 mg per antigen. Preferably, the vaccine is administered to a subject repeatedly, preferably at least three times, or at least four, five, or more times. Preferably, the vaccine can be used in one or more treatment cycles, each comprising at least two or three consecutive administrations at intervals of at least one or two weeks, for example, within a period of one year. Preferably, the treatment cycle may be repeated 1×, 2×, 3×, or 4× or more times, for example, within a period of five years or less.

[0048] According to a further particular embodiment, the present invention provides a method of producing the vaccines described herein by mixing and / or conjugating the antigens described herein, or DNA or RNA encoding said antigens, with a pharmacologically acceptable human carrier to obtain a human adjuvant formulation.

[0049] Antigens can also be delivered as antigen-encoding RNA or DNA formulations, preferably as mRNA vaccines with mRNA molecules having the following structure: 5'UTR-signal peptide-encoded antigen or epitope-3'UTR (or other, e.g., replicase-providing). Such formulations have been used in human clinical trials for both local and systemic injection. DETAILED DESCRIPTION OF THE INVENTION

[0050] 4. General Description of the Invention A current challenge in the field of P. acnes-associated pathologies is to define a specific prophylactic or immunotherapeutic product in terms of its design and detailed description of how many and which antigens or antigenic epitopes should be included, how these should be combined and produced, to which antigenic epitopes and against which P. acnes strains the immune response should be directed to ensure the desired effect of immune effector cells against the bacteria, and how the product should be manufactured and administered to provide optimal protective and / or therapeutic effects in susceptible hosts.

[0051] The present invention provides a solution to these problems by providing the precise composition of antigens and antigenic epitopes required within a vaccine material to induce a protective response against P. acnes, and provides instructions on how the product should be produced and administered. In addition, the present invention provides a solution to the problem of which P. acnes strains should be targeted by immunotherapeutic and prophylactic approaches: these discoveries also , leading to different conclusions about optimal treatment strategies and products, resulting in vaccines with antigenic compositions that ensure significantly broader strain coverage than currently suggested in the prior art and bring a further level of innovation to the current state of knowledge in the field.

[0052] In particular, the discovery of vaccines and immune-based therapies has been hampered by the difficulty of studying host immune responses that are regulated by both central and skin immunity, and that are induced and embodied by both colonization and infection.

[0053] Active vaccination can be based on monovalent or multivalent subunit vaccines or whole-cell vaccines. In the case of whole-cell vaccines, bacterial cells are inactivated by fixation with various chemical reagents or killed by heat or cell lysis, and then administered orally or subcutaneously repeatedly. In contrast to subunit vaccines, whole-cell vaccines contain a poorly characterized mixture of many different proteins contained within or secreted by the cell wall of microorganisms (only some of which will be involved in protection; however, their effectiveness depends on the level of natural epitope expression and conservation among all different strains with pathogenic potential). In particular, autologous vaccines prepared using autologous bacterial strains add an additional level of uncertainty to the quality of the immune response because the effects of chemical processing on natural structures and epitopes can vary greatly between strains isolated from different individuals, and the level of pre-existing immunity to a particular autologous bacterial strain can vary greatly between different patients. For all these reasons, the efficiency of immunization with whole-cell vaccines can vary greatly in different subjects, and, being associated with increased safety risks, whole-cell vaccines are no longer a preferred choice of treatment and have been banned from use in many Western societies. Nevertheless, reports of beneficial effects of autologous vaccine therapy using autologous bacterial strains of several commensal opportunistic pathogens, including P. acnes (Zaluga 1998; Loveckova and Havlikova 2002), and recent technological advances allowing the rapid sequencing of large numbers of bacterial strains, have led to a resurgence of research in the field of prevention and immunotherapy of P. acnes-associated diseases.

[0054] Over the past 20 years, considerable effort has been spent to identify antigens that can be used as vaccines. Many putative P. acnes virulence factors have been identified by different research groups, and various hypotheses about acne pathogenesis have been proposed. However, because P. acnes expresses and secretes hundreds of different proteins, many of which have been proposed to be putative virulence factors, and because humans are colonized by the specific signatures of various P. acnes strains that are unique to each individual, no specific indication or evidence has been provided of what a protective preventative or therapeutic product should look like to be protective against disease caused by P. acnes, and whether it has the potential to provide therapeutic benefit in a larger percentage of susceptible individuals. Thus, based on the data provided herein, the present invention can identify and specify that the current challenge in the art is to define which, if any, antigens are best selected for a vaccine combination, which P. acnes strains should be targeted by the vaccine, to which antigenic epitopes an immune response should be directed, and how the product should be produced and administered to provide an optimal protective or therapeutic immunomodulatory effect that achieves protection against a high proportion of strains and reduces the chance of escape mutants, which are more likely with vaccines containing a single antigen. The state of the art is illustrated in recent publications that review what is known about the role of P. acnes in acne vulgaris and other pathological conditions, highlighting the challenges associated with discovering protective therapeutic products (O'Neill and Gallo 2018; Bruggemann 2019; McLaughlin et al. 2019). These challenges are currently being addressed in the treatment of P. acnes-induced infections. The idea has shifted away from developing broadly cross-reactive vaccines against various pathogens, but instead to design products that should act only against one specific phylotype; for example, in the case of acne vulgaris, phylotype IA1 is considered the major virulence factor. The difficulty of developing vaccine-based strategies has led to new directions in the field, focusing on non-immune agents, such as probiotics, phototherapy, and other approaches, that should act to support the growth of strains considered harmless commensals.

[0055] The prior art and most relevant background to the invention that is the subject of this patent application can be summarized as follows:

[0056] In 2001 and 2003, two patent applications filed by Corixa Corporation were published (WO 2001 / 81581A2 and WO 2003 / 033515A1) disclosing the results of serological screening of phage display libraries of P. acnes using antibodies and T cells isolated from human donors. WO 2001 / 81581A2 disclosed a series of P. acnes proteins and immunogenic fragments thereof. WO 2003 / 033515A1 disclosed antigenic compositions and vaccines against P. acnes. These two applications contained numerous polypeptide sequences and immunogenic fragments, including a whole range of proteins that could be used as immunotherapeutic or diagnostic products. Candidate antigens reactive with human sera, designated immunogenic P. acnes proteins, are summarized in Table 5 of WO 2001 / 81581A2. Potential antigen candidates were considered to be predicted to have biological functions that may be relevant in host defense based on bioinformatics analysis and homology to proteins expressed by other microbial pathogens. These include, among others, several different transferases, enterotoxins, lipoproteins, permeases, proteases, membrane proteins, secreted proteins, adhesins, transporters, hemolysins, penicillin-binding proteins, sialidases, siderophores, zinc-, iron-, and manganese-binding proteins (Table 6, WO 2001 / 81581A2). Many of the antigens disclosed in this application were also later identified and studied in more detail by other research groups. However, only a few have been evaluated in vitro and in vivo for their suitability as potential preventative or therapeutic vaccine candidates (Nakatsuji et al., 2008; Liu et al., 2011; Wang et al., 2018). The most promising antigen candidates include sialidase, lipase, and CAMP factor 2, which were protected by U.S. Patent No. 9,340,769 B2 (U.S. Patent Application Publication No. 2011 / 0243960 A1, filed as WO 2010 / 065735 A2).

[0057] In 2006, Lodes et al. characterized in more detail four of the immunoreactive P. acnes proteins disclosed in International Publication Nos. WO 2001 / 81581 A2 and WO 2003 / 033515 A1. Two proteins were related to the Corynebacterium diphtheria htaA gene (PA-21693 and PA-4687); the other two (PA-5541 and PA-25957) were found to share some similarity with streptococcal M-like proteins and to be 68% similar to each other. Based on bioinformatics analysis, the authors predicted that PA-21693 would be more conserved among various clinical isolates, and its expression was found to be dependent on iron availability. However, the other three proteins were found to be highly variably expressed by different strains due to numerous frameshifts and mutations identified within the DNA sequences. Furthermore, in strains that were thought to be able to express the protein, differences in cellular location were predicted, such that the protein could be located on the cell surface or secreted. Secretion is dependent on the presence of a specific cell wall binding motif, termed the LPTG(X) domain, where X can be any amino acid. These results suggest that the proteins are highly antigenic. Bioinformatics and immunoblot analysis led the authors to conclude that the identified proteins have the potential for both phase variation (expression and non-expression) and antigenic variation (expression of different antigenic variants of the same protein) among various P. acnes isolates. Additionally, the proline-threonine (PT) repeat region located toward the C-terminus of proteins PA-5541 and PA-25957 was predicted to be highly antigenic. By analyzing the reactivity of human antibodies with an N-terminal protein fragment lacking PT repeats (NH2-25957) compared with a C-terminal fragment containing PT repeats (PT-25957), the authors concluded that antibodies in acne-negative sera (defined as those with a history of mild or no acne) reacted specifically with any of the four tested IgG subclasses to the C-terminal fragment containing PT repeats, and that this response was of the IgG2 / 3 type, whereas little or no reactivity was detected to the N-terminal fragment. In contrast to these conclusions, the authors found that antibodies from acne-positive sera (defined as those with a history of moderate to severe acne) were directed against the N-terminal portion of the protein and were of the IgG1 / 4 subtype. Furthermore, the hydrophilic repeat regions identified in PA-21693 and PA-4687 were predicted to be highly antigenic. However, the reactivity of human antibodies from acne-negative and acne-positive subjects to these regions was not studied. It has been suggested that the differences in antibody type and binding between different fragments of PA-25957 and PA-5541 are likely responsible for deregulation of immune responses in acne-prone individuals. However, the authors did not provide any indication of whether acne patients can be effectively vaccinated or how they should be treated to induce a more regulated immune response. The authors also did not provide any indication of which of the studied proteins or sequence regions (epitopes) should be included in the vaccine (only the C-terminal PT repeat region was mentioned as antigenic in healthy individuals and potentially relevant for healthy skin).Additionally, the expression of the antigens PA-25957, PA-5541, and PA-4687 was suggested to be highly variable. However, no indication was provided of how to overcome the expected antigenic and expression variations to ensure vaccine protection across different P. acnes strains and genotypes colonizing different individuals, which of these strains should be targeted, or how to ensure action against disease-associated strains and functional epitopes. Questions regarding specific antigen composition, epitope selection, product design, and application were all left open, and the authors suggested that these should be investigated in future research studies. Thus, the authors conclude from their study that their findings regarding the four proteins PA-21693, PA-4687, PA-5541, and PA-25957 may also be important for selecting antigens for therapeutic vaccines and for selecting key epitopes of antigens that can drive desired immune responses, although none of the four proteins has been suggested as suitable vaccine antigens. Quite to the contrary, the authors conclude that a deregulated immune response to these variably expressed P. acnes antigens may arise in individuals with severe inflammatory acne, and this possibility remains unexplored in the process of selecting such antigens for therapeutic vaccines.

[0058] In contrast to these very early attempts to file patent applications purely based on primary sequences, (rough) bioinformatics, detection of denatured proteins by SDS-PAGE, and testing of only four strains (two of type IA and two of type II), the strategy according to the present invention was based on a real-live approach and quantitative measurements based on the actual binding and recognition of specific antigens by antibodies on the surface of many different P. acnes strains and phylotypes, and the ability of antigen-specific antibodies to actually trigger bacterial killing by the human immune system. Thus, flow cytometry was used with antibodies raised against many different antigens, and these antibodies were incubated with live P. acnes to determine which antigens, which parts of the sequences (peptides and fragments) were exposed on the surface and accessible to antibodies on the surface of live bacteria. Thus, the strategy according to the present invention provided "real-life" evidence that these antigens are accessible to antibody binding on the surface of P. acnes cells, that the antibodies are highly cross-reactive, and that they can confer protection from the majority of over 100 tested strains. This is very different from detecting peptides expressed by Escherichia coli (E. coli) bacteria during phage library screening, in which any peptide recognized by an antibody will be detected regardless of its actual location on live P. acnes bacteria and its accessibility to the antibody. In addition, immune sera raised by the antigens were tested in antibody-dependent opsonophagocytic killing assays, which provide a "real-life" assessment of the functional immune consequences of antibodies binding to specific surface-exposed epitopes. (Particularly in the human setting, when a vaccine is injected into a patient's skin or muscle, it is taken up by antigen-presenting cells, which then process the antigen and stimulate T and B cells to increase the production of opsonophagocytic killing antibodies against epitopes within specific fragments / sequence regions; helping to further opsonize and act against bacteria that breach the skin barrier (which are the etiological agents of disease or infection).) While many antigens were immunogenic (capable of raising antibodies when used as a vaccine), there was wide variation in the ability of these antigens to raise immunologically relevant antibodies that can opsonize bacteria, leading to phagocyte-mediated killing of P. acnes.

[0059] WO 2011 / 149099A1 discloses two short antigenic peptides, designated "PepA" and "PepD," having lengths of 14 and 13 amino acids, derived from the amino acid sequence of Genbank / EMBL / DDBJ accession number YP_056445, as well as vaccines containing these peptides or nucleotides encoding these peptides.

[0060] In 2010, Holland et al. attempted to identify acne-associated virulence factors by analyzing proteins secreted by P. acnes during anaerobic culture. Many putative virulence-related factors were identified in this study: protein PPA1939 was found to be most strongly secreted by all isolates, and PPA0816 was secreted by strains IB, II, and III and was suggested to be a surface protein that may contribute to adhesion and virulence. Other putative adhesins were also identified, such as PPA1715, which contains a dipeptide proline-threonine (PT) repeat, as described for PPA2127 and PPA2210 by Lodes et al. (2006) and Holland et al. (2010). However, only PPA2127, secreted by type IA strain 266, was detected; type IB strains (KPA171202 and P6) did not express this protein due to mutations and frameshifts in the DNA sequence. Additional pathogenesis-related factors were suggested to be PPA2175 (a hypothetical protein likely to be the endocytic peptidoglycan transglycosylase RlpA) and PPA0687 (CAMP2, a member of the five-member CAMP factor superfamily). The authors suggested that future investigations should focus on a more detailed investigation of secreted virulence factors and their importance in pathogenicity. According to the authors, characterization of the function of secreted factors will require the development of appropriate tools, such as mutagenesis approaches to generate P. acnes knockout mutants, and elucidation of the molecular basis for the observed differences in virulence among various P. acnes clinical isolates. Thus, although many different proteins have been suggested to potentially act as virulence factors, no specific direction was given regarding the selection of specific proteins or specific uses of products for the prevention or treatment of P. acnes-associated pathologies.

[0061] In 2011, McDowell et al. attempted to classify P. acnes strains based on their pathogenic potential by using genetic typing and antibody labeling methods.The authors generated two different monoclonal antibodies. One of them, QUBPa1, was found to bind to two antigens previously identified by Lodes et al. (2006), PA5541 / PPA2210 and PA-25957 / PPA2127 (McDowell et al., 2011). By studying the labeling patterns of the QUBPa1 monoclonal antibody on different P. acnes strains, McDowell et al. demonstrated that the identified antigens were consistently expressed by type IA strains, which are primarily isolated from acne patients. They suggested that their findings were similar to those of Holland et al., who detected secretion of PPA2127 by type IA strains but not by types II and III (Holland et al., 2010). McDowell et al. (2011) proposed that type IA strains are associated with acne pathogenesis, while types IB, II, and III are associated with other types of infection. They also confirmed the findings of Lodes et al. (2006) that antigens PA5541 / PPA2210 and PA-25957 / PPA2127 are prone to phase variation, antigenic variation, and secretion. Based on their ability to bind dermatan sulfate, the authors designated these two protein dermatan sulfate adhesins: DsA1 (PA-25957 / PPA2127) and DsA2 (PA5541 / PPA2210). This study focused on the clinical significance of strains expressing various putative virulence factors (other virulence factors were also found to be produced by IA strains, such as neuraminidase and lipase GehA) and the use of these findings as a supportive tool in the classification of various P. acnes strains. Aside from being suggested as important virulence factors, the authors did not offer any suggestions regarding possible preventive or therapeutic interventions based on these or any other described antigens. Monoclonal antibody (MAb) typing by immunofluorescence microscopy (IFM) was performed as described by McDowell et al., 2005 (McDowell et al., J. Clin. Microbiol. 43 (2005), 326-334).McDowell (2012) generated monoclonal antibodies by immunizing BALB / c mice with killed whole cells of various P. acnes strains. Hybridoma cell lines producing P. acnes-specific MAbs were cloned by limiting dilution (McDowell (2005)). Isolates were examined for reactivity with mouse monoclonal antibodies QUBPa1 and QUBPa2, which target strains within types IA1 and II, respectively. These monoclonal antibodies were used to label P. acnes strains for IFMs, which were used for phylogenetic construction (identification of type IA and type II strains). Various genes, particularly those encoding cell surface-associated antigens, were examined. These may have strong discriminatory power (McDowell (2011)). They noted that their previously used monoclonal antibody, QUBPa1, is type IA-specific (p. 10). McDowell (2011) also used a monoclonal antibody, QUBPa1, specific only for P. acnes strain IA1, for the purposes of purification on an immunoaffinity column and labeling for immunofluorescence microscopy. This is shown on page 2000: IFM analysis of type I strains with QUBPa1 reveals polar and septal labeling on the cell surface (McDowell et al., 2005). […] For type IA strains, IFM with QUBPa1 did indeed provide evidence of intrastrain phase variation in expression, but not the ability to bind to the cell surface and induce bactericidal effects against different phylotypes. Thus, this monoclonal antibody is not suggested for use in identifying major immunogenic and immunologically relevant epitopes, but rather for identifying the nature of the antigen (i.e., phase variation) and as an investigative tool for distinguishing P. acnes genotypes. Other reasons used to rationalize the association of type IA strains with acne include the expression of cell surface adhesion factors with the capacity for phase / antigenic variation and enhanced immunogenicity, as well as the production of specific virulence factors that support overgrowth and degradation of host tissue components.However, the present invention seeks to provide a vaccine that will provide broad cross-reactivity (i.e., cross-type reactivity, not limited to type IA1) and therapeutic benefit in combating P. acnes-associated disease and infection, and is directed against specific P. acnes phylotypes. It is not to identify research tools for labeling.

[0062] McLaughlin (2019) also states, "IFM analysis of skin biopsy samples with monoclonal antibodies (MAbs) demonstrated the presence of both types IA and II in the sebaceous glands of both acne and control subjects. Type II strains were detected with a different monoclonal antibody (i.e., QUBPa2 (McDowell et al., 2011)) that does not recognize DsA1 / DsA2. McLaughlin (2019) states on page 15, "Using a subsequently identified MAb (QUBPa1) to target DsA1 and DsA2 antigens on the cell surface, only strains representing types IA1, IA2, and even IC were immunoreactive and showed polarized septal labeling. We speculate that the potential of type IA to modulate interactions with the host immune system via the DsA immunogenic protein may be important in the recurrence of acne." Thus, McLaughlin (2019) refers to the labeling of polar septa of DsA1 and DsA2 by monoclonal antibodies along with the term "immunoreactive." Furthermore, they "speculate" about the reasons for acne's recurrence, without any reference to immunotherapy in conclusion, let alone any indication or motivation for how to design products to treat acne. Furthermore, McLaughlin's (2019) paper speculates on the association of various virulence factors with the importance of the pathogenicity of acne-associated strains among the long list of antigens summarized in Figure 12 and Table 2; however, they are cautious about suggesting that only strains of the IA1 phylotype should be targeted by vaccines. There was no suggestion of how to produce such a vaccine, especially one that does more than target IA1 strains.McLaughlin (2019) characterized the pathogenic potential of strain KPA171202 compared with other strains based on genes for putative tissue degradation factors, including hemolysis, cohemolytic Christie-Atkins-Munch-Peterson (CAMP) factors, sialidases, endoglycoceramidases, lipases, porphyrins, and hyaluronate lyases (HYLs), as well as genes for cell wall / cell envelope-associated proteins with inflammatory potential and proteins containing multiple proline-threonine repeats (PTRs). Another approach to defining putative virulence factors was to compare the transcriptome of KPA171202 with that of IA1 strain 266 (pleuropulmonary infection) and identify differential expression of 119 common genes during mid-logarithmic phase. These included genes for triacylglycerol lipase, endoglycoceramidase, DsA1, and HtaA / P071. However, the conclusions as to why these gene products might represent putative virulence factors were based solely on the fact that the genes were upregulated in 266, which provided no clear indication for vaccine products, the choice of antigen, and which should be used, making them feasible for human application. Furthermore, the virulence factors only indicated that strains expressing such genes were more virulent, but provided no indication whatsoever of the suitability of such factors in the context of vaccination. Thus, the prior art also points in a different direction compared to the present invention; in stark contrast, the "Vaccine Development" section of McLaughlin et al. consequently mentioned only cAMP factor 2 as a potential vaccine target.

[0063] The expression of various virulence-related factors was further studied by Brzuszkiewicz et al. (2011), who provided further insight into the activity of various virulence-related genes. Among others, they also mentioned GehA lipase (PPA2105), polyunsaturated fatty acid isomerase PAI (PPA1039), three proteins described by Lodes et al. (2006): PA-25957 / PPA2127, PA5541 / PPA2210, and iron acquisition protein HtaA (PA-4687 / PPA0786). Similar to McDowell et al. (2011) and Lodes et al. (2006), Brzuszkiewicz et al. (2011) also hypothesized that strain differences in pathogenic potential may be due to differences in the expression of virulence factors by different phylotypes, and these They suggested that other virulence factors are likely important, in addition to the phylotype-specific genome content. They also suggested that the pathogenic potential of different P. acnes strains is determined not only by phylotype-specific genome content but also by variable gene expression. The authors provided potential scenarios for how inflammatory acne lesions might manifest and which strains might be important, but did not suggest any specific direction for the selection of vaccine materials and the design of products for disease treatment or prevention. Like others, they suggested that future research efforts should be devoted to elucidating the factors responsible for the differential behavior of strains and the underlying mechanisms responsible for P. acnes-induced inflammation. They argued that hosts with an unsettled immunological response to P. acnes may be susceptible to the presence of strains with significantly higher pathogenic potential, and that strains from phylogenetic groups I-Ia, which express higher levels of adhesins and other virulence factors, are more likely to induce pathological symptoms in predisposed hosts. Mayslich et al. (Microorg. 9 (2021), 303) also reviewed the current state of research on virulence factors and P. acnes infection. Because P. acnes (as a commensal bacterium) is only weakly immunogenic, it is generally tolerated on the host's skin. This is because it is considered relevant only in a pathogenic context as an opportunistic pathogen that can lead to strong inflammatory responses in the skin and in many other internal organs when it expresses different antigenic components on its surface. While Mayslich et al. review various virulence factors (especially CAMP2), they address two remaining "major open questions": first, whether specific P. acnes types can become more virulent in response to environmental changes with respect to the expression of specific virulence factors; and second, how P. acnes interactions with the skin microbiota affect virulence (in an overview and conclusions about the state of the art). Both questions, and how to address this virulence progression, of course, remain unanswered. In 2011, Liu et al. published a study using a monoclonal antibody against CAMP2.One of the antigens was previously identified by Corixa through a serological screen and subsequently by Holland et al. (2010) through secretome proteomics. This protein has also been identified as a virulence factor secreted by other bacteria, which has been reported to function as a hemolytic factor and pore-forming toxin. The effectiveness of monoclonal antibodies against the CAMP2 antigen suggested that neutralization of the secreted CAMP2 virulence factor is important for attenuating its virulence. The authors specifically suggested that attenuation of P. acnes virulence by targeting secreted virulence factors, rather than proteins located on the P. acnes cell surface, should be the goal of acne immunotherapy. With this new approach, they abandoned the previously selected vaccine candidate, the cell surface protein P. acnes sialidase. Indeed, the authors of all recent reviews conclude that promising new therapeutic approaches should focus on "modulating the population of C. acnes strains on the skin without inducing a negative response" and aim for products designed to act directly against P. acnes and avoid disrupting the microbiome. Therefore, the introduction of "healthy" commensal strains of P. acnes, as vaccine approaches do, is considered preferable to acting against "pathogenic" strains (see, e.g., O'Neill and Gallo 2018; Bruggemann et al. 2019; Cong et al. 2019; Mayslich et al. 2021; Petronelli 2021). In summary, all these recent reviews suggest new therapeutic approaches to address the pathology of P. acnes-associated infections by moving away from vaccine approaches (which were suggested as promising nearly 20 years ago) or by raising extreme caution / warnings against them ("without inducing a negative response"; Mayslich et al. 2021).

[0064] In 2014, Bek-Thomsen et al. attempted to study the pathological processes involved in acne formation by proteomic analysis of sebaceous follicular casts extracted from healthy and acne-prone individuals (Bek-Thomsen et al., 2014). They found that the sebaceous follicular casts in acne-prone skin and They searched for proteins that were differentially expressed in healthy skin pores compared with acne-affected follicular samples. However, they were unable to identify evidence for differential expression of any virulence factors. Instead, they concluded that both healthy and acne-affected follicular samples contained the same protein composition. The most abundant P. acnes proteins identified in both acne-affected and healthy follicular samples were the dermatan sulfate adhesins DsA1 (PA-25957 / PPA2127) and DsA2 (PA5541 / PPA2210), CAMP factors 1 and 2, and an uncharacterized lipase (PPA1796). Other proteins they considered important were myeloperoxidase, lactotransferrin, neutrophil elastase inhibitor, and vimentin. Contrary to McDowell et al. (2011), they concluded that lipase GehA (glycerol-ester hydrolase A, PPA2105) is probably not an important virulence factor because it was found in only a small percentage of healthy skin samples and not in any of the acne-affected skin samples. Instead, the authors suggested that a novel, uncharacterized lipase, PPA1796, may be more important in vivo. They proposed a new gene name, GehB, for this potential virulence factor. The most important biological process in acne was found to be the "response to bacteria," which was concluded based on the identification of high levels of human host proteins known to be involved in immune response, tissue remodeling, and healing. In addition, the authors concluded that both healthy and acne-affected sebaceous follicles were colonized by the same strains, suggesting they were type IA, based on high levels of expression of two adhesins, DsA1 and DsA2, that are not expressed by types IB, II, and III. They also suggested that there may be important differences in pathogenic potential among type IA strains, which should be investigated in future studies.Although this study presented potentially interesting data on the proteomic abundance of pilosebaceous glands in acne compared with healthy skin, the authors acknowledged that the study had many limitations that made it difficult to draw more specific conclusions, and they did not provide any guidance on how to use the knowledge to develop any specific products for acne treatment. Instead, they argued that certain pathogenic strains of P. acnes may exploit the presence of specific human host molecules identified in this study, such as vimentin, to invade skin cells and induce inflammation, and that future research should be directed toward identifying the origin of vimentin expression in pilosebaceous follicular casts.

[0065] In 2015, Achermann et al. investigated potential vaccine candidates by examining P. acnes proteins produced in vivo during biofilm infection with one specific P. acnes strain and testing animal sera for the presence of antibodies against proteins found in P. acnes cell wall-associated and membrane-associated fractions separated by two-dimensional electrophoresis. They identified 23 immunogenic proteins that presented as potential P. acnes vaccine candidates. Of these, the most promising vaccine candidate was suggested to be glyceraldehyde-3-phosphate dehydrogenase (GADPH), identified by accession number G7U8Y4, which is the same protein previously identified as PPA0816 by Holland et al. (2010) and Bek-Thomsen et al. (2014). The ABC transporter (D4HAH2) was found to be particularly important; among the best candidates, malate dehydrogenase (Q6A6Z5), DnaK chaperone (W4TZS5), methylmalonyl-CoA mutase (E4D8Y8), and several other proteins were mentioned. The detection of proteins analyzed by Western blot in cell fractions isolated under denaturing conditions does not provide any evidence that the proteins were exposed on the bacterial cell surface and accessible to antibody binding. Furthermore, no single antigen was tested as vaccine material for its ability to induce antigen-specific and functional antibodies, neither against the P. acnes strain used in this study nor against any other strains from the many other known P. acnes genotypes. It is unclear whether these antigens are capable of inducing antigen-specific and functional antibodies in the human host. Neither were tested to be immunogenic in humans nor detectable by human antibodies. Nevertheless, the authors suggested that further studies should be performed to discover the potential of these candidates to prevent chronic P. acnes biofilm-mediated infection or to be used in diagnostic tests.

[0066] Concurrent with efforts to identify factors responsible for the pathogenesis of P. acnes-associated diseases, new technological developments in the field have introduced more complex studies of the entire microbial population on human skin, and in addition, attempts have been made to subclassify P. acnes strains into those that are more or less pathogenic and to identify health-associated or true commensal strains.

[0067] Phylogenetic studies based on multilocus gene sequencing and whole-genome analysis of isolates from the Human Microbiome Project (HMP) have provided valuable insights into the genetic population structure of P. acnes. Multilocus sequence typing (MLST) generates distinct sequences, defined as prominent alleles, which are subsequently used to generate sequence types (STs) for each P. acnes isolate. Two different MLST typing schemes have been developed that divide P. acnes into closely related clusters IA1, IA2, IB, IC, II, and III (McDowell et al., 2012) or I-1a, I-1b, I-2, II, and III (Lomholt and Kilian, 2010).

[0068] More recent genomic studies have led to the identification of further subdivisions based on whole-genome sequencing of all known P. acnes strains. This has led to the single-locus sequence typing (SLST) scheme, which is useful, among other things, for identifying P. acnes ST diversity in mixed microbial communities (Scholz et al., 2014). An alternative single-locus approach based on 16S rRNA profiling has been used to investigate the pathogenic role of P. acnes in acne and other P. acnes-related pathologies. Certain P. acnes strains have been associated with acne, while others have been associated with healthy skin or have been implicated in other types of infection (Fitz-Gibbon et al., 2013; O'Neill and Gallo 2018; McLaughlin et al., 2019).

[0069] Although these studies largely relied on the analysis of strains isolated from different individuals and had limitations related to skin sampling and analytical methods, they provided direction for follow-up research and influenced the current state of knowledge in the field. These developments directed research efforts toward the identification and analysis of proteins expressed by selected P. acnes genotypes considered to be pathogenic, or by analyzing proteins found in different types of clinical material.

[0070] Following the novel discovery of the importance of specific ribotypes in acne pathogenesis, Yu et al. (2015; 2016) used proteomic analysis to analyze the expression of various proteins by P. acnes strains classified according to specific ribotypes thought to be associated with acne or healthy skin. These authors found that the expression of several proteins was variable among different ribotypes. Because these ribotypes have previously been associated with disease versus health and induced different amounts of cytokine secretion by human cells, the authors proposed that these proteins be investigated as potential vaccine candidates. In particular, they noted proteins corresponding to GI 50843388 (PPA1939), GI 50843565 (PA-25957), GI 50843218 (PPA1758), and several other proteins of unknown function. The authors also suggested that the search for vaccine candidates should not be directed toward finding antigens that induce antibodies, but instead toward those that induce strong Treg cell responses. Thus, they deviated from the suggestion by Lodes et al. and others that antigen candidates should be both antigenic and reactive with human antibodies. This direction would suggest that P. acnes may be a symbiotic agent that induces inflammation. This study was based on the assumption that optimal vaccine antigens should act to reduce, rather than enhance, adaptive immune response activation (Yu et al., 2015). Additionally, the authors removed PA-5541 (gi 50843645) from the list of potential vaccine candidates due to the discovery that it was only expressed by acne-associated strains or strains of neutral significance, but not by health-associated strains (II RT6) (Yu et al., 2016). In summary, many of the proteins disclosed in these studies have also been previously identified by others, and the authors did not offer solutions or specific suggestions for vaccine composition and product design, but only suggested future research directions for disease pathogenesis research in the discovery of optimal vaccines.

[0071] Further publications in 2016 presented research findings on several proteins previously identified by others: PPA1939 (Allhorn et al., 2016) and PA-25957 (Grange et al., 2017). These two proteins were found to be interesting based on their putative additional biological functions: PPA1939 was proposed to be related to heme binding and antioxidant capacity, and PA-25957 was suggested to play a role in fibrinogen binding. The pathogenic potential of PA-25957 was also supported based on its interaction with fibrinogen and its expression restricted to specific disease-associated P. acnes phylotypes. These authors did not present how this protein could be used as a vaccine product, but only hypothesized its role as a virulence factor that, in addition to binding dermatan sulfate, could act by contacting fibrinogen and forming a "clump" inside the hair follicle. They also suggested that future investigations should focus on developing tools to test the ability of different protein fragments to bind fibrinogen, and that the protein is likely to show great variability in the levels of secretion and expression on the surface of different strains, which should be investigated by future studies to gain more understanding of its potential significance.

[0072] Recently, CAMP2 immunotherapy product development has shifted from monoclonal antibodies toward therapeutic vaccines that should act to induce antibodies that act against inflammation by reducing P. acnes cytotoxicity and preventing cytokine release by keratinocytes and phagocytes (Wang et al., 2018). Notably, the negative effect of P. acnes on phagocyte survival was presented as key evidence of CAMP2 pathogenicity, supporting the argument for a beneficial effect of the immune response induced by CAMP2 vaccines. A limitation of this study is that the effect of CAMP2 antibodies on phagocyte viability in the presence of P. acnes was not tested, nor was it compared with the effects of antibodies against other antigen candidates and a larger panel of P. acnes strains in the same assay, making it difficult to assess the significance of these findings.

[0073] Confusion in the field regarding optimal vaccine candidates persists due to constantly evolving and changing hypotheses about the importance of various P. acnes strains in acne pathogenesis, the potential role of other microorganisms that are part of the human skin microbiome, and unresolved controversies surrounding the functional importance of the human immune response to bacteria. Caution in selecting antigen candidates for various vaccination approaches was expressed in a recent review, which emphasized that it remains unclear which P. acnes strains are pathogenic and should be targeted by vaccines, and that it remains unclear whether acne severity is driven by the patient's underlying innate immune condition or by the level of P. acnes virulence, which should be taken into account when designing immunotherapy protocols, particularly when determining the choice of target antigens (Contassot 2018). The authors rationalized that specific inhibition of secreted virulence factors could limit the risk of unwanted targeting of nonpathogenic bacteria and overcome the possible selection of resistant bacteria. More importantly, The authors suggested that vaccines should be highly specific based on surface antigens to avoid "off-target" effects and activity against P. acnes strains that may be beneficial to the host. However, if acne severity is driven by the patient's innate immune status rather than by specific pathogenic strains, this should have consequences for immunotherapeutic approaches, particularly with regard to the type of antigens selected for the vaccine.

[0074] Although many different molecules and proteins have been studied and proposed as potential virulence factors, only a few of them have been tested as potential vaccine or therapeutic product candidates, and the current state of the art makes it impossible to draw firm conclusions about the potential of every molecule described as a suitable acne vaccine product candidate.Some virulence factors have been found to be secreted, and some are expressed within the cell membrane, but their accessibility and functional immunological relevance on the bacterial cell surface have not been studied, and some have been found to be expressed only by specific strains whose pathogenic potential is not fully understood.Some potential virulence factors have been suggested to be expressed by specific phylotypes that are considered pathogenic, but due to high antigenic diversity and diverse expression, specific presentations used in specific products have not been suggested or demonstrated, if possible.In addition, all studies have contained significant limitations that make it impossible to draw firm conclusions about the immunologically relevant advantages of different antigens.For example, the ability of identified antigens to induce immune responses in the form of vaccines has not been tested, or conclusions have been based mainly on the results obtained with a single antigen using suboptimal methods. For example, bioinformatics analysis provides limited information regarding the likelihood of expression of a particular gene product and its cellular location. Proteomic analysis can provide more valuable information regarding the actual level of gene expression within specific bacterial cells within a defined genetic background, but it is prone to error and is not useful for evaluating the potential of a particular gene product as an immunotherapy target. Factors such as the stability of expressed proteins during preparation of biological material, their behavior during SDS-PAGE separation, ease of solubilization, and extraction from the cell membrane can all affect the results of the analysis, potentially leading to suboptimal conclusions. Furthermore, proteomic analysis does not provide any information regarding the accessibility of proteins and their immune-protective epitopes on the bacterial cell surface.In addition, many studies have focused on testing only a few selected P. acnes strains; the clinical samples used for analysis were obtained and collected from a few donors, introducing additional mutations that make it difficult to draw more reliable conclusions about the suitability of various molecules or their derivatives as product candidates. Coupled with the most recent developments in the field, which add further conflicting information about the importance of various factors in mediating P. acnes-induced pathology or factors that may provide protection, those skilled in the art have not been able to reliably predict, based on the current state of the art, which antigens or epitopes, and to what extent, are needed to design an optimal vaccine product (if such a product acts to induce, reduce, or modulate an immune response against a specific protein or its epitope), how to design it to be protective against all strains with pathogenic potential, or which strains should be targeted to achieve the greatest benefit to the host.

[0075] The prior art lacks systematic immunological studies involving many genetically distinct P. acnes strains and comparing various antigens and fragments with each other using the same immunological vaccine-related methods. Progress has also been hampered by a lack of understanding of host-bacterial interactions and why P. acnes is associated with disease pathology only in certain individuals. This has also made the research community wary of developing P. acnes-based immunotherapy products. Because there is no consensus yet on which strains of P. acnes a product should act against, some researchers are very hesitant when considering vaccination approaches due to concerns about adverse effects of vaccines on strains that may be beneficial to human health. This caution stems, inter alia, from a limited understanding of the host factors that contribute to disease. Because P. acnes is typically thought to be resistant to phagocytic killing by immune cells (Webster et al., 1985), it has also been reported that it can evade phagocytic killing even after being internalized by phagocytes (Fischer et al., 2013). Additionally, various intracellular pathways of P. acnes trafficking have been described, one of which involves autophagy, a nonspecific response that is not dependent on P. acnes antibodies, varies depending on the cell type and conditions, may occur in conjunction with the innate immune response, and may not lead to the activation of adaptive immunity. Therefore, homeostatic or protective adaptive immune responses are activated depending on how the host immune system perceives the outcome of bacterial interactions with the host and the amount of risk the bacteria pose to the host.

[0076] Progress in identifying suitable vaccines has been slow, in part due to a lack of available methods and models for vaccine antigen identification. Contrary to more typical infectious diseases, all human immune systems are already primed to respond to P. acnes, and antibodies to this bacterium can be found in the blood of both patients and healthy individuals. Because the immune system is stimulated not only by bacteria in the skin but also by bacteria in other colonized sites (e.g., the oral cavity, the intestine), the level of immune response to P. acnes is also influenced by the interplay of individual-specific factors that may result in the immune system perceiving the stimulus as more or less "dangerous": the mere presence of antibodies in a particular individual's blood is not an indication of actual clinical efficacy or significance. Therefore, selecting antigen candidates based on serological screening in the absence of any functional assays is constrained by the same limitations as determining the pathogenicity of a particular P. acnes strain by its mere presence and isolation from the skin of different individuals. Without knowing anything about the antibacterial activity of antibodies against P. acnes, it is difficult to distinguish which antigen-specific antibodies may assist the immune system in controlling bacterial growth and invasive behavior and which may lead to the production of antibodies that are merely a by-product of continuous colonization, secretion, and antigen presentation during the process of normal cell turnover.

[0077] Thus, current challenges in identifying P. acnes immunoprotective antigens are multifold, encompassing both a lack of suitable disease models and a lack of reliable functional in vitro assays for antigen screening. In other typical vaccine discovery projects, even if an animal model of disease is not available, functional in vitro assays can be used to select antigens suitable for protective vaccines. For example, the discovery of a commercial vaccine against Neisseria meningitidis was made possible by studying the effect of antigens on inducing bactericidal antibodies in animals tested in serum bactericidal assays (Giuliani et al., 2006); vaccine discovery against Streptococcus pneumoniae was facilitated by studying the opsonophagocytic killing effects of antibodies in in vitro opsonophagocytic killing assays (van Westen et al., 2013); and several antiviral vaccines have been discovered based on the neutralizing effect of antibodies on viral replication as measured in antibody neutralization assays (Vanblargan et al., 2016). Such functional in vitro assays have not been developed for P. acnes vaccine research. Furthermore, in the current state of the art, P. acnes is believed to be resistant to phagocyte killing and perpetuates inflammation due to widespread immune stimulation resulting from the expression and secretion of numerous putative virulence factors. Which of these factors may have the potential to attenuate or resolve inflammation by assisting host immune cells in reducing bacterial activity and density in inflamed tissues is currently a highly controversial question, and no satisfactory answer has yet been provided. It remains to be seen whether, in acne vulgaris, strains derived from phylotype IA1 may be more virulent than others, and ideal acne vaccine candidates should be investigated among those expressed by such strains (O'Neill and Gallo 2018; McLaughlin et al. 2019). This suggests that phylotypes other than IA1 may be more virulent than P. acnes itself. The significance of the discovery that this phylotype is frequently isolated from other types of P. acnes-associated infections (e.g., implant infections) and that this phylotype also colonizes human skin and can infect susceptible individuals during surgical procedures remains unsatisfactory. The possibility that the same strain may be able to cause disease in different contexts, depending on the host's opportunity and susceptibility, has not been adequately addressed in the prior art. Instead, efforts have focused on associating specific P. acnes phylogenetic types with specific diseases, but this approach has led to many unsatisfactory or even contradictory findings and much debate among various research groups (Fitz-Gibbon et al., 2013; Eady and Layton, 2013; Alexeyev and Zouboulis, 2013).

[0078] The most novel concept that has recently evolved in this field and influenced researchers is that of the skin microbiome. Accordingly, recent research has focused on identifying pathogenic factors that may result from a disruption in the balance of interactions within the total microbial community colonizing a specific area of ​​skin affected. The term "microbiota dysbiosis" was introduced to describe such a possibility (O'Neill and Gallo 2018). Therefore, current efforts by researchers are focused on studying the mutual influence of various microbial species and developing products that address "microbiota dysbiosis." Although this highly novel concept lacks a description of what a "healthy" microbiome should look like in each individual, numerous researchers have already developed next-generation acne treatment products based on mixtures of specific bacterial strains or their products as skin probiotics and prebiotics. Recent reviews of progress in the field of therapeutic product development still identify many different pathogenic factors that likely contribute to pathogenesis. However, only a handful of suggested virulence factors have actually been tested as potential vaccine candidates: CAMP2 and the group of antigens identified by Acherman et al., 2015 (O'Neill and Gallo, 2018); however, even these antigens have not been systematically tested and compared in the same assay using a larger panel of strains that would allow for unbiased selection of the most suitable candidate (McLaughlin et al., 2019). Therefore, even among these candidates, it is difficult to predict which, if any, will be most protective and suitable as a vaccine candidate without further testing.

[0079] Over the past 15 years, numerous different proteins have been proposed to contribute to P. acnes virulence, and their biological and potential clinical significance have been studied by many different research groups. Their cellular location, secretion, and expression by various bacterial strains in various environments (various growth media, various in vitro growth conditions, or human skin) have been investigated. In some cases, antibodies recognizing these antigens have been detected in the serum of acne patients and in healthy individuals. However, the significance of these discoveries cannot be extrapolated to designing vaccine products for treating acne or other P. acnes-associated pathologies. Because many proteins have been studied by multiple research groups and their biological and pathological significance is still under investigation in current public databases (e.g., UniProt: https: / / www.uniprot.org / ), many of the proteins are still designated as "putative" or "uncharacterized," or named according to their suggested hypothetical biological function, e.g., "putative, conserved protein for Fe-transport." To facilitate understanding of the data presented in this invention, the sequences and nomenclature used in public sequence databases as of the filing date of this invention are summarized in Table 1.

[0080] 5. Detailed Description of the Invention For the purposes of the present invention, the following names and abbreviations are used for the P. acnes polypeptides referred to in the present invention (in public databases, with annotations in the literature): P002 (SEQ ID NO: 1): example for CAMP factor, also known as Christie-Atkins-Munch-Petersen (CAMP) factor (Liu et al., 2011; Wang et al., 2018); P005 (SEQ ID NO: 2): Example for glyceraldehyde-3-phosphate dehydrogenase; also described by Holland et al. (2010) and suggested as a potential vaccine candidate by Achermann et al. (2015). P018 (SEQ ID NO: 3): example for "repressor"; P022 (SEQ ID NO: 4): an example of an "uncharacterized protein" and described in the published literature as "host cell surface adhesion protein PA25957" (Lodes et al., 2006) and "dermatan sulfate-binding adhesin / DsA1" (McDowell et al., 2011). P027 (SEQ ID NO: 8): an example for an "uncharacterized protein", also known as "host cell surface adhesion protein PA5541" (Lodes et al., 2006) and "dermatan sulfate-binding adhesin / DsA2"; P028 (SEQ ID NO: 13): Example for "putative, conserved protein for Fe-transport", also known as PA21693 (Lodes et al., 2006), P032 (SEQ ID NO: 21): Example for translation initiation factor IF-2; P035 (SEQ ID NO: 22): Example for malate dehydrogenase identified by Achermann et al. (2015); Bek-Thomsen et al. (2014), identified as 50843200 by Yu et al. (2016). P042 (SEQ ID NO: 23): Example for a "TED domain-containing protein", also identified by Holland et al. (2010) as PPA1715 and by Yu et al. (2016) as gi 50843175. P046 (SEQ ID NO: 24): Example for "probable endocytic peptidoglycan transglycosylase RlpA"; also identified by Holland et al. (2010) as PPA2175 P068 (SEQ ID NO: 25): An example for an "uncharacterized protein" in protein sequence databases - this protein is also described as PPA1939 by Holland et al., 2010; Yu et al. (2015) and Yu et al. (2016) 50843388 and described as RoxP by Allhorn et al. (2016) ; P069 (SEQ ID NO: 26): Example for an "outer membrane lipoprotein", which is identified as gi 50843218 by Yu et al. (2015). P070 (SEQ ID NO: 27): Example for an "ABC transporter, ATP-binding protein," suggested as a potential vaccine candidate by Achermann et al. (2015). P071 (SEQ ID NO: 28): Example for the protein described by Lodes as PA-4687, which at the time of filing this application is referred to in public databases as an "HtaA-like surface protein"; this same name has also been used by other authors who consider this protein among important virulence factors (Brzuszkiewicz et al., 2011; McLaughlin et al., 2019).

[0081] In the present invention, protein P022 is referred to as an example of a (natural) DsA1 polypeptide, P027 is referred to as an example of a (natural) DsA2 polypeptide, and P028 is referred to as an example of a (natural) putative iron transport protein (PITP) polypeptide of P. acnes.

[0082] Rationale for the choice of DsA1 and / or DsA2 as antigens: The current problem in the field is the identification of compounds that have been studied in the prior art and suggested to be potentially relevant as pathogenic factors or as potential materials in developing therapeutic or preventative treatments. The challenge is to select the most suitable antigen as a vaccine material from the many different candidates that have been developed. Making a final selection is complicated by the lack of systematic comparative studies in which the actual performance of vaccine candidates is analyzed and compared with each other in their ability to induce a protective immune response against bacteria.

[0083] During the course of this invention, numerous proteins were tested that were suggested to be involved in P. acnes pathogenesis and / or considered potential candidates for immunotherapeutic products. However, while all prior art antigens were able to induce an immune response in mouse immunization studies according to the invention, as evidenced by their ability to induce antigen-specific antibodies detectable in ELISA (e.g., Figure 2), this did not translate into comparable levels of surface binding and opsonophagocytic activity.

[0084] To address one or more of the objects of the present invention, there is provided a method for producing a vaccine for use in treating or preventing P. acnes associated infections, comprising the steps of: - selecting at least one antigen comprising at least one P. acnes epitope, wherein the P. acnes epitope is an epitope that is directly accessible to human serum antibodies when presented by viable P. acnes bacteria, and wherein specific binding of the epitope by the human immune system leads to a reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof; Binding of the epitope by the immune system, in particular by the human immune system, is demonstrated by using a flow cytometry assay, preferably a fluorescence-activated cell sorting (FACS) assay; The method is provided wherein the reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof, particularly the reduction in bacterial numbers (due to the direct effect of the antibody), is demonstrated by an opsonophagocytic killing assay (OPK) in the presence of an antibody specific for at least one antigen comprising at least one P. acnes epitope.

[0085] A method for producing a vaccine for use in treating or preventing a P. acnes associated infection according to the present invention comprises: - selecting at least one antigen comprising at least one P. acnes epitope, wherein the P. acnes epitope is an epitope that is directly accessible to human serum antibodies when presented by viable P. acnes bacteria, and wherein specific binding of the epitope by the human immune system leads to a reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof; Binding of the epitope by the immune system, in particular by the human immune system, is demonstrated by antibodies that bind to P. acnes bacteria using a flow cytometry assay, preferably by a fluorescence-activated cell sorting (FACS) assay, and by binding of antigen-specific antibodies to live P. acnes bacteria in the flow cytometry assay; Reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof is demonstrated by an opsonophagocytic killing assay (OPK), which uses a polyclonal human antibody specific for at least one antigen comprising at least one P. acnes epitope, or at least two two-fold serial dilutions of human serum or antigen-induced polyclonal antibody serum containing such an antibody, starting with a complement-inactivated serum dilution of at least 1 / 200, after incubation for at least 24 hours or longer at 37°C in the presence of 5% CO2. 50 It exhibits at least a 50% reduction in bacterial cell count in the reaction sample compared to the negative control at at least a two-fold higher dilution used in the opsonophagocytic killing assay for titration.

[0086] This method identifies and provides the first "real world" P. acnes antigens, providing a completely new understanding of the nature of human P. acnes microbiota interactions with the host, which Based on this, a new and effective "real-world" strategy is provided to combat and prevent the damage caused by the pathological consequences of human colonization by P. acnes bacteria. In contrast to other strategies presented in the prior art, the identification of antigens according to the present invention provides information on the relevance of the antigen and the specific epitopes that can be tested by antigen-induced polyclonal antibodies.

[0087] The present invention safeguards the relevance of real-world vaccine candidates by actually testing whether the antigen can actually induce antibodies capable of binding to the surface of many different strains (as opposed to inferring based on indirect evidence / bioinformatics or methods that only look at protein expression in cell lysates, e.g., by Western blot or MasSpec; or relying on unrelated models that do not accurately represent human disease (e.g., cAMP and sialidase studies in rabbit ear inflammation); notably, no flow cytometry studies have been done to assess antigen expression in P. acnes (as the only quantitative method to assess differences in surface accessibility of the antigen and its epitopes to antibodies raised by immunization). During the course of this invention, a systematic study of many different strains and phylotypes (over 100 strains, not just 1-6 as done by others) was performed. Furthermore, clinically relevant material was isolated from pustules (inflamed lesions) of acne patients after sterilizing the skin surface and analyzed separately (samples were taken from the skin surface from areas not typically affected by acne, and during analysis, data from all individuals was analyzed as a group (e.g., acne vs. healthy) (however, each individual is unique with respect to its microbiota, and putting all data "in the same bag" would only skew the results and lead to erroneous conclusions, as opposed to the prior art). Additionally, real-world relevance was shown regarding the importance of the human immune response (increased binding to P. acnes surfaces leads to increased bactericidal efficacy of phagocytes; for this, antibody binding must be directed to the correct epitope). This approach also showed how effective it is in humans (surface binding and OPK data correlated when human serum was tested). Finally, the features of the OPK method used in the course of the present invention demonstrated, inter alia, the importance for adaptive immunity, whereas the prior art only examined innate immunity, based on the interaction of P. acnes and human cells in the absence of antibodies.

[0088] Indeed, the present invention overcomes limitations in the field related to the methods used to identify and select the best antigens and epitopes for inclusion in vaccines.

[0089] The present invention provides "real world" evidence of differences in surface expression and accessibility of different antigens and antigenic epitopes across a large number of P. acnes strains (Table 2); and "real world" evidence of the functional consequences of binding to induce an effective antigen-specific adaptive immune response (which is a major criterion for vaccine antigen selection), based on quantitative assessment of binding and its functional consequences, using methods superior to others in the prior art: Flow cytometry Opsonophagocytosis SPR / Biacore

[0090] Flow cytometry: A systematic evaluation of a large number of different P. acnes strains and antigens has not previously been done in the prior art for P. acnes with the aim of quantitatively assessing and confirming the actual expression of different antigens on the bacterial cell surface and the accessibility of antigenic epitopes to the binding of antigen-specific antibodies. Instead, antigen expression was assessed indirectly using suboptimal methods and only a few P. acnes strains (e.g., Western blot in Lodes et al., 2006). None of these methods quantitatively assessed the level of protein expression on the cell surface (detection of proteins after separation by SDS-PAGE and Western blot confirmed that the proteins were indeed present on the cell surface). (These methods do not mean that an antigenic epitope is expressed and sufficiently accessible for antibody binding on the surface of P. acnes. These methods do not allow for comparison of levels of expression across different strains and different antigens.) To be suitable as a vaccine candidate, an antigenic epitope must not only be surface accessible, but also sufficiently conserved among a large percentage of strains (a vaccine should be able to provide protection to as many patients as possible, and therefore should induce antibodies that can recognize all strains that can become pathogenic and colonize different hosts).

[0091] Other researchers have studied antigen expression in cell lysates, cell secretory fractions, and / or membrane fractions analyzed by Western blot or mass spectrometry (Holland et al., 2010; Yu et al., 2016; Bek-Thomsen et al., 2014; Achermann, 2015; Lodes, 2006), or evaluated the immunogenicity (ability to induce antibodies against recombinant proteins) of various antigens expressed by only one P. acnes strain during infection of rabbit bone tissue (Achermann et al., 2015). All of these methods had similar limitations: a small number of strains were tested, and the analyzed proteins were denatured and separated by SDS-PAGE; therefore, the proteins were removed from the context of living cells, altering their native epitope structure. For example, Achermann et al., 2015, did not identify any of the antigens described in this invention.

[0092] Even without the results presented in this invention, one skilled in the art could not help but conclude that the most effective vaccine against P. acnes would need to be highly cross-reactive, and that the combination of DsA1 and DsA2, either as single antigens or in a hybrid in combination with P028 (PITP), would provide significantly broader cross-reactivity than products incorporating P022 and P027, each as single antigen vaccines or in combination without P028. This is particularly relevant considering the individual-specific and unique profile of strains colonizing each individual, and the expected variability in in vivo expression of a single antigen in the context of the various pathological processes and virulence mechanisms used by the bacterium.

[0093] Opsonophagocytic injury method: The OPK method allows assessment of the functional importance of antigen-induced antibody responses in mobilizing adaptive immune defenses against bacteria, specifically reducing bacterial cell numbers and proliferation at the site of pathogenesis.

[0094] Because P. acnes does not colonize or pathogenicize animals, pharmacodynamic studies typically performed on vaccines (e.g., protection from challenge) are not relevant in animal models. Published animal models, in which P. acnes was used to induce inflammation in rabbit ears (e.g., Liu et al., 2011), have not been accepted by the scientific community as relevant for acne vulgaris or for identifying vaccine antigen candidates: only one single antigen (Camp2) was studied because the model did not allow for objective comparison of performance against other P. acnes antigens (O'Neil and Gallo, 2018; McLaughlin, 2019). Furthermore, Liu et al. (2011) did not even consider bactericidal activity as a desired vaccine mode of action. Instead, they stated that "neutralization of bacterial-induced virulence and inflammation without directly killing bacteria would be an excellent immunotherapy for the treatment of acne vulgaris" (Liu et al., 2011; p. 3, paragraph 1).

[0095] The ability of serum samples to opsonize bacteria can be measured by various in vitro opsonophagocytic killing (OPK) assays, which have been shown to be superior to the simple opsonophagocytic absorption (without killing) OPA assay and the best functional correlate of protection.

[0096] Although peripheral blood-derived granulocytes (neutrophils, basophils, and eosinophils) can be used as a source of phagocytes for opsonization assays, it is more convenient and reproducible to use cell lines as phagocytes. Promyelocytic leukemia cell lines, such as HL60, can be induced to differentiate into granulocyte-like cells, and differentiation can be monitored by surface antigen expression.

[0097] These assays are not trivial, and assay development requires accounting for the unique properties of a particular bacterial species and the nature of its interaction with the human immune system. Despite the success of the OPK and OPA assays in assessing vaccine antigen-induced responses against Streptococcus pneumoniae, and more recently, a similar assay has been developed for Streptococcus pyogenes (S. pyogenes), no such assay has been developed for P. acnes. The difficulty in establishing such an assay for P. acnes stems from, among other things, its unique cell biology, cell wall properties, and its inherent interactions with the human immune system as a commensal bacterium that is an opportunistic pathogen.

[0098] Further studies have not attempted to evaluate the role of antibodies as a key adaptive immune defense against P. acnes-associated infections, which could lead to bactericidal effects. Instead, over the last two decades, the scientific community has studied the general phenomenon of P. acnes interactions with various cell types, independent of antibodies, and attempted to understand the behavior of P. acnes immediately after internalization or invasion of these cells. While these studies have only provided insight into innate immune defenses against P. acnes, they have not resolved the questions of how adaptive immune defenses can prevent or resolve infection and / or pathogenic damage caused by this bacterium when innate immune responses are unable to stop it, or which antigens can induce antibodies with bactericidal activity against P. acnes.

[0099] The main differences of the OPK assay developed for P. acnes in the course of this invention are, for example: (1) The preferred use of the most relevant cells for the assay: granulocytes, and especially neutrophils (HL60 cells differentiated into granulocytes). Granulocytes, and especially neutrophils, play a key role in early host defense against bacterial invasion. In response to infection, neutrophils are recruited from the bloodstream and migrate toward local sites of inflammation, where they assist local innate immune defenses to stop or prevent the spread of infection.

[0100] The particularly preferred cell type used, HL60 cells, are non-adherent cells that are easily mixed in solution, which allows the assay to be run with a consistent number of cells in every well, which significantly reduces assay variability and makes it difficult to compare the performance of multiple test samples simultaneously. (2) The preferred use of antibodies specifically raised against different antigens to investigate their role in inducing phagocytic killing by the cells used in the assay (e.g., preferably HL60 cells differentiated into granulocytes). (3) Phagocyte killing is preferably assessed under conditions that do not involve antigen-nonspecific killing, which can occur in the absence of antibodies in studies where antibiotics or complement are used. This ensures an unbiased evaluation of the potential of different antigens and antigenic epitopes to induce antibodies with bactericidal activity. (4) The phagocyte method is preferably optimized to ensure optimal P. acnes growth and survival of both extracellular P. acnes and HL60 phagocytes over long incubation periods of greater than 24 hours; in other methods, the time during the killing phase is less than 24 hours; if longer times are allowed, antibiotics are used to prevent growth of extracellular bacteria, or assay variability increases, making systematic evaluation of multiple samples in the same assay difficult.

[0101] SPR / Biacore: is preferably used in accordance with the present invention as a further quantitative method for antigen selection (in surface binding and opsonophagocytosis assays) including assessment of antibody binding levels and affinity (stability of interaction) with various antigens selected based on their immune relevance.

[0102] The skin is a complex and dynamic ecosystem inhabited by the skin microbiota: bacteria, archaea, fungi, and viruses. Skin-resident bacteria are not just passive residents; they actively interact with host immunity through the intact skin barrier, activating specific immune cell populations in a species- and strain-dependent manner.

[0103] Over recent decades, it has become clear that the skin possesses its own cutaneous immune system (SIS), and that although most antibodies act systematically by blood distribution throughout the body and diffusion into tissues, tissue titers can be enhanced by localized antibody production by B cells present in the skin. In mammalian skin, B cells are localized in the dermis. In human skin, IgA is secreted in eccrine sweat glands and appears in sweat and sebum, and antibodies of the IgG and IgM isotypes reach the skin surface by an undefined mechanism.

[0104] Despite these findings, other work in the prior art has not attempted to address the need to increase local concentrations of antibodies to P. acnes by stimulating production and increasing bactericidal activity. Instead, the field has suggested that increasing antibody levels to bacteria may be more harmful than beneficial, and caution has been raised about unwanted effects if such a strategy is considered, leading to the belief that secreted, non-surface proteins may be more optimal targets.

[0105] According to the present invention, the quantity and quality of P. acnes-specific antibodies secreted locally and diffusing from blood vessels into hair follicles are increased. These antibodies strengthen the local skin immune defenses, increasing their efficiency, so that inflammatory processes are prevented or significantly reduced (Figures 24A and 24B).

[0106] The pathogenesis of acne is triggered by the interactive influence of several factors, including genetics, hormonal activity, the skin environment that contributes to P. acnes virulence, and the resulting immune response. This is particularly relevant in light of the commensal versus pathogenic role of skin-colonizing bacteria. The complex interactions between various environmental, bacterial, and host factors result in distinct clinical manifestations of disease and responsiveness to currently available treatments. Other P. acnes-associated pathological conditions and diseases involve similarly complex interactions, as mere colonization by a specific strain is not sufficient to induce infection, inflammation, and disease.

[0107] For this reason, over recent decades, the scientific community has mainly focused on isolating P. acnes from infected tissues and lesions to draw conclusions about their pathogenic importance and then on understanding the pathogenic processes leading to different clinical manifestations when comparing the expression of different virulence factors expressed by “pathogenic” strains.

[0108] Indeed, prior art studies have only shown evidence of antigen expression by specific phylotypes, compared the expression levels of various proteins inside acne versus healthy skin pores, or investigated their ability to interact with human proteins and various components of human immune cells and tissues; however, to ensure benefit to patients without causing unwanted effects by compromising more "health-related" P. acnes strains, , leaving open many questions regarding optimal antigen selection and design of vaccines.

[0109] Scientific research in this field has yet to answer the essential question of how to design a vaccine suitable for P. acnes-associated pathologies. In fact, the scientific community has even stopped considering vaccines for treating P. acnes-associated diseases as a viable approach. The biological function and expression of DsA1 and DsA2 in the context of human disease have been studied by many groups. However, there are still no vaccines proposed, let alone vaccines based on these two proteins designed to induce antibody responses targeting specific epitopes of these proteins. Furthermore, numerous cautions have been raised about vaccine approaches targeting P. acnes surface-associated proteins. In summary, unlike the prior art's identification of "virulence factors," which have only shown that these proteins are involved in interactions with the host immune system leading to inflammation, the present invention provides an "immune-related" vaccine. In contrast to such "virulence factors," the "immune-related" vaccine according to the present invention refers to its relevance as a vaccine candidate that, upon immunization, induces antibodies that significantly increase the ability of phagocytes against the bacteria, preventing their growth and pathogenic behavior. Thus, the "immune relevant" antigens of the present invention do not induce inflammation, but rather act against it by reducing it; and therefore have a protective effect as a vaccine in the host.

[0110] Also, in contrast to the OPK assays of the prior art, the OPK assay according to the present invention preferably - in the presence and / or absence of antibiotic compounds added to the assay, and / or - without the presence and / or addition of complement and / or complement factors; performed in an OPK assay to allow an unbiased analysis of the quality of the antigen itself for effective killing of P. acnes bacteria without the systematic influence of antibiotics or complement (innate immune system) on killing.

[0111] While the previous studies mentioned screen for the presence or absence of surface-based antigens, the present invention uses flow cytometry as a first step to select the most suitable and best candidates, which are then further subjected to OPK assay. Nakatsuji et al., 2008, used flow cytometry to study the binding of recombinantly expressed sialidase to sebocytes. However, it was never demonstrated that sialidase is expressed on the surface of P. acnes and is accessible to antibodies induced by immunization. In fact, in the prior art, only bioinformatics predictions were used to select antigens that may be associated with the cell wall. It is worth mentioning that even the group of Nakatsuji et al., 2008, later changed their strategy and concluded that targeting surface proteins was not the best approach for P. acnes. Because they did not want to target the entire bacterium, which (because it is a commensal) could have negative health consequences, they continued to pursue CAMP2 (a secreted protein) as the preferred vaccine antigen (Liu et al., Vaccine 29 (2011), pp. 3230-3238): Regarding previous proposals for using CAMP as a vaccine target, the authors concluded that "vaccines targeting surface sialidase or bacterial particles have shown preventative efficacy against P. acnes, but the lack of therapeutic activity and inability to neutralize secreted virulence factors motivates the generation of novel immunotherapeutic approaches." Consequently, in the Liu et al., 2011 paper, the scientists skipped CAMP2 as an active vaccine target and developed immunotherapeutic antibodies against the secreted CAMP factor of P. acnes.

[0112] The OPK assay according to the present invention can be carried out with any cell type that is capable of absorbing and killing P. acnes bacteria, preferably granulocytes (especially neutrophils and The OPK assay is typically performed with basophils (polymorphonuclear cells), but polymorphonuclear cells purified from the blood of healthy volunteers, as well as certain types of macrophages, monocytes, and other phagocyte lines, may also be used. Preferably, the OPK assay is performed with a mixture of phagocytes (granulocytes) in the presence of (polyclonal) serum or other tissue fluids containing, inter alia, P. acnes-specific antibodies. Preferred cell lines used in the OPK assay according to the present invention are HL-60 cells, human myeloblasts, and promyelocytic leukemia cells, which differentiate into granulocytes (including neutrophils, the major cell type that responds to infection and migrates from the blood to sites of local inflammation; a process that depends on cytokine immune signals released by local resident cells and macrophages in response to tissue damage when innate immune mechanisms are unable to clear and prevent the spread of infection). Neutrophils and / or granulocytes are the most preferred cell types to be used in the present OPK assay.

[0113] Antibodies induced by DsA1 and DsA2 consistently bind to the P. acnes cell surface. This is surprising and in contrast to other prior art proteins, which have been suggested to be important virulence factors, immunogenic, exposed on the cell surface, and / or secreted (P002, P005, P035, P042, P046, P068, P069, P070, P071). Indeed, none of these prior art antigens was detected by our flow cytometry-based surface binding assay on representative strains of all six MLST phylotypes, or the surface binding of antibodies induced by these antigens was extremely low (e.g., Figure 1A).

[0114] Despite the high secretion potential, variable expression, and antigenic sequence differences suggested in the prior art (Lodes et al., 2006; Holland et al., 2010; Yu et al., 2016; McLaughlin et al., 2019), our surface binding experiments demonstrate that DsA1 and DsA2, although expressed only within cell wall fractions or on the cell surface of a limited number of strains or phylotypes, are highly accessible to antibodies raised by immunization when used as vaccine candidates, and this binding is shown to be much stronger and more cross-reactive / cross-type-reactive than other proteins described in the prior art (O'Neill and Gallo, 2018; McLaughlin et al., 2019) (e.g., Figure 1A, Figure 4). The expression and accessibility of these antigens on the cell surface of viable P. acnes is not only greater on the cell surface of phylotype IA1 (McLaughlin et al., 2019), but the level of expression on the bacterial cell surface of phylotypes IA2, II, and IC is also far superior compared to other antigens described in the prior art (Holland et al., 2010; Brzuszkiewicz et al., 2011; Bek-Thomsen et al., 2014; Achermann et al., 2015; Yu et al., 2016; O'Neill and Gallo, 2018; McLaughlin et al., 2019).

[0115] Furthermore, the importance of antibody surface binding can be confirmed in the present invention by opsonophagocytic killing assays using human phagocytes, which demonstrate that only antibodies acting against antigens DsA1 and DsA2 are able to induce significant opsonophagocytic killing of P. acnes, while antibodies induced by immunization with other prior art antigens are clearly inferior to antigens DsA1 and DsA2 when examined against a panel of genetically distinct strains representing the most common MSLT groups (e.g., Figure 1B), i.e., IA1, IA2, IB, IC, II, and III, thereby providing evidence of cross-reactivity and cross-type reactivity.

[0116] Thus, similar to other suggested virulence factors that are located on the cell surface of specific phylotypes or that can be secreted and transiently expressed within the bacterial cell wall, only DsA1 and DsA2 are likely to be major protective immunogens and vaccine candidates. Although not characterized, they were able to induce antibodies capable of binding to the surface of viable P. acnes bacteria in sufficient quantities and of the requisite quality to induce significant opsonophagocytic killing.

[0117] Both proteins represent virulence factors in P. acnes that are required for the infectious P. acnes cycle. They have been suggested to bind to specific host factors, such as dermatan sulfate (Lodes et al., 2006) and fibrinogen (Grange et al., 2017). However, given the current state of the art, those skilled in the art are still unable to draw conclusions about how to design and develop protective therapeutic products using either of these proteins. Instead, caution is particularly heightened regarding antigens associated with the cell surface of most P. acnes strains. Such a vaccine would be considered dangerous because it induces unwanted effects against "commensal" strains, as prior art teachings dictate selecting among antigens expressed primarily by MLST group IA1 (McLaughlin et al., 2019, p. 23); or selecting antigens that are secreted and not associated with the bacterial cell surface (Contassot 2018; Keshari et al., 2019), and these authors did not even mention DsA1 and DsA2 among the various antigen candidates to be evaluated in future studies to determine their potential as possible vaccine antigen candidates.

[0118] DsA1 and DsA2 are homologous to each other and are differentially expressed on strains within phylotypes IA1, IC, and II, with some expressing more of one than the other homolog (e.g., Figures 16A-16D). It is also possible that one can take over the function of the other when targeted separately. Thus, providing at least one of the two, or even better, both, in a vaccine can reduce the chance of immune defense evasion and increase vaccine potential against strains expressing lower levels of one of the two.

[0119] Various virulence functions are also attributed to other proteins suggested as important by different authors (Brzuszkiewicz et al., 2011; Achermann et al., 2015; O'Neill and Gallo, 2018; McLaughlin et al., 2019); since numerous factors expressed by bacteria contribute to pathogenic potential. Thus, what makes the present invention unique is not the identification of a protein as one of many different virulence factors that contribute to the invasive potential of a particular P. acnes genotype and interact with the host, but the evidence that the combined action of DsA1- and DsA2-specific antibodies induced by immunization with them as vaccine material can recruit host defenses (phagocytes) and specifically induce and increase killing capacity toward a large number of bacterial strains and phylotypes, prevent or heal damage to skin hair follicles, or prevent further spread within the organism if they manage to breach the skin barrier. The induction of such a strong opsonophagocytic damaging effect obtained in the course of the present invention makes these two proteins prime candidates for vaccine production, unlike many other virulence factors that have been suggested as potential candidates in the prior art.

[0120] Rationale for the selection of PITP as a vaccine antigen Antigen P028 or PITP (PA-21693) was described by Lodes et al. (2006) as one of two putative P. acnes proteins (PA-21693 and PA-4687) similar to the product of C. diphtheriae htaA and involved in the mechanism of iron uptake by P. acnes.

[0121] The present data suggest that under iron-restricted conditions, the expression of PITP, but not other surface proteins, is significantly increased. We demonstrated that expression of the htaA-like iron-binding protein remained unchanged under the same conditions; expression of a second htaA-like iron-binding protein, also suggested by Lodes et al. (2006) as one of three variably expressed immunogenic P. acnes proteins, P071 (PA-4687), was only slightly increased under iron-limited conditions in some strains (e.g., Figure 3). Iron is essential for bacterial replication, especially under inflammatory conditions, because iron absorption plays a role in bacterial survival in host tissues.

[0122] Under iron-limited conditions, PITP was detected in variable amounts on the surface of six MLST phylotypes (types IA1, IA2, IB, IC, II, and III) (e.g., Figure 1A). PITP was highly immunogenic, and the induced immune response recognized and specifically bound to strains belonging to all six MLST types. Furthermore, the induced immune response was able to induce significant killing by phagocytes in OPK assays against at least two or more different MLST phylotypes (e.g., types IA1 and IA2, or IA1 and IB, or IA1, IA2, and IB, or IA1, IA2, IB, IC, and / or II, and / or III) (e.g., Figure 1B). Thus, PA-21693 (Lodes et al., 2006) (PITP) as a vaccine antigen was also suggested during the present study and evaluation as a highly immunoreactive P. acnes surface protein (Lodes et al., 2006) and was considered among the important virulence factors and potential immunotherapy candidates against IA1 strains (McLaughlin et al., 2019). Contrary to other similar iron-binding proteins, such as P071 and the Hta-like protein PA-4687 (Lodes et al., 2006), it demonstrated clear advantages in its ability to induce cross-reactive and cross-type-reactive P. acnes antibodies that bind to the cell surface of a wide range of MLST phylotypes (e.g., Figure 1A) and induce phagocytic killing (e.g., Figure 1B).

[0123] Due to the fact that IB strains are isolated from the skin of both acne patients and healthy individuals, their pathological significance has not been recognized in the prior art (O'Neill and Gallo, 2018; McLauglin et al., 2019), and IB strains in acne vulgaris are considered among "commensal" or "neutral" strains because they occur equally frequently in both healthy and acne-affected skin. However, sporadic clinical findings suggest that this may not always be the case. For example, strains found to cause acne treatment failure due to antibiotic resistance were of the IB phylotype, but a strain considered "pathogenic" (IA1) was sensitive to the antibiotic used in treatment; acne symptoms were reduced only when the antibiotic was replaced with one effective against IB strains (Sadhasivam et al., 2016). Thus, although IB strain types are frequently isolated from other types of P. acnes infections, the clinical significance of the IB phylotype in acne remains unresolved in the art. However, the present invention points out that IB strains are also isolated from inflamed acne lesions (e.g., Figures 14A and 14B), and that humans make significant levels of antibodies against IB strains, which can reach opsonophagocytic serum titers (K50) of over one million (e.g., Figure 13B).

[0124] Furthermore, although type III strains are not frequently isolated in association with acne (McLaughlin et al., 2019), they have pathogenic potential, as evidenced by their isolation from implant-associated infections and other types of P. acnes infections, and have been suggested to be specifically associated with progressive patchy amelanosis (Barnard et al., 2016; Dagnelie et al., 2018). The reason for the less common isolation of type III strains from the skin of acne patients is that they tend to be attracted to different environments and colonize more restricted areas of the face and body: in published studies, type III strains were only identified on the forehead and forearms of some individuals (Dekio et al., 2012) and on the oral mucosa (Scholz et al., 2014). Furthermore, data generated during the course of this study indicate that type III strains may prefer to colonize more restricted areas of the body. Why? This is because type III strains were not isolated from healthy individuals or acne patients from the facial areas sampled in the study according to the present invention. In addition, type III strains also grow more slowly than other phylotypes, and it has been noted that isolated clinical material can be easily lost when grown on plates prior to sequencing, depending on the sampling method and the type of area sampled.

[0125] Therefore, PITP is a suitable surface antigen to increase the effect of DsA1 and DsA2 on additional phylotypes that are expressed at lower levels or are downregulated to target additional P. acnes types with pathogenic potential (e.g., IB and / or III) (e.g., Figure 4).

[0126] Furthermore, the interaction of the induced humoral immune response with PITP, in addition to attracting immune cells, may lead to the inhibition or reduction of iron absorption by bacteria during combat with host immune cells or interference with biofilm dispersion and spread of infection.

[0127] Rationale for the combination of DsA1+DsA2+PITP in a single vaccine product The antigen PITP, due to its surface accessibility and OPK activity profile, best complements the immune response induced by antigens DsA1 or DsA2: it is accessible on the cell surface and can induce antibodies capable of phagocytic killing of P. acnes type IB, and to a lesser extent type III (which do not express the other two antigens). On strains where PITP surface expression may be lower than on DsA1 and DsA2 (P. acnes types IA2 and II), antibodies induced by the other two antigens can compensate for this (e.g., Figure 1A, Figure 1B; Figure 4, Figure 5).

[0128] The functional importance of opsonophagocytic killing (OPK) is a key inventive step of the present invention, stemming from the fact that many other proteins have the ability to act as virulence factors but are unable to induce antibodies with the same effect on bacteria when tested in comparison with each other in the same immunological assay. For example, CAMP2 and its homologs possess hemolytic activity capable of lysing human cells; it was recently reported that CAMP2 can induce phagocyte killing in an in vitro assay of co-culture with P. acnes (Wang et al., 2018). Expression and secretion of CAMP protein by various P. acnes strains has been suggested as one of the factors contributing to P. acnes resistance to opsonophagocytic killing. However, as demonstrated in the OPK assay performed according to the present invention, killing of P. acnes was consistently detectable in the presence of opsonizing antibodies against DsA1, DsA2, and PITP antigens.

[0129] Studies performed for the present invention in flow cytometry-based surface binding assays showed that the best effect could be achieved by combining antigens DsA1 and / or DsA2 with antigen PITP (e.g., Figure 5). This could be confirmed by the opsonophagocytic injury that antibodies against PITP and against DsA1 and DsA2-induced antibodies could induce, for example, by antibodies against strains from phylogenetic groups that do not induce MLST phylotypes IB and III (e.g., Figures 1B and 1D). Thus, the combination of DsA1 and / or DsA2 with PITP achieves broader cross-reactivity / cross-type reactivity than products incorporating only DsA1 and DsA2 used as single antigens or in combination. This is especially true in the context of various pathologies in which bacteria are or may be involved: various types of acne (acne vulgaris, acne conglobata, cystic acne), implant infections, sarcoidosis, progressive patchy amelanosis, and other types of conditions, taking into account the individual-specific and unique profiles of strains colonizing different individuals and of strains with different pathogenic potential. As evidenced by the examples of this application, vaccine products according to the invention will induce antibodies with broader cross-reactivity and cross-type reactivity profiles directed against virulence factors expressed on a greater number of phylotypes, and have a significantly higher potential to be protective in a higher percentage of individuals at risk and in various pathological conditions.

[0130] This particular combination of PITP with DsA1 or DsA2 allows us to target not only strains classified according to ribotypes suggested by others to be pathogenic in acne vulgaris (RT4, RT5, and RT8) (Fitz-Gibbon et al., 2013; O'Neill and Gallo, 2018), but also strains that have the same ability to express these virulence factors within other ribotypes (e.g., Figure 4: RT1 and RT3), but whose pathogenic importance has not been recognized until now due to the fact that they are found at high frequency on healthy skin (e.g., RT1 and RT3) or have not been isolated to a significant extent in the context of infection (e.g., Figure 4: RT16 and RT532).

[0131] Although ribotyping and other similar genetic typing schemes may be correlated with immunological data in some cases, this correlation does not imply a causal relationship. The data obtained by the present invention demonstrate that the surface accessibility of selected vaccine candidates tends to be significantly lower on ribotypes not commonly isolated from acne-prone skin (RT2 and RT6) and higher on strains implicated in acne pathogenesis (RT4, RT5, RT8); however, exceptions were detected during the course of the present invention, in which completely different surface expression patterns were detected on strains classified according to the same ribotype (e.g., Figure 4, strains 79 and 87 in RT1, or strain 80 in RT5).

[0132] Additionally, a vaccine targeting three different antigens is a strategy to reduce the occurrence of potential escape and resistance mechanisms that the bacterium may employ by downregulating one or two of the proteins during its infection cycle.

[0133] Rationale for Producing Fragments or Hybrid Molecules of DsA1 and / or DsA2 The goal of the preferred strategy according to the present invention was to find a hybrid protein capable of inducing antibodies with surface binding and functionality as comparable as possible to those induced by the combination of both full-length proteins DsA1 and DsA2, with the aim of simplicity of production and lower cost, in an opsonophagocytic killing assay.

[0134] Several fragments of DsA1 have been generated and used for immunization studies. Surprisingly, a fragment of DsA1 that primarily occupies the C-terminus of the protein (e.g., the fragment designated "F4") induced antibodies that did not sufficiently bind to the P. acnes cell surface (e.g., Figures 6 and 18A) and were unable to induce bactericidal activity in opsonophagocytosis assays (Figures 18B and 18C). This is in stark contrast to authors who emphasized the high immunogenicity and immunological importance of the C-terminus containing the PT repeat domain of DsA1 (Lodes et al., 2006; Grange et al., 2017).

[0135] Bioinformatics analysis revealed a three-domain structure of the protein (referred to herein as "CSD1," "CSD2," and "CSD3") that was used in the course of this invention as a tool to optimize the design of fragments and hybrids to be tested in immunological studies based on sequence regions that offer some flexibility regarding the start and end of specific fragments after determining the biological / clinical and immune-related importance of the target (e.g., Figure 7A / C). The "swap sites" used, i.e., the DsA1 and DsA2 sequence regions (in protein crossover) were chosen to optimize the design of the fragments and hybrids to be tested in immunological studies based on sequence regions that offer some flexibility regarding the start and end of specific fragments (e.g., Figure 7A / C). The positions to be switched (such that the nucleotide sequence is switched) were chosen within the swap region, a region with minimal likelihood of interfering with structural elements, particularly the alpha helix (e.g., Figure 7B).

[0136] Based on insights from immunological studies (e.g., Figure 6) and bioinformatics analyses, additional fragments and hybrids of DsA1 and DsA2 have been designed to further explore the most important epitopes of the proteins used as immunogens in order to elicit antibodies capable of binding specifically to both DsA1 and DsA2 equally well. For example, serum antibodies induced by hybrid H4 molecules containing functionally more relevant regions, primarily restricted to the N-terminus and central portion of the DsA1 protein molecule (amino acids 29-145; 278-333), and only the central portion of the DsA2 protein sequence (amino acids 190-321) (Figure 12A), were capable of specifically binding to both full-length proteins in ELISA assays (e.g., Figure 8). Similarly, another example, the hybrid molecule H2, which contains the two central and homologous regions of DsA1 (amino acids 146-277) and DsA2 (190-321), induced antibodies with comparable abilities to recognize both full-length DsA1 and DsA2 proteins (e.g., Figure 8). Immunization with hybrid H4 induced antibodies that exhibited the most balanced specific binding to both DsA1 and DsA2 proteins. This contrasts with antibodies induced by immunization with the full-length proteins and other fragment and hybrid versions, which bound most highly specifically to themselves, albeit to a much lesser extent than to the second antigen (homologue). Similar conclusions were reached based on analysis of the strength and stability of the interaction by surface plasmon resonance (SPR) technology performed on a Biacore (Figures 19A and 19B).

[0137] In surface binding assays involving a collection of strains from various phylotypes, antibodies induced by immunization with hybrid H4 had the broadest and most balanced cross-reactivity and cross-type reactivity among the exemplary hybrids tested according to the present invention (e.g., Figure 9).

[0138] Antibodies induced by immunization with other versions of the hybrid construct (e.g., H3 and H5), as well as newly designed and additionally optimized C-terminal fragments (F12, F13) and N-terminal fragment (F10), when tested as single vaccines or within the design of other hybrid molecules, such as hybrid H3, were significantly less cross-reactive than antibodies induced by immunization with hybrid H4 (e.g., Figure 9). This also contrasts with prior art knowledge: although studies of similar proteins found in other bacteria, such as protein M (Fischetti, 1989), demonstrated the crucial importance of the C-terminus for the induction of immune responses with high levels of cross-reactivity among many different serotypes and strains, data obtained in the course of this invention showed that the C-termini of DsA1 and DsA2 are not immunologically relevant, despite their similarity to streptococcal proteins. This is because fragments containing primarily the C-terminal portion of the protein sequence performed inferiorly compared to fragments and hybrids containing more central portions of the molecule.

[0139] Multiple sequence alignments of DsA1 (native) polypeptides from various P. acnes sources revealed that DsA1, while generally considered a hypervariable protein, is in fact highly conserved and essentially invariant. Of the existing variability, only PT variability is understood as true protein variability. This is particularly because the transient nature of the observed genomic frameshifts has not yet been determined, and the effect of N-terminal frameshifts is fundamentally non-expressive. Another exception could be the rare C-terminal loss of an anchorage motif, which, according to current data, is very rare or limited to genes that are not expressed as proteins. Importantly, this invariance is This does not suggest or favor any part of the protein over another. Specifically, careful consideration of assembly errors in public repositories can exaggerate the fairly typical PT length variability. Furthermore, the length variability of PT regions can be understood as the influence of immunological pressures and has been interpreted as such in the literature. In the course of this invention, we have shown that this is inaccurate, and indeed, the experimental results disclosed herein demonstrate the opposite: PTs are largely immunologically irrelevant. They can be understood as flexible, presumably glycosylated, linkers tethering the N-terminal domain to the cell surface. This further solidifies that sequence variability clearly does not suggest any particular part of the DsA1 sequence as a vaccine candidate, and that such an interpretation (in the case of PTs) is indeed inaccurate.

[0140] Thus, contrary to the prior art, it was found during the course of this invention that structural epitopes and epitopes within the more central regions of the DsA1 (amino acids 146-277) and DsA2 (amino acids 190-321) sequences are highly immunologically relevant as vaccine antigens or epitopes, since fragments containing these regions, when used as vaccine antigens, induced highly cross-reactive / cross-type reactive antibodies that bound significantly better to the surface of P. acnes (e.g., Figure 9: H4, F11, H2, F9, P027-F1) compared to fragments and hybrids lacking or containing only this portion of the sequence (e.g., Figure 6: F3, F4; and Figure 9: F10, F12, F13). When the various hybrids and constructs were compared with each other in opsonophagocytosis assays against type IA1 strains, fragments and hybrids containing the more central regions of DsA1 and DsA2 induced very high levels of opsonophagocytic injury at almost all serial dilutions. Hybrid H3, containing the N-terminal sequence regions of DsA1 (amino acids 29-145) and DsA2 (amino acids 72-189), induced significant opsonophagocytic injury at serum dilutions up to 1 / 40,000, whereas the C-terminal fragment, F4, was negative at all dilutions tested (Figures 18B and 18C). Furthermore, hybrid H4 induced ≥95% opsonophagocytic injury at all dilutions, even at 1 / 1,280,000, demonstrating even better serum antibody responses to the full-length DsA1 and DsA2 proteins (e.g., Figures 18B and 18C).

[0141] Despite significantly higher levels of conservation than suggested by the prior art, DsA1 and DsA2 exhibit variable amounts of expression on some strains; in some cases, DsA2 is expressed instead of DsA1, or vice versa; thus, for example, DsA2-induced antibodies bind less well to strains expressing more DsA1 (e.g., Figures 9, 16A, 16C, and 16D), and DsA1-induced antibodies bind less well to strains expressing more surface epitopes of DsA2 (e.g., Figures 16A, 16B, and 16E). For this reason, the use of hybrid constructs, such as hybrid H4, helps maintain consistent, balanced antibody levels that bind to multiple strains, regardless of whether DsA1 or DsA2 is more highly expressed (e.g., Figures 16A-F).

[0142] Furthermore, the biochemical properties of hybrid polypeptides such as H4 are even better than those of the full-length DsA1 and DsA2 proteins alone, making them suitable for manufacturing and vaccine administration. Protein H4 exhibits increased stability when compared to both full-length proteins DsA1 and DsA2. Compared to protein DsA2, protein H4 is expressed as a single-band protein and, in contrast to DsA1, is resistant to proteolysis (e.g., Figures 10A and 10B).

[0143] Thus, hybrid polypeptides such as H4 are not only immunologically more consistent and simpler molecules that can exert the effects of both full-length proteins. , which typically have improved stability and purity profiles compared to the parent version.

[0144] Rationale for designing a vaccine containing the protein PTIP and hybrid H4 The combination of the protein PITP and a DsA1 / DsA2 hybrid molecule, such as hybrid H4, makes it possible to target all P. acnes strains associated with acne vulgaris pathology (e.g., Figures 11A-C). Vaccines containing PITP and a DsA1 / DsA2 hybrid molecule, such as hybrid H4, induce immune responses that demonstrate clear superiority in terms of cross-reactivity and cross-type reactivity over the immune responses induced even after vaccination with either single protein alone (e.g., Figure 9, Figures 16A-E).

[0145] The use of a DsA1 / DsA2 hybrid molecule, such as hybrid H4, instead of DsA1 and DsA2 has the obvious additional advantage that only one protein is needed instead of two, which expedites the production process. This becomes important when one additional antigen, PITP, is included in the vaccine, as it significantly simplifies the production process and reduces costs.

[0146] Relevance of functional assays to human subjects The importance of functional assays has been demonstrated and developed in the course of this invention. These functional assays have been essential for determining vaccine antigen composition (e.g., selecting the three proteins DsA1, DsA2, and PITP as vaccine candidates) and optimizing vaccine design, particularly to provide an effective cross-reactive vaccine for administration to human individuals at risk or exposed to pathologically relevant P. acnes infection.

[0147] All human subjects contain pre-existing antibodies against P. acnes that can be detected in bodily fluids such as serum. These antibodies target antigens expressed by viable P. acnes, and if the epitopes of those antigens are accessible on the P. acnes cell surface, they can induce opsonophagocytic killing of the bacteria (e.g., Figures 13A and 13B). Indeed, according to data obtained for the present invention, opsonophagocytic killing is the most important functional activity of surface-bound antibodies, as evidenced by the significant increase in OPK K50 titers with increasing surface binding of human antibodies to P. acnes. Furthermore, the efficiency of opsonophagocytic killing is affected not only by the amount of antibody that binds to P. acnes, but also by the specific antigens expressed on the surface and targeted by antibodies generated by each individual against a particular P. acnes strain, as evidenced by the avidity of polyclonal antibodies directed against specific antigens and common isotypes and subtypes of antibodies generated in the human host during immune interactions and responses to the bacteria.

[0148] There is a need to produce vaccines that induce cross-reactive / cross-type reactive antibodies The present invention poses a challenge currently faced in the prior art and provides an answer to the question, "Which bacterial phylotype should be targeted by a vaccine to provide the greatest benefit to the host?"

[0149] The current state of the art is to move away from products that target a large percentage of P. acnes strains entirely, because only a small number of phylotypes are considered pathogenic and there is concern that by inducing an immune response against a large percentage of P. acnes, this could lead to the complete elimination of P. acnes and pose a threat to humans.

[0150] Based on the conclusions from these and other studies, different phylotypes were studied with regard to the frequency of isolation from patients compared to healthy individuals, and the current indications in the prior art are unclear. Any therapeutic product for P. acnes-induced pathologies, such as acne, should only act against strains of a specific phylogenetic group considered "pathogenic," and the current state of the art teaches that only strains of the MLST8IA1 type should be targeted (McLaughlin et al., 2019, p. 23; O'Neill and Gallo 2018, p. 4, column 2, end of the first long paragraph). Because these are commonly isolated from the skin of acne patients, others can be found on both acne-prone and healthy skin. However, the current state of knowledge in the art does not take into account that the human host immune system plays an important role in regulating the colonization density, pathogenic behavior, and phylogenetic profile of strains that colonize different individuals and body regions, and that the results of these interactions are host-specific. Additionally, additional factors, such as hormonal activity, skin barrier integrity, and the environment within the site where infection occurs, are unique to each individual and may contribute to factors that promote versus limit the pathogenic potential of different strains.

[0151] However, data according to the present invention indicate that not only IA1 but also other phylotypes are equally capable of inducing acne, since direct isolation and analysis of material from inflamed acne lesions (pustules) revealed that in many patients it was not IA1 but other phylotypes that were predominantly found in this material (e.g., Figure 14A).

[0152] Additionally, data according to the present invention indicate that the skin surface and skin pores of acne patients are colonized by the IA1 phylotype, but the patients do not develop acne lesions primarily due to the IA1 strain. This is because acne lesions are also enriched in P. acnes phylotypes different from IA1, and in some cases, non-IA1 phylotypes were identified at significantly higher abundances. For example, a P. acnes strain (CR086) isolated from an inflamed lesion of a particular patient was typed according to the SLST scheme and found to be F4, belonging to the IA2 phylotype (e.g., Figure 14A); however, strains detected in material collected from skin pores in the same facial region of the same patient contained IA1, IA2, and IC phylotypes, plus one phylotype not yet included in public SLST databases (so that MLST classification could not be assigned to this particular strain) (e.g., Figure 14B). Similarly, the most abundant strain CR078 detected in the patient's inflamed acne lesions was of SLS types G1 (phylotype IC) and K1 (phylotype II) (e.g., FIG. 14A), while some strains colonizing this patient's skin pores contained SLS types A1, C1, and C2 (phylotype A1) (e.g., FIG. 14B). This further confirms the need addressed by the present invention to provide cross-reactive and cross-type reactive vaccines to combat P. acnes infection.

[0153] The appearance and size of lesions typically vary on the skin, with some being smaller, some larger, some more inflamed, and some less inflamed. These differences are typically attributed to different stages of acne development or the environment on the skin; however, the data obtained by the present invention demonstrates that not only the type of strain isolated from the skin surface or skin pores, but also the phylotypic composition of the strains dominating the inside of the inflamed lesions, and the amount and specificity of antibodies reaching each individual's skin and pilosebaceous glands, play a crucial role in determining host outcome: bacterial interaction, and affect the extent to which acne lesions develop, become visible on the skin surface, and progress to clinical symptoms. Therefore, knowledge of the amount of antibodies, antigen specificity, and effectiveness in inducing phagocytic killing of various P. acnes strains colonizing the skin of different individuals must be taken into consideration when designing immunotherapy products and selecting antigens to be included in vaccines.

[0154] Novelty over the prior art regarding the clinical significance of P. acnes strains Prior art studies have collected samples for analysis from a single location on the skin, which often does not represent the typical location of acne lesions. For example, Fitz-Gibbon's study (Fitz-Gibbon et al., 2013) received some negative reviews from experts in the field. This was due to the fact that they only collected material from skin pores on the skin covering the nose, which is not typically affected by acne, and the methodology they used was not considered in accordance with acceptable standards (Eady and Layton, 2013; Alexeyev and Zouboulis, 2013). The findings of the present invention show that it is essential to sample not only skin pores in affected and non-affected areas, but also inflamed lesions, because the strains found on the skin surface or in pores may not necessarily be the same strains that persist in inflamed lesions. Because only a subset of strains colonizing each individual has pathogenic potential in that particular individual, strain identity cannot be universally estimated by combining results from various studies and looking for the most common strains isolated from many different individuals. Rather, an analysis and comparison of both bacterial- and host-specific factors operative in each particular patient must be performed instead.

[0155] Thus, the present invention also demonstrates that data obtained from the analysis of many different individuals can be grouped and analyzed together to demonstrate that the relevance of findings for one individual, whose acne may not be the most common among those identified in other studies, can nevertheless be attributed to a phylotype that is a virulence factor in that particular individual. Instead of assessing the pathogenic potential of different strains by analyzing the frequency of isolation across many different individuals, individual-specific studies, along with analysis of the immune status, are significantly more beneficial because each individual inherits a specific signature of colonizing strains, only some of which may be pathogenic in a particular human host. The data provided by the present invention also provide evidence for the differential role of specific P. acnes phylotypes in the pathogenesis of acne vulgaris, which is individual-specific. This provides important evidence for a vaccine that is broadly cross-protective and not directed solely at the specific phylotype identified as most frequent in studies involving many different patients.

[0156] Furthermore, different sampling areas are also colonized by different strains because they are attracted to different environments in the skin. As demonstrated in the course of this invention, in some patients, similar strain compositions appear in samples collected from the forehead and cheek skin punctures, while in others significant differences were found.

[0157] Without information about the strains found inside the inflamed lesions and the immune status (antibodies to the strains identified in the lesions and their specificity), it is impossible to confirm the actual relevance of the strains to the acne pathology in each individual. For example, an individual patient's skin pores (CR078) were colonized by MLST types A1, C1, and C2 (MLST phylotype IA1) and by SLS types G1 (phylotype IC) and K1 (phylotype II). However, SLS types G1 (phylotype IC) and K1 (phylotype II) were detected as the most common inside the inflamed acne lesions of the same patient. Similarly, SLS type F4 (phylotype IA2) was identified as the most common in the inflamed acne lesions of patient CR086, but at least two additional phylotypes were identified in the skin pores surrounding the same sampled area (e.g., Figure 14B).

[0158] Supportive evidence for the selection of hybrid molecules of H4 species for use in human vaccines. H4 contains functionally relevant epitopes of both Dsa1 and DsA2. H4-specific antibodies (i.e., at least one (relevant) DsA1 and one (relevant) DsA2) ) antibodies induced by hybrid molecules containing the DsA2 epitope) appear to be more abundant in patients with severe acne compared to DsA1 and DsA2, indicating that the patient's immune response in the skin is driven toward functionally relevant epitopes of both proteins that are well represented by hybrid antigens, such as the H4 construct (e.g., Figure 15).

[0159] The present invention provides the results of the comparison of a large number of suggested potential candidates and epitopes using a series of functional assays, and selects the key components (immunologically relevant antigens and epitopes) that must be included in a protective vaccine product. In addition, the present invention provides a new direction in the field by revealing the actual relevance of the expression of different virulence factors for the immune response of the human host, leading to the conclusion that vaccine products should be designed to induce immune responses not only against specific phylogenetic groups, but also against as many as possible, preferably the most relevant, especially all bacterial strains that can express virulence factors and acquire pathogenic traits, and are effectively targeted by antibodies raised by host immunization.

[0160] Human subjects already have an established and ongoing immune interaction with P. acnes, and during the course of this interaction, they respond differently to P. acnes antigens. The immune system's response to a specific antigen (the quality of the immune response and the quantity of antibodies) varies individually depending on the location and context in which the specific antigen is encountered. For example, antigens are encountered in various areas colonized by P. acnes (not just the skin), in the context of inflammatory versus healthy / tolerogenic states (e.g., normal versus inflamed hair follicles, normal versus damaged skin barriers, etc.), in contact with bacterial biofilms versus planktonic cells, as part of damaged or dying bacterial cells, in tissue secretions. Thus, intracellular proteins, secreted proteins, or membrane-bound proteins can all induce immune responses and elicit antibody development. However, only a portion of these proteins have the potential to induce a protective immune response that acts against bacteria. Thus, in accordance with the present invention, immunologically relevant antigens and epitopes have been selected that elicit the most protective response against bacteria and that are most suitable for use as vaccine material (e.g., optimized for production, formulation, and human use).

[0161] 5.1 Definition and Description of the Preferred Embodiment In the context of describing the present invention (particularly in the context of the claims and embodiments which follow), the use of the terms "a," "an," and "the," and similar referents, should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context.

[0162] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including") unless otherwise specified. For purposes of the present invention, the term "consisting of" "Of" is considered to be a preferred embodiment of the term "comprising of." Hereinafter, if a group is defined to comprise at least a certain number of embodiments, this is also defined to encompass a group that preferably consists of only these embodiments.

[0163] The term "adjuvant" refers to any substance that, when administered in conjunction with an antigen or epitope, augments and / or redirects the immune response to the antigen or epitope. Adjuvants can augment the immune response by several mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Adjuvants can bind to antigens by covalent bonds, electrostatic interactions, or adsorption. The antigen and adjuvant activity may be combined by genetically (recombinantly) fusing the coding regions, or portions thereof, of the antigen and adjuvant; thus, the immunogen may contain only one component or component. Adjuvants may be artificial or naturally occurring.

[0164] In the present context, adjuvants include emulsion-based adjuvants, aluminum hydroxide gels, solid-phase adsorbents, nanospheres, and encapsulating materials such as liposomes. The vaccines described herein particularly use "human adjuvant formulations," which are understood to be specifically compatible with the human immune system. Human vaccine formulations in particular do not contain Freund's incomplete adjuvant (nor, of course, Freund's complete adjuvant or similar formulations), which are often used solely for generating animal immune sera. Contrary to non-human adjuvant formulations, such as those used to generate rabbit immune sera, human adjuvant formulations are particularly characterized by the use of adjuvants that are approved for human use to ensure a high level of safety (not inducing undesirable local or systemic effects) and promote the required immune response in the vaccine target population via the optimal vaccine administration route, while not eliciting an immune response against the human host itself.

[0165] Exemplary adjuvants include metal salts (e.g., aluminum or calcium salts), high molecular weight molecules, cationic peptides, CpG oligonucleotides, and squalene-based adjuvants (e.g., MF59). Metal salts include alum (potassium aluminum sulfate), aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, aluminum hydroxyphosphate, calcium phosphate, cerium nitrate, zinc sulfate, colloidal iron hydroxide, and calcium chloride. Several aluminum adjuvants with different physical properties are commercially available and approved for human use.

[0166] In the present context, an adjuvant may also be any suitable high molecular weight molecule, typically a protein or high (i.e., generally greater than 6000 kD) molecule of sufficient molecular complexity to elicit an immune response to the antigen or epitope to which it is covalently linked. A category of suitable high molecular weight adjuvants is exemplified by any mutant cross-reactive material of toxins, toxoids, or toxins derived from tetanus, diphtheria, pertussis, Pseudomonas species, Escherichia coli, Staphylococcus species, and Streptococcus species. Such toxins or toxoids include tetanus toxoid, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa; bacterial outer membrane proteins, such as outer membrane protein complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C. difficile enterotoxin (toxin A) and cytotoxin (toxin B), or Haemophilus influenzae. In addition, polypeptides according to the present invention can also be formulated as virus-like particles bound to (monoclonal) antibodies or nanoparticles (recently reviewed in Malonis et al., Chem. Rev. 120 (2020), pp. 3210-3229). The latter can be used particularly for conformational epitopes. Higher-order structure antibodies, for example, DsA1 R87-K90+S234-G250, R87-K90+L246-A260, R87-K90+A256-E270, R87-K90+R266-T277, A310-D313+V289-K296, A310-D313+V289-K296, A310-D313+T285-T300, A The DsA1 polypeptides, particularly DsA1 fragments or DsA1 derivatives according to the present invention, may be presented in their essentially natural environment. Alternatively, these conformational epitopes may be presented in an artificial scaffold. Established techniques for such scaffold presentation include those in which virus-like particles are conjugated to (monoclonal) antibodies, nanoparticles, alphabodies, protein A, protein G, designed ankyrin-repeat domains (DARPins), fibronectin type III repeats, anticalins, knottins, or engineered CH2 domains (nanobiodies) (see, e.g., Malonis et al., 2020, U.S. Patent Application Publication No. 2019 / 0383829A1, WO 2019 / 123262A1).

[0167] In certain embodiments, the vaccines described herein contain an adjuvant, which is a heterogeneous chemical or biological material or substance commonly used to enhance an active immune response after vaccination with a vaccine antigen.Typically, the adjuvant is alum, such as a phosphate or hydroxide, a TLR agonist, such as CpG or monophosphorylated lipid A.Cytokines such as IL-1 and IL-2 can also be used as adjuvants.

[0168] As used herein, the terms "heterologous" or "exogenous" with respect to adjuvants refer to molecules derived from a source other than a P. acnes antigen. A heterologous adjuvant may refer to an artificial, inorganic, or organic compound or substance, possibly derived from a different bacterium or a different P. acnes strain or subtype, or from an unrelated source (e.g., a different pathogen, a chemically synthesized material, or an organic or inorganic material).

[0169] Alternatively, selected vaccine antigens can be used in unadjuvanted form and presented in physiological solutions or formulations suitable for skin-specific immunization. Intradermal, transdermal, or subcutaneous administration can be performed using a variety of methods, including intradermal injection applicators, microneedles, transdermal laser devices, skin patches, or other suitable skin-compatible application procedures (Engelke et al., 2015).

[0170] As used herein, the term "polypeptide" refers to larger polypeptides (proteins), such as DsA1, DsA2, and PITP, and larger fragments and derivatives thereof, as well as shorter polypeptides (oligopeptides), such as epitopes, fragments or derivatives of DsA1, DsA2, and PITP.

[0171] The term "antigen" as used herein refers to a whole molecule or a fragment of such a molecule in its natural environment or as an isolated antigen, and also includes recombinant antigens or single antigens produced by genetic engineering of host cells transformed with a recombinant heterologous nucleotide sequence, which are specifically bound by an antibody binding site. Specifically, the term also includes substructures of antigens, such as polypeptide and / or carbohydrate structures commonly referred to as "epitopes" (e.g., B-cell epitopes or T-cell epitopes, preferably B-cell epitopes), which are immunologically relevant. An antigen can be immunogenic, and if it is poorly immunogenic, it can be made immunogenic by appropriate engineering or formulation. The terms "immunogen" and "antigen" as used herein also encompass epitopes and are used interchangeably.

[0172] As used herein, the term "epitope" is intended to specifically refer to a molecular structure that can completely make up a specific binding partner or that can be part of a specific binding partner to the binding site of an antibody or cognate T-cell receptor. Epitopes can be any molecular structure, including carbohydrate, peptide structures, , fatty acids, organic, biochemical, or inorganic substances, or derivatives thereof, and any combination thereof. If an epitope is composed of a peptide structure, e.g., a peptide, polypeptide, or protein, it will typically contain at least six amino acids, preferably at least 7, 8, 9, or 10, and up to 40 amino acids, more preferably 6-35, 7-30, 8-25, and especially 10-20 amino acids. Epitopes can be linear or conformational. A linear epitope is composed of a single segment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be contiguous or overlapping. A conformational epitope is composed of amino acids or carbohydrates grouped together by folding a polypeptide to form a tertiary structure, where the amino acids are not necessarily adjacent to each other in the linear sequence.

[0173] Epitopes of a given antigen can be identified using several epitope mapping techniques well known in the art. See, for example, *Epitope Mapping Protocols in Methods in Molecular Biology* (Morris, 2005). For example, linear epitopes can be determined by synthesizing multiple peptides, e.g., peptides corresponding to portions of protein molecules, in parallel on a solid support and reacting the peptides with antibodies while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871. Alternatively, instead of epitopes (linear, conformational, or both), mimotopes (linear, conformational, or both) can be used in vaccines according to the present invention, which mimic the structure of the epitope and induce an immune response similar to that elicited by the epitope. Antibodies for a given epitope antigen will recognize mimotopes that mimic the epitope. Mimotopes are typically obtained by biopanning from phage display libraries. Mimotope analysis has been widely used to map epitopes (Smith and Petrenko, 1997) and develop therapeutics (Macdougall et al., 2009) and vaccines (Knittelfelder et al., 2009). A "mimotope" is a polypeptide that differs from the polypeptides disclosed herein by one or more amino acids but mimics the three-dimensional structure of the wild-type polypeptide / epitope. Generally, mimotopes, in the context of a larger protein scaffold called a carrier, can stimulate the host's immune system to produce an antibody antigen-specific response. The host produces antibodies that specifically bind to the mimotopes disclosed herein and the corresponding wild-type epitope. Mimotopes may have a primary amino acid sequence capable of eliciting a T-cell effector response and / or the three-dimensional structure necessary to bind to B cells and result in the maturation of an adaptive immunological response in animals, particularly humans. An antibody for a given epitope antigen will recognize a mimotope that mimics that epitope.Thus, a "mimotope" is a polypeptide that mimics a protein, carbohydrate, or lipid epitope and can be generated, for example, by phage display technology. When bound to a carrier or displayed in the form of multiple antigenic peptides, the mimotope achieves immunogenicity and induces an epitope-specific antibody response upon vaccination. A mimotope can be a polypeptide, e.g., a peptide with an amino acid sequence length of at least about 8 to about 25 amino acids or more.

[0174] Exemplary algorithms and programs based on mimotope-based predictive models include, for example, MimoPro (http: / / informatics.nenu.edu.cn / MimoPro), PepSurf (http: / / pepitope.tau.ac.il), and EpiSearch (http: / / curie.utmb.edu / episearch.html). Additionally, sequence-based predictive models that rely only on the primary sequence of the antigen, such as BEST and EpiSearch, reviewed in Sun et al., 2013, are also available. In addition, binding site prediction models that infer methods focused on binding site prediction for protein-protein interactions, antigen and antibody interactions, e.g., ProMate, ConSurf, PINUP, and PIER, can be used.

[0175] Similarly, conformational epitopes may be identified by determining spatial conformation of amino acids by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., epitope mapping protocols (supra).

[0176] As used herein, the term "immunologically relevant" with respect to an antigen or epitope refers to the ability to be recognized by the immune system of a recipient organism and to induce antibodies in the recipient that are cross-binding and / or cross-reactive, especially cross-type reactive, and have antibacterial or pathologic ameliorating activity.

[0177] The specific antigens described herein are any of the proteins identified by UniProt accession numbers Q6A5X9, Q6A5P9, and Q6A9N1 of P. acnes strain KPA171202, or similar proteins, particularly homologous proteins of any of the proteins identified by UniProt accession numbers Q6A5X9, Q6A5P9, and Q6A9N1 of different P. acnes strains. Any reference herein to a sequence database shall, in case of doubt, refer to the version of the database as of the priority date of the present invention. This is also reflected herein, for example, in Figure 12 or in the sequence listing.

[0178] Proteins Q6A5X9, Q6A5P9, and Q6A9N1 are also referred to in the present invention as examples of naturally occurring "DsA1," "DsA2," and "PITP" polypeptides, particularly in the Examples section (as preferred examples ("P022," "P027," and "P028")) and in the Figures. Also, as further outlined below, in accordance with the present invention, "DsA1 polypeptide," "DsA2 polypeptide," and "PITP polypeptide" include naturally occurring, functional polypeptides of P. acnes strains that include all regions and domains defined below. "DsA1 polypeptide," "DsA2 polypeptide," and "PITP polypeptide" further include fragments and derivatives of DsA1, DsA2, and PITP, respectively. "DsA1 fragment," "DsA2 fragment," and "PITP fragment" refer to fragments of native, functional DsA1 polypeptides, DsA2 polypeptides, or PITP polypeptides of P. acnes strains that comprise a specified minimum amino acid length having at least 7, preferably at least 8, more preferably at least 9, and especially at least 10 consecutive amino acids of the DsA1 polypeptide, DsA2 polypeptide, or PITP polypeptide, respectively (i.e., fragments having the native consecutive polypeptide sequence of these functional P. acnes polypeptides). Preferably, the "DsA1 fragment," "DsA2 fragment," and "PITP fragment" comprise at least an epitope of the DsA1 polypeptide, DsA2 polypeptide, or PITP polypeptide, respectively, that can be recognized by the human immune system and can elicit functional anti- P. acnes antibodies reactive to this epitope (i.e., via T cells (without the appropriate antibody) or by antibodies that remove / inactivate secreted forms of the polypeptide, i.e., effective in opsonization assays disclosed herein or effective in promoting a pathology-ameliorating effect). The "DsA1 fragment," "DsA2 fragment," and "PITP fragment" also comprise deletions of the native polypeptide (i.e., composed of two or more fragments of the native polypeptide).A notable example of a typical fragment is one that includes an N-terminal methionine but lacks part of the N-terminal portion of the polypeptide following the N-terminal methionine. According to the definition herein, a fragment that merely lacks the signal peptide (but may include the N-terminal methionine) is a fragment of (intact or native) DsA1 / DsA2 / P. The fragments are considered to be ITP polypeptides. Fragments containing further deletions, such as the N-terminus at the NSR of DsA1 / DsA2 and the N-terminus at the ENFD of PITP, but still containing an N-terminal methionine, are referred to herein as "fragments." N-terminal extensions resulting from DNA-level frameshifts and replacement of the signal peptide with an alternative frame sequence are equally treated as falling within the definition of a fragment of a native polypeptide herein, since the actual polypeptide is "functional" within the meaning of this invention. "DsA1 derivative," "DsA2 derivative," and "PITP derivative" refer to a polypeptide derived from a DsA1 polypeptide, a DsA2 polypeptide, or a PITP polypeptide, or a DsA1 fragment, a DsA2 fragment, or a PITP fragment (having at least the number of amino acid residues of a "fragment" according to the invention (i.e., at least 7 amino acids) and containing at least one amino acid exchange / deletion / insertion compared to the native DsA1 / DsA2 / PITP sequence (i.e., polypeptide / fragment), but still capable of being recognized by the human immune system and having at least a functional reactivity against the DsA1 polypeptide epitope, DsA2 polypeptide epitope, or PITP polypeptide epitope). Each of the DsA1, DsA2, and PITP polypeptides contains an epitope capable of inducing anti-P. acnes antibodies (i.e., is effective in the opsonization assays disclosed herein). As used herein, "derivative" can be derived from one or more of DsA1, DsA2, and PITP; i.e., polypeptides containing DsA1 and DsA2 epitopes can be referred to as "DsA1 derivatives" and "DsA2 derivatives." Preferred derivatives according to the present invention contain at least an epitope of a DsA1 polypeptide, a DsA2 polypeptide, or a PITP polypeptide, i.e., an epitope that is identical to an amino acid sequence within a native (wild-type) P. acnes DsA1 / DsA2 / PITP polypeptide.Also, as defined below, the epitopes described herein are polypeptide sequences that, when administered to a human individual, are capable of eliciting antibodies with antibacterial activity as determined in a functional assay (preferably the opsonization assays disclosed below, particularly the opsonization assays disclosed in the Examples section). Further preferred derivatives are combinations that include one or more DsA1 / DsA2 / PITP fragments (i.e., one or more naturally occurring polypeptide fragments of these P. acnes proteins).

[0179] The protein identified by UniProt accession number Q6A5P9 is specifically characterized by the following features: hypothetical and uncharacterized protein (locus tag = PPA2210). The protein was found by Holland et al. to be upregulated during stationary phase (Holland et al., 2010) and variably expressed among P. acnes strains with different genetic phylotypes (Lodes et al., 2006; Yu et al., 2016; McLaughlin et al., 2019).

[0180] The protein identified by UniProt accession number Q6A5X9 is specifically characterized by the following features: a putative adhesion or S-layer protein (locus tag = PPA2127) with an N-terminal sequence in place of a signal peptide from amino acids 1 to 28, and a mature polypeptide chain comprising amino acids 29 to 405. The N-terminal sequence is thought to be the result of a genomic frameshift event and may not be part of the mature protein. Expression of this protein has been found to be highly variable among P. acnes isolates using genetic and proteomic analysis techniques (Lodes et al., 2006; Brzuszkiewicz et al., 2011; Yu et al., 2016).

[0181] Q6A5X9 (PA-25957) was described by Lodes et al. (Lodes et al., 2006) as having slight similarity to M-like proteins found in other bacterial species, possessing a common cleavable signal sequence, hydrophilic proline-rich repeats near the carboxy terminus, and an LPXTG motif, and containing a transmembrane helix in the central region. M-like proteins are generally involved in the complement system and in glycosylation of proteins containing dermatan sulfate. It has been found to interact with various binding forms of aminoglycans. Streptococcal M proteins are known to be more conserved toward the C-terminus, and cross-reactive epitopes have been identified: antibodies recognizing the C-terminal portion of the protein cross-react with 30 different serotypes (Fischetti, 1989). Lodes et al. also suggested that the C-terminus of PA-25957 (later named DsA1 (McDowell et al., 2011)), which contains PT repeats, is highly antigenic and associated with "health," because higher reactivity of "acne-negative" sera was detected against this region compared with the rest of the P022 sequence. The PT region has also been found to be more variable, potentially contributing to differences in expression across different strains.

[0182] In addition, Lodes et al. suggested that the cleavable LP(X)TG domain identified in the sequences of both PA-25957 (Q6A5X9) and PA-5541 (Q6A5P9) is involved in membrane anchoring and is absent in some P. acnes isolates; this suggests that PA-25957 DsA1 is not only found on the cell membrane but is also secreted by some strains, which further contributes to the variability in protein expression.

[0183] According to prior art, these proteins are primarily expressed by type IA strains, which are primarily isolated from acne-affected skin (Bek-Thomsen et al., 2014; McLaughlin et al., 2019). However, specific protein expression signatures were not detected in acne-prone sebaceous follicles compared to healthy skin (Bek-Thomsen et al., 2014), and the study had many methodological limitations described in the publication, leaving many questions open regarding their actual importance in disease pathology and their relevance for acne treatment. For this reason, P022 (DsA1) and P027 (DsA2) are currently considered virulence factors rather than vaccine candidates (O'Neill and Gallo, 2018). Furthermore, the prior art as of today is very cautious about using vaccine-based approaches, instead focusing on strategies that do not act directly against P. acnes but target secreted factors (Contassot 2018; Keshari et al. 2019), or alternative therapies, such as probiotics (O'Neill and Gallo 2018; Bruggemann 2019; McLaughlin et al. (2019) ("We can speculate that the potential of type IA to modulate interactions with the host immune system via the DsA immunogenic protein may be important in the recurrence of acne"; this is a very vague statement, but is consistent with the data available at the time of this invention). In fact, McLaughlin et al. (2019) did not even mention these antigens: in this paper, it was shown that type IA expresses a number of different virulence factors in addition to the DsA protein, and it was concluded that systematic research in the field is lacking to compare all of their potential with each other and determine which should be selected. Furthermore, Keshari et al. (2019) emphasized that among the various virulence factors expressed by P. acnes, vaccines should only include secreted ones that are not associated with the cell surface, again warning that P. acnes is a commensal bacterium and that the bacterium itself should not be targeted by vaccines.

[0184] Yu et al. ( Yu et al., 2015 ; Yu et al., 2016 ) used proteomic analysis to analyze the expression of various proteins by acne-associated strains and found that, among the proteins studied, the expression of proteins corresponding to Q6A5X9 (gi 50843565) and Q6A5P9 (gi 50843645) was highly variable among P. acnes strains from different genetic groups and phylotypes, whereas Q6A5P9 was absent or barely expressed by P. acnes ribotypes considered “pathogenic.”

[0185] According to a preferred embodiment of the present invention, modified P022 (DsA1) and / or P027 (DsA2) polypeptides are provided (DsA1 fragments, DsA1 derivatives, DsA2 fragments, and DsA2 derivatives) that have advantageous properties compared to the wild-type P022 and P027 proteins from P. acnes and are particularly suitable for vaccination purposes. The novel use of the DsA1 / DsA2 proteins (e.g., P022 and P027) according to the present invention for use in interfering with P. acnes (i.e., preventing and / or treating pathologies caused thereby) is based on advantageous properties (revealed by the present invention) regarding their immunogenic properties and their handling characteristics (which allow for easier large-scale recombinant expression and production). Both advantages emerged in the course of the present invention and are surprising in light of the knowledge in the art.

[0186] According to a preferred embodiment, the present invention also relates to fragments or derivatives of DsA1 / DsA2 polypeptides. A DsA1 / DsA2 "fragment" is a portion of a naturally occurring DsA1 or DsA2 protein; a DsA1 / DsA2 "derivative" is a non-naturally occurring polypeptide comprising a DsA1 / DsA2 fragment containing at least an antigenic epitope (i.e., an epitope that is immunogenic and accessible to antibody binding on the surface of P. acnes). A DsA1 / DsA2 fragment or derivative preferably has a length of at least 15, preferably at least 20, more preferably at least 30, and especially at least 50 amino acids of a naturally occurring DsA1 or DsA2 protein. Preferred DsA1 or DsA2 fragments or derivatives according to the present invention have a shortened PT repeat region, preferably having only 1, 2, 3, 4, or 5 PT repeats. Although the PT repeat region has been considered "highly antigenic" in the prior art (Lodes et al., 2006) and therefore found to be immunogenic in principle, evaluation of this protein region using patient antibodies in the course of the present invention has shown that this region is not essential for providing an appropriate acne vaccine. More specifically, DsA1 or DsA2 polypeptides lacking most of the PT repeat region surprisingly exhibit significant immunogenic advantages compared to DsA1 or DsA2 polypeptides containing only one or a few PT repeats. Furthermore, DsA1 or DsA2 peptides with fewer PT repeats induced stronger cross-reactive / cross-reactive antibodies compared to DsA1 or DsA2 peptides containing the complete (wild-type) PT repeat region. It is even possible to omit the entire PT sequence; however, preferred embodiments of the present invention contain at least one PT repeat.

[0187] With respect to the terminology used herein, all references herein to sequences, fragments, etc. always refer to contiguous amino acids (unless explicitly stated otherwise). For example, a DsA1 / DsA2 fragment of at least 15 amino acids always refers to at least 15 contiguous amino acid residues of the DsA1 / DsA2 polypeptide. The term "contiguous" means that a given amino acid is at a given position within the alignment of Figure 12. Of course, exchanges between different DsA1 / DsA2 homologs are included, provided that positions are maintained (i.e., no deletions, unless explicitly foreseen in Figure 12).

[0188] For example, the UniProt amino acid sequence Q6A5X9 refers to the protein "PPA2127" of P. acnes strain DSM 16379 / KPA171202, i.e., a polypeptide having the following sequence: MLSLLRRLLR WSDQSQQSPP PPPPQAEASS NRPRSVAQAA IATDGKGIID KDCRDAVIND AKLRAAIAGA LVKAGFSSAD AVALAPRIAK EMAKEGVLLI NHHKLKALIG AQLGLLTDAK IQRAAAAVDL GIKATLAATI IPNALHSAAF KDAVVANLVA AGVDKKLAKA TAVAIAATAL NPALGPIAKT EAIKAEIAAQ AALLVGRGVH LKKAAIEHII GRSFDAAVAT AIVSSPILNA RIVTHLVRAG IDKSLAVQIA PRIIDRLAKE PLLALNTAKL MKNITRQIVD VITADKAIKT AEQLEKELPA LDDLVKKACS CPKPTPTPTP TPTPTPKPTP TPTPKPTPTP KPKPTPAPAP TSGA TSDEST SRSGGHSQGG SGTHYIHHGV APVLTHSSDL PSTGF

[0189] In this wild-type DsA1 polypeptide, the PT repeat region extends from proline 324 (P324) to threonine 361 (T361) and contains 19 PT repeats. The UniProt amino acid sequence Q6A5P9 refers to the protein "PPA2210" of P. acnes strain DSM 16379 / KPA171202. In this wild-type DsA2 polypeptide, the PT repeat region extends from proline 367 (P367) to threonine 420 (T420) and contains 27 PT repeats. Within the meaning of the present invention, the term "PT repeat" is defined as a section within the primary amino acid sequence of the DsA1 / DsA2 protein that has a repetitive proline-containing dipeptide. It typically follows the consensus sequence DX1LVX2KACX3(C)PX4 in P. acnes DsA1 (and also in DsA2), where X1 is typically D (in DsA1) or G (in DsA2); X2 is typically K (in DsA1) or Q (in DsA2); X3 is typically S (in DsA1) or T (in DsA2); and X4 is typically K (in DsA1) or E or D (in DsA2). By this definition in accordance with the present invention, the initial PK (in DsA1) or "PE" or "PD" (in DsA2) dipeptide stretch sequence is not defined as part of the PT repeat region beginning with the first proline residue after the "PK," "PE," or "PD" dipeptide. The proline-containing dipeptides within the PT repeat region are primarily proline-threonine (PT) dipeptides, but may also be proline-alanine (PA), proline-asparagine (PN), or proline-lysine (PK) dipeptides; however, the PT repeat region is primarily composed of PT dipeptides. Alternatively to the series of PT dipeptides, polar / acidic / negatively charged peptides can be found, such as "SDTDTDSNPNADADTDA." These polar / acidic / negatively charged peptides are composed of "SD" and "TD" dipeptides, but "Sn," "PN," or "AD," and less frequently, "AP," are also possible.For example, the PT repeat region in Q6A5X9 consists of 19 repeating proline dipeptide stretches with 13 PT, 4 PK, and 2 PA dipeptides; the PT repeat region in Q6A5P9 consists of 27 repeating proline dipeptide stretches with 22 PT and 5 PA dipeptides. In some cases, the PT repeat region is also glycosylated.

[0190] A "P. acnes DsA1 polypeptide" according to the present invention is a naturally occurring DsA1 protein ("native DsA1") from a P. acnes strain, and includes all of the following domains (N-terminus to C-terminus): the N-terminal swapping region ("NSR"), the first conserved subdomain ("CSD1"), the first swapping region ("SR1"), the second conserved subdomain ("CSD2"), the second swapping region ("SR2"), the third conserved subdomain ("CSD3"), the Pro-Thr repeat-containing region ("PT repeat region"), and the C-terminal region ("CTR"; often with an LPXTG motif near the C-terminus), or optionally, a shortened PT repeat region. The PT repeat region located between CSD3 and CTR is shortened or absent altogether (compared to naturally occurring DsA1 proteins) in preferred DsA1 polypeptides according to the present invention. The sequences of naturally occurring DsA1 polypeptides contained within sequence databases (see, e.g., Figure 12) are highly identical, differing only in the length and composition of the PT repeat region (besides point mutations and rare exceptions, particularly the presence of the terminal LPXTG motif).

[0191] Alignment of selected, naturally occurring, representative DsA1 variants shows the more or less ubiquitous invariance of the protein. There are three notable exceptions. First, frequently, the gene is actually a pseudogene resulting from a frameshift at the C-terminus of the signal peptide. The gene is not expected to be expressed adjacent to the signal peptide. However, in gene prediction, the variant appears with an N-terminal extension that is not expected to be expressed but is rather an artifact of gene prediction. Strain KPA171202 has been cited as a representative example. It is possible, but not likely, that the frameshift is reversible under certain conditions (a form of phase-variable regulation). Second, the length of the PT repeat varies considerably between isolates. This PT repeat also appears to be a frequent source of misassembly, creating the impression of a C-terminal deletion. However, when considering the entire genome, the C-terminus appears to be more or less universally conserved, at least within expression-competent proteins, and the reported variant is most likely a sequencing artifact. There is a rare alternative frameshift mutation (approximately 10% of cases) that removes the last 10 amino acids at the C-terminus. This is intriguing because the variant lacks the C-terminal LPXTG motif, which is thought to be important for cell wall anchoring of the protein. However, this particular frameshift appears to be limited to proteins that are also frameshifted at the N-terminus (these are likely not expression-competent). Therefore, this variant is likely not generated in vivo. In any way, this variant is not defined as a "DsA1 polypeptide" within the meaning of the present invention, but as a "DsA1 derivative," because it does not contain all domains / regions of DsA1, as defined above. Taken together, this indicates that DsA1, while generally considered a hypervariable protein, is in fact highly conserved. Among the existing variabilities, only PT variability can be understood as true protein variability, particularly because the transient nature of the observed genomic frameshift has not yet been determined, and the effect of the N-terminal frameshift is essentially non-expression. Another exception could be the rare C-terminal loss of an anchorage motif, but this, according to current data, is very rare or restricted to genes that are not expressed as proteins (i.e., a "DsA1 derivative," not a "DsA1 polypeptide").(Because the sequence does not contain all domains / regions of DsA1, as defined above, these data demonstrate the DsA1 polypeptide as essentially invariant. Indeed, when considering DsA1 sequence variants within the region CSD1-SR1-CSD2-SR2-CSD3, the maximum observed difference between sequences has previously been determined as 4-0-4-3-2 amino acids, with dissimilarities (i.e., different amino acids) determined as 1-0-4-2-2. Similarly, when considering DsA2 sequence variants within the region CSD1-SR1-CSD2-SR2-CSD3, the maximum observed difference between sequences has previously been determined as 4-1-9-0-4 amino acids, with dissimilarities (i.e., different amino acids) determined as 3-1-6-0-1. DsA1 and DsA2 are clearly homologs, but are distinct, with minimal amino acid differences between the CSD1-SR1-CSD2-SR2-CSD3 regions of 25-6-22-1-23 and 15-4-8-1-14 different residues, respectively.

[0192] Importantly, this considerable invariance of DsA1 does not suggest or favor any part of the protein over another. Specifically, careful consideration of assembly errors in public repositories can make the fairly typical PT length variability appear unusually large. Furthermore, length variability in the PT region could be interpreted as an effect of immunological pressure, and has been interpreted as such in the literature. This is shown to be inaccurate by the data provided by this invention. In fact, the results of this experiment demonstrate the opposite: the PT is largely immunologically irrelevant. The present data clearly show that it is a linker connecting the N-terminal domain to the cell surface. This further solidifies that sequence variability clearly does not suggest any particular part of the DsA1 sequence as a vaccine candidate, and that such an interpretation (in the case of the PT) is indeed inaccurate.

[0193] Some sequence entries do not contain a CTR; such entries are likely (sequencing) artifacts or are not DsA1 polypeptides within the meaning of the present invention. i.e., they do not function as DsA1 proteins in P. acnes. Therefore, these sequences are also referred to as "DsA1 derivatives." The sequence numbering used in accordance with the present invention is based on the Q6A5X9 numbering, which means, for example, that the proline-lysine dipeptides preceding the PT region amino acids are always referred to as "P322" and "K323," even if DsA1 polypeptides (or fragments or derivatives) of different lengths (i.e., P322 / K323 are at different amino acid numbers within this particular polypeptide) are involved. In addition, DsA1 mRNA encodes an N-terminal signal sequence (starting with the N-terminal methionine residue and ending with a proline-glutamine-alanine-glutamic acid-alanine sequence), which is not part of the mature polypeptide. Thus, the P. acnes DsA1 polypeptide according to the invention starts at serine residue 29 (S29) and ends at phenylalanine 405 (F405) of the amino acid sequence Q6A5X9 in the UniProt database.

[0194] Thus, the functional domains of the DsA1 polypeptide (as referred to herein) are defined as follows, based on Q6A5X9 numbering (see also, e.g., Figures 12A and 12B): NSR: S29 to 148; CSD1: I49 to L130; SR1: G131 to S147; CSD2: A148 to L267; SR2: A268 to T277; CSD3: A278 to K323; PT repeat region: P324 to T361; CTR: S362 to F405. The signal peptides (SPs) M1 through A28 are typically not included in the final polypeptide (vaccine) (other than an N-terminal methionine, which may be present). Thus, the SP is not part of the mature (functional) sequence, is not part of the antigen, and is not part of the production set-up for the vaccine formulations disclosed herein; however, when produced by some form of continuous fermentation (i.e., the cells are not harvested and broken open), secretion into the medium using a signal peptide may be used with an appropriate SP.

[0195] A "P. acnes DsA2 polypeptide" according to the present invention is a naturally occurring DsA2 protein of a P. acnes strain ("native DsA2") and includes all of the domains (N-terminus to C-terminus): NSR, CSD1, SR1, CSD2, SR2, CSD3, PT repeat region, and CTR, as defined above for DsA1. The sequences of naturally occurring DsA2 polypeptides contained in sequence databases (see, e.g., Figures 12A and 12C) are highly identical, differing only in the length and composition of the PT repeat region (besides point mutations). Some sequence entries do not contain NSR, CSD1, and CTR; these entries are likely (sequencing) artifacts or pseudogenes and are not DsA2 polypeptides within the meaning of the present invention, i.e., do not function as DsA2 proteins in P. acnes; nevertheless, they have been included in the alignments in the figures to illustrate possible sequence variations within the reported sequences in the databases.

[0196] The term "amino acid numbering corresponds to the amino acid sequence Q6A5X9 in the UniProt database" means that the numbering in the claims and embodiments generally refers to the numbering of the amino acid sequence of the DsA1 protein. This indicates that the corresponding sequence of DsA2, if applicable, is intended to correspond to the relevant position in the DsA1 protein. This means that the sequence numbering for DsA2 used in accordance with the present invention is based on the numbering of Q6A5P9 (unless explicitly stated otherwise), which means, for example, that the proline-glutamic acid dipeptides preceding the PT region amino acids are always referred to as "P365" and "E366," even if DsA2 polypeptides (or fragments or derivatives) of different lengths are involved (i.e., e.g., P365 / E366 are at different amino acid numbers within this particular polypeptide). Furthermore, DsA2 mRNA contains an N-terminal signal sequence (starting from the N-terminal methionine residue, followed by proline-leucine-proline-alanine-aspartate). The P. acnes DsA2 polypeptide according to the invention thus begins at alanine residue 72 (A72) and ends at alanine 463 (A463) of the amino acid sequence Q6A5P9 in the UniProt database.

[0197] Thus, the functional domain of the DsA2 polypeptide (referred to herein as Q6A5P9: MIIFVRVGNR HMRKRRAVLV VAFQLVNRRL AITKFPTPRR NDASNKNHCG SCDSNCLNRL TISYHPLPAN AASNGNSSIT QSAAFSPRAT TKISEDCRKA IINDLKLRGA IVGALVKAGL SAADAAALAP RIAAEMAAEG TLTINHHRLK VLVASQLGLV ADAAVQHAAA AIDLSFKAIL GASIIPNALG SAAFKNAVIA NLVAAGIDKH LARATAVAIV ATALNPALGP IAKFELIKAE IAAQAALLIR RGVHLQKAAI EHVIGRAFDA AVATAIISSP ILSARIVTHL VRAGIDKSIA ISLAPHIVKR LAKEPLLAFN TAKLVKDIAR QIVDIRNTQE AIAVYKQLKA ELPTLDGLVQ KACTPEPTPT PTPTPTPTPT PAPTPTPAPT PTPAPTPAPT PTPAPTPTPT PTPTPTPTPT HGATTTTPIS RTTDRHNLGS HHTRIAAPAL IHAKALPATG TGA Based on the numbering in and their sequences, they are defined as follows (also see, for example, Figures 12A and 12C): NSR: A72 to K92, CSD1, I93 to L174, SR1, S175 to S191, CSD2, A192 to L311, SR2, A312 to T321, CSD3, A322 to E366, PT repeat region, P367 to T420, CTR, H421 to A463. The signal peptides (SPs) M1 to A71 are typically not contained in the final polypeptide (vaccine) (other than an N-terminal methionine that may be present). Thus, the SP is not part of the mature (functional) sequence, is not part of the antigen, and is not part of the production set-up for the vaccine formulations disclosed herein; however, when produced by some form of continuous fermentation (i.e., the cells are not harvested and broken open), secretion into the medium using a signal peptide may be used with an appropriate SP.

[0198] In this context, it is important to recall that the DsA1 and DsA2 proteins are homologs (paralogs) with a typical sequence identity of 60-71%, depending on which region of the protein is aligned. However, excluding the PT region, length polymorphisms reveal a high degree of amino acid identity, typically >90%, within the intact DsA1 and DsA2 proteins, respectively. DsA1 and DsA2 are clearly very similar. Therefore, it is important to effectively distinguish these proteins. Thus, a sequence is preferentially classified as DsA1 if a local alignment of that sequence with the Q6A5X9 (characteristic DsA1 template) binding region CSD1-SR1-CSD2-SR2-CSD3 includes at least 70% of the length of the Q6A5X9 CSD1-SR1-CSD2-SR2-CSD3 region, where the amino acid identity is higher than in a local alignment matching the new sequence to Q6A5P9 and including at least 70% of the Q6A5X9 CSD1-SR1-CSD2-SR2-CSD3 region.

[0199] A sequence is preferentially classified as DsA2 if a local alignment of the sequence with the Q6A5P9 (characteristic DsA2 template) binding region CSD1-SR1-CSD2-SR2-CSD3 includes at least 70% of the length of the Q6A5P9 CSD1-SR1-CSD2-SR2-CSD3 region and the amino acid identity is higher than in a local alignment matching the new sequence to Q6A5P9 and including at least 70% of the Q6A5X9 CSD1-SR1-CSD2-SR2-CSD3 region.

[0200] What has been said about the invariance rather than apparent variability of DsA1 is largely true for DsA2 as well (in a manner that would be expected given that they are fairly close homologues). However, many more DsA2 variants than DsA1 variants are known, and this suggests that P. acnes strains commonly express DsA2 variants, sometimes as apparent pseudogenes. However, assuming that each type encodes one protein, it shows relatively high variability. As of November 6, 2019, 100 DsA1 variants can be found in the NCBI protein database, compared to 173 DsA2 variants (the database source for the sequences used in the present invention is usually the NCBI protein database (https: / / www.ncbi.nlm.nih.gov / protein)). In addition, in contrast to DsA1, some strains (mainly MLST type II) show a distinct PT repeat region that is strongly composed of polar, negatively charged (acidic) amino acids. However, even here, there are a few variants of this negatively charged stretch, which serve a functionally distinct role as a spacer (e.g., potentially compensating for the lack of PT glycosylation in affected strains) and are generally not hypervariable regions. In a sense, negative substitutions combined with a small number of variants of the sequence type in question argue against hypervariability (negative charge, which suspected bacterial glycosylation of PT repeats can usually provide) rather than for a clear functional role requiring a specific physicochemical setup.

[0201] For example, the N-terminus of Q6A5P9 contains specific amino acid exchanges compared to other P. acnes DsA2 polypeptides contained in sequence databases (see Figures 12A and 12C). These exchanges, which primarily involve the N-terminal signal sequence (besides the PT region), do not have any impact on the DsA2 polypeptides used as vaccines according to the present invention (because they are usually provided in their mature form, i.e., without a signal sequence; the major exception is when a nucleic acid vaccine is provided, which usually contains the coding sequence for a signal sequence (although the signal sequence may be compatible with the cell in which the nucleic acid vaccine is designed to be expressed)). Due to this difference in the N-terminus, several DsA2 sequences exist in the database in which the N-terminal alanine residue (A72) of the NSR has been shifted to position 31 (A31 in EGR92169.1) or 21 (A21 in EFS48378.1) (but still have the same sequence thereafter (up to the PT region)).

[0202] Major differences in the DsA2 sequence within the CTR are either due to sequencing bias (attributed to the PT coding region) resulting in (presumably artificial) truncations (with expected loss of function) or involve specific exchanges, e.g., the R444H exchange.

[0203] A "fragment of a DsA1 or DsA2 polypeptide in accordance with the present invention" is a shortened version of a naturally occurring version of a P. acnes DsA1 or DsA2 polypeptide (see the definition of "fragment" above). Preferably, a fragment in accordance with the present invention comprises or consists of at least a CSD2 fragment, which is (1) the contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184), (2) the contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), or (3) the contiguous polypeptide sequence from histidine 218 (H218) to leucine 267 (L267) (as well as the corresponding fragments of DsA2, i.e., F194-L228, F194-L311, and H262-L311, respectively). According to a preferred embodiment of the present invention, these three peptides may be defined as "minimal peptides" that define the minimum length of a polypeptide to be used in a vaccine according to the present invention while still providing immunogenic protection. The shorter the peptide, the less reliable it may be in eliciting an adequate immune response. On the other hand, the polypeptide should be as short as possible. Although shorter peptides are known to exhibit convenient properties in terms of production and handling, the protection provided by such shortened versions of natural immunogens such as DsA1 or DsA2 is unpredictable and unlikely.

[0204] Therefore, vaccines comprising the N-terminal fragments provided by the present invention, which lack the CTR containing the LPXTG motif, are protective for the treatment and prevention of P. acnes infections. It was surprising that the CSD2 fragments of the present invention contain at least the CSD2 fragment disclosed above and, if present, the shortened PT repeats defined above, i.e., 0, 1, 2, 3, or 5 PT repeats. Fragments may also contain additional domains, e.g., one or more of the NSR, CSD1, SR1, CSD2, SR2, and CTR; complete domains or portions thereof. Fragments of the present invention may also consist of multiple fragments, e.g., a polypeptide containing a CSD2 fragment, CDS1, SR2, CSD3, and a shortened PT repeat.

[0205] "Derivatives of DsA1 polypeptides of the present invention" include fragments of DsA1 or DsA2 polypeptides of the present invention and additional non-naturally occurring amino acid sequences (e.g., non-DsA1 or DsA2 sequences). Derivatives of DsA1 or DsA2 polypeptides of the present invention contain at least a DsA1 or DsA2 fragment derived from the wild-type sequence of P. acnes DsA1 or DsA2 protein, and additionally contain at least one amino acid or amino acid sequence that, in combination with the fragment derived from the native sequence, defines a non-naturally occurring derivative series. For example, if a fragment is derived from a native DsA1 protein, the "derivative" may contain additional non-DsA1 sequence, such as a sequence derived from a DsA2 protein. According to a preferred embodiment, the additional sequence in these derivatives of the present invention further contains an immunogenic region. Preferred derivatives of the present invention may contain, in addition to the CSD2 of the present invention, additional P. acnes sequences, particularly additional sequences encoding at least one (non-DsA1 or non-DsA2) antigen or epitope of P. acnes. For example, derivatives of the present invention derived from DsA1 (i.e., fragments of the native sequence by deletion) can contain at least one epitope of P. acnes DsA2. Preferably, derivatives of the present invention, if the fragment is derived from a wild-type DsA1 polypeptide, contain one or more of the NSR, CSD1, SR1, CSD2, SR2, CSD3, and CTR of the P. acnes DsA2 polypeptide; other preferred derivatives of the present invention, if the fragment is derived from a DsA2 polypeptide, contain one or more of the NSR, CSD1, SR1, CSD2, SR2, CSD3, and CTR of the P. acnes DsA1 polypeptide. Thus, these preferred derivatives contain DsA1 and DsA2 sequences. Derivatives containing at least one DsA1 epitope and at least one DsA2 epitope provide the most balanced vaccination.This is because such a combined immunogen can recognize both DsA1 and DsA2 equally well; thus, in strains of phylotype IC, it induces antibodies that can induce the most balanced reactivity to both DsA1 and DsA2 (where more DsA1 than DsA2 can be detected on the surface by flow cytometry, as opposed to the full-length proteins and other fragments, which react most highly to themselves but recognize the second antigen to a much lesser extent). Thus, the broadest and most balanced cross-reactivity profile for a large collection of strains covering a variety of phylotypes can be achieved by using such hybrid DsA1 / DsA2 polypeptides (e.g., hybrid H4 molecules). Therefore, such hybrid polypeptides, especially if they contain the functionally / immunologically most relevant regions restricted primarily to the central and N-terminal portions of the polypeptides (as in the example of H4, which contains these portions of DsA1 (29-145; 278-333) and the central portion of DsA2 (amino acids 190-321) and showed the ability to bind to both full-length proteins in ELISA assays (see the Examples section, especially Figure 8)), are not only immunologically more consistent and simpler molecules capable of exerting the effects of both full-length proteins, but also have significantly more promising stability and purity profiles compared to the parental versions and are preferred development candidates.

[0206] In a preferred embodiment of the invention, the vaccine fragment or derivative comprises at least - a consecutive polypeptide sequence from phenylalanine 150 (F150) to isoleucine 193 (I193), - a contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), or - a continuous polypeptide sequence from histidine 218 (H218) to leucine 267 (L267) It comprises or consists of:

[0207] Preferably, a fragment or derivative according to the invention does not further comprise CSD1 of DsA1 or DsA2, SR1 of DsA1 or DsA2, SR2 of DsA1 or DsA2, CSD3 of DsA1 or DsA2, or a PT repeat region of DsA1 or DsA2, provided that the fragment or derivative does not further comprise CSD1 of DsA1 or DsA2, SR1 of DsA1 or DsA2, SR2 of DsA1 or DsA2, CSD3 of DsA1 or DsA2, or a PT repeat region of DsA1 or DsA2.

[0208] Preferred fragments in a vaccine according to the invention (based on the numbering of DsA1, but also extending to the corresponding fragments of DsA2) are: (1) a continuous polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus); (2) a contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the C-terminus); (3) a contiguous polypeptide sequence from histidine 218 (H218) to leucine 267 (L267) (extended by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the N-terminus and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus); (4) a contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus); (5) a contiguous polypeptide sequence from alanine 148 (F148) to leucine 271 (L271) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the C-terminus); (6) a contiguous polypeptide sequence from histidine 146 (H146) to threonine 277 (T277) (extended by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the N-terminus and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus); (7) a contiguous polypeptide sequence from leucine 238 (L238) to leucine 272 (L272) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus); (8) a continuous polypeptide sequence from leucine 145 (L145) to alanine 201 (A201) (extended by 1, 2, 3, or 4 amino acids at the N-terminus and / or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the C-terminus); and / or (9) A continuous polypeptide sequence from leucine 145 (L145) to leucine 280 (L280) (at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids at the N-terminus and / or 1, 2, 3, 4 amino acids at the C-terminus) , 5, 6, 7, 8, 9, or 10 additional amino acids) is.

[0209] Preferably, a fragment according to the invention has a length of at least 35 amino acids, preferably at least 40 amino acids, especially at least 50 amino acids.

[0210] A preferred vaccine according to the invention comprises at least one additional polypeptide having an amino acid sequence containing or consisting of at least one additional P. acnes antigen or epitope that is not a DsA1 or DsA2 antigen or a DsA1 or DsA2 epitope, preferably at least a PITP polypeptide of P. acnes or at least one additional amino acid sequence containing at least one epitope of a PITP polypeptide of P. acnes, wherein the PITP polypeptide (see also below) comprises, from the N-terminus to the C-terminus, a signal peptide ("SP"; SP means It is not part of the mature (functional) sequence, is not part of the antigen, and is not part of the production setup for the vaccine formulations disclosed herein; however, when produced by some form of continuous fermentation (i.e., when the cells are not harvested and opened), secretion into the medium using a signal peptide (an appropriate SP may be used), an extended neocarzinostatin family domain ("ENFD"), a first swapping region ("SR1"), a heme-binding domain ("HbD"), a second swapping region ("SR2") comprising a C-terminal LPXT(G) domain, and a hydrophobic C-terminal region ("hLAR"). Preferably, the PITP derivative comprises at least one additional sequence from a PITP polypeptide, preferably a sequence comprising at least one of ENFD, SR1, SR2, and HbD, preferably selected from ENFD and HbD.

[0211] Because DsA1 / DsA2 and PITP contain spacer or swapping regions ("SRs"), the terms "spacer region" or "swapping region" as used herein always refer to the SRs of the proteins involved; SRs have various sequences but share the property of linkage ("spacing" two structural domains of a given protein and usually being less ordered (intrinsically unordered) compared to the structural domains of proteins such as CSD, ENFD, or HbD, which can be combined by "swapping" structural domains while appropriately manipulating the "spacer / swapping regions" (i.e., maintaining the same length (number of amino acids)). It is usually clear which SR is being referred to herein; however, in case of doubt, the term "SR" (unspecified) shall apply to all SRs, i.e., SR1 and SR2 of DsA1 and DsA1, and SR1 and SR2 of PITP.

[0212] Another preferred derivative within the vaccine according to the invention comprises at least one fragment of DsA1 and at least one fragment of DsA2.

[0213] Preferably, in a fragment or derivative of DsA1 / DsA2, at least five PT repeats, preferably at least 10 PT repeats, and especially at least 15 PT repeats are deleted compared to the naturally occurring wild-type DsA1 / DsA2 polypeptide ("native DsA1 / DsA2"), and preferably at least one, more preferably at least two, more preferably at least three, even more preferably at least four, and especially five PT repeats are present.

[0214] According to a preferred embodiment of the present invention, the derivatives in the vaccine further comprise at least NSR, CSD1, SR1, CSD2, SR2, CSD3, and / or CTR from at least one other DsA1 and / or DsA2 or from at least one P. acnes strain different from the strain from which the CSD2 fragment was derived.

[0215] Preferred DsA1 / DsA2 fragments or derivatives lack the NSR, CTR, and / or LPXTG motifs. DsA1 / DsA2 and their fragments and derivatives according to the present invention may be used in the present invention in soluble or membrane-bound form.

[0216] In a preferred embodiment, the vaccine according to the invention further comprises (in addition to the CSD2 fragment or CSD2-containing fragment or DsA1 polypeptide) another P. acnes antigen or epitope, preferably an antigen selected from DsA2 and PITP and / or DsA2 epitopes and / or PITP epitopes, in particular an epitope-containing fragment of another P. acnes polypeptide, preferably an epitope-containing fragment of DsA2 and PITP.

[0217] Because computer predictions are usually biased, epitope validation (so that the epitope is a valid epitope with a reliable likelihood of functioning in an immunological environment, particularly for vaccine use) relies on "real-world" wet biochemical data and the actual immunogenic counterparts functioning in the appropriate validation system. According to the present invention, epitopes were analyzed using appropriate anti-DsA1, -DsA2, and anti-PITP antibodies in linear, conformational, and MS epitope mapping, with further antigen fragment mapping by ELISA and dot blot. This also provided the epitopes disclosed in the Examples section.

[0218] Therefore, further preferred fragments are those that contain or consist of immunogenic epitopes, such as those identified by the present invention, namely R32-I41, Q38-K51, R32-K51, T43-K51, Q38-K51, R87-K90+T43-K51, R87-K90+T117-I132, and R87-K90+S234-G250, R87-K90+L246-A260, R87-K90+A256-E270, R87-K90+R266-T277, T117-I132, T117-A127, V128-I132, A144-N157, H146-A160, A148-N15 7, A156-A170, K166-L180, A176-T190, P186-A198, N181-E191, I216-F2 24, I216-D225, A226-A240, S234-G250, I251-I263, I264-P271, P236-G 250, L246-A260, A256-E270, S234-G250, I251-I263, I251-L267, A268- L280, R266-T277, T285-R286+I216-F224, T285-R286+I264-P271, T285- R286+V289-K296, T285-R286+V289-K296, T285-R286+A144-N157, A310 -D313+T285-R286, T285-D290, T285-D290+V291-T300, T285-D290+A30 1-E307, V291-T300, A301-E307, T285-T300, A301-E307, R286-D290+V2 91-T300, R286-D290+A301-E307, R286-T300, V289-K296, A310-D313+I2 16-F224, A310-D313+I264-P271, A310-D313+T285-R286, A310-D313+R 286-D290, A310-D313+V289-K296, A310-D313+V289-K296, A310-D313+T 285-T300, A310-D313+A144-N157, A310-D313+T285-R286, A310-D313+ T285-D290, A310-D313+T293-E307, T285-D290, V291-T300, T293-E307,A301-E307(DsA1);L152-Q166、G190-P230、I199-D208、A218-I237、P230-Q244、I231-A270、H254-A270、A271-S279、A271-R310、L311-T321、L311-K323、V333-Q347、A218-P230、I231-I237、H254-H、 262, Q256-H262, E261-D269, D269-S279, K313-K323(DsA2); D79-T90, E73-D85, R43-I50, P68-Y75, P86-E92, I39-G45, Y84-D89, F81-D89 , D79-T90, T37-E44, E73-W98, E73-F81, D89-T90, P72-F81, A129-F138, D120-Q134, F111-D120, F132-G147, D152-E165, R115-F123, D120- K128, P131-F138, N181-E191, T143-T159, P116-T124, P131-D137, P131-D137, T175-C231, Q198-K203, P179-K185, G200-Q210, K174-A188 , K174-K185, P201-Q209, P183-P201, P183-K191, K185-P195, R164-S180, E165-S180, K185-S190, V193-N202, V193-G200, K203-P208, R21 6-T225, R216-R224, P173-K191, K197-K203, P168-T175, K185-K203, R164-K174, T175-V193, S250-N261, D287-S300, K340-V347, D338-F3 52, D338-D348, S285-P288+G305-L314, S285-P288+H306-L314, S285-P288+T342-T351, S285-P288+D338-D348, D287-S300, T342-T351, D 338-D348, H306-L314, G305-L314, G364-K375, R382-E399, V367-G373, A383-L390, T342-T351, M387-T395, E385-T392, V401-V410, N404-A409, G416-L427, L396-V410, T406-I415, D417-G424, V407-D418, V407-V414, K421-V429, S419-T430, D408-I415, T406-V414(PITP).

[0219] Thus, according to a further aspect, the present invention also provides a polypeptide comprising an epitope of DsA1 and / or DsA2 and / or PITP, P.and R87-K90+S234-G250, R87-K90+L246-A260, R87-K90+A256-E270, R87-K90+R266-T277, T117-I132, T117-A127, V128-I132, A144-N157, H146-A160, H146-A177, H146-A180, H146-A190, H157-A260, H157-A277, H157-A280, H157-A290, H160-A290, H160-A290, H17 ... A148-N157, A156-A170, K166-L180, A176-T190, P186-A198, N181-E191, I216-F224, I216-D225, A226-A240, S234-G250, I251-I263, I264-P271, P 236-G250, L246-A260, A256-E270, S234-G250, I251-I263, I251-L267, A2 68-L280, R266-T277, T285-R286+I216-F224, T285-R286+I264-P271, T28 5-R286+V289-K296, T285-R286+V289-K296, T285-R286+A144-N157, A31 0-D313+T285-R286, T285-D290, T285-D290+V291-T300, T285-D290+A301 -E307, V291-T300, A301-E307, T285-T300, A301-E307, R286-D290+V291 -T300, R286-D290+A301-E307, R286-T300, V289-K296, A310-D313+I216- F224, A310-D313+I264-P271, A310-D313+T285-R286, A310-D313+R286- D290, A310-D313+V289-K296, A310-D313+V289-K296, A310-D313+T285-T 300, A310-D313+A144-N157, A310-D313+T285-R286, A310-D313+T285-D2 90, A310-D313+T293-E307, T285-D290, V291-T300, T293-E307, A301-E3. 07(DsA1);L152-Q166,G190-P230,I199-D208,A218-I237,P230-Q244,I231-A270,H254-A270,A271-S279,A271-R310,L311-T321,L311-K323,V333-Q347,A218-P230,I231-I237,H254-H262,Q256-H262,E261-D269,D269-S279,K313-K323(DsA2);D79-T90, E73-D85, R43-I50, P68-Y75, P86-E92, I39-G45, Y84-D89, F81-D8 9, D79-T90, T37-E44, E73-W98, E73-F81, D89-T90, P72-F81, A129-F138, D1 20-Q134, F111-D120, F132-G147, D152-E165, R115-F123, D120-K128, P131 -F138, N181-E191, T143-T159, P116-T124, P131-D137, P131-D137, T175-C2 31, Q198-K203, P179-K185, G200-Q210, K174-A188, K174-K185, P201-Q209 , P183-P201, P183-K191, K185-P195, R164-S180, E165-S180, K185-S190, V 193-N202, V193-G200, K203-P208, R216-T225, R216-R224, P173-K191, K19 7-K203, P168-T175, K185-K203, R164-K174, T175-V193, S250-N261, D287-S 300, K340-V347, D338-F352, D338-D348, S285-P288+G305-L314, S285-P28 8+H306-L314, S285-P288+T342-T351, S285-P288+D338-D348, D287-S300, T342-T351, D338-D348, H306-L314, G305-L314, G364-K375, R382-E399, V3 67-G373, A383-L390, T342-T351, M387-T395, E385-T392, V401-V410, N404- A409, G416-L427, L396-V410, T406-I415, D417-G424, V407-D418, V407-V414, K421-V429, S419-T430, D408-I415, T406-V414 (PITP), preferably the epitope comprises at least one additional amino acid residue at the N- or C-terminus of the DsA1, DsA2, or PITP sequence, especially the epitope comprises at least two additional amino acid residues at the N- or C-terminus of the DsA1, DsA2, or PITP sequence;The polypeptide is preferably covalently linked to a carrier molecule or embedded in a scaffold molecule, particularly a carrier polypeptide. Again, the preferred maximum length of the epitope-bearing polypeptide is as disclosed herein; for example, less than 400 amino acid residues, preferably less than 350 amino acid residues, particularly less than 300 amino acid residues (e.g., if more than one or two epitopes of different proteins (DsA1, DsA2, PITP)) are shuffled within a single polypeptide; or less than 250 amino acid residues, preferably less than 200 amino acid residues, particularly less than 150 amino acid residues (if only one or more epitopes of the same protein (DsA1, DsA2, PITP)) are present on the polypeptide.

[0220] Another aspect of the invention is the therapeutic treatment of, preferably, acne vulgaris caused by P. acnes, keratitis, synovitis acne pustulosis ossificans osteitis (SAPHO) syndrome, endocarditis, prosthetic joint infections, surgical wound infections, vascular graft infections, anaerobic arthritis, cardiovascular device-associated infections, e.g., prosthetic valve endocarditis; ophthalmic implant infections, breast implant disease, sciatica, conjunctivitis, shunt-related and / or spinal hardware, and central nervous system infections, among others. Infections, shunt-related central nervous system infections, sarcoidosis, endophthalmitis / osteomyelitis, allergic alveolitis, rheumatoid arthritis, infectious arthritis, chronic juvenile arthritis, chronic destructive oligoarthritis, degenerative disc disease, dental infections, ulcerative colitis / hyperthermia, brain abscess, subdural empyema, peritonitis, periodontitis, endodontic infections, endophthalmitis, keratitis, chronic sinusitis, folliculitis, keratitis, corneal ulcer, endophthalmitis, prostatic inflammation, chronic prostatitis, primary biliary cirrhosis, hidradenitis suppurativa, pulmonary vasculitis, and ulcerative colitis. The present invention relates to DsA1 and / or DsA2 and / or PITP, and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP according to the present invention, for use in the treatment or prevention of P. acnes-associated infections selected from the group consisting of: acne vulgaris, progressive patchy amelanosis, acne conglobata, atherosclerosis, prostate cancer, and medical implant biofilm infections. In particular, DsA1 and / or DsA2 and / or PITP according to the present invention, and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP, for use in the treatment or prevention of P. acnes-associated infections in human patients suffering from P. acnes-associated infections and pathological conditions associated with either type I, type II or type III P. acnes, or for use as cross-reactive vaccines, especially cross-type reactive vaccines, against a combination of at least two phylotypes of types I, II and III, or at least two ribotypes of P. acnes, preferably P. acnes, are provided, especially for the treatment or prevention of infections in human patients suffering from P. acnes-associated infections and pathological conditions associated with types IB and III of P. acnes.

[0221] According to a preferred embodiment, the vaccine according to the present invention further comprises a P. acnes PITP polypeptide and specific fragments and variants thereof.

[0222] A "P. acnes PITP polypeptide" according to the present invention is a naturally occurring PITP protein ("native PITP") from a P. acnes strain, which PITP polypeptide comprises, from N-terminus to C-terminus, an extended neocarzinostatin family domain ("ENFD"), a first swapping region ("SR1"), a heme-binding domain ("HbD"), a second swapping region ("SR2") comprising a C-terminal LPXT(G) domain, and a hydrophobic C-terminal region ("hLAR").

[0223] Thus, the functional domains of the PITP polypeptide (referred to herein as Q6A9N1: MTTSAMRKIV ASVLGAILAL TGVLITPAAW AAGPTVTVIP VGREGGDITI SGKGFSTTGF GVYVAVAPAS VPEFYGNSDK FYGYDPSKDT TESPSTIWVY TPSQKAIGSR FAQGRPMNND GSFTITMKAP PFEQGKDFVV LTTKAHGVGK TDHSDDTRTP VTYREATPAP TGPKTPIAPS KQPSKQAAPS KQVKPSKQAG PNKQSTTPQQ KTAEHRSQTP AAHRTMTKQV CTIGASKVTS GSLTWGIRTS FTSYLRGPIA NGSWKLSGGA NWNGSAFTFP LTSGSFDPAT KSGSLKYSGS VHMTGHHGIL DMTLAEPSLQ IKGSTGHLYL DVKSSSMDGK KTNYGRVDFA TFGVSVSGNA AIKGSPVKLT ATGAKAFAGF YRAGEPMNPL STNLTLSAEK VCHNVTVDAV TGKVIGDDSG KGAGRGLPVT GAEGPSSDEI DLGIVGGLAL TAVVSTVVVC RRYAARI Based on the numbering in and their sequences, they are defined as follows (also see, for example, Figure 12D): ENFD, A32 to R164; SR1, E165 to K237; HbD, V238 to L396; SR2 (containing the C-terminal LPXT(G) motif (i.e., not including G, but including LPXT)), S397 to T430; and hLAR, G431 to I467. The signal peptides (SPs) of M1 to A31 are typically not contained within the final polypeptide (in the vaccine) (other than an N-terminal methionine that may be present).

[0224] Accordingly, an embodiment of the present invention relates to vaccines comprising a PITP polypeptide and / or a fragment and / or derivative of PITP, wherein the fragment and / or derivative comprises or consists of at least a PITP epitope.

[0225] Vaccines of the present invention preferably contain a PITP polypeptide or a fragment or derivative thereof. A PITP "fragment" is a portion of the naturally occurring PITP protein; a PITP "derivative" is a PITP fragment that contains at least an antigenic epitope (i.e., an epitope that is immunogenic and accessible to antibody binding on the surface of P. acnes). The present invention also provides a non-naturally occurring polypeptide comprising at least 20 amino acids, preferably at least 30 amino acids, and particularly at least 50 amino acids in length of a naturally occurring PITP protein. Particularly preferred central domains useful as vaccines are the ENFD and HbD domains. Accordingly, particularly preferred fragments and derivatives include sequences derived from the ENFD and / or HbD domains, e.g., fragments of at least 10, preferably at least 20, more preferably at least 30, and particularly at least 50 amino acids, of the naturally occurring PITP ENFD and / or HbD domains. A preferred ENFD fragment comprises the peptide from A32 to T143; a preferred HbD fragment comprises the peptide from V238 to N393.

[0226] For the avoidance of doubt, it is also clear that all references herein to sequences, fragments, etc., in connection with PITP fragments and derivatives always refer to contiguous amino acids (unless expressly stated otherwise). For example, a PITP fragment of at least eight amino acids always refers to at least eight contiguous amino acid residues of a PITP polypeptide. The term "contiguous" means that a given amino acid is at a given position within the alignment of Figure 12. Of course, exchanges between different PITP homologs are included, provided that positions are maintained (i.e., there are no deletions, unless expressly foreseen in Figure 12).

[0227] In addition to the SR, adjacent portions of the ENFD and HbD domains may also be used as spacers or linkers between fragments, for example, within hybrid molecules. For example, the region from K144 to T227 (or the entire SR1 from K144 to K237; or G147 to K228; or any fragment thereof at least 10 amino acids long) can be used as an intermediate / swapping / spacer region (unordered linker bond, e.g., ENFD and HbD). hLAR is defined to start after the LPXT(G) motif according to the present invention, and the terminal G of the LPXTG motif is already included within hLAR, because it makes the hydrophobic portion of the protein removed upon anchoring to the cell wall. Therefore, for practical reasons, hLAR is defined for the present invention as starting from the G of the LPXTG motif.

[0228] The UniProt amino acid sequence Q6A9N1 refers to the protein "PPA0779" of P. acnes strain DSM 16379 / KPA171202. In this wild-type PITP polypeptide, the LPXT(G) motif is defined by amino acids 427-431 (L427 to G431; however, G431 already forms part of the hLAR), which therefore begins with a glycine at position 431 (G431) and ends with a C-terminal isoleucine at position 467 (I467). hLAR has a fairly hydrophilic region (at the N-terminus) comprising four acidic amino acids (E433, D438, E439, and D441), which ends with an aspartic acid residue at position 441 (D441), followed by a hydrophobic region starting with a leucine at position 442 (L442) and extending to the C-terminal isoleucine. According to a preferred embodiment of the invention, hLAR may be completely or partially deleted. Partial deletions preferably concern the entire hydrophobic region (i.e., L442 to I467). This allows for preferred immunogenic fragments of the protein with improved properties, particularly with regard to handling and manufacturing.

[0229] A preferred PITP fragment according to the present invention is a PITP in which the C-terminal region (hLAR) has been shortened or completely deleted, while including all of the other domains (N-terminus to C-terminus): ENFD, SR1, HbD, and SR2, which contain the LPXT(G) motif (i.e., LPXT without G). The hLAR, which is located at the C-terminus, is shortened or completely absent in a preferred PITP polypeptide fragment according to the present invention (compared to the native (i.e., naturally occurring) PITP protein from P. acnes). The sequence of a naturally occurring PITP polypeptide contained in a sequence database (see, e.g., Figure 12D) can be used. The Q6A9N1 sequences (references) are highly identical and differ only at the N-terminus or C-terminus (besides point mutations). However, this difference(s) in some database entries at the C-terminus may be a sequencing artifact or may not represent a native PITP polypeptide within the meaning of the present invention, i.e., they do not function as a PITP protein in P. acnes. The sequence numbering used in accordance with the present invention is based on the Q6A9N1 numbering, which means, for example, that the LPXTG motif preceding hLAR is always referred to as extending from L427 to G431, even if a PITP polypeptide (or fragment or derivative) of different length (i.e., for example, L427 to G431 is at a different amino acid number within this particular polypeptide) is involved. Furthermore, the PITP mRNA encodes an N-terminal signal sequence (starting with an N-terminal methionine residue and ending with a proline-alanine-alanine-tryptophan-alanine sequence), which is not part of the mature polypeptide. Thus, the P. acnes PITP polypeptide according to the invention starts at alanine residue 32 (A32) and ends at isoleucine 467 (I467) of the amino acid sequence Q6A9N1 in the UniProt database.

[0230] For example, the N-terminus of Q6A9N1 contains specific amino acid exchanges compared to other P. acnes PITP polypeptides contained in sequence databases (see Figure 12D). These exchanges primarily involve the N-terminal signal sequence and therefore do not have any impact on the PITP polypeptide used as a vaccine according to the present invention (because it is usually provided in its mature form, i.e., without a signal sequence; the major exception is when a nucleic acid vaccine is provided, which usually contains the coding sequence for a signal sequence (however, the signal sequence may be compatible with the cell in which the nucleic acid vaccine is designed to be expressed)). The difference in hLAR may be due to sequencing bias.

[0231] Preferably, the PITP fragment and / or derivative is a PITP polypeptide in which hLAR is deleted, replaced by a hydrophilic C-terminal region, or partially deleted, the partial deletion resulting in the loss of hLAR other than the N-terminal 12 amino acids of hLAR, preferably other than the N-terminal 11 amino acids of hLAR, and particularly other than the N-terminal 10 amino acids of hLAR; or a fragment or derivative thereof containing at least the amino acids corresponding to Proline 34 to Glutamic Acid 73, or Proline 94 to Threonine 143 of ENFD, or Valine 238 to Asparagine 393 of HbD, within the amino acid sequence Q6A9N1 in the UniProt database. The fact that such deletion fragments and derivatives of PITP exhibit enhanced physicochemical properties, which are particularly suitable for vaccination purposes and recombinant vaccine production, was further unexpected because the LPXTG motif is not automatically detected, meaning that it is a motif not described or annotated in public databases for this sequence. In the prior art, it has not been experimentally demonstrated that PITP is actually anchored to the cell wall and can serve as a suitable substrate for transpeptidases. In the course of this invention, we demonstrated that the presence of LPXTG in the sequence does not affect the surface accessibility of PITP on different strains, as PITP could be consistently detected regardless of whether the LPXTG motif was present. Finally, the corresponding sortase enzyme is not clearly relevant in P. acnes. Another surprising property of PITP fragments or derivatives containing deletions in hLAR or lacking hLAR is their enhanced expression in many recombinant expression systems, particularly in high-yield systems. In many such systems, the expression product is present in the soluble fraction rather than in inclusion bodies. As a further advantage, PITP fragments or derivatives containing deletions in hLAR or lacking hLAR exhibit improved binding to typical purification columns used for polypeptide purification, such as SP Sepharose or Superdex 200 columns. Finally, the resolution of the target polypeptide from (further) truncated forms or of other expression artifacts is also improved.The effect is even greater with the substitution of cysteine ​​residues within the polypeptide, particularly C231 and C4. The O2 substitution may be even more pronounced.

[0232] The PITP according to the invention, and fragments and derivatives thereof, can be used in the present invention in soluble form or in membrane-bound form.

[0233] Preferably, a DsA1 / DsA2 derivative according to the invention additionally comprises a PITP fragment and / or the derivative is a PITP polypeptide as defined herein, in particular wherein hLAR has been deleted, replaced by a hydrophilic C-terminal region or partially deleted, the partial deletion resulting in the loss of hLAR other than the N-terminal 12 amino acids of hLAR, preferably other than the N-terminal 11 amino acids of hLAR, in particular other than the N-terminal 10 amino acids of hLAR; or a fragment or derivative thereof comprising at least the amino acids corresponding to Proline 34 to Glutamic Acid 73, or Proline 94 to Threonine 143 of ENFD, or Valine 238 to Asparagine 393 of HbD, within the amino acid sequence Q6A9N1 in the UniProt database.

[0234] Preferably, the PITP fragment or derivative comprises at least - the consecutive polypeptide sequence from proline 34 to glutamic acid 73 of ENFD, - a contiguous polypeptide sequence from proline 94 to threonine 143 of ENFD, preferably from proline 94 to glycine 147, or - The continuous polypeptide sequence from valine 238 to asparagine 393 of HbD Comprises or consists of.

[0235] According to a preferred embodiment, the PITP fragment or derivative contains an ENFD and / or HbD fragment of the PITP polypeptide at least 8 amino acid residues, preferably at least 10 amino acid residues, and especially at least 15 amino acid residues in length. Alternative preferred embodiments include longer PITP fragments or derivatives at least 35 amino acids in length, preferably at least 40 amino acids in length, and especially at least 50 amino acids in length.

[0236] Preferably, the PITP fragment or derivative comprises or consists of at least an epitope of ENFD and / or an epitope of HbD, and preferably, the fragment or derivative comprises or consists of at least an epitope of ENFD and an epitope of HbD. Thus, preferred embodiments of the PITP polypeptide, PITP fragment, or PITP derivative comprise a porphyrin-binding domain.

[0237] Preferably, the PITP derivative further comprises at least ENFD and / or HbD from at least one other PITP fragment or fragment from at least one P. acnes strain different from the strain from which the derivative is derived.

[0238] According to a preferred embodiment, the fragment or derivative of the PITP polypeptide lacks at least one of the SRs corresponding to serine 180 (S180) to glutamine 198 (Q198) within the amino acid sequence Q6A9N1 in the UniProt database, preferably at least the amino acid sequence corresponding to proline 179 (P179) to threonine 207 (T207), in particular the amino acid sequence corresponding to glutamic acid 165 (E165) to lysine 237 (K237) (for SR1), or valine 401 (V401) to threonine 430 (T430), preferably serine 397 (S397) to T430, in particular leucine 394 to T430 (for SR2).

[0239] Preferably, the fragment or derivative of the PITP polypeptide lacks at least the amino acid sequence corresponding to leucine 427 (L427) to glycine 431 (G431) according to the numbering in the amino acid sequence Q6A9N1 in the UniProt database. Preferably, at least the amino acid sequence corresponding to proline 179 (P179) to glycine 431 (G431) is deleted, and particularly, at least the amino acid sequence corresponding to threonine 392 (T392) to glycine 431 (G431) is deleted.

[0240] A preferred fragment or derivative of the PITP polypeptide is a polypeptide lacking at least the amino acid sequence corresponding to serine 180 (S180) to glutamine 198 (Q198) and / or phenylalanine 74 (F74) to serine 93 (S93) within the amino acid sequence Q6A9N1 in the UniProt database, preferably at least the amino acid sequence corresponding to proline 179 (P179) to threonine 207 (T207), particularly the amino acid sequence corresponding to threonine 159 (T159) to threonine 219 (T219).

[0241] Preferred fragments or derivatives of the PITP polypeptide according to the present invention consist of the following amino acids: A32 to T430, A32 to G426, A32 to Q198, A32 to T143, A32 to K400, A32 to T159, A32 to I177, A32 to Q204, A32 to G234, A32 to R164, A32 to S391, A32 to P179, A32 to R158, A32 ~G147, A32~E73, and P94~G147; P34~T430, P34~G426, P34~Q198, P34~T143, P34~K400, P34~T159, P34~I177, P34~Q204, P34~G234, P34~R164, P34~S391, P34~P179, P34~R158, P34~G147, P34~E73, and P94~G147;S240~S391, A32~D441, A32~I440, A32~E439, A32~D438, A32~S437, A32~S436, A32~P435 , A32~G434, A32~E433, A32~A432, A32~G431; P34~D441, P34~I440, P34~E439, P34~D438, P34~S437, P34~S436, P34~P435, P34~G434, P34~E433, P34~A432, P34~G431; S240~D441, S240~I440, S240~E43 9, S240~D438, S240~S437, S240~S436, S240~P435, S240~G434, S240~E433, S240~A432, S240~G431;A32~T430, A32~V429, A32~P428, A32~L427, A32~G426, A32~R425, A32~G424, A32~A423, A32~G422, A32~K421, A32~G420, A32~ S419, A32~D418, A32~D417, A32~G416, A32~I415, A32~V414, A32~K413, A32~G412, A32~T411, A32~V410, A32~A409, A32~D408 A32~V407, A32~T406, A32~V405, A32~N404, A32~H403, A32~C402, A32~V401, A32~K400, A32~E399, A32~A398, A32~S397, A32 ~L396, A32~T395, A32~L394, A32~N393, A32~T392;P34~T430, P34~V429, P34~P428, P34~L427, P34~G426, P34~R425, P34~G424 、P34~A423、P34~G422、P34~K421、P34~G420、P34~S419、P34~D 418、P34~D417、P34~G416、P34~I415、P34~V414、P34~K413、P34 ~G412、P34~T411、P34~V410、P34~A409、P34~D408、P34~V407、P 34~T406、P34~V405、P34~N404、P34~H403、P34~C402、P34~V401 、P34~K400、P34~E399、P34~A398、P34~S397、P34~L396、P34~T 395、P34~L394、P34~N393、P34~T392;G172~T430、G172~V401、G 172~K400, G172~L396, G172~N393, A199~T430, A199~V401, A199~K400, A199~L396, A199~N393, H223~T430, H223~V401, H223; ~K400, H223~L396, H223~N393, T232~T430, T232~V401, T232~K400, T232~L396, T232 ~N393, G234~T430, G234~V401, G234~K400, G234~L396, G234~N393, V238~T430, V238~ V401, V238~K400, V238~L396, V238~N393, S240~T430, S240~V429, S240~P428, S240~L 427, S240~G426, S240~R425, S240~G424, S240~A423, S240~G422, S240~K421, S240~G4 20, S240~S419, S240~D418, S240~D417, S240~G416, S240~I415, S240~V414, S240~K4 13, S240~G412, S240~T411, S240~V410, S240~A409, S240~D408, S240~V407, S240~T40 6, S240~V405, S240~N404, S240~H403, S240~C402, S240~V401, S240~K400, S240~E399 , S240~A398, S240~S397, S240~L396, S240~T395, S240~L394, S240~N393, S240~T392.

[0242] According to a preferred embodiment, the present invention relates to PITP fragments of at least 8 amino acids, and derivatives containing such fragments. Preferably, fragments or derivatives containing such fragments are at least 9 amino acids long, especially at least 10 amino acids long. The following list includes preferred 10-mer fragments (alternative amino acid annotations are used below; however, it is clear that, for example, the term "39I" is "I39" and "isoleucine 39" as used elsewhere in this specification): 39I-48I、40P-49T、41V-50I、42G-51S、43R-52G、44E-53K、68P-77N、69A-78S、70S-79D、71V-80K、72P-81F、73E-82Y、74F-83G、75Y-84Y、76G-85D、77N-86P、78S-87S、79D-88K、80K-89D、81F-90T、82Y-91T、83G-92E、84Y-93S、85D-94P、86P-95S、87S-96T、88K-97I、89D-98W、90T-99V、91T-100Y、92E-101T、93S-102P、94P-103S、95S-104Q、96T-105K、97I-106A、98W-107I、99V-108G、100Y-109S、101T-110R、102P-111F、103S-112A、104Q-113Q、105K-114G、106A-115R、107I-116P、108G-117M、109S-118N、110R-119N、111F-120D、112A-121G、125I-134Q、126T-135G、127M-136K、128K-137D、144K-153H、145A-154S、146H-155D、147G-156D、148V-157T、149G-158R、150K-159T、151T-160P、152D-161V、153H-162T、154S-163Y、155D-164R、156D-165E、157T-166A、158R-167T、159T-168P、160P-169A、161V-170P、162T-171T、163Y-172G、164R-173P、165E-174K、166A-175T、167T-176P、168P-177I、169A-178A、170P-179P、171T-180S、172G-181K、173P-182Q、174K-183P、175T-184S、176P-185K、177I-186Q、178A-187A、179P-188A、180S-189P、181K-190S、182Q-191K、183P-192Q、184S-193V、185K-194K、186Q-195P、187A-196S、188A-197K、189P-198Q、190S-199A、191K-200G、192Q-201P、193V-202N、194K-203K、195P-204Q, 196S-205S, 197K-206T, 198Q-207T, 199A-208P, 200G-209Q, 201P -210Q, 202N-211K, 203K-212T, 204Q-213A, 205S-214E, 206T-215H, 207T-21, 6R, 208P-217S, 209Q-218Q, 210Q-219T, 211K-220P, 212T-221A, 213A-222A, 214E-223H, 215H-224R, 216R-225T, 217S-226M, 21 8Q-227T, 219T-228K, 220P-229Q, 221A-230V, 222A-231C, 223H-232T, 224R-233I, 225T-234G, 226M-235A, 227T-236S, 228K-237 K, 229Q-238V, 230V-239T, 231C-240S, 232T-241G, 233I-242S, 266L-275S, 267S-276A, 268G-277F, 282T-291K, 334S-343N, 353G -362I, 354V-363K, 355S-364G, 356V-365S, 357S-366P, 358G-367V, 359N-368K, 377F-386P, 378A-387M, 379G-388N, 380F-389P, 396L-405V, 397S-406T, 398A-407V, 399E-408D, 400K-409A, 401V-410V, 402C-411T, 403H-412G, 404N-413K, 405V-414V, 406T- 415I, 407V-416G, 408D-417D, 409A-418D, 410V-419S, 411T-420G, 412G-421K, 413K-422G, 414V-423A, 415I-424G, 416G-425R, 4 17D-426G, 418D-427L, 419S-428P, 420G-429V, 421K-430T, 422G-431G, 423A-432A, 424G-433E, 425R-434G, 426G-435P, 427L-436S, 428P-437S, 429V-438D, 430T-439E, 431G-440I, 432A-441D, 433E-442L, 434G-443G, 435P-444I, 436S-445V, and 437S-446G.

[0243] Preferred fragments and derivatives of PITP include at least the following PITP fragments: I39 to K53, P68 to G121, I125 to D137, K144 to S242, L266 to F277, T282 to K291, S334 to N343, G353 to K368, F377 to P389, and L396 to G446.

[0244] Particularly preferred fragments and derivatives of PITP comprise or consist of at least the following PITP fragments: A32 to R164, A32 to Q198, A32 to T143, A32 to V148, A32 to T171, P34 to R164, A32 to T159, A32 to I177, A32 to Q204, A32 to G234, A32 to K400, A32 to S391, V238 to K400, A199 to T430, V238-T395, G234-K400, H223-K400, T232-V401, V238-T392, V238-N393, V238-L394, V238-T395, V238-L396, T232-T430, G172-K400, and G172-G234, and in particular fragments A32-T430, A32-I467, A32-S391 (with deletion of K174-T239), and A32-S391. These fragments contain at least one epitope effective to elicit an appropriate cross-reactive immune response in humans when used as a vaccine. Furthermore, these fragments can be efficiently produced by recombinant expression systems and refined into final pharmaceutical vaccine formulations.

[0245] According to particularly preferred embodiments, fragments and derivatives of PITP comprise the complete or nearly complete HbD domain, i.e., fragments comprising at least amino acids V238 to T392, or at least amino acids V238 to N393, or at least amino acids V238 to L394, or more preferably at least amino acids V238 to T395, or at least amino acids V238 to L396. Such fragments are stable and suitable for expression in upscaled formats. HbD fragments lacking T392 to T395, N393 to T395, L394 to T395, or T395 at the C-terminus of the domain may be less stable with respect to expression and epitope presentation. .

[0246] According to particularly preferred embodiments, fragments and derivatives of PITP include fragments containing the complete HbD domain, i.e., at least amino acids V238 to T392 and up to L396. Such fragments are stable and suitable for expression in upscaled formats. HbD fragments lacking T392, T392 to N393, T392 to L394, or T392 to L395 may be less preferred for certain purposes, such as large-scale production.

[0247] According to a preferred embodiment of the present invention, a modified PITP polypeptide is provided that has advantageous properties compared to the wild-type PITP protein derived from P. acnes, and is particularly suitable for vaccination purposes. The novel use of the PITP protein according to the present invention, which is used to interfere with P. acnes (i.e., to prevent and / or treat pathological conditions caused by it), is based on its advantageous properties (revealed by the present invention) with respect to its immunogenic properties and its handling properties (which allow for easier large-scale recombinant expression and production). Both advantages emerged in the course of the production of the present invention and are surprising in light of the knowledge of the art.

[0248] DsA1 and DsA2 can be considered fairly invariant if sequencing issues and pseudogenes are ignored, and PT length polymorphisms are examined based on the functional role of the region rather than specific amino acid-amino acid comparisons. Nevertheless, PITP (a putative iron transporter) is even more conserved. Length variants are rare, likely due to uncertainty in precisely placing the N-terminal gene start in most cases. The sequence is highly invariant, especially when ignoring the few apparently fragmented or shifted proteins. Essentially, only a few dozen positions show variability across the entire protein between known variants. One exception is GAE78839.1 (possible fusion with a downstream gene). One of the few regions showing enrichment in chemically distinct amino acid exchanges is the putative linker region connecting two predicted heme-binding domains, particularly in the region defined by the peptide TTPQQKTAEH.

[0249] According to a particularly preferred embodiment, the present invention relates to truncated fragments and variants of the P. acnes PITP polypeptide, in which the hLAR is deleted, replaced by a hydrophilic C-terminal region or partially deleted, the partial deletion resulting in the loss of more than the N-terminal 12 amino acids of hLAR, preferably more than the N-terminal 11 amino acids of hLAR, in particular more than the N-terminal 10 amino acids of hLAR; or a fragment or derivative thereof comprising at least ENFD or HbD.

[0250] Preferred derivatives of the present invention are polypeptides comprising sequence stretches of various antigens of P. acnes. Accordingly, the present invention refers to specific embodiments of polypeptides comprising at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 1 (DsA1) and at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 2 (DsA2), wherein said DsA1 and DsA2 comprise, from N- to C-terminus, an N-terminal region, a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"); and, optionally, a Pro-Leu repeat-containing region ("PT repeat region"), and a C-terminal region; The polypeptide comprises at least CSD1, CSD2, or CSD3 of DsA1 and at least CSD1, CSD2, or CSD3 of DsA2.

[0251] According to another aspect, the present invention refers to a polypeptide comprising at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 1 (DsA1) and at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 2 (DsA2), wherein said DsA1 and DsA2 comprise, from N-terminus to C-terminus, an N-terminal region, a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"); and, optionally, a Pro-Leu repeat-containing region ("PT repeat region"), and a C-terminal region; The polypeptide stretches of DsA1 and DsA2 independently have a length of at least 20 amino acid residues.

[0252] Another preferred embodiment of the present invention is - at least one antigenic polypeptide having a surface-exposed epitope of the DsA1 polypeptide or a surface-exposed epitope of the dermatan sulfate-binding adhesin 2 (DsA2 polypeptide) of P. acnes; (DsA1 or DsA2 comprises, from N-terminus to C-terminus, an N-terminal swapping region ("NSR"), a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"), and optionally, a Pro-Thr repeat-containing region ("PT repeat region"), and a C-terminal region ("CTR"); and - at least one antigenic polypeptide having a surface-exposed epitope of a PITP polypeptide; It is a vaccine containing

[0253] Another preferred embodiment of the present invention is (a) comprising at least an epitope-containing fragment or derivative of CSD1 of DsA1 and at least an epitope-containing fragment or derivative of CSD2 of DsA2; and / or (b) comprising at least a polypeptide stretch comprising a CSD1 fragment or derivative of CSD1 of DsA1 of at least 30 amino acid residues, preferably at least 40 amino acid residues, and at least a polypeptide stretch comprising a CSD2 fragment or derivative of CSD2 of DsA1 of at least 30 amino acid residues, preferably at least 40 amino acid residues; (c) At least DsA1 - a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184), - a contiguous polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), or - A consecutive polypeptide sequence from histidine 218 (H218) to leucine 267 (L267), and - a consecutive polypeptide sequence from phenylalanine 194 (F194) to leucine 228 (L228), - a consecutive polypeptide sequence from phenylalanine 194 (F194) to leucine 311 (L311), or - A consecutive polypeptide sequence from histidine 262 (H262) to leucine 311 (L311) and a vaccine comprising a polypeptide comprising:

[0254] Also in this embodiment, the polypeptide may contain, if present, the amino acids C53, C319, and C321 of DsA1 and C97 and C363 of DsA2 within the polypeptide. It is preferred to include one or more amino acid exchanges, preferably one or more of the amino acid exchanges C53S, C319S, and C321P in DsA1, and C97S and C363S in DsA2.

[0255] Also in this embodiment, at least five PT repeats, preferably at least 10 PT repeats, and especially at least 15 PT repeats of the fragment or derivative are deleted compared to the naturally occurring wild-type DsA1 / DsA2 polypeptide ("native DsA1 / DsA2"), preferably at least one, more preferably at least two, more preferably at least three, even more preferably at least four, and especially five PT repeats are present.

[0256] The DsA1 / DsA2 shuffle polypeptide preferably comprises a PITP stretch, particularly a PITP stretch having a PITP epitope. Thus, the polypeptide preferably further comprises a PITP polypeptide, or a fragment or derivative of PITP, comprising a polypeptide stretch of at least 30 amino acid residues containing at least an epitope of PITP. Preferably, the PITP polypeptide, or a fragment or derivative of PITP, comprises at least - a contiguous polypeptide sequence from proline 34 to glutamic acid 73 of ENFD or its PITP derivative, - the contiguous polypeptide sequence from proline 94 to threonine 143 of ENFD or its PITP derivative, or - a continuous polypeptide sequence from valine 238 to asparagine 393 of HbD or its PITP derivative Includes.

[0257] Again, here the PITP derivative preferably contains amino acid exchanges at positions C231 and C402.

[0258] In a similar aspect, the present invention also provides a method for producing a medicament for the treatment of a pulmonary arthritis. - at least one synthetic antigenic polypeptide having a surface-exposed epitope of a DsA1 polypeptide or a surface-exposed epitope of a DsA2 polypeptide; and - at least one synthetic antigenic polypeptide having a surface-exposed epitope of a P. acnes PITP polypeptide; The present invention relates to a vaccine comprising:

[0259] The antigens described herein, or epitopes thereof, may not be inherently immunogenic and may therefore be linked to each other and / or to an adjuvant to generate an immunogen. The link between the antigen and / or its epitope and / or adjuvant may be covalent or non-covalent, for example, by adsorptive, electrostatic, hydrophobic, or van der Waals interactions.

[0260] In some embodiments, the immunogen is a composite immunogen, engineered by linking one or more antigens of P. acnes and / or one or more epitopes of P. acnes to one another. In some embodiments, the immunogen comprises or consists of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens and / or epitopes thereof. The at least two antigens and / or epitopes thereof can be the same or different antigens and / or epitopes (e.g., two epitopes on two different antigens, two different epitopes on one antigen, two copies of the same epitope on one antigen, one antigen and one epitope on different antigens). In some embodiments, the immunogen comprises at least one antigen of P. acnes linked to an adjuvant. It comprises or consists of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigens and / or epitopes thereof, at least one of the epitopes inducing cross-reactive antibodies, especially cross-type reactive antibodies.

[0261] In some embodiments, the immunogen comprises at least two antigens and / or epitopes thereof covalently linked to one another, hi some embodiments, the immunogen comprising at least two antigens and / or epitopes thereof is further linked to an adjuvant.

[0262] In some embodiments, the immunogen comprises one antigen or epitope thereof linked to an adjuvant.

[0263] Immunogens are characterized in particular by the presence of at least one epitope that induces cross-binding and / or cross-reactive antibodies, especially cross-type reactive antibodies.

[0264] As used herein, the term "cross-binding" refers to antibodies raised by immunization with a vaccine that specifically binds to multiple antigens. A cross-binding antibody is one that, when raised with a single antigen (e.g., with a hybrid molecule such as H4), can specifically bind to two or more antigens (e.g., DsA1 and DsA2).

[0265] Cross-linking of antibodies induced after immunization with an antigen or epitope can be determined as follows. Sera from rabbits or mice immunized multiple times with a specific P. acnes antigen, alone or in combination with an adjuvant, can be evaluated for the amount of antibody binding to the antigen used for immunization, compared with other unrelated P. acnes proteins. Given the equal quality and purity of the recombinant protein antigen (immunogen), immunization with a specific P. acnes antigen leads to a substantial increase in the amount of antigen-specific antibody, as determined by EC50 titers by ELISA. Detection of an ELISA EC50 titer of animal serum raised against an unrelated P. acnes antigen not used to immunize the animal is a clear indication of cross-linking of antigen-specific polyclonal serum. In such an assay, serial dilutions of hyperimmune rabbit serum are tested by ELISA, and the EC50 titer of antibody in the serum or other body fluid is the concentration (dilution) at which half-maximal antigen-binding effect is observed (typically read as optical density (OD)). Pre-immune serum from each rabbit before immunization is used as a control for nonspecific serum effects.

[0266] The term "cross-reactive" (or "cross-reactivity") as used herein refers to antibodies raised by immunization with a vaccine that specifically binds to multiple P. acnes strains. The term "cross-reactive" (or "cross-reactive") as used herein refers to antibodies raised by immunization with a vaccine that specifically binds to multiple P. acnes phylotypes. In particular, cross-reactivity, particularly cross-reactivity, covers the specific binding of viable bacterial cells from at least two of the phylotypes or ribotypes of P. acnes strains, particularly at least two of the IA1, IA2, IB, IC, II, and III P. acnes types, as determined, for example, by reactivity with at least one strain of each type of P. acnes that expresses at least one of the antigens. For example, cross-reactivity covers the specific reactivity of an antibody that targets only one antigen of P. acnes but cross-reacts with the same or similar antigens expressed by different strains. For example, a vaccine containing DsA1, DsA2, or a fragment or derivative disclosed herein will elicit cross-reactivity to at least types IA1, IA2, IC, and II; a vaccine containing PITP, or a fragment or derivative disclosed herein will elicit cross-reactivity to at least types IA1, IA2, IB, IC, II, and III. (See e.g., Figures 1B and 1C).

[0267] The cross-binding and cross-reactivity / cross-type reactivity of vaccine-induced antibodies can be tested by surface binding assays. As used herein, the term "surface binding assay" refers to the following test procedure: sera from rabbits or mice immunized multiple times with a specific P. acnes protein, combined with or without adjuvant, can be evaluated for their ability to bind to the surface of P. acnes strains from different genotypes. Antigen surface expression and accessibility to the humoral immune system can be confirmed by the ability of antigen-specific antibodies to specifically bind to native antigens on the surface of live bacteria. The amount of antibody specifically bound to the bacterial surface is measured by a flow cytometer, which quantifies the amount of light emitted by fluorescently labeled species-specific antibodies (which are elevated in that species and recognize antibodies that bind to specific surface epitopes). Binding is expressed as median fluorescence intensity (MFI). Antigen-specific rabbit and mouse sera that show substantial binding of antibodies to multiple P. acnes isolates (the MFI positive threshold is increased by at least 3-fold, preferably at least 5-fold, relative to the MFI of a negative control (e.g., corresponding pre-immune serum, a pool of pre-immune sera, or serum generated by immunization with the same formulation without each antigen, e.g., adjuvant or physiological buffer control)) are a clear indication of P. acnes strain / type cross-reactivity.

[0268] The cross-reactivity and cross-type reactivity of antibodies induced by vaccines can also be tested by functional assays. As used herein, the term "functional assay" refers to an assay that uses a pathogenic cell structure specifically bound by an antibody and a readout that determines the effect of the antibody binding to the pathogenic cell structure. As used herein, such a readout can be cell death (e.g., to determine antibacterial activity), growth inhibition, and / or neutralization of mammalian cell invasion. Thus, each functional assay can be an assay for determining pathogen cell death, inhibition and / or neutralization of cell growth, host cell invasion, reduction of biofilm formation ability, or some other disease-related function. This allows the antibacterial activity of antibody-containing serum fractions to be tested as described herein. For example, sera from animals immunized multiple times with specific P. acnes proteins, with or without adjuvants, or with human serum containing P. acnes-specific antibodies can be evaluated for their ability to opsonize and induce killing of P. acnes strains of various genotypes by phagocytes, such as granulocytes, neutrophils, macrophages, monocytes, dendritic cells, mast cells, and other cells capable of absorbing and killing P. acnes bacteria, in the presence of serum or other tissue fluids containing P. acnes-specific antibodies. Preferably, mixtures containing neutrophils and granulocytes are used in these assays.

[0269] As used herein, the term "antibacterial activity" is intended to mean any effect of a compound on bacteria, directly or indirectly, e.g., eliciting an immune response, which blocks or inhibits the bacteria or the pathogenesis caused by the bacteria (e.g., bactericidal, bacteriostatic, neutralizing, or any other functional effect that reduces the pathogenic potential of the bacteria, e.g., interfering with the growth or adhesion to human cells, reducing biofilm formation, or reducing the secretion of bacterial proteins).

[0270] Antibacterial activity is evidenced by a reduction in bacterial cell counts (colony forming units, CFU) recovered at the end of an opsonophagocytic killing assay (OPK; preferably as performed in the Examples section of the present invention below), e.g., a reduction of more than at least 50% compared to a negative control (immune serum from an animal immunized with buffer containing no antigen, and / or corresponding pre-immune serum taken before the first immunization with each antigen, and / or a sample containing all assay reaction components other than serum) (reaching at least 50% of the CFU reduction at a dilution of at least 1 / 200 to 1 / 1000 (e.g., 1 / 800). The number of bacterial cells in the reaction sample after at least 24 hours or more of incubation using at least two two-fold serial dilutions of antibody or serum, starting from a 1 / 200 dilution, preferably at least four to a maximum of seven serial dilutions (1 / 200 to 1 / 25,600), more preferably at least eight or nine serial dilutions (starting from 1 / 200 to 1 / 128,000), and most preferably ten or more serial dilutions (e.g., 1 / 200 to 1 / 204,800), is at least two-fold (or at least 60%, or at least 70%, or at least 80%, or at least 90%) higher dilutions used in the opsonophagocytic killing assay for K50 titer determination. It is understood that antibacterial activity should be demonstrated when the percentage of killing falls from >90% to >50% in at least two dilution steps, more preferably five or more dilution steps, or remains above 50% in at least two dilution steps, with the end of the serial dilution where the % of OPK activity remains above 50% before falling being defined as the K50 titer of the antibody or serum sample being tested (see the preferred performance of the OPK assay detailed in the Examples section below, according to which OPK activity according to the present invention is determined in case of doubt). As a positive control, serum obtained from a vaccinated individual or an individual who has developed antibodies against P. acnes may be used, as well as polyclonal, e.g., animal or human, serum generated against whole bacteria or bacterial lysates containing antibodies capable of killing, inhibiting, or neutralizing the bacteria. Negative controls may be corresponding pre-immune serum or non-immune serum (e.g., serum raised by immunization with physiological buffer or adjuvant without the addition of antigen).

[0271] Exemplary functional assays for determining antibacterial activity are the serum bactericidal assay (SBA) (Taylor, 1983) or the opsonophagocytic killing assay (OPKA or OPK assay) (Gordon, 2016). For example, serum antibodies or sera with the ability to opsonize bacteria for uptake and killing by phagocytes, indicating protective efficacy, can be tested by an opsonophagocytic killing assay.

[0272] Vaccine-induced antibodies can function in a process known as opsonization. Opsonization is the process by which microbial pathogens are targeted for ingestion by phagocytes of the immune system. Binding of opsonins attracts phagocytes, resulting in the destruction of the bacterial pathogen. Phagocytosis is mediated by macrophages, granulocytes, or other cells capable of killing bacteria and involves the ingestion and digestion of microorganisms, damaged or dead cells, cellular debris, insoluble particles, and activated clotting factors. Opsonins are agents that promote the phagocytosis of the aforementioned foreign substances. Opsonic antibodies, therefore, are antibodies that perform the same function.

[0273] Exemplary functional assays for determining bacteriostatic function are growth inhibition assays, bacteriostatic assays, or disk diffusion susceptibility methods that measure the bacteriostatic range (zone of inhibition) of a tested agent.

[0274] Exemplary functional assays for determining bacterial neutralization function are neutralization of cell / tissue adhesion, neutralization of bacterial binding to host molecules (e.g., neutralization of binding complement, fibrinogen, or other plasma proteins), neutralization of toxins, enzymes and enzymatic activities that regulate growth, tissue invasion and spread, or neutralization of cell-cell interactions, and biofilm formation.

[0275] In certain embodiments, functional assays provide a characterization of antibodies by their effect on pathogens that differs from function determined by simple binding assays, because binding assays only determine the (specific) binding properties of an antibody. Thus, functional assays can differentiate between protective antibodies and simple specific binders.

[0276] Any immunologically relevant target antigen or epitope described herein can be characterized by its ability to elicit antibodies with antibacterial activity, as determined by a functional assay. Such a functional assay uses, for example, a specific amount of the target antigen as an isolated molecule or structure, a pathogen with a cell surface that expresses the target antigen, and an antibody directed against the target antigen. In the functional assay, the effect of the antibody on the pathogen is determined in the presence and absence of the isolated target antigen. If the target antigen or epitope is immunologically relevant, the antibacterial function of an antibody against this target antigen or epitope will be significantly inhibited by the presence of a competing amount of the isolated target antigen or epitope or by pre-incubating the antibody with the target antigen or epitope.

[0277] Exemplary functional assays for determining the inhibition of protective antibodies include bactericidal inhibition assay and antibody depletion assay.In these assays, target antigens are used to inhibit or deplete bactericidal antibodies from the tested serum or purified antibody sample, thereby demonstrating that the selected antigen is actually the target of bactericidal antibodies, and therefore a good vaccine candidate.Antigens derived from pathogens that can infect animals and have good animal models can also be evaluated in vivo (for example, if the serum is pre-incubated with the same antigen to deplete antigen-specific antibodies, it can inhibit or reduce the protection caused by serum transfer).

[0278] Generally, the bactericidal activity of hyperimmune P. acnes serum or antibodies induced after vaccination with a protective vaccine is determined as the K50 titer, which refers to the highest dilution that shows a greater than 50% reduction in bacterial counts compared to a negative control at the same dilution. Adsorption of antigen-specific antibodies in serum to a selected protein leads to their removal, resulting in a reduction in the bactericidal activity of the serum compared to a non-adsorbed sample at the same dilution. This reduction in bactericidal activity can then be used as a direct correlation for the degree of antibacterial activity of antibodies in each human serum against the target antigen being evaluated.

[0279] Other functional assays that can be used to test the protective function of antibodies induced after immunization with a vaccine containing an immunologically relevant antigen or epitope include serum assays that measure the ability of antibodies to inhibit bacterial growth, adhesion, biofilm formation, inhibition of nutrient acquisition, secretion of toxins or immunomodulatory signaling molecules (e.g., those that inhibit complement activation or cytokine function).

[0280] The term "variant," as used herein with respect to a protein that is an antigen or contains one or more epitopes as described herein, refers to either an equivalent or non-parent protein that has substantially the same functional activity. Particularly preferred variants are referred to herein as "derivatives," particularly in the embodiments and claims section. Variants may, for example, be proteins of the same type as the equivalent, but derived from or originating from different bacterial strains or analogous proteins. Variants may, for example, be derivatives of natural proteins that serve as parent proteins for generating variants and derivatives, respectively. In particular, the variant is derived from or related to the P. acnes protein identified by UniProt Accession Nos. Q6A5X9, Q6A5P9, Q6A9N1 or an analog protein or fragment of any of the foregoing, or comprises at least about 90% amino acid sequence identity, preferably at least 95%, more preferably at least 98%, more preferably at least 99% or at least 99.5%, or at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the amino acid sequence of such a protein identified by, for example, UniProt Accession Nos. Q6A5X9, Q6A5P9, Q6A9N1. It may also be an analogous protein or an artificial proteinaceous substance or protein that does not occur in nature.

[0281] Sequence identity is preferably determined (in cases of any uncertainty or concern) by using the Clustal Omega multiple sequence alignment software algorithm, version 1.2.4, available from EMBL-EBI. Clustal Omega is suitable software for determining global sequence alignments. It does not produce a sequence identity graph, i.e., an alignment rather than a number. However, this alignment portion is a critical step, since determining identity can be done manually or with simple scripts that compare character identity. Whatever software or algorithm is used instead, global rather than local alignments should be performed when proteins are to be compared. Local algorithms such as BLAST exclude non-corresponding regions in the flanks (in the N- and C-termini). Specifically, an artificial N-terminus from a frameshifted sequence may result in differences between proteins. In this context, it also makes sense to explicitly define alignment gaps as mismatches (rather than disregarding them in identity counts). Therefore, blastp from the NCBI BLAST+ package (e.g., version 2.9.0) is also a suitable tool. It creates local alignments, usually ignoring artificial N-termini and focusing on regions of actual similarity. Identity and similarity values ​​are also reported directly. These local alignments are more meaningful (taking into account pseudogenes and sequencing artifacts). However, if sequences should be compared in their entirety (as is, so to speak), Clustal Omega is the tool of choice.

[0282] Preferred variants of epitopes can be used that incorporate one or more point mutations in the epitope or epitope region, such as at least 1, 2, 3, 4, and up to 5 point mutations in the amino acid sequence by, for example, insertion, deletion, and / or substitution of amino acid residues. Suitable point mutations are those that are already present in other protein variants (e.g., as shown in Figures 12B, 12C, and 12D) and are then introduced into the native protein as a derivative according to the invention with a different amino acid residue at this position.

[0283] The reference sequences Q6A5X9 and Q6A5P9 were compared to both variants. This data shows amino acid positions that are unique for CSD1, CSD2, and CSD3 in DsA1 compared to their respective CSD sequences in DsA2 (i.e., amino acid positions at which it can be unambiguously concluded that a particular sequence is derived from CSD1, CSD2, or CSD3 from DsA1 (or DsA2)).

[0284] To calculate the percentage, the count of a particular variant is compared to all sequences defined at this position. This may also include gaps, as long as they are not in the contiguous region. If gaps are in the contiguous region, they should not be considered / counted as differences (they are a sign that the first sequence is simply short / incomplete). Gaps within a sequence are considered differences.

[0285] E366D may be biased by C-terminally truncated translation, i.e., the process that generates matched sequences after genomic BLAST / search. Many of the BLAST-derived sequences appear to lack the PT region (indeed already CTPEPTPT, so slightly before the end of CSD3), which is likely a systematic technical issue. For this reason, E366D as well as the baseline "E" variant are very likely not quantified correctly, i.e., either species may be arbitrarily more abundant. In addition to this reason for truncation, In addition, the reasons may be of a technical nature or sequencing issues.

[0286] The "alternative variant percentage" referred to herein below is the percentage of analyzed sequences that contain a particular variant that is not present in the reference sequence. This analysis was performed on the sequences disclosed in Figure 12 and the following additional sequences: After the EP priority date (including database version / status): GenBank entry IDs (DsA1): VBYU01000003.1:466799-467896 (May 22, 2019) and QJIR01000003.1:466818-467915 (June 3, 2019), QJII01000011.1:3-1160 (June 3, 2019) and VBYK01000011.1:3-1151 (May 22, 2019). GenBank entry IDs (DsA2): MVCC01000003.1:313005-313898 (October 17, 2017), BFFM01000002.1:c62117-61161 (May 16, 2019) and LKVC01000009.1:231840-232958 (May 16, 2019), LKVC01000009.1 (October 16, 2017) and GCA_000145535.1_ASM14553v1 (August 16, 2010) or GCA_000342585.1_PropiAcnFZ1_2_0_1.0 (March 1, 2013). The following intra-DsA1, intra-DsA2 and intra-DsA1-DsA2 amino acid variations are present in DsA1 and DsA2: Position 50 of CSD1 of DsA1 (reference = D (7.6%) and variant = N (79.6%)) corresponds to position 94 of DsA2 (reference = S (81.5%) and variant = D (10.9%)). Position 51 of CSD1 of DsA1 (reference = K (87.2%)) corresponds to position 95 of DsA2 (reference = E (81.5%) and variant = A (10.9%)). Position 53 of CSD1 of DsA1 (reference = C (85.5%) and variant = Y (1.6%)) corresponds to position 97 of DsA2 (reference = C (92.4%)). Position 55 of CSD1 of DsA1 (reference = D (87.2%)) corresponds to position 99 of DsA2 (reference = K (92.4%)). Position 57 of CSD1 of DsA1 (reference = V (85.5%) and variant = I (1.6%)) corresponds to position 101 of DsA2 (reference = I (92.4%)). Position 61 of CSD1 of DsA1 (reference = A (87.2%)) corresponds to position 105 of DsA2 (reference = L (92.4%)). Position 65 of CSD1 of DsA1 (reference = A (87.2%)) corresponds to position 109 of DsA2 (reference = G (92.4%)). Position 68 of CSD1 of DsA1 (reference = A (87.2%)) corresponds to position 112 of DsA2 (reference = V (92.4%)). Position 71 of CSD1 of DsA1 (reference = L (87.2%) and variant = M (2.5%)) corresponds to position 115 of DsA2 (reference = L (92.4%)). Position 76 of CSD1 of DsA1 (reference = F (89.8%)) corresponds to position 120 of DsA2 (reference = L (92.4%)). Position 78 of CSD1 of DsA1 (reference = S (89.8%)) corresponds to position 122 of DsA2 (reference = A (92.4%)). Position 82 of CSD1 of DsA1 (reference = V (89.8%) and variant = M (10.1%)) corresponds to position 126 of DsA2 (reference = A (92.4%)). Position 86 of CSD1 of DsA1 (reference = P (96.6%) and variant = S (2.5%) and variant = R (0.8%)) corresponds to position 130 of DsA2 (reference = P (93.2%)). Position 90 of CSD1 of DsA1 (reference = K (20.3%) and variant = R (79.6%)) corresponds to position 134 of DsA2 (reference = A (94.1%)). Position 94 of CSD1 of DsA1 (reference = K (100%)) corresponds to position 138 of DsA2 (reference = A (98.3%)).Position 97 of CSD1 of DsA1 (reference = V (100%)) corresponds to position 141 of DsA2 (reference = T (98.3%)). Position 99 of CSD1 of DsA1 (reference = L (100%)) corresponds to position 143 of DsA2 (reference = T (98.3%)). Position 100 of CSD1 of DsA1 (reference = I (100%)) corresponds to position 144 of DsA2 (reference = I (84.8%) and variant = L (13.4%)). Position 104 of CSD1 of DsA1 (reference = I (100%)) corresponds to position 144 of DsA2 (reference = I (84.8%) and variant = L (13.4%)). Variant 1 (reference = K (100%)) corresponds to position 148 of DsA2 (reference = R (98.3%)). Variant 106 of CSD1 of DsA1 (reference = K (100%)) corresponds to position 150 of DsA2 (reference = K (94.9%) and variant = E (3.3%)). Variant 107 of CSD1 of DsA1 (reference = A (100%)) corresponds to position 151 of DsA2 (reference = V (98.3%)). Variant 109 of CSD1 of DsA1 (reference = I (100%)) corresponds to position 153 of DsA2 (reference = V (98.3%)). Variant 110 of CSD1 of DsA1 (reference = G (100%)) corresponds to position 154 of DsA2 (reference = A (98.3%)). Position 111 of CSD1 of DsA1 (reference = A (100%)) corresponds to position 155 of DsA2 (reference = S (98.3%)). Position 113 of CSD1 of DsA1 (reference = L (5%) and variant = V (94.9%)) corresponds to position 157 of DsA2 (reference = L (98.3%)). Position 114 of CSD1 of DsA1 (reference = G (100%)) corresponds to position 158 of DsA2 (reference = G (94.9%) and variant = S (3.3%)). Position 116 of CSD1 of DsA1 (reference = L (100%)) corresponds to position 160 of DsA2 (reference = V (98.3%)). Position 117 of CSD1 of DsA1 (reference = T (100%)) corresponds to position 161 of DsA2 (reference = A (98.3%)). Position 120 of CSD1 of DsA1 (reference = K (100%)) corresponds to position 164 of DsA2 (reference = A (98.3%)). Position 121 of CSD1 of DsA1 (reference = I (100%)) corresponds to position 165 of DsA2 (reference = V (98.3%)). Position 123 of CSD1 of DsA1 (reference = R (100%)) corresponds to position 167 of DsA2 (reference = H (98.3%)). Position 124 of CSD1 of DsA1 (reference = A (100%)) corresponds to position 168 of DsA2 (reference = A (97.4%) and variant = T (0.8%)). Position 128 of CSD1 in DsA1 (reference = V (100%)) corresponds to position 172 of DsA2 (reference = I (98.3%)).

[0287] From these sequences, it follows that the following CSD1 derivatives are preferred embodiments of the present invention due to their conservative nature: In view of the CSD1 of Q6A5X9: CSD1 derivatives with one or more, preferably one, two or three, in particular one, of the following amino acid exchanges: D50N, D50S, K51E, K51A, C53Y, D55K, V57I, A61L, A65G, A68V, L71M, F76L, S78A, V82M, V82A, L84M, P86R, P86S, K90R, K90A, K94A, V97T, L99T, I100L, K104R, K106E, A107V, I110L, K111R, K112R, K113E, A114V, I115L, K116R, K117R, I118L, K119R, K120R, K121R, K122R, K123R, K124R, K125R, K126R, K127R, K128R, K129R, K130R, K131R, K132R, K133R, K134R, K135R, K136R, K137R, K138R, K139R, K140R, K141R, K142R, K143R, K144R, K145R, K146R, K147R, K148R, K149R, K150R, K151R, K152R, K153R, K154R, K155R, K156R, K157R, K158R, K159R, K160R, K161R, K162R, K163R, K164R, K165R, K166R, K167R, K168R, K169R, K170R, K171R, In view of CSD1 of Q6A5P9, G110A, G111S, L113V, G114S, L116V, T117A, K120A, I121V, R123H, A124T, V128I, and Q6A5P9: CSD1 derivatives having one or more, preferably one, two or three, particularly one, of the following amino acid exchanges: S94D, S94N, E95A, E95K, C97Y, K99D, I101V, L105A, G109A, V112A, L115M, L120F, A122S, A126M, A126V, L128M, P130R, P130S, A134K, A134R, A138K, T141V, T143L, I144L, R148K, K150E, V151A, V153I, A154G, S155A, L157V, G158S, V160L, A161T, A164K, V165I, H167R, A168T, I172V in DsA2.

[0288] Position 149 of the CSD2 of DsA1 (reference = A (100%)) corresponds to position 193 of DsA2 (reference = A (94.9%) and variant = T (3.3%)). Position 152 of the CSD2 of DsA1 (reference = D (100%)) corresponds to position 196 of DsA2 (reference = N (84.8%) and variant = S (13.4%)). Position 155 of the CSD2 of DsA1 (reference = V (100%)) corresponds to position 199 of DsA2 (reference = I (98.3%)). Position 163 of the CSD2 of DsA1 (reference = V (100%)) corresponds to position 207 of DsA2 (reference = I (94.9%) and variant = V (3.3%)). Position 166 of CSD2 (reference = K (100%)) corresponds to position 210 of DsA2 (reference = H (97.4%) and variant = P (0.8%)). Position 168 of CSD2 of DsA1 (reference = A (98.3%) and variant = T (1.6%)) corresponds to position 212 of DsA2 (reference = A (98.3%)). Position 169 of CSD2 of DsA1 (reference = K (100%)) corresponds to position 213 of DsA2 (reference = R (98.3%)). Position 171 of CSD2 of DsA1 (reference = T (100%)) corresponds to position 215 of DsA2 (reference = T (94.9%) and variant = A (3.3%)). Position 173 of the CSD2 of DsA1 (reference = V (100%)) corresponds to position 217 of DsA2 (reference = V (98.3%) and variant = M (1.6%)). Position 176 of the CSD2 of DsA1 (reference = A (100%)) corresponds to position 220 of DsA2 (reference = V (100%)). Position 183 of the CSD2 of DsA1 (reference = A (98.3%) and variant = T (1.6%)) corresponds to position 227 of DsA2 (reference = A (100%)). Position 190 of the CSD2 of DsA1 (reference = T (100%)) corresponds to position 234 of DsA2 (reference = F (95.7%) and variant = I (4.2%)). Position 191 of CSD2 of DsA1 (reference = E (100%)) corresponds to position 235 of DsA2 (reference = E (99.1%) and variant = K (0.8%)). Position 192 of CSD2 of DsA1 (reference = A (100%)) corresponds to position 236 of DsA2 (reference = L (99.1%) and variant = F (0.8%)). Position 198 of CSD2 of DsA1 (reference = A (20.3%) and variant = G (79.6%)) corresponds to position 242 of DsA2 (reference = A (100%)). Position 199 of CSD2 of DsA1 (reference = A (100%)) corresponds to position 243 of DsA2 (reference = A (99.1%) and variant = T (0.8%)). Position 202 of CSD2 of DsA1 (reference = A (100%)) corresponds to position 246 of DsA2 (reference = A (95.7%) and variant = T (4.2%)). Position 205 of CSD2 of DsA1 (reference = V (100%)) corresponds to position 249 of DsA2 (reference = I (100%)).Position 206 of CSD2 of DsA1 (reference = G (6.7%) and variant = N (1.6%) and variant = S (91.5%)) corresponds to position 250 of DsA2 (reference = R (84%) and variant = Q (15.9%)). Position 212 of CSD2 of DsA1 (reference = K (100%)) corresponds to position 256 of DsA2 (reference = Q (100%)). Position 214 of CSD2 of DsA1 (reference = A (97.4%) and variant = T (2.5%)) corresponds to position 258 of DsA2 (reference = A (100%)). Position 219 of CSD2 of DsA1 (reference = I (100%)) corresponds to position 263 of DsA2 (reference = V (95.7%) and variant = A (4.2%)). Position 223 of CSD2 of DsA1 (reference = S (100%)) corresponds to position 267 of DsA2 (reference = A (100%)). Position 225 of CSD2 of DsA1 (reference = D (100%)) corresponds to position 269 of DsA2 (reference = D (99.1%) and variant = N (0.8%)). Position 233 of CSD2 of DsA1 (reference = V (100%)) corresponds to position 277 of DsA2 (reference = I (100%)). Position 235 of CSD2 of DsA1 (reference = S (99.1%) and variant = F (0.8%)) corresponds to position 279 of DsA2 (reference = S (100%)). Position 239 of CSD2 of DsA1 (reference = N(100%)) corresponds to position 283 of DsA2 (reference = S(100%)). Position 255 of CSD2 of DsA1 (reference = L(100%)) corresponds to position 299 of DsA2 (reference = I(100%)). Position 257 of CSD2 of DsA1 (reference = V(100%)) corresponds to position 301 of DsA2 (reference = I(100%)). Position 258 of CSD2 of DsA1 (reference = Q(100%)) corresponds to position 302 of DsA2 (reference = S(100%)). Position 259 of CSD2 of DsA1 (reference = I(100%)) corresponds to position 303 of DsA2 (reference = L(100%)). Position 262 of CSD2 of DsA1 (reference = R(100%)) corresponds to position 306 of DsA2 (reference = H(100%)). Position 264 of CSD2 of DsA1 (reference = I(100%)) corresponds to position 308 of DsA2 (reference = V(100%)). Position 265 of CSD2 of DsA1 (reference = D(100%)) corresponds to position 309 of DsA2 (reference = K(100%)).

[0289] From these sequences, it follows that the following CSD2 derivatives are preferred embodiments of the present invention due to their conservative nature: In view of the CSD2 of Q6A5X9: CSD2 derivatives having one or more, preferably one, two or three, especially one of the following amino acid exchanges: A149T, D152N, D152S, V155I, V163I, K166H, K166P, A16 8T, K169R, T171A, V173M, A176V, A183T, T190F, T190I, E191K, A192L, A192F, A198G, A199T, A202T, V205I, G20 6N, G206S, G206R, G206Q, K212Q, A214T, I219V, I219A, S223A, D225N, V233I, S235F, N239S, L255I, V257I, Q25 In view of the CSD2 of 8S, I259L, R262H, I264V, D265K and Q6A5P9: CSD2 derivatives having one or more, preferably one, two or three, especially one of the following amino acid exchanges: A193T, N196D, N196S, I199V, I207V, H210K, H210P, A212T, R213K, T215A, V217M, V220A in DsA2; A227T, F234T, F234I, E235K, L236A, L236F, A242G, A243T, A246T, I249V, R250G, R250S, R250N, G250Q, Q256K, A258T, V263I, V263A, A267S, D269N, I277V, S279F, S283N, I299L, I301V, S302Q, L303I, H306R, V308I, K309D.

[0290] Position 281 of CSD3 of DsA1 (reference = M (100%)) corresponds to position 325 of DsA2 (reference = V (100%)). Position 283 of CSD3 of DsA1 (reference = N (100%)) corresponds to position 327 of DsA2 (reference = D (100%)). Position 285 of CSD3 of DsA1 (reference = T (100%)) corresponds to position 329 of DsA2 (reference = A (100%)). Position 286 of CSD3 of DsA1 (reference = R (100%)) corresponds to position 330 of DsA2 (reference = R (95.7%) and variant = Q (4.2%)). Position 289 of CSD3 of DsA1 (reference = V (98.3%) and variant = A (1.6%)) corresponds to position 333 of DsA2 (reference = V (100%)). Position 291 of CSD3 of DsA1 (reference = V (100%)) corresponds to position 335 of DsA2 (reference = I (100%)). Position 292 of CSD3 of DsA1 (reference = I (100%)) corresponds to position 336 of DsA2 (reference = R (100%)). Position 293 of CSD3 of DsA1 (reference = T (100%)) corresponds to position 337 of DsA2 (reference = N (100%)). Position 294 of CSD3 of DsA1 (reference = A (100%)) corresponds to position 338 of DsA2 (reference = T (100%)). Position 295 of CSD3 of DsA1 (reference = D (100%)) corresponds to position 339 of DsA2 (reference = Q (95.7%) and variant = K (4.2%)). Position 296 of CSD3 of DsA1 (reference = K (100%)) corresponds to position 340 of DsA2 (reference = E (99.1%) and variant = K (0.8%)). Position 298 of CSD3 of DsA1 (reference = I (100%)) corresponds to position 342 of DsA2 (reference = I (72.2%) and variant = V (27.7%)). Position 299 of CSD3 of DsA1 (reference = K (100%)) corresponds to position 343 of DsA2 (reference = A (100%)). Position 300 of CSD3 of DsA1 (reference = T (100%)) corresponds to position 344 of DsA2 (reference = V (86.5%) and variant = I (13.4%)). Position 301 of CSD3 of DsA1 (reference = A (100%)) corresponds to position 345 of DsA2 (reference = Y (100%)).From position 301 onwards in the CSD3 of DsA1, reference = GAP (99.1%) and variant = D (0.8%) correspond to positions 345 onwards in DsA2, which are GAP (100%). Position 302 on the CSD3 of DsA1 (reference = E (100%)) corresponds to position 346 on DsA2 (reference = K (100%)). Position 305 on the CSD3 of DsA1 (reference = E (100%)) corresponds to position 349 on DsA2 (reference = K (100%)). Position 306 on the CSD3 of DsA1 (reference = K (100%)) corresponds to position 350 on DsA2 (reference = A (100%)). Position 310 on the CSD3 of DsA1 (reference = A (100%)) corresponds to position 354 on DsA2 (reference = T (100%)). Position 313 of the CSD3 of DsA1 (reference = D (100%)) corresponds to position 357 of DsA2 (reference = G (100%)). Position 316 of the CSD3 of DsA1 (reference = K (100%)) corresponds to position 360 of DsA2 (reference = Q (100%)). Position 320 of the CSD3 of DsA1 (reference = S (100%)) corresponds to position 364 of DsA2 (reference = T (99.1%)). Position 321 of the CSD3 of DsA1 (reference = C (100%)) corresponds to gap (100%) of DsA2. Position 322 of the CSD3 of DsA1 (reference = P (100%)) corresponds to position 365 of DsA2 (reference = P (97.4%) and variant = L (1.6%)). Position 323 of CSD3 in DsA1 (reference = K (100%)) corresponds to position 366 in DsA2 (reference = E (86.5%) and variant = D (12.6%)).

[0291] From these sequences, it follows that the following CSD3 derivatives are preferred embodiments of the present invention due to their conservative nature: In view of the CSD3 of Q6A5X9: CSD3 derivatives with one or more, preferably one, two or three, especially one of the following amino acid exchanges: In view of the CSD3 of M281V, N283D, T285A, R286Q, V289A, V291I, I292R, T293N, A294T, D295K, D295Q, K296E, I298V, K299A, T300V, T300I, A301Y, gap between 301 and 302D, E302K, E305K, K306A, A310T, D313G, K316Q, S320T, C321-, P322L, K323D, K323E and Q6A5P9 in DsA1: CSD3 derivatives having one or more, preferably one, two or three, especially one of the following amino acid exchanges: In DsA2, V325M, D327N, A329T, R330Q, V333A, I335V, R336I, N337T, T338A, Q339K, Q339D, E340K, I342V, A343K, V344T, V344I, Y345A, gap between 345 and 346D, K346E, K349E, A350K, T354A, G357D, Q360K, T364S, gap between 364 and 365C, P365L, E366D, E366K.

[0292] Variants include, for example, proteins with one or more added or deleted amino acid residues at the N- or C-terminus, as well as within one or more internal domains. Certain variants as described herein include additional amino acids at the N-terminus and / or C-terminus to extend the antigen sequence as described herein, e.g., to extend the sequence of the epitope or epitope region within the protein by at least one amino acid residue, preferably by fewer than three amino acids, particularly fewer than five or fewer than ten amino acids. Furthermore, a variant may be a fusion protein in which the antigen sequence as described herein is extended by additional amino acid residues of another polypeptide or protein. According to a preferred embodiment, the vaccines of the present invention include derivatives, particularly those in which a DsA1 / DsA2 fragment containing the DsA1 / DsA2 epitope and / or a PITP fragment containing the PITP epitope is extended by additional amino acid residues of another polypeptide or protein, preferably by one or more immunologically relevant epitopes, as fusion proteins, in which the derivatives contain a His-tag at the N- or C-terminus containing at least four, preferably at least five, and in particular at least six histidine residues. Generally, a His-tag may contain at least two to ten or more histidine residues. His-tags are preferably included in derivatives intended for experimental purposes and are not necessarily included in vaccines intended for human use. Therefore, any sequence containing a His-tag as used herein should also be considered as disclosed without the His-tag.

[0293] Variants may, for example, have one or more cysteines replaced, which may result in reduced product-related impurities and microheterogeneity, protein stability, folding, or Other types of proteins that result in improved biochemical properties are included (Figure 10b, Table 2).

[0294] According to a preferred embodiment, the derivatives of the present invention are "Cys-replacement derivatives" in which one or more naturally occurring cysteine ​​residues are replaced by different amino acid residues. For example, the following cysteines may be replaced: DsA1: C53, C319, C321; DsA2: C97, C363 (at the N-terminus of some DsA2 proteins, there are even more cysteines that may be replaced; see, for example, SEQ ID NO: 8); PITP: C231, C402, C460. Of course, the absolute amino acid numbering in a given polypeptide (e.g., in a fragment or derivative) varies with the length of the fragment / derivative; however, this numbering of cysteines (DsA1: C53, C319, C321; DsA2: C97, C363; PITP: C231, C402, C460) allows for the absolute identification of the cysteines to be replaced. For example, in the DsA1 fragments / derivatives in the sequence listing, cysteines C53, C319 and C321 correspond to C53, C325 and C327 in SEQ ID NO:4; C71, C337 and C339 in SEQ ID NO:5; C26, C292 and C294 in SEQ ID NO:7; C119 and C121 in SEQ ID NO:31; C56 and C58 in SEQ ID NO:32; C53, C325 and C327 in SEQ ID NO:34; C231 and C233 in SEQ ID NO:35; C38 and C304 in SEQ ID NO:36; C32, C298 and C300 in SEQ ID NO:37; C32 in SEQ ID NO:38, etc. In the DsA2 fragments / derivatives in the Sequence Listing, cysteines C97 and C363 correspond to C67 and C333 in SEQ ID NO:8; C97 and C363 in SEQ ID NO:9; C74 and C340 in SEQ ID NO:10; C33 and C299 in SEQ ID NO:11; C27 and C293 in SEQ ID NO:12, etc. In the PITP fragments / derivatives in the Sequence Listing, cysteines C231, C402, C460 correspond to C201, C372, and C430 in SEQ ID NO:15; C201 in SEQ ID NO:17; C372 in SEQ ID NO:18; C201 in SEQ ID NO:19, etc.

[0295] Preferably, the replacement is carried out with an amino acid that is similar in size and charge / polarity to cysteine ​​but does not have any sulfur group. This means that one or more cysteines are preferably not methionine, arginine, histidine, lysine, tryptophan, aspartic acid, or glutamic acid. Therefore, preferred replacements are replacements of cysteine(s) with serine, proline, alanine, threonine, asparagine, glutamine, valine, isoleucine, leucine, phenylalanine, tyrosine, and glycine, preferably with serine, proline, alanine, threonine, asparagine, glutamine, valine, isoleucine, leucine, and especially with serine, proline, alanine, threonine, asparagine, and glutamine. In a given derivative of DsA1, DsA2, or PITP, or a fragment thereof, preferably at least two cysteines are replaced, and the replacement of two cysteines in each of these fragments / derivatives is particularly preferred.

[0296] A preferred embodiment of the present invention is a derivative of DsA1 in which C53 has been exchanged, in particular a C53S exchange (as in SEQ ID NOs: 48 and 49). Another preferred embodiment of the present invention is a derivative of DsA1 in which C319 has been exchanged, in particular a C319S exchange. Another preferred embodiment of the present invention is a derivative of DsA1 in which C321 has been exchanged, in particular a C321P exchange. A particularly preferred embodiment of the present invention is a derivative of DsA1 in which two cysteines have been exchanged, preferably all three cysteines have been exchanged, in particular a C53S, C319S and C321P exchange. Another preferred embodiment of the present invention is a derivative of DsA2 in which C97 has been exchanged, in particular a C97S exchange. Another preferred embodiment of the present invention is a derivative of DsA2 in which C363 has been exchanged, in particular a C363S exchange. Another preferred embodiment of the present invention is a derivative of PITP in which C231 is exchanged, in particular in which there is a C231S exchange. Another preferred embodiment of the present invention is a derivative of PITP in which C402 is exchanged, in particular in which there is a C231S exchange. , a derivative of PITP in which there is a C402S exchange. Another preferred embodiment of the present invention is a derivative of PITP in which C460 is exchanged, in particular in which there is a C460S exchange.

[0297] However, other preferred DsA derivatives according to the invention can also be designed in the other direction, i.e., two or more amino acid residues in the fragment or derivative are changed to cysteine ​​(Cys, C) residues to further stabilize the fragment or derivative by their ability to form disulfide bonds, preferably the following amino acid pairs in which the natural amino acids are replaced with cysteines: PHE150-GLY185, LEU167-ALA199, ASN157-ALA192, LYS166-ALA199, ALA153-GLY185, ASP164-ALA199, VAL154-ALA179, PHE150-LEU184, VAL 154-ALA188, ALA153-ALA188, ALA179-GLU191, THR178-GLU191, ALA161-ALA195, IL E175-ALA195, ILE175-GLU191, LEU158-ALA176, ASN157-ALA188, ASN157-ALA179, A SP164-GLU196, VAL154-LEU180, ALA153-LYS189, ASN157-LYS189, ALA161-ALA192, LEU167-ALA198, LEU158-ALA179, ALA160-ALA192, LYS165-ALA199, LYS166-GLN200;VAL205-ALA260, ILE232-ARG266, SER235-ILE263, SER235-ARG262, ILE232-LEU267, PRO236-ILE263, ALA231-ARG266, ASN239-ILE259, ILE232- ILE263, VAL209-PRO236, ALA201-ALA256, SER235-ILE259, VAL228-ARG266, ALA202-ALA256, ALA201-VAL257, GLY206-VAL243, GLY208-ILE264, G Derivatives containing at least one of LY208-ILE263, VAL205-ASN239, LEU238-ILE259, GLY208-ALA260, ALA201-LYS253, ALA202-VAL243, LEU204-ALA260, VAL205-ALA256, VAL205-ILE259, SER235-ARG266, ASN239-ALA260, VAL209-ILE263, or VAL209-ASN239 (with respect to the DsA1 sequence (also applicable to the DsA2 sequence; Figure 12A));

[0298] Specifically, preferred cysteine ​​replacement fragments and derivatives of PITP include those fragments of PITP in which at least one of the following substitutions is replaced: C231, C402, and C460; preferably, at least two of C231, C402, and C460 are replaced; in particular, C231 and C402 are replaced; A32-R164, A32-Q198, A32-T143, A32-V148, A32-T171, P34-R164, A32-T159, A32-I177, A32-Q204, ... and G172 to G234, A32 to K400, A32 to S391, V238 to K400, A199 to T430, V238 to T395, G234 to K400, H223 to K400, T232 to V401, V238 to T392, V238 to N393, V238 to L394, V238 to T395, V238 to L396, T232 to T430, G172 to K400 and G172 to G234, in particular comprising or consisting of the fragments A32-T430, A32-I467, A32-S391 (with K174-T239 deleted), A32-S391. Preferably, the cysteine ​​exchange is also with serine, proline, alanine, threonine, asparagine, glutamine, valine, isoleucine, leucine, phenylalanine, tyrosine and glycine, preferably with serine. Therefore, for these fragments and derivatives, the presence of C231S, C402S and / or C460S exchanges, especially the presence of C231S and C402S, is preferred. These fragments contain at least one effective epitope that induces an appropriate cross-reactive immune response when provided as a vaccine in humans. Furthermore, these fragments can be efficiently produced by recombinant expression systems. This allows for the final pharmaceutical vaccine formulation.

[0299] In addition to cysteine ​​replacement, other amino acid replacements that allow for higher stability, increased solvation, and / or increased pH stability of the polypeptide are preferred in the derivatives of the present invention. For example, the replacement of a specific lysine with an arginine can stabilize unstructured regions in the polypeptide or further stabilize already structured regions (the latter is particularly preferred because it can further stabilize already structured epitopes). Amino acid residues with a side chain solvent accessibility of 20% or less can be replaced (preferably with polar or charged amino acid residues, particularly aspartic acid or glutamic acid, lysine, or arginine residues, to increase stability and solubility, as long as these replacements do not have a detrimental effect on the antigenic profile).

[0300] After identifying the epitopes of the polypeptides according to the invention, amino acid exchanges in the polypeptide sequences were analyzed based on the behavior of these polypeptides at varying pH, with residues that exert a destabilizing effect (called buried acidic residues). For example, exchanges of acidic residues focused on buried residues, since they are destabilizing at higher pH.

[0301] In particular, histidine-to-lysine exchanges focus on solvent-exposed residues and solvation. A distinction is made between stability and solubility, as exchanges can affect one, the other, or both, sometimes resulting in a trade-off. Biochemically, a mixture of the two has been observed, largely a matter of the assay used: there can be pH-driven insolubility (if the protein concentration is too high, this can, in turn, lead to complex formation and possible loss of structure due to hydrophobic interactions), or there can be temperature- or denaturant-induced loss of structure, which can also (but not necessarily) lead to complex formation due to exposed hydrophobic moieties.

[0302] Preferred exchanges were provided that also have a stabilizing effect, extending exchange by reducing buried acidic residues. Exchanges that benefit overall stability in addition to pH-dependent stability are particularly preferred. pH stability can be measured, for example, using Prometheus technology using differential scanning fluorometry (Chattopadhyay et al., Prot. Sci. 28 (2019), 1127-1134; Martin et al., 2014 - NanoTemper Technologies GmbH - Application Note NT-PR-001 - Thermal Unfolding; Krakowiak et al., J. Biol. Phys. 45 (2019), pp. 161-172) (see Examples section). Differential scanning fluorimetry allows for the measurement of thermal unfolding or chemical denaturation under native, label-free conditions ...

Claims

1. 1. A method of producing a vaccine for use in treating or preventing a P. acnes associated infection, comprising: selecting at least one antigen comprising at least one P. acnes epitope, the P. acnes epitope being an epitope that is directly accessible to human serum antibodies when presented by viable P. acnes bacteria, and specific binding of the epitope by the human immune system leads to a reduction in bacterial numbers, fitness, growth, virulence or a combination thereof, in particular a reduction in bacterial numbers; Binding of the epitope by the immune system, in particular by the human immune system, is demonstrated by using a flow cytometry assay, preferably a fluorescence activated cell sorting (FACS) assay; The method, wherein the reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof is demonstrated by an opsonophagocytic killing assay (OPK) in the presence of an antibody specific for at least one antigen comprising at least one P. acnes epitope.

2. 1. A method for producing a vaccine for use in treating or preventing a P. acnes associated infection, comprising: selecting at least one antigen comprising at least one P. acnes epitope, the P. acnes epitope being an epitope that is directly accessible to human serum antibodies when presented by viable P. acnes bacteria, and specific binding of the epitope by the human immune system leads to a reduction in bacterial numbers, fitness, growth, virulence or a combination thereof, in particular a reduction in bacterial numbers; Binding of the epitope by the immune system, particularly the human immune system, is demonstrated by antibody binding to P. acnes bacteria using a flow cytometry assay, preferably by a fluorescence-activated cell sorting (FACS) assay, and by binding of antigen-specific antibodies to viable P. acnes bacteria in the flow cytometry assay; Reduction in bacterial numbers, fitness, growth, virulence, or a combination thereof is demonstrated by an opsonophagocytic killing assay (OPK), which uses polyclonal human antibodies specific for at least one antigen comprising at least one P. acnes epitope, or at least two serial two-fold dilutions of human serum or antigen-induced polyclonal antibody serum containing such antibodies, starting with a complement-inactivated serum dilution of at least 1 / 200, in a 5% CO atmosphere. ２ After incubation at 37° C. for at least 24 hours or longer in the presence of K ５０ The method shows at least a 50% reduction in bacterial cell count in the reaction sample compared to a negative control at at least a two-fold higher dilution used in the opsonophagocytic killing assay for titer determination.

3. The OPK assay - without the presence and / or addition of antibiotic compounds, and / or - without the presence and / or addition of complement and / or complement factors, The method according to claim 1 or 2, wherein

4. The method according to any one of claims 1 to 3, wherein the OPK assay is carried out using phagocytes, preferably neutrophils, granulocytes, macrophages, monocytes and / or other cells capable of engulfing and killing P. acnes bacteria, in particular neutrophils or granulocytes.

5. and a vaccine comprising the P. acnes dermatan sulfate-binding adhesin 1 (DsA1 polypeptide) and / or the P. acnes dermatan sulfate-binding adhesin 2 (DsA2 polypeptide) and / or a fragment and / or derivative of DsA1 or DsA2, wherein DsA1 and DsA2 contain, from the N-terminus to the C-terminus, an N-terminal swapping region ("NSR"). ), a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"), a Pro-Thr repeat-containing region ("PT repeat region") and a C-terminal region ("CTR"), wherein the fragment and / or derivative comprises or consists of at least a CSD2 fragment, with the proviso that the CSD2 fragment preferably does not comprise the NSR and the CTR; - a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184), - a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), - a contiguous polypeptide sequence from leucine 238 (L238) to leucine 272 (L272), or - a consecutive polypeptide sequence from histidine 218 (H218) to leucine 267 (L267) where the amino acid numbering corresponds to the amino acid sequence Q6A5X9 in the UniProt database, The DsA1 domains are defined as follows: NSR in the sequence of Q6A5X9: S29-I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, PT repeat region from P324 to T361, and CTR from S362 to F405, or any other functional DsA1 sequence containing all of these domains, particularly the DsA1 sequence of Figure 12 which contains all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence Q6A5P9: A72 to K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420 and CTR from H421 to A463 or any other functional DsA2 sequence comprising all of these domains, in particular the DsA2 sequence of Figure 12 comprising all of these domains, said vaccine.

6. A fragment or derivative must be at least a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184), preferably a consecutive polypeptide sequence from phenylalanine 150 (F150) to isoleucine 193 (I193), - a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), - a contiguous polypeptide sequence from leucine 238 (L238) to leucine 272 (L272), or - a consecutive polypeptide sequence from histidine 218 (H218) to leucine 267 (L267) 6. The vaccine of claim 5, comprising or consisting of:

7. A vaccine comprising a P. acnes putative iron transport protein (PITP polypeptide) and / or a fragment and / or derivative of PITP, wherein PITP comprises, from N-terminus to C-terminus, an extended neocarzinostatin family domain ("ENFD"), a first swapping region ("SR1"), a heme-binding domain ("HbD"), a second swapping region ("SR2") comprising a C-terminal LPXT(G) motif, and a hydrophobic C-terminal region ("hLAR"). The fragment and / or derivative comprises or consists of at least a PITP epitope, wherein the PITP domain is defined as follows in the sequence of Q6A9N1: ENFD from A32 to R164; SR1 from E165 to K237; HbD from V238 to L396; SR2 from S397 to T430 (including a C-terminal LPXT(G) motif (i.e., L PXT but not G) and hLAR from G431 to I467 or any other functional PITP sequence containing all of these domains, in particular the PITP sequence of Figure 12 containing all of these domains, said vaccine.

8. 8. The vaccine of claim 7, wherein the fragment and / or derivative is a PITP polypeptide in which hLAR has been deleted, replaced by a hydrophilic C-terminal region, or partially deleted, resulting in the loss of hLAR except for the N-terminal 12 amino acids of hLAR, preferably except for the N-terminal 11 amino acids of hLAR, in particular except for the N-terminal 10 amino acids of hLAR; or a fragment or derivative thereof comprising at least the amino acids corresponding to proline 34 to glutamic acid 73 of ENFD or proline 94 to threonine 143 of valine 238 to asparagine 393 of HbD in the amino acid sequence Q6A9N1 in the UniProt database.

9. 9. The vaccine of claim 7 or 8, wherein the PITP fragment and / or derivative is a hLAR deleted PITP polypeptide.

10. 10. The vaccine of any one of claims 7 to 9, wherein the PITP derivative comprises amino acid exchanges at positions Y63, H146, H153, F74, L141, F81, P72, K144, T143, Y75, W98, D156 and R158.

11. 11. The vaccine of any one of claims 7 to 10, wherein the PITP derivative comprises amino acid exchanges at positions C231 and C402.

12. A vaccine comprising at least two antigenic polypeptides having surface-exposed P. acnes epitopes, wherein preferably at least one epitope is a surface-exposed epitope of P. acnes dermatan sulfate-binding adhesin 1 (DsA1 polypeptide), a surface-exposed epitope of P. acnes dermatan sulfate-binding adhesin 2 (DsA1 polypeptide), or a surface-exposed epitope of P. acnes putative iron transport protein (PITP polypeptide); DsA1 or DsA2 comprises, from N-terminus to C-terminus, an N-terminal swapping region ("NSR"), a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"), a Pro-Thr repeat-containing region ("PT repeat region"), and a C-terminal region ("CTR"); and The PITP polypeptide comprises, from N-terminus to C-terminus, an extended neocarzinostatin family domain ("ENFD"), a first swapping region ("SR1"), a heme-binding domain ("HbD"), a second swapping region ("SR2") containing a C-terminal LPXT(G) motif, and a hydrophobic C-terminal region ("hLAR"); The DsA1 domains are defined as follows: NSR in the sequence of Q6A5X9: S29-I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, PT repeat region from P324 to T361, and CTR from S362 to F405, or any other functional DsA1 sequence containing all of these domains, in particular the DsA1 sequence of Figure 12 which contains all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence of Q6A5P9: A72-K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420 and CTR from H421 to A463 or any other functional DsA2 sequence containing all of these domains, in particular the DsA2 sequence of Figure 12 containing all of these domains; and / or the PITP domains are defined as follows: ENFD from A32 to R164 in the sequence of Q6A9N1; SR1 from E165 to K237, HbD from V238 to L396, SR2 from S397 to T430 (containing a C-terminal LPXT(G) motif (i.e., containing LPXT but not G)) and hLAR from G431 to I467 or any other functional PITP sequence containing all of these domains, in particular the PITP sequence of Figure 12 containing all of these domains, said vaccine.

13. A vaccine, at least one antigenic polypeptide having a surface-exposed epitope of a DsA1 polypeptide or a surface-exposed epitope of a dermatan sulfate-binding adhesin 2 (DsA2 polypeptide) of P. acnes, DsA1 or DsA2 is the at least one antigenic polypeptide comprising, from N-terminus to C-terminus, an N-terminal swapping region ("NSR"), a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3") and optionally a Pro-Thr repeat-containing region ("PT repeat region") and a C-terminal region ("CTR"); and at least one antigenic polypeptide having an epitope of a PITP polypeptide exposed on its surface, The DsA1 domains are defined as follows: NSR in the sequence of Q6A5X9: S29-I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, PT repeat region from P324 to T361 and CTR from S362 to F405 or any other functional DsA1 sequence containing all of these domains, in particular the DsA1 sequence of Figure 12 containing all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence Q6A5P9: A72-K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420, and CTR from H421 to A463, or any other functional DsA2 sequence containing all of these domains, in particular the DsA2 sequence of Figure 12 containing all of these domains, said at least one antigenic polypeptide. The vaccine comprising:

14. (a) comprising at least an epitope containing a fragment or derivative of CSD1 of DsA1 and at least an epitope containing a fragment or derivative of CSD2 of DsA2; and / or (b) comprising at least a polypeptide stretch comprising a CSD1 fragment or derivative of CSD1 of DsA1 of at least 30 amino acid residues, preferably at least 40 amino acid residues, and at least a polypeptide stretch comprising a CSD2 fragment or derivative of CSD2 of DsA1 of at least 30 amino acid residues, preferably at least 40 amino acid residues; (c) at least DsA1 - a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 184 (L184), a consecutive polypeptide sequence from phenylalanine 150 (F150) to leucine 267 (L267), or - a consecutive polypeptide sequence from histidine 218 (H218) to leucine 267 (L267), and DsA2 - A consecutive polypeptide of phenylalanine 194 (F194) to leucine 228 (L228) peptide sequence, a consecutive polypeptide sequence from phenylalanine 194 (F194) to leucine 311 (L311), or - A vaccine according to any one of claims 5 to 13, comprising a polypeptide comprising the consecutive polypeptide sequence from histidine 262 (H262) to leucine 311 (L311).

15. 15. The vaccine of any one of claims 5 to 14, wherein the polypeptide comprises amino acid exchanges at one or more of C53, C319 and C321 of DsA1 and C97 and C363 of DsA2, if present in the polypeptide, preferably one or more of the amino acid exchanges C53S, C319S and C321P of DsA1 and C97S and C363S of DsA2.

16. 16. A vaccine according to any one of claims 5 to 15, wherein in the fragment or derivative at least 5 PT repeats, preferably at least 10 PT repeats, in particular at least 15 PT repeats are deleted compared to the naturally occurring wild-type DsA1 / DsA2 polypeptide ("native DsA1 / DsA2"), and preferably at least 1, more preferably at least 2, more preferably at least 3, even more preferably at least 4, in particular 5 PT repeats are present.

17. The polypeptide further includes a PITP polypeptide or a fragment or derivative of PITP comprising a polypeptide stretch of at least 30 amino acid residues comprising at least an epitope of PITP, preferably the PITP polypeptide or fragment or derivative of PITP comprises at least - a contiguous polypeptide sequence from proline 34 to glutamic acid 73 of ENFD or its PITP derivative, - the contiguous polypeptide sequence from proline 94 to threonine 143 of ENFD or its PITP derivative, or - a continuous polypeptide sequence from valine 238 to asparagine 393 of HbD or its PITP derivative The vaccine of any one of claims 5 to 16, comprising:

18. 18. The vaccine of claim 17, wherein the PITP derivative comprises amino acid exchanges at positions C231 and C402.

19. A vaccine, - at least one synthetic antigen polypeptide having a surface-exposed epitope of a DsA1 polypeptide or a surface-exposed epitope of a DsA2 polypeptide; and - at least one synthetic antigenic polypeptide having a surface-exposed epitope of a P. acnes PITP polypeptide; The vaccine comprising:

20. A polypeptide comprising at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 1 (DsA1) and at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 2 (DsA2), wherein said DsA1 and DsA2 comprise, from N-terminus to C-terminus, an N-terminal region, a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"); and optionally, a Pro-Leu repeat-containing region ("PT repeat region") and a C-terminal region; the polypeptide comprises at least an epitope of CSD1, CSD2 or CSD3 of DsA1 and at least an epitope of CSD1, CSD2 or CSD3 of DsA2, preferably the polypeptide comprises at least CSD1, CSD2 or CSD3 of DsA1 and CSD1, CSD2 or CSD3 of DsA2; The DsA1 domains are defined as follows: NSR in the sequence of Q6A5X9: S29-I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, PT repeat region from P324 to T361, and CTR from S362 to F405, or any other functional DsA1 sequence containing all of these domains, particularly the DsA1 sequence of Figure 12 which contains all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence of Q6A5P9: A72 to K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420 and CTR from H421 to A463, or any other functional DsA2 sequence containing all of these domains, in particular the DsA2 sequence of Figure 12 containing all of these domains, said polypeptide.

21. A polypeptide comprising at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 1 (DsA1) and at least one polypeptide stretch of P. acnes dermatan sulfate-binding adhesin 2 (DsA2), wherein said DsA1 and DsA2 comprise, from N-terminus to C-terminus, an N-terminal region, a first conserved subdomain ("CSD1"), a first swapping region ("SR1"), a second conserved subdomain ("CSD2"), a second swapping region ("SR2"), a third conserved subdomain ("CSD3"); and optionally, a Pro-Leu repeat-containing region ("PT repeat region") and a C-terminal region; The DsA1 and DsA2 polypeptide stretches independently have a length of at least 20 amino acid residues; The DsA1 domains are defined as follows: NSR in the sequence of Q6A5X9: S29-I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, PT repeat region from P324 to T361, and CTR from S362 to F405, or any other functional DsA1 sequence containing all of these domains, particularly the DsA1 sequence of Figure 12 which contains all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence of Q6A5P9: A72 to K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420 and CTR from H421 to A463, or any other functional DsA2 sequence containing all of these domains, in particular the DsA2 sequence of Figure 12 containing all of these domains, said polypeptide.

22. 1. A vaccine for use in treating or preventing P. acnes-associated infections, comprising a polypeptide comprising an epitope of DsA1 and / or DsA2 and / or PITP, wherein the epitope is selected from the group consisting of R32-I41, Q38-K51, R32-K51, T43-K51, Q38-K51, R87-K90+T43-K51, R87-K90+T117-I132 and R87-K90+S234-G250, R87-K90+L2 46-A260, R87-K90+A256-E270, R87-K90+R266-T277, T117-I132, T117-A127, V128-I132, A144-N157, H146-A160, A148- N157, A156-A170, K166-L180, A176-T190, P186-A198, N181-E191, I216-F224, I216-D225, A226-A240, S234-G250, I251- I263、I251-L267、I264-P271、P236-G250、L246-A260、A256-G270、S234-G250、I251-L267、A268-G280、R266--277、T285- - - 7. R285-R286、A310-R313+R286-D290、A310-D313+V289-K296,A310-D313+V289-K296,A310-D313+T285-D313+ - 01-E307;DsA2のL152-Q166、G190-P230、I199-2208、A218--237、P230-Q244、I231-A270、H254--S27- 0.PITP's D79-T90, E73-D85, R43-I50, P68-Y75, P86-E92, I39-G45, Y84-D89, F81-D89, D79-D89, T37-E44, E73-W98, E73-F81, D79-T90, P72-F81, A129-F 138, D120-Q134, F111-D120, F132-G147, D152-E165, R115-F123, D120-K12 8, P131-F138, N181-E191, T143-T159, P116-T124, P131-D137, P131-D137, T175-C231, Q198-K203, P179-K185, G200-Q210, K174-A188, K174-K185, P2 01-Q209, P183-P201, P183-K191, K185-P195, R164-S180, E165-S180, K185 -S190, V193-N202, V193-G200, K203-P208, R216-T225, R216-R224, P173-K 191, K197-K203, P168-T175, K185-K203, R164-K174, T175-V193, S250-N26 1, D287-S300, K340-V347, D338-F352, D338-D348, S285-P288+G305-L314, S285-P288+H306-L314, S285-P288+T342-T351, S285-P288+D338-D348, D2 87-S300, T342-T351, D338-D348, H306-L314, G305-L314, G364-K375, R382 -E399, V367-G373, A383-L390, T342-T351, M387-T395, E385-T392, V401-V 410, N404-A409, G416-L427, L396-V410, T406-I415, D417-G424, V407-D418, V407-V414, K421-V429, S419-T430, D408-I415, T406-V414, preferably the epitope comprises at least one additional amino acid residue at the N- or C-terminus of the DsA1, DsA2 or PITP sequence, in particular the epitope comprises at least two additional amino acid residues at the N- or C-terminus of the DsA1, DsA2 or PITP sequence;The polypeptide is preferably covalently linked to a carrier molecule or embedded in a scaffold molecule, in particular a carrier polypeptide; The DsA1 domain is defined as follows: NSR in the sequence of Q6A5X9: S29 to I48, CSD1 from I49 to L130, SR1 from G131 to S147, CSD2 from A148 to L267, SR2 from A268 to T277, CSD3 from A278 to K323, P3 any other functional DsA1 sequence containing the PT repeat region from T24 to T361 and the CTR from S362 to F405 or all of these domains, in particular the DsA1 sequence of Figure 12 containing all of these domains; and / or the DsA2 domains are defined as follows: NSR in the sequence of Q6A5P9: A72-K92, CSD1 from I93 to L174, SR1 from S175 to S191, CSD2 from A192 to L311, SR2 from A312 to T321, CSD3 from A322 to E366, PT repeat region from P367 to T420 and CTR from H421 to A463 or any other functional DsA2 sequence containing all of these domains, in particular the DsA2 sequence of Figure 12 containing all of these domains; and / or the PITP domains are defined as follows: ENFD from A32 to R164 in the sequence of Q6A9N1; SR1 from E165 to K237, HbD from V238 to L396, SR2 from S397 to T430 (containing a C-terminal LPXT(G) motif (i.e., containing LPXT but not G)) and hLAR from G431 to I467 or any other functional PITP sequence containing all of these domains, in particular the PITP sequence of Figure 12 containing all of these domains, said vaccine.

23. The epitopes are R32-I41, Q38-K51, T43-K51, Q38-K51, R87-K90+S234-G250, R87-K90+L246-A260, R87-K90+A256-E270, R87-K90+R266-T277, T117-I132, T117-A127, A144-N157, H146-A160, A148-N157, A156- A170, K166-L180, A176-T190, P186-A198, N181-E191, I216-F224, A226-A240, S234-G250, I251-I267, I 264-P271, P236-G250, L246-A260, A256-E270, S234-G250, I251-L267, A268-L280, R266-T277, T285-D2 90, T285-D290+V291-T300, T285-D290+A301-E307, V291-T300, A301-E307, T285-T300, R286-D290+V2 91-T300, R286-D290+A301-E307, R286-T300, V289-K296, A310-D313+V289-K296, A310-D313+V289-K29 6, A310-D313+T285-T300, A310-D313+A144-N157, A310-D313+T285-R286, A310-D313+T285-D290, A310-D313+T293-E307, T285-D290, V291-T300, T293-E307, A301-E307; DsA2 L152-Q166, G190-P230, I199- D208, P230-Q244, L311-T321, L311-K323, H254-H262, Q256-H262, E261-D269, D269-S279, K313-K323;PITP's D79-T90, E73-D85, P86-E92, F81-D89, E73-W98, E73-F81, P72-F81, A129-F138, D120-Q134, P116-T124, P131-D137, Q198-K203, K174-A188, K174-K185, P1 83-P201, P183-K191, K185-P195, R216-T225, K197-K203, P168-T175, R164-K17 4, S285-P288+G305-L314, S285-P288+H306-L314, S285-P288+T342-T351, S285- 23. The vaccine for use according to claim 22, wherein the epitope is selected from the group consisting of P288+D338-D348, D287-S300, T342-T351, D338-D348, H306-L314, T406-I415, preferably the epitope comprises at least one additional amino acid residue at the N- or C-terminus of the DsA1, DsA2 or PITP sequence, in particular the epitope comprises at least two additional amino acid residues at the N- or C-terminus of the DsA1, DsA2 or PITP sequence; and the polypeptide is preferably covalently linked to a carrier or scaffold molecule, in particular a carrier polypeptide.

24. 24. DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP according to any one of claims 5 to 23, optionally contained in a vaccine or pharmaceutical formulation, comprising an N-terminal methionine amino acid residue.

25. For use in therapeutic treatment, preferably, in particular acne vulgaris, keratitis, synovitis acne pustulosis ossificans osteitis (SAPHO) syndrome, endocarditis, prosthetic joint infections, surgical wound infections, vascular graft infections, anaerobic arthritis, cardiovascular device-related infections such as prosthetic valve endocarditis; eye infections, breast implant disease, sciatica, conjunctivitis, shunt-related and / or spinal hardware central nervous system infections, shunt-related central nervous system infections, sarcoidosis, endophthalmitis, osteomyelitis, P. acnes is a P. acnes-infected organism selected from the group consisting of allergic alveolitis, rheumatoid arthritis, infectious arthritis, chronic juvenile arthritis, chronic destructive oligoarthritis, degenerative disc disease, dental infections, ulcerative colitis hyperthermia, brain abscess, subdural empyema, peritonitis, periodontitis, endodontic infections, endophthalmitis, keratitis, chronic sinusitis, folliculitis, keratitis, corneal ulcer, endophthalmitis, prostatitis, chronic prostatitis, primary biliary cirrhosis, hidradenitis suppurativa, pulmonary vasculitis, acne inversus, progressive patchy hypomelanosis, acne conglobata, atherosclerosis, prostate cancer, and medical implant biofilm infections caused by P. acnes. DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP according to any one of claims 5 to 24 for use in the treatment or prevention of acne-related infections.

26. 25. DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP according to any one of claims 5 to 24, for use in the treatment or prevention of P. acnes associated infections in human patients suffering from pathologies associated with either P. acnes types I, II or III, or with at least two phylotypes, or with a combination of at least two ribotypes, of P. acnes types I, II and III, preferably for use as a cross-reactive vaccine against P. acnes, in particular a cross-type reactive vaccine, in particular for the treatment or prevention of infections in human patients suffering from P. acnes associated infections and with pathologies associated with P. acnes types IB and III.

27. 27. The vaccine or polypeptide according to any one of claims 5 to 26, wherein DsA1 and / or DsA2 and / or PITP and / or a fragment or derivative of DsA1 or DsA2 or PITP is provided as DNA or RNA encoding the antigen or epitope containing the fragment or derivative thereof, preferably as an mRNA vaccine, in particular as an mRNA vaccine formulated with a cationic polymer, the mRNA molecule having the following structure: 5'UTR-antigen or epitope encoded by signal peptide-3'UTR; or as a vector-based vaccine, in particular using an adenovirus vector, adeno-associated virus vector, vesicular stomatitis virus vector, Newcastle disease virus vector, alphavirus vector, baculovirus vector or retrovirus vector; and / or wherein at the DNA level the coding sequence has the initial coding DNA sequence ATGGTG; in particular wherein the mRNA comprises a sequence as transcribed from SEQ ID NO: 67 to 76.

28. A pharmaceutical preparation comprising a vaccine or a polypeptide according to any one of claims 5 to 27, comprising a polypeptide according to any one of these claims, i.e. DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP according to any one of claims 1 to 27 or mixtures thereof, and a pharmaceutically acceptable excipient or carrier. The pharmaceutical preparation comprising

29. 28. A method for the treatment or prevention of P. acnes associated infections in human patients suffering from P. acnes associated infections and pathologies associated with any of types I, II or III P. acnes, or with a combination of at least two phylotypes of P. acnes types I, II and III, or with at least two ribotypes, preferably for use as a cross-reactive vaccine against P. acnes, in particular a cross-type reactive vaccine, for the treatment or prevention of infections in human patients suffering from P. acnes associated infections and pathologies associated with types IB and III P. acnes, comprising the administration to a patient in need thereof of an effective amount of DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP or a mixture thereof according to any one of claims 5 to 27.

30. 30. The method according to claim 29, wherein the administration is carried out by intradermal, subcutaneous (s.c.), parenteral, intramuscular (i.m.), mucosal, transdermal or topical administration, preferably by intradermal or intramuscular administration, in particular by syringe or by microneedling device.

31. 29. Methods for producing the polypeptides, vaccines and formulations according to any one of claims 5 to 28, wherein a polypeptide comprising at least one P. acnes epitope according to these claims is expressed in host cells, extracted from these host cells, purified, and optionally formulated and finished into a pharmaceutical preparation, in particular a vaccine, for use in the treatment or prevention of P. acnes-associated infections in human patients.

32. Use of DsA1 and / or DsA2 and / or PITP and / or fragments and / or derivatives of DsA1 and / or DsA2 and / or PITP or mixtures thereof according to any one of claims 5 to 27 for the manufacture of a medicament for the treatment or prevention of P. acnes associated infections in human patients suffering from pathologies associated with any of types I, II or III P. acnes, or with at least two phylotypes, or a combination of at least two ribotypes, of types I, II and III of P. acnes, preferably for use as a cross-reactive vaccine against P. acnes, in particular a cross-type reactive vaccine, in particular for the treatment or prevention of infections in human patients suffering from P. acnes associated infections and with pathologies associated with types IB and III of P. acnes.

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