Composition for use in the treatment of acne

Abstract

The present invention relates to compositions (e.g., immunogenic compositions) that can be used to immunize against C. acnes. The compositions include C. acnes antigens and combinations of antigens, used in the form of nucleic acids encoding antigen proteins (e.g., mRNA) or recombinant protein antigens.

Description

[Technical Field]

[0001] The present invention relates to the treatment and prevention of Cutibacterium acnes (C. acnes) infections, such as acne vulgaris. In particular, the present invention relates to antigens and antigen combinations that can be used to immunize against C. acnes, used in the form of nucleic acids (e.g., mRNA) encoding antigenic proteins or recombinant protein antigens. [Background technology]

[0002] Cutibacterium acnes (C. acnes, formerly known as Propionibacterium acnes) is a Gram-positive bacterium known to be involved in skin diseases such as acne vulgaris. C. acnes is a symbiotic bacterium of the skin, mainly residing in sebaceous hair follicles, which provide a unique environment rich in lipids due to sebum secretion. In addition to the skin, C. acnes is also found in the conjunctiva, respiratory tract, urogenital tract, and gastrointestinal tract of humans and other animals.

[0003] Acne is a disease of the follicular sebaceous glands of the skin, affecting over 85% of adolescents, with over 20% of the population continuing to experience symptoms well into their teens. Acne vulgaris can manifest in varying degrees of severity: mild, moderate, and severe. Moderate and severe acne accounts for more than one-third of all cases and requires medical treatment. Acne can also appear as a symptom in adulthood after puberty, often associated with hormonal fluctuations, which are more common in women. C. acnes is also thought to be involved in other severe forms of acne, such as acne clusters, fulminant acne, and cystic acne. In addition to skin conditions, C. acnes is also found in corneal ulcers and is a common cause of chronic endophthalmitis after cataract surgery. Postoperative infections related to artificial implants and devices (implant-associated infections), endocarditis, sarcoidosis, osteomyelitis, allergic alveolitis, pulmonary vasculitis, SAPHO syndrome (synovitis, acne, pustulosis, hyperossification, osteitis), and various other inflammatory diseases, including inflammation of the lumbar nerve roots causing sciatica, have been associated with C. acnes. More recent studies have also suggested a potential pathogenic role of C. acnes in non-infectious diseases such as prostate cancer, due to its ability to persist within cells and potentially lead to altered gene expression.

[0004] Phylogenetic studies based on multilocus gene sequencing, as well as whole-genome analyses of isolates from the Human Microbiome Project (HMP), provide valuable insights into the genetic population structure of C. acnes. Multilocus sequence typing (MLST) generates different nucleotide sequences defined as individual alleles, which are then used to generate a sequence type (ST) for each C. acnes isolate. Two different MLST typing schemes have been developed to divide C. 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), according to the MLST8 or MLST9 typing schemes, respectively. Regarding therapeutics, some have suggested that only strains of type IA1 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 strains of type IA1 are most commonly isolated from the skin of acne patients, while other strains can be found in both acne-prone and healthy skin. However, other lineages have also been shown to be associated with acne (International Publication No. 2021 / 165543). For example, C. acnes strains derived from lineages IA2, IC, or II have been isolated from inflammatory lesions in human subjects (International Publication No. 2021 / 165543).

[0005] C. acnes strains can be grouped using genomic sequencing of 16S rDNA sequences, known as ribotypes (RT). This system allows for individual comparison of C. acnes strain populations based on 16S rDNA sequences. While the top 10 major ribotypes are extremely abundant, a considerable number of rare ribotypes were also identified. Analysis of the top 10 ribotypes allowed for the identification of both disease-specific and health-specific associations (Fitz-Gibbon et al. 2013, Tomida et al. 2013, McLaughlin et al. 2019). The three most abundant ribotypes (RT1, RT2, and RT3) were fairly evenly distributed between acne-prone and normal individuals. However, ribotypes RT4, RT5, RT7, RT8, RT9, and RT10 were mostly found in acne patients, while RT6 was strongly associated with normal skin. Phylogenetic trees based on the locations of unique single nucleotide polymorphisms in the core genomes obtained from these 71 C. acnes genomes showed that 16S rDNA ribotypes represent phylogenetic relationships to a considerable extent, suggesting that 16S rDNA sequences are useful molecular markers for identifying major C. acnes lineages (Fitz-Gibbon et al. 2013, Tomida et al. 2013). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Current treatments target one or two steps in the pathogenesis of acne and include benzoyl peroxide, topical retinoids, and topical or oral antibiotics. Nevertheless, antibiotic resistance is a concern in acne. In addition, oral isotretinoin, which is recommended for more severe cases, has been associated with adverse effects, the most serious of which is teratogenicity. An effective C. acnes vaccine is needed. [Means for solving the problem]

[0007] The present inventors have provided C. acnes antigens and combinations of antigens that can be used to immunize against C. acnes.

[0008] In particular, the inventors have shown that antigens derived from C. acnes CAMP2, DsA1, DsA2, and / or PITP polypeptides, either individually or in combination, induce an antibody response when delivered by mRNA encoding the relevant antigen or in the form of recombinant polypeptides.

[0009] Accordingly, the present invention provides C. acnes CAMP2 polypeptide, modified C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide and chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide, as well as nucleic acids comprising nucleotide sequences encoding such polypeptides.

[0010] The polypeptide antigens described herein may be delivered by nucleic acids (e.g., mRNA) containing the nucleotide sequence encoding the polypeptide, i.e., in that form, or as recombinant proteins, i.e., in the form of recombinant proteins.

[0011] CAMP2 Christie-Atkins-Münch-Petersen (CAMP) factor 2, or CAMP2, is a polypeptide belonging to the CAMP factor superfamily, whose members are known for their co-hemolytic and cytotoxic properties. C. acnes CAMP factors 1-5 (or CAMP1-5) are thought to be the causative agents of the co-hemolytic reaction between C. acnes and both sheep and human erythrocytes (Choudhury, 1978; Soerensen et al, Journal of Microbiological Methods (2010) 83(2):211-216). The amino acid sequences of C. acnes CAMP factors 1-5 have a low degree of sequence identity among them (for example, the amino acid sequences of CAMP2 and CAMP4 are about 50%, and even lower for other amino acid sequences). The naturally occurring C. acnes CAMP2 polypeptide is a secreted protein.

[0012] CAMP2 has been identified as a putative acne-associated virulence factor (Holland et al (2010)). Expression and secretion of CAMP protein by different C. acnes strains have been suggested as one of the factors contributing to C. acnes' resistance to opsonin phagocytic injury. CAMP2 has been reported to induce phagocytic cell injury in in vitro assays, including co-culture with C. acnes (Wang et al. 2018). Furthermore, anti-CAMP2 neutralizing antibodies have been shown to significantly reduce C. acnes-induced inflammation in a mouse ear model (Liu et al. 2011, Vaccine, 29;3230-3238).

[0013] The inventors recognized that the C. acnes CAMP2 polypeptide can be used to induce an immune response to C. acnes infection. In particular, the inventors demonstrated that the C. acnes CAMP2 polypeptide induces an antibody (e.g., IgG) response. Notably, the C. acnes CAMP2 polypeptide induced an antibody (e.g., IgG) response not only when delivered as antigen-encoding mRNA but also when delivered in the form of a recombinant protein. Furthermore, the inventors demonstrated that an antibody (e.g., IgG) response is induced when mRNA encoding the C. acnes CAMP2 polypeptide is delivered in lipid nanoparticles (LNPs). Antibodies induced by the C. acnes CAMP2 polypeptide reduced the co-hemolytic activity of CAMP2. Therefore, C. acnes CAMP2 polypeptide can induce antibodies in a target that can neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2.

[0014] Accordingly, the inventors have demonstrated that the C. acnes CAMP2 polypeptide (delivered, for example, in the form of antigen-encoding mRNA or as a recombinant protein) is a suitable vaccine antigen that can be used alone or in combination with one or more of the other C. acnes antigens described herein, such as the C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide and chimeric C. acnes DsA1 / DsA2 / PITP polypeptide or chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide (delivered, for example, in the form of antigen-encoding mRNA or as a recombinant protein) described herein. CAMP2 polypeptides induce antibodies that neutralize the inflammatory activity of CAMP2, while immunization with one or more of these other C. acnes antigens induces antibodies that bind to the surface of C. acnes bacteria, thereby recruiting effector cells (e.g., effector cells of the immune system, such as phagocytes, or the innate immune system).

[0015] Thus, in one aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide. Typically, the nucleic acid is messenger RNA (mRNA). In a preferred embodiment, the nucleic acid is a non-naturally occurring nucleic acid, such as non-naturally occurring mRNA. Thus, the nucleic acid can be a synthetic nucleic acid (e.g., synthetic mRNA). In a further aspect, the present invention provides a polypeptide comprising the amino acid sequence of a C. acnes CAMP2 polypeptide. For example, the C. acnes CAMP2 polypeptide for use in the present invention, as delivered as mRNA and / or in LNP, can induce antibodies in a subject. Such antibodies can neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2. Such antibodies can neutralize the co-hemolytic activity of the CAMP2 polypeptide. Neutralization of the inflammatory activity of CAMP2 can be determined as a decrease in pro-inflammatory cytokines such as IL-1b and / or IL-6. Neutralization of the co-hemolytic activity can be determined by an in vitro co-hemolytic assay.

[0016] The amino acid sequence of the native C. acnes CAMP2 antigen is shown in SEQ ID NO: 202.

Chemical formula

[0017] The native signal peptide sequence corresponds to residues 1-28 of the amino acid sequence of CAMP2 of C. acnes (SEQ ID NO: 202) and is underlined. Residues 29-267 of SEQ ID NO: 202 correspond to the full-length CAMP2 polypeptide of mature C. acnes (excluding the native signal peptide sequence).

[0018] The N-terminal and C-terminal domains of C. acnes CAMP2 are shown in bold (N-terminal domain) and bold and underlined (C-terminal domain). The N-terminal domain of the C. acnes CAMP2 polypeptide corresponds to amino acid residues 29-176 of the C. acnes CAMP2 polypeptide in SEQ ID NO: 202. The C-terminal domain of the C. acnes CAMP2 polypeptide corresponds to amino acid residues 189-267 of the C. acnes CAMP2 polypeptide in SEQ ID NO: 202. A linker domain is located between the N-terminal and C-terminal domains. The linker domain corresponds to amino acid residues 177-188 of SEQ ID NO: 202.

[0019] "C. acnes CAMP2 polypeptide" includes the full-length, mature form of the natural C. acnes CAMP2 polypeptide and its immunogenic variants, without its native signal peptide sequence. Immunogenic variants of the natural C. acnes CAMP2 polypeptide can induce an immune response (e.g., antigen-specific immune response), such as an antibody response, in a target. Immunogenic variants of the natural C. acnes CAMP2 polypeptide include immunogenic fragments of the natural C. acnes CAMP2 polypeptide. Immunogenic C. acnes CAMP2 fragments include fragments of the natural C. acnes CAMP2 polypeptide with an amino acid length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 225 amino acids.

[0020] Exemplary C. acnes (C. acnes) CAMP2 polypeptide sequences include the natural C. acnes (C. acnes) CAMP2 polypeptide sequence of SEQ ID NO: 203, which lacks the natural signal peptide sequence of the C. acnes (C. acnes) CAMP2 polypeptide. Other exemplary C. acnes (C. acnes) polypeptide sequences are presented in SEQ ID NOs: 313–338. These are naturally occurring CAMP2 polypeptides derived from different strains of C. acnes (C. acnes) and contain the natural secretion signal peptide sequence of the C. acnes (C. acnes) CAMP2 polypeptide. Corresponding sequences of CAMP2 polypeptides that do not contain the natural secretion signal peptide sequence are shown in SEQ ID NOs: 339–363.

[0021] Analysis of 430 CAMP2 polypeptide sequences derived from naturally occurring strains of C. acnes revealed that when the naturally occurring secretory signal peptide sequence was included in the alignment, only 27 distinct CAMP2 sequences (SEQ ID NOs. 202 and 313-338) existed. When the naturally occurring secretory signal peptide sequence was excluded, only 26 distinct CAMP2 sequences (SEQ ID NOs. 203 and 339-363) existed. Sequence mutations among these CAMP2 polypeptides were concentrated in specific residues (Figure 44 and Table 1).

[0022] [Table 1]

[0023] [Table 2]

[0024] In some embodiments, the C. acnes CAMP2 polypeptide contains amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 203. In some embodiments, the C. acnes CAMP2 polypeptide contains residues T6 (e.g., T6I), A9 (e.g., A9T), S11 (e.g., S11A), S18 (e.g., S18N), D19 (e.g., D19E or D19Y), R21 (e.g., R21H), I24 (e.g., I24M, I24L or I24T), A29 (e.g., A29P), H30 (e.g., H30R), V37 (e.g., V37A), D48 (e.g., D48N), R61 (e.g., R61H), E65 (e.g., E65D), A68 (e.g., A68T), D76 (e.g., D76N), V87 (e.g., V87A), I91 (e.g., I91V), D92 (e.g., D92G), T98 (e.g., T98K), T100 (e.g., T100I), R106 (e.g., R106S), K118 (e.g., K118N), S128 (e.g., S128T), A138 (e.g., A138T), R143 (e.g., R143H), E145 (e.g., E145D or E145K), T154 (e.g., T154A) The amino acid substitution includes one or more amino acid substitutions at the positions corresponding to ), K169 (e.g., K169R), N177 (e.g., N177D), D179 (e.g., D179N or D179H), A189 (e.g., A189E), N207 (e.g., N207D or N207A), E221 (e.g., E221K), and / or L223 (e.g., L223F).

[0025] In some embodiments, the C. acnes CAMP2 polypeptide includes one of the sequences of SEQ ID NO: 203, SEQ ID NOs. 43-58, SEQ ID NOs. 1-4, SEQ ID NOs. 10-16, or SEQ ID NOs. 339-363 (for example, SEQ ID NO: 203), or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Typically, sequence mutations are located at the positions listed above, namely residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of sequence number 203. Accordingly, in some embodiments, the C. acnes CAMP2 polypeptide contains amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 203. In some embodiments, the C. acnes CAMP2 polypeptide contains a sequence having at least 90% identity with any one of SEQ ID NOs: 203, SEQ ID NOs: 43-58, SEQ ID NOs: 1-4, SEQ ID NOs: 10-16, or SEQ ID NOs: 339-363 (e.g., SEQ ID NO: 203). In some embodiments, the C. acnes CAMP2 polypeptide comprises a sequence having at least 95% identity with one of sequence numbers 203, 43-58, 1-4, 10-16, or 339-363 (e.g., sequence number 203).Typically, the C. acnes CAMP2 polypeptide contains a sequence having at least 85% identity with one of the following: SEQ ID NO: 203, SEQ ID NOs: 43-58, SEQ ID NOs: 1-4, SEQ ID NOs: 10-16, or SEQ ID NOs: 339-363 (e.g., SEQ ID NO: 203). In some embodiments, the C. acnes CAMP2 polypeptide contains, is essentially derived from, or consists of (e.g., contains) the amino acid sequence of SEQ ID NO: 203.

[0026] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding the C. acnes CAMP2 polypeptide comprises a nucleotide sequence of any one of SEQ ID NOs. 153-167, SEQ ID NOs. 87-89, or SEQ ID NOs. 95-101, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 75% identical to any one of SEQ ID NOs. 153-167, SEQ ID NOs. 87-89, or SEQ ID NOs. 95-101.

[0027] Modified CAMP2 The inventors have generated a modified C. acnes (C. acnes) CAMP2 polypeptide containing a C. acnes (C. acnes) CAMP2 polypeptide sequence and a heterologous transmembrane domain (TMB) sequence. The inclusion of a heterologous TMB sequence may be advantageous because, when the antigen is expressed in cells, such as eukaryotic cells (e.g., the target cell), the sequence can localize the antigen to the cell membrane. By including a heterologous TMB sequence, intracellular localization of the antigen can be reduced compared to antigens without a TMB sequence. The inventors have shown that such a modified C. acnes (C. acnes) CAMP2 polypeptide containing a TMB domain sequence induces an antibody (e.g., IgG) response. Antibodies induced by the modified C. acnes (C. acnes) CAMP2 polypeptide reduced the co-hemolytic activity of the C. acnes (C. acnes) CAMP2 polypeptide. C. acnes CAMP2 polypeptide can induce antibodies in a target that can neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2.

[0028] Furthermore, the modified C. acnes (C. acnes) CAMP2 polypeptide induced higher antibody titers compared to the C. acnes (C. acnes) CAMP2 polypeptide lacking the TMB domain. Therefore, the inclusion of the TMB sequence may enhance the immunogenicity (e.g., antibody response) of the C. acnes (C. acnes) CAMP2 polypeptide in a target. By including the TMB sequence in the modified C. acnes (C. acnes) CAMP2 polypeptide, a stronger antibody response, such as a higher antibody titer, may be induced in a target compared to the CAMP2 polypeptide without the TMB sequence. Surface-expressed antigens (e.g., modified C. acnes (C. acnes) CAMP2 polypeptide) can enhance B cell activation in a target, thereby enhancing the antigen-specific B cell response of the target (e.g., enhanced antigen-specific antibody response, such as a higher antibody titer).

[0029] Accordingly, the inventors have demonstrated that these modified C. acnes CAMP2 polypeptides are suitable vaccine antigens that can be used alone or in combination with one or more of the other C. acnes antigens described herein, such as the C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, and chimeric C. acnes DsA1 / DsA2 / PITP polypeptide. When used alone or in combination with one or more of the other antigens described herein, these modified C. acnes CAMP2 polypeptides can induce an immune response to infection. Modified C. acnes CAMP2 polypeptides, as described herein, can be delivered by nucleic acids containing a nucleotide sequence encoding the modified C. acnes CAMP2 polypeptide.

[0030] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a modified C. acnes (C.acnes)CAMP2 polypeptide, wherein the modified C. acnes (C.acnes)CAMP2 polypeptide comprises an amino acid sequence comprising a C. acnes (C.acnes)CAMP2 polypeptide sequence and a heterologous transmembrane domain sequence. The heterologous TMB sequence can localize the antigen to the cell membrane when the antigen is expressed in a cell (e.g., a eukaryotic cell such as a target cell). This can enhance the immunogenicity (e.g., antibody response such as antibody titer) induced in the target compared to, for example, an antigen lacking the TMB sequence.

[0031] In a further embodiment, the present invention provides a polypeptide comprising the amino acid sequence of a modified C. acnes CAMP2 polypeptide.

[0032] In certain embodiments, the heterogeneous transmembrane domain sequence is a TMB sequence (eukaryotic transmembrane sequence) of a eukaryotic transmembrane polypeptide, a TMB sequence (prokaryotic transmembrane sequence) of a prokaryotic transmembrane polypeptide, or a TMB sequence (viral transmembrane domain sequence) of a viral transmembrane protein. In certain embodiments, the heterogeneous transmembrane domain is a TMB sequence of a viral transmembrane protein.

[0033] In certain embodiments, the heterologous TMB sequence is located at the N-terminus of the modified C. acnes CAMP2 polypeptide. In other embodiments, the heterologous TMB sequence is located at the C-terminus of the modified C. acnes CAMP2 polypeptide.

[0034] Typically, the nucleic acids described herein that encode a modified C. acnes CAMP2 polypeptide containing a transmembrane domain also include nucleotide sequences that encode a secretion signal peptide sequence.

[0035] Further details regarding TMB domains suitable for use in the present invention are given below in this specification. Exemplary TMB sequences are described below in this specification.

[0036] In certain embodiments, the heterologous transmembrane domain includes a hemagglutinin (HA) transmembrane domain sequence derived from influenza A or influenza B virus, preferably influenza A virus.

[0037] In certain embodiments, the transmembrane domain (TMB) sequence is directly fused to the C. acnes CAMP2 polypeptide described herein (i.e., in the modified C. acnes CAMP2 polypeptide described herein, there is no linker, such as an amino acid linker, connecting the TMB sequence to the C. acnes CAMP2 polypeptide). In other embodiments of the modified C. acnes CAMP2 polypeptide described herein, the TMB sequence of this disclosure is linked to the C. acnes CAMP2 polypeptide described herein by a linker. Preferred linkers are presented in the “Linkers” section below.

[0038] In some embodiments, the modified C. acnes (C. acnes) CAMP2 polypeptide includes a C. acnes (C. acnes) CAMP2 polypeptide sequence comprising a sequence such as SEQ ID NO: 203, SEQ ID NOs: 43-58, SEQ ID NOs: 1-4, SEQ ID NOs: 10-16, or SEQ ID NOs: 339-363 (e.g., SEQ ID NO: 203), or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Typically, mutations in the C. acnes (C. acnes) CAMP2 polypeptide sequence may be located at the positions presented above herein. Accordingly, C. acnes CAMP2 polypeptide sequence mutations can typically be located at the positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 203, as presented above herein. In some embodiments, the C. acnes CAMP2 polypeptide sequence contains amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 203.In some embodiments, the C. acnes CAMP2 polypeptide contains residues T6 (e.g., T6I), A9 (e.g., A9T), S11 (e.g., S11A), S18 (e.g., S18N), D19 (e.g., D19E or D19Y), R21 (e.g., R21H), I24 (e.g., I24M, I24L or I24T), A29 (e.g., A29P), H30 (e.g., H30R), V37 (e.g., V37A), D48 (e.g., D48N), R61 (e.g., R61H), E65 (e.g., E65D), A68 (e.g., A68T), D76 (e.g., D76N), V87 (e.g., V87A), I91 (e.g., I91V), D92 (e.g., D92G), T98 (e.g., T98K), T100 (e.g., T100I), R106 (e.g., R106S), K118 (e.g., K118N), S128 (e.g., S128T), A138 (e.g., A138T), R143 (e.g., R143H), E145 (e.g., E145D or E145K), T154 (e.g., T154A) The modified C. acnes CAMP2 polypeptide includes amino acid substitutions at one or more positions corresponding to ), K169 (e.g., K169R), N177 (e.g., N177D), D179 (e.g., D179N or D179H), A189 (e.g., A189E), N207 (e.g., N207D or N207A), E221 (e.g., E221K), and / or L223 (e.g., L223F). In some embodiments, the modified C. acnes CAMP2 polypeptide includes a C. acnes CAMP2 polypeptide sequence having at least 90% identity with one of SEQ ID NOs. 203, SEQ ID NOs. 43-58, SEQ ID NOs. 1-4, SEQ ID NOs. 10-16, or SEQ ID NOs. 339-363 (e.g., SEQ ID NO. 203). In some embodiments, the modified C. acnes CAMP2 polypeptide includes a C. acnes CAMP2 polypeptide sequence having at least 95% identity with one of sequence numbers 203, 43-58, 1-4, 10-16, or 339-363 (e.g., sequence number 203).Typically, a modified C. acnes CAMP2 polypeptide contains a C. acnes CAMP2 polypeptide sequence that has at least 85% identity with one of the following sequences: SEQ ID NO: 203, SEQ ID NOs: 43-58, SEQ ID NOs: 1-4, SEQ ID NOs: 10-16, or SEQ ID NOs: 339-363 (e.g., SEQ ID NO: 203).

[0039] In some embodiments, the modified C. acnes CAMP2 polypeptide includes the sequence represented by SEQ ID NO: 207 or any one of SEQ ID NOs 5-9 (e.g., SEQ ID NO: 207) or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Typically, sequence mutations are located at the positions shown above herein, namely at residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 207. Accordingly, in some embodiments, the modified C. acnes CAMP2 polypeptide includes amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 with respect to the residue numbering of SEQ ID NO: 207. In some embodiments, the modified C. acnes CAMP2 polypeptide includes a sequence having at least 90% identity with any one of SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NOs: 43-58, SEQ ID NOs: 1-16, or SEQ ID NOs: 339-363 (e.g., SEQ ID NO: 207). In some embodiments, the modified C. acnes CAMP2 polypeptide contains a sequence having at least 95% identity with one of sequence numbers 203, 207, 43-58, 1-16, or 339-363 (e.g., sequence number 207).In some embodiments, the modified C. acnes CAMP2 polypeptide contains a sequence having at least 85% identity with one of sequence numbers 203, 207, 43-58, 1-16, or 339-363 (e.g., sequence number 207).

[0040] In some embodiments, nucleic acids comprising nucleotide sequences encoding modified C. acnes CAMP2 polypeptide include nucleotide sequences from any one of SEQ ID NOs. 153-167, SEQ ID NOs. 87-89, or SEQ ID NOs. 95-101, or sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a modified C. acnes CAMP2 polypeptide comprises a nucleotide sequence of any one of SEQ ID NOs. 90-94 or SEQ ID NOs. 391-392 (e.g., SEQ ID NO. 91) or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 75% or 85% (e.g., 75%) identical to any one of SEQ ID NOs. 153-167, SEQ ID NOs. 391-392, or SEQ ID NOs. 87-101 (e.g., SEQ ID NO. 91).

[0041] The sequences of SEQ ID NOs. 90, 91, 391, and 392 were aligned. Mutations were found at the indicated positions in SEQ ID NOs. 398 and 399. In some embodiments, nucleic acids containing nucleotide sequences encoding the modified C. acnes CAMP2 polypeptide include the nucleotide sequence according to SEQ ID NO. 398, with nucleotides at positions 66, 78, 81, 87, 102, 120, 150, 222, 232, 234, 261, 298, 321, 339, 351, 367, 420, 423, 435, 438, 456, 462, 468, 478, 516, 528, 541, 546, 574, 583, 648, 664, 684, 706, 711, 735, and 834. The nucleotides at positions 480, 660, and 849 are independently selected from A, C, or G, respectively, the nucleotides at positions 171, 282, 318, 375, 432, 675, and 738 are independently selected from A, C, or T, respectively, the nucleotides at positions 6, 57, 93, 114, 168, 246, 300, 363, 384, 396, 405, 447, 459, 543, 585, 591, 609, 666, 708, 714, and 857 are independently selected from A or G, respectively, and 73 and 9 The nucleotides at positions 7, 103, 118, 169, 181, 190, 283, 433, 493, 553, 745, 769, 775, 793, 805, 808, 826, 841, and 850 were independently selected from A or T, respectively, and the nucleotides at positions 18, 27, 54, 98, 119, 153, 162, 170, 182, 191, 210, 213, 240, 264, 284, 312, 369, 390, 408, 434, 441, 483, 494, 554, 573, 603, 690, 746, 770, 774, 776, 794, 806, and 809 were selected. The nucleotides at positions 827, 842, 851, and 855 were independently selected from C or G, respectively, and the nucleotides at positions 30, 33, 36, 48, 51, 108, 111, 117, 123, 135, 177, 183, 186, 189, 207, 219, 225, 243, 255, 258, 267, 270, 276, 279, 285, 297, 327, 372, 378, 402, 426, 429, 448, 453, 465, 477, 501, 507, 519, 522, 531, 537, 549, 564, 582, 594, 615, and 624,The nucleotides at positions 636, 672, 693, 696, 702, 729, 741, 750, 753, 771, 780, 795, 804, 807, 810, 828, 837, 840, and 852 are independently selected from C or T, respectively. In some embodiments, a nucleic acid containing a nucleotide sequence encoding a modified C. acnes CAMP2 polypeptide includes the nucleotide sequence according to SEQ ID NO: 399, where the nucleotides at positions 66, 81, 87, 102, 222, 232, 234, 261, 298, 321, 339, 351, 420, 438, 456, 462, 468, 478, 516, 528, 541, 546, 574, 583, 648, 660, 664, 684, 706, 711, 735, and 834 are A or C. Each of these is independently selected, the nucleotide at position 849 is selected from A, C, or G, the nucleotides at positions 318, 432, and 675 are independently selected from A, C, or T, the nucleotides at positions 6, 57, 93, 168, 300, 363, 384, 396, 405, 447, 459, 480, 543, 585, 609, 666, 708, 714, and 857 are independently selected from A or G, and the nucleotides at positions 73, 97, 103, 118, 169, 181, 283, 433, 493, 745, and 76 The nucleotides at positions 9, 793, 805, 808, 826, 841, and 850 were independently selected from A or T, respectively, and the nucleotides at positions 18, 27, 54, 98, 119, 153, 170, 182, 213, 264, 284, 312, 369, 390, 408, 434, 483, 494, 573, 603, 746, 770, 774, 794, 806, 809, 827, 842, 851, and 855 were independently selected from C or G, respectively, and the nucleotides at positions 30, 33, 36, 48, 117, 123, and 13 The nucleotides at positions 5, 171, 177, 183, 186, 189, 225, 243, 255, 258, 267, 270, 276, 282, 285, 297, 327, 372, 375, 378, 426, 453, 465, 477, 501, 519, 522, 531, 549, 564, 582, 624, 636, 672, 693, 696, 738, 741, 750, 753, 771, 780, 795, 807, 810, 828, 837, 840, and 852 are independently selected from C or T, respectively. Typically,Nucleic acids containing the nucleotide sequence of SEQ ID NO: 398 or 399 encode the sequence of SEQ ID NO: 6.

[0042] In some embodiments, the nucleic acid contains a nucleotide sequence that is at least 75% or 85% (e.g., 75%) identical to one of the sequence numbers 153-167, 391-392, or 87-101 (e.g., sequence number 91).

[0043] In one embodiment, the nucleic acid of the present invention is mRNA comprising or consisting of the following structural elements (for example, consisting of the following): (i) A 5' cap having the following structure: [ka] (ii) The 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO. 265, (iii) A protein coding region having a nucleic acid sequence by SEQ ID NO: 90, SEQ ID NOs: 391-392, or SEQ ID NO: 91 (for example, SEQ ID NO: 91), (iv) The 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides.

[0044] In some embodiments, the mRNA is chemically modified, and this modification includes N1-methylpseudridine instead of any uridine. The mRNA can be encapsulated in LNPs.

[0045] DsA1 Dermatan sulfate-adhesin 1 (DsA1) of C. acnes, also known as P22 or P022, has been identified as a putative pathogenic factor of C. acnes (Lodes et al, 2006 (Microbiology (Reading). 2006 Dec;152(Pt 12):3667-3681), McDowell et al., 2011). This protein is abundant in both acne-affected and healthy hair follicle samples (Bek-Thomsen et al., 2014). The C. acnes DsA1 polypeptide, its derivatives and fragments are described in International Publication No. 2021 / 165543.

[0046] The inventors demonstrated that mRNA encoding different C. acnes (C. acnes) DsA1 polypeptides and recombinant C. acnes (C. acnes) DsA1 protein induces an antibody (e.g., IgG) response. Antibodies induced by the C. acnes (C. acnes) DsA1 polypeptide were shown to bind to the surface of C. acnes bacteria and recruit effector cells (e.g., immune system effector cells such as phagocytic cells). Antibodies induced by the C. acnes (C. acnes) DsA1 polypeptide were shown to induce opsonin phagocytic injury in vitro.

[0047] The C. acnes DsA1 polypeptide of the present invention can induce an immune response (cross-reactive immune response) to a wide range of different C. acnes strains, lineages, and variants that cause disease.

[0048] The inventors have demonstrated that C. acnes (C. acnes) DsA1 polypeptide is a suitable vaccine antigen that can be used in combination with one or more other C. acnes antigens capable of inducing an antibody response in a target, such as the C. acnes (C. acnes) DsA2 polypeptide described herein, the C. acnes (C. acnes) PITP polypeptide described herein, and the C. acnes (C. acnes) CAMP2 polypeptide described herein. The C. acnes (C. acnes) CAMP2 polypeptide can induce antibodies in a target, such as antibodies capable of neutralizing the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2.

[0049] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA1 polypeptide. In a further embodiment, the present invention provides a polypeptide comprising the amino acid sequence of the C. acnes (C. acnes) DsA1 polypeptide. For example, the C. acnes (C. acnes) DsA1 polypeptide for use in the present invention, delivered as mRNA or recombinant protein, can induce antibodies in a subject. Such antibodies can opsonize C. acnes bacteria. Opsonization allows immune effector cells, such as phagocytic cells, to target C. acnes bacteria. This results in damage to C. acnes bacteria by such immune effector cells (e.g., phagocytic damage). Antibody binding to the cell surface of C. acnes (C. acnes) can be measured, for example, using an in vitro surface binding assay. An in vitro opsonization-phagocytic injury assay can be used to evaluate the ability of antibodies to induce opsonization and injury of different genotypes of C. acnes strains using phagocytic cells.

[0050] The amino acid sequence (excluding the signal peptide sequence) of the full-length C. acnes DsA1 polypeptide in its natural mature form is shown in SEQ ID NO: 204. [ka]

[0051] Naturally mature C. acnes (C. acnes) DsA1 polypeptide typically contains an N-terminal swap region ("NSR"), a first conserved subdomain ("CSD1"), a first swap region ("SR1"), a second conserved subdomain ("CSD2"), a second swap region ("SR2"), a third conserved subdomain ("CSD3"), a Pro-Thr repeat-containing region ("PT repeat region," shown in bold above), and a C-terminal region ("CTR," often containing an LPXTG motif near the C-terminus). The C-terminal LPXTG motif is considered crucial for the protein's anchoring to the cell wall.

[0052] For example, in the natural C. acnes DsA1 polypeptide of sequence number 204, the N-terminal swap region ("NSR") corresponds to amino acid residues 1-20, the first conserved subdomain ("CSD1") corresponds to amino acid residues 21-102, the first swap region ("SR1") corresponds to amino acid residues 103-119, and the second conserved subdomain ("CSD2") corresponds to amino acid residues 120-239. Accordingly, the second swap region ("SR2") corresponds to amino acid residues 240-249, the third conserved subdomain ("CSD3") corresponds to amino acid residues 250-295, the Pro-Thr repeat-containing region ("PT repeat region") corresponds to amino acid residues 296-333, and the C-terminal region ("CTR") corresponds to amino acid residues 334-377 (including the LPXTG motif corresponding to amino acid residues 372-376).

[0053] The sequences of naturally occurring DsA1 polypeptides are largely highly identical, differing only in the length and composition of the PT repeat region (with the exception of point mutations and rare exceptions, specifically the presence of terminal LPXTG motifs). A more detailed sequence analysis of DsA1 is described elsewhere (International Publication No. 2021 / 165543). As is well known in the art, the locations of the NSR, CSD1, SR1, CSD2, SR2, CSD3, PT repeat region, and CTR within other naturally occurring C. acnes (C. acnes) DsA1 polypeptides can be determined by aligning the polypeptide sequence of a given DsA1 polypeptide with the polypeptide sequence of SEQ ID NO: 204 and identifying the regions of the DsA1 polypeptide that have high sequence identity with the polypeptide sequences of the NSR, CSD1, SR1, CSD2, SR2, CSD3, PT repeat region, and CTR of SEQ ID NO: 204.

[0054] "C. acnes DsA1 polypeptide" includes the full-length, mature form of the natural DsA1 polypeptide of C. acnes, without its native signal peptide sequence, and its immunogenic variants. Immunogenic variants of the natural C. acnes DsA1 polypeptide can induce an immune response (e.g., antigen-specific immune response), such as an antibody response, in a target. Immunogenic variants of the natural C. acnes DsA1 polypeptide include immunogenic fragments of the natural C. acnes DsA1 polypeptide. The immunogenic C. acnes (C. acnes) DsA1 fragments include fragments of the native C. acnes (C. acnes) DsA1 polypeptide having an amino acid length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 amino acids. In some embodiments, the immunogenic C. acnes (C. acnes) DsA1 fragments include the CSD2 domain of the C. acnes (C. acnes) DsA1 polypeptide. In some embodiments, the immunogenic variants exclude sequence motifs found in the proteome of the subject (e.g., human), such as sequence motifs with amino acid lengths of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more (e.g., 8 or more). The excluded subject proteomic sequence motifs are typically 8 or longer. This helps minimize undesirable cross-reactivity caused by homology between the antigen and the self-protein.

[0055] The exemplary C. acnes (C. acnes) DsA1 polypeptide sequence includes the native C. acnes (C. acnes) DsA1 polypeptide sequence of SEQ ID NO: 204, which lacks the native secretory signal peptide sequence of the C. acnes (C. acnes) DsA1 polypeptide.

[0056] In some embodiments, the C. acnes (C. acnes) DsA1 polypeptide includes a sequence that is at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to it. In some embodiments, the C. acnes (C. acnes) DsA1 polypeptide includes a sequence that is at least 90% identical to

[0057] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding the C. acnes DsA1 polypeptide comprises a nucleotide sequence corresponding to one of SEQ ID NOs. 102-104 or SEQ ID NOs. 168-170, or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 75% identical to one of SEQ ID NOs. 102-104 or SEQ ID NOs. 168-170.

[0058] DsA2 Dermatan sulfate-adhesin 2 (DsA2) of C. acnes is also known as P27 or P027. DsA2 has been identified as a putative pathogenic factor of C. acnes (Lodes et al, 2006 (Microbiology (Reading). 2006 Dec;152(Pt 12):3667-3681), McDowell et al., 2011). This protein is found in both acne-affected and healthy hair follicle samples (Bek-Thomsen et al., 2014). The DsA1 and DsA2 proteins are homologs (paralogs) with 60-71% typical sequence identity depending on which regions of the protein are aligned. Aside from polymorphisms in the length of the PT region, high sequence identity of typically over 90% can be observed within intact DsA1 and DsA2 proteins. The C. acnes DsA2 polypeptide, its derivatives and fragments are described in International Publication No. 2021 / 165543.

[0059] The inventors demonstrated that different C. acnes (C. acnes) DsA2 polypeptides and mRNA encoding recombinant C. acnes (C. acnes) DsA2 protein induce antibody (e.g., IgG) responses. Antibodies induced by C. acnes (C. acnes) DsA2 polypeptides were shown to bind to the surface of C. acnes bacteria and recruit effector cells (e.g., immune system effector cells such as phagocytic cells). Antibodies induced by C. acnes (C. acnes) DsA2 polypeptides were shown to induce opsonin phagocytic injury in vitro.

[0060] The C. acnes DsA2 polypeptide of the present invention can induce an immune response (cross-reactive immune response) to a wide range of different C. acnes strains, lineages, and variants that cause disease.

[0061] The inventors have demonstrated that C. acnes (C. acnes) DsA2 polypeptide is a suitable vaccine antigen that can be used in combination with one or more other C. acnes antigens capable of inducing an antibody response in a target, such as the C. acnes (C. acnes) DsA1 polypeptide described herein, the C. acnes (C. acnes) PITP polypeptide described herein, and the C. acnes (C. acnes) CAMP2 polypeptide described herein. The C. acnes (C. acnes) CAMP2 polypeptide can induce antibodies in a target, such as antibodies capable of neutralizing the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2.

[0062] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA2 polypeptide. In a further embodiment, the present invention provides a polypeptide comprising the amino acid sequence of the C. acnes (C. acnes) DsA2 polypeptide. For example, the C. acnes (C. acnes) DsA2 polypeptide for use in the present invention, delivered as mRNA or recombinant protein, can induce antibodies in a subject. Such antibodies can opsonize C. acnes bacteria. Opsonization allows immune effector cells, such as phagocytic cells, to target C. acnes bacteria. This results in damage to C. acnes bacteria by such immune effector cells (e.g., phagocytic damage). Antibody binding to the cell surface of C. acnes (C. acnes) can be measured, for example, using an in vitro surface binding assay. An in vitro opsonization-phagocytic injury assay can be used to evaluate the ability of antibodies to induce opsonization and injury of different genotypes of C. acnes strains using phagocytic cells.

[0063] The amino acid sequence (excluding the signal peptide sequence) of the full-length C. acnes DsA2 polypeptide in its natural mature form is shown in SEQ ID NO: 205. [ka]

[0064] Naturally mature C. acnes (C. acnes) DsA2 polypeptides typically contain an N-terminal swap region ("NSR"), a first conserved subdomain ("CSD1"), a first swap region ("SR1"), a second conserved subdomain ("CSD2"), a second swap region ("SR2"), a third conserved subdomain ("CSD3"), a Pro-Thr repeat-containing region ("PT repeat region"), and a C-terminal region ("CTR", often containing an LPXTG motif near the C-terminus). The C-terminal LPXTG motif is thought to be crucial for the protein's anchoring to the cell wall.

[0065] For example, in the natural C. acnes DsA2 polypeptide of sequence number 205, the N-terminal swap region ("NSR") corresponds to amino acid residues 1-21, the first conserved subdomain ("CSD1") corresponds to amino acid residues 22-103, the first swap region ("SR1") corresponds to amino acid residues 104-120, and the second conserved subdomain ("CSD2") corresponds to amino acid residues 121-240. Accordingly, the second swap region ("SR2") corresponds to amino acid residues 241-250, the third conserved subdomain ("CSD3") corresponds to amino acid residues 251-295, the Pro-Thr repeat-containing region ("PT repeat region") corresponds to amino acid residues 296-349, and the C-terminal region ("CTR") corresponds to amino acid residues 350-392 (including the LPXTG motif corresponding to amino acid residues 385-389).

[0066] The sequences of naturally occurring DsA2 polypeptides are largely highly identical, differing only in the length and composition of the PT repeat region (with the exception of point mutations and rare exceptions, specifically the presence of terminal LPXTG motifs). A more detailed sequence analysis of DsA2 is described elsewhere (International Publication No. 2021 / 165543). As is well known in the art, the locations of the NSR, CSD1, SR1, CSD2, SR2, CSD3, PT repeat region, and CTR in other naturally occurring C. acnes (C. acnes) DsA2 polypeptides can be determined by aligning the polypeptide sequence of a given DsA2 polypeptide with the polypeptide sequence of SEQ ID NO: 205 and identifying the regions of the DsA2 polypeptide that have high sequence identity with the polypeptide sequences of the NSR, CSD1, SR1, CSD2, SR2, CSD3, PT repeat region, and CTR of SEQ ID NO: 205.

[0067] "C. acnes DsA2 polypeptide" includes the full-length, mature form of the natural DsA2 polypeptide of C. acnes (C. acnes) and its immunogenic variants, without its native signal peptide sequence. Immunogenic variants of the natural C. acnes DsA2 polypeptide can induce an immune response (e.g., antigen-specific immune response), such as an antibody response, in a target. Immunogenic variants of the natural C. acnes DsA2 polypeptide include immunogenic fragments of the natural C. acnes DsA2 polypeptide. The immunogenic C. acnes (C. acnes) DsA2 fragments include fragments of the native C. acnes (C. acnes) DsA2 polypeptide having an amino acid length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, or at least 375 amino acids. In some embodiments, the immunogenic C. acnes (C. acnes) DsA2 fragments include the CSD2 domain of the C. acnes (C. acnes) DsA2 polypeptide. In some embodiments, the immunogenic variants exclude sequence motifs found in the proteome of the subject (e.g., human), such as sequence motifs with amino acid lengths of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more (e.g., 8 or more). The proteomic sequence motifs that are excluded are typically 8 amino acids or longer. As further explained above, this helps minimize undesirable cross-reactivity resulting from homology between the antigen and the self-protein.

[0068] The exemplary C. acnes (C. acnes) DsA2 polypeptide sequence includes the native C. acnes (C. acnes) DsA2 polypeptide sequence of Sequence ID No. 205, which lacks the native secretory signal peptide sequence of the C. acnes (C. acnes) DsA2 polypeptide.

[0069] In some embodiments, the C. acnes (C. acnes) DsA2 polypeptide includes a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. In some embodiments, the C. acnes (C. acnes) DsA2 polypeptide includes a sequence having at least 90% identity with

[0070] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding the C. acnes DsA2 polypeptide comprises a nucleotide sequence of any one of SEQ ID NOs. 105-112 or SEQ ID NOs. 171-178 or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 75% identical to any one of SEQ ID NOs. 105-112 or SEQ ID NOs. 171-178.

[0071] PITP The putative iron transport protein (PITP) of C. acnes, also known as P28 or P028, is a protein involved in the mechanism of iron uptake by C. acnes (Lodes et al., 2006). Iron uptake plays a role in the survival of bacteria in host tissues. Under iron-restricted conditions, PITP expression increases on the cell surface (Lodes et al., 2006). C. acnes PITP polypeptide, its derivatives and fragments are described in International Publication No. 2021 / 165543.

[0072] The C. acnes (C. acnes) PITP polypeptide of the present invention can induce immune responses to a wide range of different C. acnes (C. acnes) strains, lineages, and variants that may cause disease. The C. acnes (C. acnes) PITP polypeptide has been shown to bind to the surface of C. acnes bacteria and induce antibodies that recruit effector cells (e.g., effector cells of the innate immune system, such as phagocytic cells). Combining the C. acnes (C. acnes) PITP polypeptide of the present invention with C. acnes (C. acnes) DsA1 and / or DsA2 polypeptides (delivered as mRNA or in the form of recombinant proteins) may induce immune responses to a greater number of C. acnes (C. acnes) strains or lineages compared to using individual antigens.

[0073] The inventors demonstrated that mRNA encoding different C. acnes (C. acnes) PITP polypeptides and recombinant C. acnes (C. acnes) PITP proteins induces an antibody (e.g., IgG) response. Antibodies induced by C. acnes (C. acnes) PITP polypeptides were shown to bind to the surface of C. acnes bacteria. Antibodies induced by C. acnes (C. acnes) PITP polypeptides were shown to induce opsonization phagocytosis injury in vitro. Antibodies induced by C. acnes (C. acnes) PITP polypeptides were also shown to cross-react across various C. acnes strains.

[0074] The inventors have demonstrated that C. acnes (PITP) polypeptide is a suitable vaccine antigen that can be used in combination with one or more other C. acnes antigens capable of inducing an antibody response in a target, such as the C. acnes (DsA1) polypeptide described herein, the C. acnes (DsA2) polypeptide described herein, and the C. acnes (CAMP2) polypeptide described herein. The C. acnes (CAMP2) polypeptide can induce antibodies in a target, such as antibodies capable of neutralizing the biological activity of the CAMP2 polypeptide, including the inflammatory activity of CAMP2.

[0075] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide. In a further embodiment, the present invention provides a polypeptide comprising an amino acid sequence of a C. acnes (C. acnes) PITP polypeptide. For example, a C. acnes (C. acnes) PITP polypeptide for use in the present invention, delivered as mRNA or recombinant protein, can induce antibodies in a subject. Such antibodies can opsonize C. acnes bacteria. Opsonization allows immune effector cells, such as phagocytic cells, to target C. acnes bacteria. This results in damage to C. acnes bacteria by such immune effector cells (e.g., phagocytic damage). Antibody binding to the cell surface of C. acnes (C. acnes) can be measured, for example, using an in vitro surface binding assay. An in vitro opsonization-phagocytic injury assay can be used to evaluate the ability of antibodies to induce opsonization and injury of different genotypes of C. acnes strains using phagocytic cells.

[0076] The amino acid sequence (excluding the signal peptide sequence) of the full-length C. acnes PITP polypeptide in its natural mature form is shown in SEQ ID NO: 206. [ka]

[0077] Naturally mature C. acnes (C. acnes) PITP polypeptides typically contain an elongated neocardinostatin family domain ("ENFD"), a first swap region ("SR1"), a heme-binding domain ("HbD"), a second swap region ("SR2", containing the first four N-terminal residues of the LPXTG motif (i.e., containing LPXT but not G)), and a hydrophobic C-terminal region ("hLAR", containing the C-terminal Gly residue of the LPXTG motif). The LPXTG motif is thought to be crucial for the protein's anchoring to the cell wall.

[0078] For example, in the natural C. acnes PITP polypeptide of Sequence ID No. 206, the elongated neocardinostatin family domain ("ENFD") corresponds to amino acid residues 1-133, the first swap region ("SR1") corresponds to amino acid residues 134-206, the heme-binding domain ("HbD") corresponds to amino acid residues 207-365, the second swap region ("SR2") corresponds to amino acid residues 366-399, and the hydrophobic C-terminal region ("hLAR") corresponds to amino acid residues 400-436. The LPXTG motif corresponds to amino acid residues 396-400.

[0079] The sequences of naturally occurring PITP polypeptides are largely highly identical, differing only in the N-terminus and C-terminus (except for point mutations). A more detailed sequence analysis of DsA1 is described elsewhere (International Publication No. 2021 / 165543). As is well known in the art, the positions of ENFD, SR1, HbD, SR2, and hLAR in other naturally occurring C. acnes PITP polypeptides can be determined by aligning the polypeptide sequence of a given PITP polypeptide with the polypeptide sequence of SEQ ID NO: 206 and identifying the regions of the PITP polypeptide that have high sequence identity with the polypeptide sequences of ENFD, SR1, HbD, SR2, and hLAR of SEQ ID NO: 206, respectively.

[0080] "C. acnes PITP polypeptide" includes the full-length, mature form of the natural PITP polypeptide of C. acnes (C. acnes) and its immunogenic variants, without its native signal peptide sequence. Immunogenic variants of the natural C. acnes PITP polypeptide can induce an immune response (e.g., antigen-specific immune response), such as an antibody response, in a target. Immunogenic variants of the natural C. acnes PITP polypeptide include immunogenic fragments of the natural C. acnes PITP polypeptide. Immunogenic C. acnes (C. acnes) PITP fragments include fragments of the natural C. acnes (C. acnes) PITP polypeptide having an amino acid length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, or at least 425 amino acids. In some embodiments, the immunogenic C. acnes (C. acnes) PITP fragment includes the ENFD and / or HbD (e.g., ENFD) domain of the C. acnes (C. acnes) PITP polypeptide. In some embodiments, the immunogenic variant excludes sequence motifs found in the proteome of the subject (e.g., human), such as sequence motifs with an amino acid length of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more (e.g., 8 or more). The proteomic sequence motifs that are excluded are typically 8 amino acids or longer. As further explained above, this helps minimize undesirable cross-reactivity resulting from homology between the antigen and the self-protein.

[0081] Exemplary C. acnes (C. acnes) PITP polypeptide sequences include the native C. acnes (C. acnes) PITP polypeptide sequence of SEQ ID NO: 206, which lacks the native secretory signal peptide sequence of the C. acnes (C. acnes) PITP polypeptide.

[0082] In some embodiments, the C. acnes (C. acnes) PITP polypeptide includes a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. In some embodiments, the C. acnes (C. acnes) PITP polypeptide includes a sequence having at least 90% identity with one of the sequence numbers 206, 31-37, or 73-79. In some embodiments, the C. acnes (C. acnes) PITP polypeptide includes a sequence having at least 95% identity with one of the sequence numbers 206, 31-37, or 73-79. In some embodiments, the C. acnes (C. acnes) PITP polypeptide contains, consists of, or is essentially derived from the amino acid sequence of SEQ ID NO: 73. Typically, the C. acnes (C. acnes) PITP polypeptide contains the amino acid sequence of SEQ ID NO: 73 or a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0083] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding the C. acnes PITP polypeptide comprises a nucleotide sequence of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence of at least 75% identity with at least 75% identity with at least 115, at least 117–121, and at least 182–188 (e.g., at least 182 or at least 115, e.g., at least 182).

[0084] In one embodiment, the nucleic acid of the present invention is mRNA comprising or consisting of the following structural elements (for example, consisting of the following): (i) A 5' cap having the following structure: [ka] (ii) The 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO. 265, (iii) Protein coding region having nucleic acid sequence according to Sequence ID No. 115, (iv) The 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides.

[0085] In some embodiments, the mRNA is chemically modified, and this modification includes N1-methylpseudridine instead of any uridine. The mRNA can be encapsulated in LNPs.

[0086] Chimera DsA1 / DsA2 The chimeric C. acnes DsA1 / DsA2 polypeptide has been previously described (International Publication No. 2021 / 165543).

[0087] C. acnes DsA1 and DsA2 proteins are homologs (paralogs) with 60–71% typical sequence identity depending on which regions of the protein are aligned. Aside from polymorphisms in the length of the PT region, high sequence identity of over 90% can typically be observed within intact DsA1 and DsA2 proteins. C. acnes DsA1 and C. acnes DsA2 are expressed differently in C. acnes strains within the IA1, IC, and II lineages. Some strains express more DsA1 polypeptide than DsA2 polypeptide, while others express more DsA2 polypeptide than DsA1 polypeptide. Providing both C. acnes DsA1 polypeptide and C. acnes DsA2 polypeptide may be advantageous because it allows for targeting a wider range of C. acnes strains. The DsA1 and DsA2 polypeptides may take over each other's functions when targeted individually. Therefore, providing both DsA1 and DsA2 polypeptides can reduce the possibility of immunodefence evasion. Providing both DsA1 and DsA2 polypeptides may induce immune (e.g., antibody) responses to a greater number of C. acnes strains compared to using either DsA1 or DsA2 polypeptide alone (e.g., an antibody response to C. acnes strains expressing one of the two polypeptides at a lower level). Therefore, providing both DsA1 and DsA2 polypeptides may increase immunogenicity. By providing the chimeric C. acnes DsA1 / DsA2 polypeptide as a single molecule, rather than providing the DsA1 polypeptide and DsA2 polypeptide as separate polypeptides, it becomes possible to facilitate the production of polypeptides and / or nucleic acids for use in the present invention.

[0088] The chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide of the present invention can be used to induce an immune response against C. acnes infection. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide have been shown to bind to the surface of C. acnes bacteria. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide have been shown to induce opsonization phagocytosis injury in vitro. These antibodies may exhibit superior cross-reactivity compared to antibodies induced by individual (non-chimeric) C. acnes (C. acnes) DsA1 polypeptides or C. acnes (C. acnes) DsA2 polypeptides. Chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptides can induce antibodies that specifically bind to both full-length C. acnes (C. acnes) DsA1 polypeptide and C. acnes (C. acnes) DsA2 polypeptide, as determined, for example, by ELISA assays. Antibodies induced by either the DsA1 polypeptide or the DsA2 polypeptide may cross-reactive to various C. acnes (C. acnes) strains. Furthermore, antibodies induced by chimeric DsA1 / DsA2 polypeptides may cross-reactive to various C. acnes (C. acnes) strains. This invention demonstrates that chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptides induced antibody (e.g., IgG) responses not only when delivered as antigen-encoding mRNA but also when delivered in the form of recombinant proteins.

[0089] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide, wherein the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises (i) (a) CSD2 of the C. acnes (C. acnes) DsA1 polypeptide and (b) CSD1 and / or CSD3 of the C. acnes (C. acnes) DsA2 polypeptide, or (ii) (a) CSD2 of the C. acnes (C. acnes) DsA2 polypeptide and (b) CSD1 and / or CSD3 of the C. acnes (C. acnes) DsA1 polypeptide. The nucleic acid may be messenger RNA (mRNA). In further embodiments, the present invention provides a chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide having an amino acid sequence comprising (i)(a) CSD2 of the C. acnes (C.acnes)DsA1 polypeptide and (b) CSD1 and / or CSD3 of the C. acnes (C.acnes)DsA2 polypeptide, or (ii)(a) CSD2 of the C. acnes (C.acnes)DsA2 polypeptide and (b) CSD1 and / or CSD3 of the C. acnes (C.acnes)DsA1 polypeptide. For example, a chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide for use in the present invention, delivered as mRNA or recombinant protein, may induce an immune response (e.g., an antibody response) in a subject. Such an antibody may be a cross-reactive antibody that specifically binds to both the native C. acnes (C.acnes)DsA1 polypeptide and the native C. acnes (C.acnes)DsA2 polypeptide. Such antibody responses may be cross-reactive to various C. acnes lineages (e.g., lineages IA1, IA2, IB, IC, II, and III).

[0090] In some embodiments, the chimeric C. acnes DsA1 / DsA2 polypeptide is (i) NSR of C. acnes DsA1 polypeptide or NSR of C. acnes DsA2 polypeptide (for example, NSR of C. acnes DsA1 polypeptide), (ii) CSD1 of C. acnes DsA1 polypeptide or CSD1 of C. acnes DsA2 polypeptide (for example, CSD1 of C. acnes DsA1 polypeptide), (iii) optionally, SR1 (or a portion thereof) of the C. acnes DsA1 polypeptide and / or SR1 (or a portion thereof) of the C. acnes DsA2 polypeptide, (iv) CSD2 of C. acnes DsA1 polypeptide or CSD2 of C. acnes DsA2 polypeptide (for example, CSD2 of C. acnes DsA2 polypeptide), (v) optionally, SR2 (or a portion thereof) of the C. acnes DsA1 polypeptide and / or SR2 (or a portion thereof) of the C. acnes DsA2 polypeptide, and (vi) CSD3 of C. acnes DsA1 polypeptide or C. acnes DsA2 polypeptide (for example, CSD3 of C. acnes DsA1 polypeptide) Includes.

[0091] As described in International Publication No. 2021 / 165543, the SRSs within the C. acnes (C. acnes) DsA1 and C. acnes (C. acnes) DsA2 polypeptides are typically regions rich in essentially irregular polypeptide sequences (i.e., they are largely unstructured). SRs (Structural Links) share the common property of linking ("spaced") two structural domains, such as CSDs (e.g., CSD1, CSD2, and CSD3). Therefore, CSDs derived from C. acnes (C. acnes) DsA1 and C. acnes (C. acnes) DsA2 polypeptides can be combined by appropriately genetically engineering the SRs so that their lengths (number of amino acid residues) are preserved. Advantageously, this design approach allows for the preservation of the structural integrity of the individual structural domains (e.g., CSDs) in the resulting chimeric DsA1 / DsA2 polypeptide.

[0092] In some embodiments, the chimeric C. acnes DsA1 / DsA2 polypeptide of the present invention comprises (for example, from the N-terminus to the C-terminus) amino acid residues 21-102 of SEQ ID NO: 204, amino acid residues 121-240 of SEQ ID NO: 205, and amino acid residues 250-295 of SEQ ID NO: 204.

[0093] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences assigned to SEQ ID NOs: 28-30, SEQ ID NOs: 39, SEQ ID NOs: 70-72, or SEQ ID NOs: 81. Preferably, the chimeric C. acnes DsA1 / DsA2 polypeptide includes the amino acid sequence according to SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0094] In some embodiments, the chimeric C. acnes DsA1 / DsA2 polypeptide contains, consists of, or is essentially derived from the amino acid sequence of Sequence ID No. 70.

[0095] In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide include nucleotide sequences such as SEQ ID NOs. 113-114, SEQ ID NOs. 179, SEQ ID NOs. 123, or SEQ ID NOs. 190 (e.g., SEQ ID NOs. 179), or sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide include nucleotide sequences that are at least 85% identical to SEQ ID NOs. 113-114, SEQ ID NOs. 179, SEQ ID NOs. 123, or SEQ ID NOs. 190 (e.g., SEQ ID NOs. 179). In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises or consists of a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence as described herein) and the nucleotide sequence according to SEQ ID NO: 179. In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises or consists of the nucleotide sequence according to SEQ ID NO: 113.

[0096] In one embodiment, the nucleic acid of the present invention is mRNA comprising or consisting of the following structural elements (for example, consisting of the following): (i) A 5' cap having the following structure: [ka] (ii) The 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO. 265, (iii) Protein coding region having nucleic acid sequence according to Sequence ID No. 113, (iv) The 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides.

[0097] In some embodiments, the mRNA is chemically modified, and this modification includes N1-methylpseudridine instead of any uridine. The mRNA can be encapsulated in LNPs.

[0098] Chimera DsA1 / DsA2 / PITP The chimeric C. acnes DsA1 / DsA2 / PITP polypeptide has been previously described (International Publication No. 2021 / 165543).

[0099] DsA1 and DsA2 polypeptides can be expressed in C. acnes lineages IA1, IA2, IC, and II. Antibodies induced by DsA1 and DsA2 polypeptides may not be able to bind to C. acnes MLST lineages IB and III. On the other hand, antibodies against C. acnes PITP polypeptide may be able to bind to C. acnes type IB and C. acnes type III. Therefore, PITP may be an antigen that can complement immune responses induced by other C. acnes antigens, such as DsA1 and / or DsA2. Combining the C. acnes (C. acnes) PITP polypeptide with the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide (delivered as mRNA or in the form of recombinant protein) may induce an immune response against a greater number of C. acnes (C. acnes) strains or lineages compared to using the individual antigens. Combining DsA1 and / or DsA2 delivered as mRNA or in the form of recombinant protein with PITP may provide a cross-reactive immune response (e.g., an antibody response) against a greater number of C. acnes (C. acnes) strains or lineages compared to using DsA1 and / or DsA2 alone. The C. acnes (C. acnes) PITP polypeptide may be provided as separate polypeptide molecules (delivered as mRNA or in the form of recombinant protein) or as part of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide (delivered as mRNA or in the form of recombinant protein).

[0100] The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide of the present invention (delivered as mRNA or in the form of recombinant protein) can be used to induce an immune response against C. acnes infection. Compared to using individual antigens (e.g., DsA1, DsA2, or PITP), the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide (delivered as mRNA or in the form of recombinant protein) can induce an immune response against a greater number of C. acnes strains, such as a cross-reactive immune response. Rather than providing C. acnes (C.acnes)DsA1 polypeptide, C. acnes (C.acnes)DsA2 polypeptide, and C. acnes (C.acnes)PITP polypeptide (or chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide and C. acnes (C.acnes)PITP polypeptide) as individual polypeptides, providing the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide makes it possible to facilitate the production of polypeptides and / or nucleic acids for use in the present invention.

[0101] The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide can induce cross-reactive antibody responses to various C. acnes (C. acnes) strains. This invention demonstrates that the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide induced antibody (e.g., IgG) responses not only when delivered as antigen-encoding mRNA but also when delivered in the form of a recombinant protein. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide were shown to bind to the surface of C. acnes bacteria.

[0102] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide, wherein the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide is the ENFD of C. acnes (C. acnes) PITP polypeptide, and (i)(a) CSD2 of C. acnes DsA1 polypeptide and (b) CSD1 and / or CSD3 of C. acnes DsA2 polypeptide, or (ii) (a) CSD2 of C. acnes DsA2 polypeptide and (b) CSD1 and / or CSD3 of C. acnes DsA1 polypeptide Includes.

[0103] Nucleic acids can be messenger RNA (mRNA).

[0104] In a further embodiment, the present invention relates to the ENFD of C. acnes (C. acnes) PITP polypeptide, and (i)(a) CSD2 of C. acnes DsA1 polypeptide and (b) CSD1 and / or CSD3 of C. acnes DsA2 polypeptide, or (ii) (a) CSD2 of C. acnes DsA2 polypeptide and (b) CSD1 and / or CSD3 of C. acnes DsA1 polypeptide This provides a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide containing the following:

[0105] For example, C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptides for use in the present invention, delivered as mRNA or recombinant protein, can induce an immune response (e.g., antibodies) in a subject. Such antibodies may include antibodies that specifically bind to the native C. acnes (C. acnes) DsA1 polypeptide, antibodies that specifically bind to the native C. acnes (C. acnes) DsA2 polypeptide, and / or antibodies that specifically bind to the native C. acnes (C. acnes) PITP polypeptide. Such antibodies may cross-reactive to various C. acnes (C. acnes) lineages (e.g., lineages IA1, IA2, IB, IC, II, and III).

[0106] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises CSD2 of the C. acnes (C. acnes) DsA2 polypeptide, CSD1 of the C. acnes (C. acnes) DsA1 polypeptide, CSD3 of the C. acnes (C. acnes) DsA1 polypeptide, and ENFD of the C. acnes (C. acnes) PITP polypeptide. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide is (a)(i)NSR of C. acnes DsA1 polypeptide or NSR of C. acnes DsA2 polypeptide (for example, NSR of C. acnes DsA1 polypeptide), (ii) CSD1 of C. acnes DsA1 polypeptide or CSD1 of C. acnes DsA2 polypeptide (for example, CSD1 of C. acnes DsA1 polypeptide), (iii) optionally, SR1 (or a portion thereof) of the C. acnes DsA1 polypeptide and / or SR1 (or a portion thereof) of the C. acnes DsA2 polypeptide, (iv) CSD2 of C. acnes DsA1 polypeptide or CSD2 of C. acnes DsA2 polypeptide (for example, CSD2 of C. acnes DsA2 polypeptide), (v) optionally, SR2 (or a portion thereof) of the C. acnes DsA1 polypeptide and / or SR2 (or a portion thereof) of the C. acnes DsA2 polypeptide, and (vi) CSD3 of C. acnes DsA1 polypeptide or CSD3 of C. acnes DsA2 polypeptide (for example, CSD3 of C. acnes DsA1 polypeptide), (b) ENFD of C. acnes PITP polypeptide and Includes.

[0107] As described in International Publication No. 2021 / 165543, the SRs within the C. acnes (C. acnes) DsA1 polypeptide, C. acnes (C. acnes) DsA2 polypeptide, and PITP are typically regions rich in essentially irregular polypeptide sequences (i.e., they are largely unstructured). SRs share the common characteristic of linking ("spaced") two structural domains, such as CSDs (e.g., CSD1, CSD2, and CSD3 in the C. acnes (C. acnes) DsA1 polypeptide and C. acnes (C. acnes) DsA2 polypeptide), ENFD in the C. acnes (C. acnes) PITP polypeptide, or HbD in the C. acnes (C. acnes) PITP polypeptide. Therefore, the CSD derived from the C. acnes (C. acnes) DsA1 polypeptide and the C. acnes (C. acnes) DsA2 polypeptide, the ENFD of the C. acnes (C. acnes) PITP polypeptide, and / or the HbD of the C. acnes (C. acnes) PITP polypeptide can be combined by spacing them with SRs of similar length (number of amino acid residues) to those in the natural C. acnes polypeptide. Advantageously, this design method allows for the preservation of the structural integrity of the individual structural domains (e.g., CSD, ENFD, and HbD) in the resulting chimeric DsA1 / DsA2 / PITP polypeptide.

[0108] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes an amino acid sequence of any one of SEQ ID NOs. 38, SEQ ID NOs. 40-41, SEQ ID NOs. 80, SEQ ID NOs. 82-83, or SEQ ID NOs. 367-368 (e.g., SEQ ID NOs. 80, 82, 83, or 368) or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes a sequence having at least 90% identity with any one of SEQ ID NOs. 38, SEQ ID NOs. 40-41, SEQ ID NOs. 80, SEQ ID NOs. 82-83, or SEQ ID NOs. 367-368 (e.g., SEQ ID NOs. 80, 82, 83, or 368). In some embodiments, the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide contains a sequence having at least 95% identity with one of SEQ ID NOs. 38, 40-41, 80, 82-83, or 367-368 (e.g., SEQ ID NOs. 80, 82, 83, or 368).

[0109] In some embodiments, the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide consists of the amino acid sequence of SEQ ID NOs. 80, 82, 83, or 368.

[0110] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes a nucleotide sequence by any one of SEQ ID NOs. 122, SEQ ID NOs. 124-125, SEQ ID NOs. 189, or SEQ ID NOs. 191-192 (e.g., SEQ ID NOs. 189 or 192), or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes a nucleotide sequence that is at least 85% identical to SEQ ID NOs. 122, SEQ ID NOs. 124-125, SEQ ID NOs. 189, or SEQ ID NOs. 191-192 (e.g., SEQ ID NOs. 189 or 192).

[0111] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence as described herein) and a sequence according to either SEQ ID NO: 189 or SEQ ID NOs: 191-192.

[0112] Chimera DsA1 / DsA2 / PITP / CAMP2 The inventors have designed a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide (also referred to herein as the chimeric C. acnes (C. acnes) CAMP2 / DsA1 / DsA2 / PITP polypeptide). The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide of the present invention can induce an immune (e.g., antibody) response. This response may be a protective immune (e.g., antibody) response. Chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptides can induce immune responses (e.g., antibodies) to the native C. acnes (C. acnes) DsA1 polypeptide, the native C. acnes (C. acnes) DsA2 polypeptide, the native C. acnes (C. acnes) PITP polypeptide, and / or immune responses (e.g., antibodies) to the native C. acnes (C. acnes) CAMP2 polypeptide. Such antibodies may cross-reactive to various C. acnes (C. acnes) MLST lineages (e.g., lineages IA1, IA2, IB, IC, II, and III). Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide can neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide can opsonize C. acnes bacteria. Opsonization allows immune effector cells, such as phagocytic cells, to target C. acnes bacteria. This results in damage to C. acnes bacteria by such immune effector cells (e.g., phagocytic damage). The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide can induce antibodies that can neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2.Therefore, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide can induce (a) antibodies that neutralize the biological activity of the C. acnes (C. acnes) CAMP2 polypeptide, such as inflammatory activity, and (b) antibodies that opsonize C. acnes (C. acnes) bacteria and recruit immune effector cells such as phagocytes. Accordingly, immunization with the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide of the present invention (e.g., delivered as mRNA or recombinant protein) may provide a more effective immune response (e.g., effective damage to C. acnes (C. acnes) bacteria in the target) than immunization with the chimeric DsA1 / DsA2 / PITP polypeptide or individual DsA1, DsA2, or PITP antigens or the CAMP2 polypeptide itself.

[0113] The inventors demonstrated that when the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide was delivered as the mRNA encoding this polypeptide, it induced an antibody (e.g., IgG) response to the DsA1, DsA2, PITP, and CAMP2 antigens. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide were shown to bind to the surface of C. acnes bacteria. Antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide were shown to induce opsonization phagocytosis injury in vitro. The inventors also demonstrated that antibodies induced by the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide reduce the co-hemolytic activity of the C. acnes (C. acnes) CAMP2 polypeptide.

[0114] Combining the DsA1, DsA2, PITP, and CAMP2 antigens into a single chimeric polypeptide offers advantages, such as allowing the use of smaller amounts of cationic lipids or LNPs compared to vaccine formulations containing several different mRNAs, thereby reducing the reactogenicity of the vaccine, for example, in relation to the chimeric polypeptide delivered in the form of a single mRNA. Combining the DsA1, DsA2, PITP, and CAMP2 antigens into a single chimeric polypeptide can also facilitate vaccine production. This is because it is easier, faster, and less expensive to produce a single nucleic acid or polypeptide combining four antigens than to produce separate nucleic acids or polypeptides for each different antigen.

[0115] As described above in this specification, this design scheme can leverage the structural integrity of the individual structural domains (e.g., the CSDs of DsA1 and DsA2 and the ENFD of PITP) in the resulting chimeric DsA1 / DsA2 / PITP / CAMP2 polypeptide. This design scheme can also leverage the flexible linker domain of the C. acnes CAMP2 polypeptide and / or the flexible linker-like sequence at the C-terminus of the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide.

[0116] Accordingly, in one embodiment, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide, wherein the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises C. acnes (C.acnes)DsA1 or its immunogenic fragment, C. acnes (C.acnes)DsA2 or its immunogenic fragment, C. acnes (C.acnes)PITP polypeptide or its immunogenic fragment, and C. acnes (C.acnes)CAMP2 polypeptide or its immunogenic fragment. Typically, the nucleic acid is messenger RNA (mRNA).

[0117] In a further embodiment, the present invention provides a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprising C. acnes (C. acnes) DsA1 or its immunogenic fragment, C. acnes (C. acnes) DsA2 or its immunogenic fragment, C. acnes (C. acnes) PITP polypeptide or its immunogenic fragment, and C. acnes (C. acnes) CAMP2 polypeptide or its immunogenic fragment.

[0118] In some embodiments, the immunogenic fragment of the C. acnes (C. acnes) DsA1 polypeptide is CSD2 of the C. acnes (C. acnes) DsA1 polypeptide. In some embodiments, the immunogenic fragment of the C. acnes (C. acnes) DsA2 polypeptide is CSD2 of the C. acnes (C. acnes) DsA2 polypeptide.

[0119] In some embodiments, the second conserved subdomain ("CSD2") of the C. acnes (C. acnes) DsA1 polypeptide corresponds to amino acid residues 120-239 of SEQ ID NO: 204. For a description of the other domains of the C. acnes (C. acnes) DsA1 polypeptide and their boundaries, see the "DsA1" section above. As is known in the art, the locations of CSD2 and other domains in other natural C. acnes (C. acnes) DsA1 polypeptides can be determined by aligning the polypeptide sequence of a given DsA1 polypeptide with the polypeptide sequence of SEQ ID NO: 204 and identifying the regions of the DsA1 polypeptide that have high sequence identity with the polypeptide sequences of CSD2 and other domains of SEQ ID NO: 204, respectively.

[0120] In some embodiments, the second conserved subdomain ("CSD2") of the C. acnes (C. acnes) DsA2 polypeptide corresponds to amino acid residues 121-240 of SEQ ID NO: 205. For a description of the other domains of the C. acnes (C. acnes) DsA2 polypeptide and their boundaries, see the "DsA2" section above. As is known in the art, the locations of CSD2 and other domains in other natural C. acnes (C. acnes) DsA2 polypeptides can be determined by aligning the polypeptide sequence of a given DsA2 polypeptide with the polypeptide sequence of SEQ ID NO: 205 and identifying the regions of the DsA2 polypeptide that have high sequence identity with the polypeptide sequences of CSD2 and other domains of SEQ ID NO: 205, respectively.

[0121] In some embodiments, the immunogenicity fragment of C. acnes PITP polypeptide comprises an ENFD of C. acnes PITP polypeptide.

[0122] In some embodiments, the extended neocardinostatin family domain ("ENFD") of C. acnes PITP corresponds to amino acid residues 1-133 of SEQ ID NO: 206. For a description of the other domains of the C. acnes PITP polypeptide and their boundaries, see the "PITP" section above. As is known in the art, the location of ENFD and other domains in other natural C. acnes PITP polypeptides can be determined by aligning the polypeptide sequence of a given PITP polypeptide with the polypeptide sequence of SEQ ID NO: 206 and identifying regions of the PITP polypeptide that have high sequence identity with the polypeptide sequences of ENFD and other domains of SEQ ID NO: 206, respectively.

[0123] In some embodiments, the immunogenic fragment of C. acnes (C.acnes)CAMP2 polypeptide includes the N-terminal domain of C. acnes (C.acnes)CAMP2 polypeptide. In some embodiments, the immunogenic fragment of C. acnes (C.acnes)CAMP2 polypeptide includes the N-terminal domain of C. acnes (C.acnes)CAMP2 polypeptide and the linker domain of C. acnes (C.acnes)CAMP2 polypeptide. In some embodiments, the immunogenic fragment of C. acnes (C.acnes)CAMP2 polypeptide includes the C-terminal domain of C. acnes (C.acnes)CAMP2 polypeptide. In some embodiments, the immunogenic fragment of C. acnes (C.acnes)CAMP2 polypeptide includes the linker domain of C. acnes (C.acnes)CAMP2 polypeptide and the C-terminal domain of C. acnes (C.acnes)CAMP2 polypeptide.

[0124] In some embodiments, the N-terminal domain of the C. acnes CAMP2 polypeptide corresponds to residues 29-176 of SEQ ID NO: 202. In some embodiments, the C-terminal domain of the C. acnes CAMP2 polypeptide corresponds to residues 189-267 of SEQ ID NO: 202. In some embodiments, the linker domain of the C. acnes CAMP2 polypeptide corresponds to residues 177-188 of SEQ ID NO: 202. As is known in the art, the positions of the N-terminal domain, C-terminal domain, and linker domain in other C. acnes CAMP2 polypeptides can be determined by aligning the polypeptide sequence of a given CAMP2 polypeptide with the polypeptide sequence of SEQ ID NO: 202 and identifying the regions of the CAMP2 polypeptide that have high sequence identity with the sequences of the N-terminal domain, C-terminal domain, and linker domain of SEQ ID NO: 202, respectively. Analysis of 430 CAMP2 polypeptide sequences from naturally occurring C. acnes strains revealed a high degree of sequence conservation among CAMP2 sequences (see the "CAMP2" section above). Figure 44 shows an exemplary alignment of naturally occurring CAMP2 polypeptide sequences from C. acnes strains to sequence number 202 (i.e., KPA171202_REF in Figure 44). Alignments like that in Figure 44 can be used to identify the positions of the N-terminal domain, C-terminal domain, and linker domain in sequences other than sequence number 202.

[0125] In some embodiments, two or more of the following are linked via a linker (for example, a linker as described in the "Linker" section below): (1) C. acnes DsA1 polypeptide or its immunogenic fragment, (2) C. acnes DsA2 polypeptide or its immunogenic fragment, (3) C. acnes PITP polypeptide or its immunogenic fragment, and (4) C. acnes CAMP2 polypeptide or its immunogenic fragment. In other embodiments, (1) C. acnes (C. acnes) DsA1 polypeptide or its immunogenic fragment, (2) C. acnes (C. acnes) DsA2 polypeptide or its immunogenic fragment, (3) C. acnes (C. acnes) PITP polypeptide or its immunogenic fragment, and (4) C. acnes (C. acnes) CAMP2 polypeptide or its immunogenic fragment are directly fused to one another (i.e., there are no linkers, such as amino acid linkers, connecting the DsA1, DsA2, PITP, and CAMP2 polypeptides or their immunogenic fragments to each other). In some embodiments, such a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes a linker domain of the C. acnes (C. acnes) CAMP2 polypeptide.

[0126] In one embodiment, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide is (a) For example, the chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, (b) Immunogenic fragments of C. acnes PITP polypeptide containing ENFD, and (c) C. acnes CAMP2 polypeptide or its immunogenic fragment Includes.

[0127] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises (i) (1) CSD2 of the C. acnes (C. acnes) DsA1 polypeptide and (2) CSD1 and / or CSD3 of the C. acnes (C. acnes) DsA2 polypeptide, or (ii) (1) CSD2 of the C. acnes (C. acnes) DsA2 polypeptide and (2) CSD1 and / or CSD3 of the C. acnes (C. acnes) DsA1 polypeptide. For descriptions of the domains and boundaries of the C. acnes (C. acnes) DsA1 polypeptide and the DsA2 polypeptide, see above and the sections "DsA1" and "DsA2," respectively. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide is as defined in (ii). Typically, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide contains CSD1 of the C. acnes (C. acnes) DsA1 polypeptide, CSD2 of the C. acnes (C. acnes) DsA2 polypeptide, and CSD3 of the C. acnes (C. acnes) DsA1 polypeptide.

[0128] In some embodiments, the chimeric C. acnes DsA1 / DsA2 polypeptide of (a) includes the sequence of SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0129] In some embodiments, the immunogenic fragment of the C. acnes (C. acnes) PITP polypeptide comprising the ENFD of the C. acnes (C. acnes) PITP polypeptide of (b) comprises a sequence corresponding to amino acid residues 1-133 of SEQ ID NO: 73 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of amino acid residues 1-133 of SEQ ID NO: 73. In some embodiments, the immunogenic fragment of the C. acnes (C. acnes) PITP polypeptide containing the ENFD of the C. acnes (C. acnes) PITP polypeptide of (b) contains a sequence corresponding to amino acid residues 1-146 of SEQ ID NO: 73 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of amino acid residues 1-146 of SEQ ID NO: 73. Typically, the immunogenic fragment of the C. acnes (C. acnes) PITP polypeptide containing the ENFD of the C. acnes (C. acnes) PITP polypeptide of (b) contains a sequence corresponding to amino acid residues 1-146 of SEQ ID NO: 73.

[0130] In some embodiments, (a) and (b) include sequence number 80 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. In some embodiments, (a) and (b) include sequence number 82 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. In some embodiments, (a) and (b) include sequence number 83 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it. Typically, (a) and (b) include sequence number 80.

[0131] In some embodiments, (c) is a C. acnes CAMP2 polypeptide, for example, a full-length C. acnes CAMP2 polypeptide, typically a CAMP2 polypeptide of a mature, full-length C. acnes (without a native signal peptide sequence). In some embodiments, (c) includes sequence number 203 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Typically, (c) includes sequence number 203.

[0132] In some embodiments, the immunogenic fragment of the C. acnes CAMP2 polypeptide in (c) comprises the N-terminal domain of the C. acnes CAMP2 polypeptide. In some embodiments, (c) comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 29-176 of SEQ ID NO: 202.

[0133] In some embodiments, the immunogenic fragment of the C. acnes CAMP2 polypeptide of (c) comprises the N-terminal domain of the C. acnes CAMP2 polypeptide and the linker domain of the C. acnes CAMP2 polypeptide. In some embodiments, (c) comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 29-188 of SEQ ID NO: 202.

[0134] In some embodiments, the immunogenic fragment of the C. acnes CAMP2 polypeptide in (c) comprises the C-terminal domain of the C. acnes CAMP2 polypeptide. In some embodiments, (c) comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 189-267 of SEQ ID NO: 202.

[0135] In some embodiments, the immunogenic fragment of the C. acnes CAMP2 polypeptide of (c) comprises the linker domain of the C. acnes CAMP2 polypeptide and the C-terminal domain of the C. acnes CAMP2 polypeptide. In some embodiments, (c) comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with amino acid residues 177-267 of SEQ ID NO: 202.

[0136] In some embodiments, two or more of (a), (b), and (c) are linked via a linker (e.g., a linker as described in the “Linker” section below). Typically, (a), (b), and (c) are fused directly to each other in the order specified below (i.e., there is no linker, such as an amino acid linker, connecting (a), (b), and (c)). In some embodiments, (c) comprises a linker domain of the C. acnes CAMP2 polypeptide.

[0137] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide contains (a), (b), and (c) from the N-terminus to the C-terminus. In other embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide contains (c), (a), and (b) from the N-terminus to the C-terminus. Typically, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide contains (c), (a), and (b) from the N-terminus to the C-terminus.

[0138] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes a transmembrane domain (TMB) sequence, for example, a TMB as defined herein (see the section “Heterogeneous Transmembrane Domain (TMB)” below). In certain embodiments, the TMB sequence is located at the N-terminus of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide. In other embodiments, the TMB sequence is located at the C-terminus of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide. Typically, the TMB sequence is located at the C-terminus of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide.

[0139] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence of any one of sequence numbers 373-376 (e.g., sequence number 373 or 374) or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence of sequence number 373 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto and a TMB sequence (e.g., the sequence of sequence number 84). Typically, a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes (1) the sequence of SEQ ID NO: 374 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, or (2) the sequence of SEQ ID NO: 373 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a TMB sequence.

[0140] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence such as those described herein (e.g., a secretion signal peptide sequence as described in the section “Secretion Signal Peptide (SS) Sequences” below).

[0141] In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide include a sequence of any one of SEQ ID NOs. 386-390 or SEQ ID NO. 393 (for example, SEQ ID NO. 387 or any one of sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto). In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide include the sequence according to SEQ ID NO: 384 or SEQ ID NO: 385 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a nucleotide sequence encoding the TMB sequence (for example, the nucleotide sequence encoding SEQ ID NO: 84). In some embodiments, the nucleotide sequence encoding SEQ ID NO: 84 is as defined in SEQ ID NO: 395 or 396.Typically, nucleic acids containing nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide include (1) the sequence according to SEQ ID NO: 387 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, or (2) the sequence according to SEQ ID NO: 384 or SEQ ID NO: 385 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and nucleotide sequences encoding the TMB sequence.

[0142] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a nucleotide sequence (e.g., one of sequence numbers 377-380) that is any one of sequence numbers 377-383 or 394, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a nucleotide sequence that is at least 75% identical to one of sequence numbers 377-380.

[0143] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a sequence of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide as described herein. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a sequence of the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein.

[0144] Variants of the polypeptide of the present invention Mutation of cysteine ​​residues One or more cysteine ​​residues in the polypeptides described herein can be mutated by a single amino acid substitution, for example, to serine residues. Since cysteine ​​residues are involved in the formation of disulfide crosslinks, mutating cysteine ​​can limit the polymerization of the polypeptide.

[0145] In some embodiments, the C. acnes (C.acnes)DsA1 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the cysteine ​​residues of the natural C. acnes (C.acnes)DsA1 polypeptide. In some embodiments, the C. acnes (C.acnes)DsA1 polypeptide contains one or more single amino acid substitutions at positions corresponding to C25 (e.g., C25S), C291 (e.g., C291S or C291M), and / or C293 (e.g., C293S or C293P) of SEQ ID NO: 204.

[0146] In some embodiments, the C. acnes (C. acnes) DsA2 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the cysteine ​​residues of the natural C. acnes (C. acnes) DsA2 polypeptide. In some embodiments, the C. acnes (C. acnes) DsA2 polypeptide contains one or more single amino acid substitutions at positions corresponding to C26 (e.g., C26S) and / or C292 (e.g., C292S) of SEQ ID NO: 205.

[0147] In some embodiments, the C. acnes (C. acnes) PITP polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues of the natural C. acnes (C. acnes) PITP polypeptide. In some embodiments, the C. acnes (C. acnes) PITP polypeptide contains one or more single amino acid substitutions at positions corresponding to C200 (e.g., C200S), C371 (e.g., C371S), and / or C429 (e.g., C429S) of SEQ ID NO: 206. In some embodiments, the C. acnes (C. acnes) PITP polypeptide contains a single amino acid substitution at one or both (e.g., both) positions corresponding to C200 (e.g., C200S) and C371 (e.g., C371S) of SEQ ID NO: 206. In a preferred embodiment, the C. acnes (C. acnes) PITP polypeptide having the sequence of SEQ ID NO: 73 contains serine residues at positions 200 and 371. In another embodiment, the C. acnes PITP polypeptide having the sequence of SEQ ID NO: 77 contains serine residues at positions 200 and 371.

[0148] In some embodiments, the chimeric C. acnes (C.acnes) DsA1 / DsA2 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes) DsA1 polypeptide and / or at one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes) DsA2 polypeptide. In some embodiments, the chimeric C. acnes (C.acnes) DsA1 / DsA2 polypeptide contains a single amino acid substitution at one or more (e.g., all) positions corresponding to C25 (e.g., C25S), C291 (e.g., C291S or C291M), and / or C293 (e.g., C293S or C293P) of SEQ ID NO: 204. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises one or more (e.g., all three) amino acid substitutions selected from C25S, C291S, and / or C293P, where C25, C291, and / or C293 correspond to the residues in SEQ ID NO: 204. In a preferred embodiment, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide having the sequence of SEQ ID NO: 70 comprises serine residues at positions 25 and 291 and a proline residue at position 293. In another embodiment, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide having the sequence of SEQ ID NO: 72 comprises serine residues at positions 25 and 291 and a proline residue at position 293. In another embodiment, the chimeric C. acnes DsA1 / DsA2 polypeptide having the sequence of SEQ ID NO: 81 contains a serine residue at position 18 and a proline residue at position 286. In another embodiment, the chimeric C. acnes DsA1 / DsA2 polypeptide having the sequence of SEQ ID NO: 81 contains a serine residue at position 18, a methionine residue at position 284, and a proline residue at position 286, the residues corresponding to positions 25, 291, and 293 of SEQ ID NO: 204, respectively.

[0149] In some embodiments, the chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP polypeptide described herein includes a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes) DsA1 polypeptide, one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes) DsA2 polypeptide, and / or one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes) PITP polypeptide. In some embodiments, the chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP polypeptide includes a single amino acid substitution at one or more positions corresponding to C25 (e.g., C25S), C291 (e.g., C291S or C291M), and / or C293 (e.g., C293S or C293P) of SEQ ID NO: 204. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide comprises one or more (e.g., all three) amino acid substitutions selected from C25S, C291S, and / or C293P, where C25, C291, and / or C293 correspond to the residues in SEQ ID NO: 204. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide having the sequence of SEQ ID NO: 80 or SEQ ID NO: 83 (e.g., SEQ ID NO: 83) comprises serine residues at positions 25 and 291 and a proline residue at position 293. In other embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide having the sequence of SEQ ID NO: 82 comprises a serine residue at position 25 and a proline residue at position 293. In some embodiments, the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide having the sequence of SEQ ID NO: 82 contains a serine residue at position 25, a methionine residue at position 291, and a proline residue at position 293. In other embodiments, the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide having the sequence of SEQ ID NO: 368 contains a serine residue at position 18, a methionine residue at position 284, and a proline residue at position 286.In some embodiments, the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide having the sequence of SEQ ID NO: 368 contains a serine residue at position 18, a methionine residue at position 284, and a proline residue at position 286, with these residues corresponding to positions 25, 291, and 293 of SEQ ID NO: 204, respectively.

[0150] In some embodiments, the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein includes a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes)DsA1 polypeptide, one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes)DsA2 polypeptide, and / or one or more (e.g., all) positions corresponding to cysteine ​​residues in the natural C. acnes (C.acnes)PITP polypeptide. In some embodiments, the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide includes a single amino acid substitution at one or more positions corresponding to C25 (e.g., C25S), C291 (e.g., C291S or C291M), and / or C293 (e.g., C293S or C293P) of SEQ ID NO: 204. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises one or more (e.g., all three) amino acid substitutions selected from C25S, C291S, and / or C293P, where C25, C291, and / or C293 correspond to the residues in SEQ ID NO: 204. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide having the sequence of SEQ ID NO: 373 or 374 comprises serine residues at positions 264 and 530 and a proline residue at position 532. In other embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide having the sequence of SEQ ID NO: 375 comprises serine residues at positions 185 and 451 and a proline residue at position 453. In another embodiment, the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide having the sequence of SEQ ID NO: 376 contains serine residues at positions 25 and 291 and a proline residue at position 293.

[0151] Mutation of glycosylation sites Glycosylation can occur in eukaryotic cells but typically does not occur in prokaryotic cells. As used herein, “glycosylation” refers to the addition of sugar units to a protein. In particular, N-linked glycosylation is the attachment of a glycan to the amide nitrogen of an asparagine (Asn;N) residue in a protein. N-glycosylation can occur at any asparagine residue in a protein that is reachable by a glycosylation enzyme after translation and is recognized by the glycosylation enzyme, most commonly at reachable asparagine that is part of the NXS / T motif, where the second amino acid residue following asparagine is serine (Ser;S) or threonine (Thr;T). O-linked glycosylation is the attachment of a glycan to the oxygen atom of a serine (Ser) or threonine (Thr) residue in a protein. This attachment process results in a glycosylated protein. This glycan may be a polysaccharide. Non-human glycosylation patterns can confer undesirable reactogenicity to polypeptides when used to induce antibodies. In addition, glycosylation of polypeptides that are not normally glycosylated (e.g., polypeptides naturally found in prokaryotic cells or derived from such polypeptides, as described herein) can alter their immunogenicity. For example, glycosylation may mask important immunogenic epitopes within the protein. Therefore, glycosylation can be reduced or eliminated by modifying either an asparagine residue or a serine / threonine residue, for example, by substituting it with another amino acid.

[0152] In certain embodiments, one or more of the C. acnes CAMP2 polypeptide, modified C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide and chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein comprises at least one mutated glycosylation site, preferably at least one mutated N-linked glycosylation site and / or at least one O-linked glycosylation site. In some embodiments, one or more glycosylation sites are removed from one or more of the following polypeptides as described herein: C. acnes CAMP2 polypeptide, modified C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide, and chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide. Removal of glycosylation sites can reduce the glycosylation of the polypeptide.In some embodiments, one or more of the C. acnes CAMP2 polypeptide, modified C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide, and chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein have reduced glycosylation compared to their respective natural polypeptides. Removal of glycosylation sites can eliminate glycosylation of polypeptides.

[0153] In certain embodiments, the modification includes substitution of one or more N, S, and T amino acids (e.g., in an NXS / T sequence motif), where X corresponds to any amino acid. In some embodiments, the modification includes substitution of one or more serine (Ser) or threonine (Thr) residues in the protein. In some embodiments, the N, S, or T amino acids are substituted with conservative amino acid substitutions. Typically, the N amino acid may be substituted with a Q, S, K, or A amino acid.

[0154] In some embodiments, the C.acnes(C)CAMP2 polypeptide and / or modified C.acnes(C)CAMP2 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C.acnes(C)CAMP2 polypeptide. The N-glycosylation sites in the C.acnes(C)CAMP2 polypeptide and / or modified C.acnes(C)CAMP2 polypeptide may be located at the position corresponding to residue N166 of SEQ ID NO: 203. The N-glycosylation sites may include N166, F167 and S168 with respect to the residue numbering of SEQ ID NO: 203, and have glycosylation at N166. In some embodiments, the C.acnes CAMP2 polypeptide and / or modified C.acnes CAMP2 polypeptide of the present invention include an amino acid substitution at the position corresponding to N166 (e.g., N166S) with respect to the residue numbering of SEQ ID NO: 203. The C.acnes CAMP2 polypeptide and / or modified C.acnes CAMP2 polypeptide may include the sequence of SEQ ID NO: 50, 51, or 54 having serine at position 166. The O-glycosylation site in the C.acnes CAMP2 polypeptide and / or modified C.acnes CAMP2 polypeptide may be located at the position corresponding to any S and / or T residue in SEQ ID NO: 203.

[0155] In some embodiments, the C. acnes (C.acnes)DsA1 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C.acnes)DsA1 polypeptide. The N-glycosylation site in the C. acnes (C.acnes)DsA1 polypeptide may be located at the position corresponding to residue N255 in SEQ ID NO: 204. The N-glycosylation site may include N255, I256, and T257 with respect to the residue numbering of SEQ ID NO: 204, and has glycosylation at N255. In some embodiments, the C. acnes (C.acnes)DsA1 polypeptide described herein contains an amino acid substitution at the position corresponding to N255 (e.g., N255Q or N255Q) with respect to the residue numbering of SEQ ID NO: 204. The O-glycosylation site in the C. acnes DsA1 polypeptide as described herein may be located at the position corresponding to any S and / or T residue in SEQ ID NO: 204, for example, the S292 residue.

[0156] In some embodiments, the C. acnes (C.acnes)DsA2 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation site of the natural C. acnes (C.acnes)DsA2 polypeptide. The N-glycosylation site in the C. acnes (C.acnes)DsA2 polypeptide may be located at the position corresponding to residue N5 of SEQ ID NO: 205. The N-glycosylation site may include N5, S6, and S7 with respect to the numbering of the residues in SEQ ID NO: 205, and has glycosylation at N5. In some embodiments, the C. acnes (C.acnes)DsA2 polypeptide described herein contains an amino acid substitution at the position corresponding to N5 (e.g., N5Q) with respect to the numbering of the residues in SEQ ID NO: 205. The C. acnes (C.acnes)DsA2 polypeptide may include the sequence of SEQ ID NO: 68 or 69, which has glutamine at position 5. The O-glycosylation sites in the C. acnes DsA2 polypeptide as described herein may be located at positions corresponding to any S and / or T residues in SEQ ID NO: 205.

[0157] In some embodiments, the C. acnes (C. acnes) PITP polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) PITP polypeptide. The N-glycosylation sites in the C. acnes (C. acnes) PITP polypeptide may be located at positions corresponding to residues N230, N242, N362, and N373 of SEQ ID NO: 206. The N-glycosylation sites may include (i) N230, G231 and S232 (with glycosylation at N230), (ii) N242, G243 and S244 (with glycosylation at N242), (iii) N362, L363 and T364 (with glycosylation at N362), and / or (iv) N373, V374 and T375 (with glycosylation at N373), and the amino acid numbering is relative to the residue numbering of Sequence ID No. 206. In some embodiments, the C. acnes PITP polypeptide of the present invention comprises one or more (e.g., all) amino acid substitutions at positions corresponding to N230 (e.g., N230K), S244 (e.g., S244G), N362 (e.g., N362Q), and N373 (e.g., N373S), where the amino acid numbering corresponds to the residue numbering of SEQ ID NO: 206. In some embodiments, the C. acnes PITP polypeptide as described herein comprises one or more (e.g., all) amino acid substitutions at positions corresponding to N230 (e.g., N230K), N242 (e.g., N242G), N362 (e.g., N362Q), and N373 (e.g., N373S), where the amino acid numbering corresponds to the residue numbering of SEQ ID NO: 206. The O-glycosylation site in the C. acnes PITP polypeptide may be located at a position corresponding to any S and / or T residue in SEQ ID NO: 206. In some embodiments, the C. acnes PITP polypeptide described herein includes the sequence of SEQ ID NO: 76, 77, or 79, which has lysine at position 230, glycine at position 244, glutamine at position 362, and serine at position 373.

[0158] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide described herein contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) DsA1 polypeptide and / or at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) DsA2 polypeptide. The N-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide may be located at the position corresponding to residue N255 of SEQ ID NO: 70. The N-glycosylation site may include N255, I256 and T257 with respect to the numbering of residues in SEQ ID NO: 70, and has glycosylation at N255. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes an N255Q or N255A (e.g., N255Q) substitution in SEQ ID NO: 70. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide herein includes an amino acid substitution at the position corresponding to N255 (e.g., N255Q or N255A) in relation to the residue numbering of SEQ ID NO: 70. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes the sequence of SEQ ID NO: 72 having a glutamine at position 255. The O-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide may be located at the position corresponding to any S and / or T residue in SEQ ID NO: 70. The O-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide may be located at the positions corresponding to residues S291 and / or S292 of SEQ ID NO: 70. The O-glycosylation site may include S291 and S292 with respect to the numbering of residues in SEQ ID NO: 70, and has glycosylation at S291 and / or S292. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes one or more substitutions (e.g., all three) selected from N255A, S291M, and S292G.In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes one or more (e.g., all three) amino acid substitutions at positions corresponding to N255 (e.g., N255A), S291 (e.g., S291M), and S292 (e.g., S292G) in relation to the residue numbering of SEQ ID NO: 70. The chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide may include the sequence of SEQ ID NO: 81 having alanine at position 248, methionine at position 284, and glycine at position 285. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide includes the sequence of SEQ ID NO: 81 having alanine at position 248, methionine at position 284, and glycine at position 285, where the residues correspond to positions 255, 291, and 292 of SEQ ID NO: 70, respectively.

[0159] In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptides described herein include a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) DsA1 polypeptide, one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) DsA2 polypeptide, and / or one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C. acnes) PITP polypeptide. The N-glycosylation sites in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may be located at the position corresponding to residue N255 with respect to the residue numbering of SEQ ID NO: 80 or 83. The N-glycosylation site may include N255, I256, and T257 with respect to the numbering of the residues in SEQ ID NO: 80 or 83, and has glycosylation at N255. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes an N255Q or N255A (e.g., N255A) substitution in SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide herein includes an amino acid substitution at the position corresponding to N255 (e.g., N255Q or N255A) with respect to the numbering of the residues in SEQ ID NO: 80 or 83. The O-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may be located at the position corresponding to any S and / or T residue in SEQ ID NO: 80 or 83. The O-glycosylation sites in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may be located at one or more (e.g., all) positions corresponding to residues S291, S292, T299, T301, T303, T305, T425, S428, and T436 of SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide described herein includes substitution with a GGGGG linker in the PTPTPTPT region located at positions 298-305 of SEQ ID NO: 80 or 83.In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes one or more (e.g., all) substitutions selected from S291M, S292G, P298G, T299G, P300G, T301G, P302G, T425G, S428G, and T436G. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes one or more (e.g., all) substitutions selected from S291M, S292G, T425G, S428G, and T436G for the residue numbering of SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide includes one or more (e.g., all) amino acid substitutions at positions corresponding to S291 (e.g., S291M), S292 (e.g., S292G), T425 (e.g., T425G), S428 (e.g., S428G), and T436 (e.g., T436G) with respect to the residue numbering of SEQ ID NO: 80 or 83. The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may include the sequence of SEQ ID NO: 82, which has methionine at position 291 and glycine at positions 292, 299, 301, 422, 425, and 433. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may include the sequence of Sequence ID No. 82, which has methionine at position 291 and glycine at positions 292, 298, 299, 300, 301, 302, 422, 425, and 433. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide may include the sequence of Sequence ID No. 368, which has alanine at position 248, methionine at position 284, and glycine at positions 285, 291, 292, 293, 294, 295, 415, 418, and 426.

[0160] In some embodiments, the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptides described herein include a single amino acid substitution at one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C.acnes)DsA1 polypeptide, one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C.acnes)DsA2 polypeptide, one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C.acnes)PITP polypeptide, and / or one or more (e.g., all) positions corresponding to the glycosylation sites of the natural C. acnes (C.acnes)CAMP2 polypeptide.

[0161] As described in the section "Chimera DsA1 / DsA2 / PITP / CAMP2" above, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may include the C. acnes (C. acnes) CAMP2 polypeptide or its immunogenic fragments. The N-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may be located at the position corresponding to residue N166 of SEQ ID NO: 203 (i.e., corresponding to N194 of SEQ ID NO: 202) if the residue is present in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide. In the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide, the N-glycosylation sites may be located at positions N166, F167, and S168 relative to the residue numbering in SEQ ID NO: 203, and have glycosylation at N166 (i.e., corresponding to positions N194, F195, and S196 relative to the residue numbering in SEQ ID NO: 202, and have glycosylation at N194). In some embodiments, the C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide of the present invention includes an amino acid substitution at a position corresponding to N166 (e.g., N166S) relative to the residue numbering in SEQ ID NO: 203 (i.e., corresponding to N194 (e.g., N194S) relative to the residue numbering in SEQ ID NO: 202), when present in the polypeptide. In the C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide, the O-glycosylation site, when present in the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide, may be located at a position corresponding to any S and / or T residue in SEQ ID NO: 203 (i.e., corresponding to any S and / or T residue in residues 29-267 of SEQ ID NO: 202).

[0162] As described in the section "Chimera DsA1 / DsA2 / PITP / CAMP2" above, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may include SEQ ID NOs. 80, 82, or 83. The N-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may be located at the position corresponding to residue N255 in the numbering of SEQ ID NOs. The N-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may include N255, I256, and T257 in the numbering of SEQ ID NOs. 80 or 83, and has glycosylation at N255. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptides herein include an amino acid substitution at the position corresponding to N255 (e.g., N255Q or N255A) with respect to the residue numbering of SEQ ID NO: 80 or 83. The O-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may be located at the position corresponding to any S and / or T residue in SEQ ID NO: 80 or 83. The O-glycosylation site in the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may be located at one or more (e.g., all) positions corresponding to residues S291, S292, T299, T301, T303, T305, T425, S428 and T436 in SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein includes substitution with a GGGGG linker in the PTPTPTPT region located at positions 298-305 of SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes one or more (e.g., all) substitutions selected from S291M, S292G, P298G, T299G, P300G, T301G, P302G, T425G, S428G, and T436G.In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes one or more (e.g., all) substitutions selected from S291M, S292G, T425G, S428G, and T436G for the residue numbering of SEQ ID NO: 80 or 83. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide includes one or more (e.g., all) amino acid substitutions at the positions corresponding to S291 (e.g., S291M), S292 (e.g., S292G), T425 (e.g., T425G), S428 (e.g., S428G), and T436 (e.g., T436G) for the residue numbering of SEQ ID NO: 80 or 83. The chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may contain the sequence of Sequence ID No. 82, which has methionine at position 291 and glycine at positions 292, 299, 301, 422, 425, and 433. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may contain the sequence of Sequence ID No. 82, which has methionine at position 291 and glycine at positions 292, 298, 299, 300, 301, 302, 422, 425, and 433.

[0163] Secretory signal peptide (SS) sequence The C. acnes (C. acnes) CAMP2 polypeptide, C. acnes (C. acnes) DsA1 polypeptide, C. acnes (C. acnes) DsA2 polypeptide, C. acnes (C. acnes) PITP polypeptide, chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide, chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein may contain secretory signal peptide (SS) sequences. Secretory signal peptides can be cleaved in post-translational processing of the C. acnes (C. acnes) polypeptides described herein. Therefore, mature forms of C. acnes (C. acnes) polypeptides may not contain secretory signal peptide sequences. However, in the nucleic acids described herein that encode the C. acnes polypeptide described herein, there may be nucleotide sequences that encode a secretory signal peptide sequence.

[0164] The C. acnes (C.acnes) CAMP2 polypeptide, C. acnes (C.acnes) DsA1 polypeptide, C. acnes (C.acnes) DsA2 polypeptide, C. acnes (C.acnes) PITP polypeptide, chimeric C. acnes (C.acnes) DsA1 / DsA2 polypeptide, chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein may contain the secretion signal peptide sequence of their respective native C. acnes (C.acnes) polypeptides. This may be advantageous when C. acnes (C.acnes) polypeptides are expressed as recombinant proteins in prokaryotic cells.

[0165] In some embodiments, the C. acnes (C.acnes)CAMP2 polypeptide, C. acnes (C.acnes)DsA1 polypeptide, C. acnes (C.acnes)DsA2 polypeptide, C. acnes (C.acnes)PITP polypeptide, chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide, chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein may contain secretory signal peptide sequences of a virus or a eukaryote (e.g., human). By conjugating a secretory signal peptide sequence of a virus or a eukaryote to the polypeptides described herein, many advantages can be provided to immunogenic compositions. In particular, when expressed from mRNA in eukaryotic cells, polypeptides of the present invention containing SS sequences may show increased extracellular expression compared to polypeptides without SS sequences. Increased extracellular expression can promote higher immunogenicity, which in turn can lead to better vaccine efficacy.

[0166] Viral SS sequences can be found in publicly accessible databases (e.g., NCBI or UniProt databases) that contain annotated viral polypeptide sequences and identify experimentally validated SS start and end locations.

[0167] In certain embodiments, the locations of SS sequence cleavage sites in a given known input polypeptide sequence and a SS sequence sequence can be predicted using the SignalP algorithm. The SignalP algorithm (more specifically SignalP v6.0) is described in further detail in Armenteros et al. (Nature Biotechnology. 37:420-423. 2019), Teufel et al. (Nature Biotechnology. 40:1023-1025. 2022) and https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / , each of which is incorporated herein by reference in its entirety. The strength of the prediction is assessed based on a cumulative rank score that takes into account the likelihood of detecting standard features of the signal sequence (SS likelihood score) and the likelihood of cleavage at the cleavage site (cleavage probability score). In certain embodiments, the viral secretion signal peptide has a SignalP cleavage probability score of at least 0.8, at least 0.85, at least 0.90, or at least 0.95, when determined using SignalP 6.0. In some embodiments, the viral secretion signal peptide has a SignalP signal peptide likelihood score of at least 0.8, at least 0.85, at least 0.90, or at least 0.95, when determined using SignalP 6.0.

[0168] In certain embodiments, the SS sequence is a viral SS sequence. In certain embodiments, the viral secretion signal peptide sequence is derived from a viral sequence in a virus capable of infecting humans. The terms “influenza,” “SARS-CoV-2,” “varicella-zoster virus (VZV),” “measles,” “rubella,” “rabies,” “Ebola,” and “smallpox” preceding the phrase “secretion signal peptide sequence” indicate that the secretion signal peptide is derived from the virus corresponding to its name.

[0169] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of influenza secretion signal peptide sequences, SARS-CoV-2 secretion signal peptide sequences, varicella-zoster virus (VZV) secretion signal peptide sequences, measles secretion signal peptide sequences, rubella secretion signal peptide sequences, mumps secretion signal peptide sequences, Ebola secretion signal peptide sequences, rabies secretion signal peptide sequences, and smallpox secretion signal peptide sequences. These specific signal peptides are derived from viral sequences in viruses that have been administered to humans as vaccines (attenuated, inactivated, or mRNA) with a proven robust safety profile.

[0170] In certain embodiments, the viral secretion signal peptide is selected from the group consisting of influenza hemagglutinin (HA) secretion signal peptide sequences, SARS-CoV-2 spike secretion signal peptide sequences, VZV gB secretion signal peptide sequences, VZV gE secretion signal peptide sequences, VZV gI secretion signal peptide sequences, VZV gK secretion signal peptide sequences, measles F protein secretion signal peptide sequences, rubella E1 protein secretion signal peptide sequences, rubella E2 protein secretion signal peptide sequences, mumps F protein secretion signal peptide sequences, Ebola GP protein secretion signal peptide sequences, rabies virus glycoprotein (rabies G) secretion signal peptide sequences, and smallpox 6kDa IC protein secretion signal peptide sequences.

[0171] In certain embodiments, the viral secretion signal peptide includes an HA secretion signal peptide sequence derived from influenza A or influenza B, preferably from influenza A.

[0172] The amino acid sequences of the exemplary viral secretion signal peptides of this disclosure are shown in Table 2 below. The amino acid sequences of the exemplary viral secretion signal peptides derived from influenza A or influenza B of this disclosure are shown in Table 3 below.

[0173] [Table 3]

[0174] [Table 4]

[0175] [Table 5]

[0176] [Table 6]

[0177] [Table 7]

[0178] [Table 8]

[0179] The secretion signal peptide sequence may be located at the N-terminus or C-terminus (e.g., the N-terminus) of the polypeptide described herein.

[0180] In certain embodiments, the SS amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0181] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence in a virus capable of infecting humans.

[0182] In certain embodiments, the viral secretion signal peptide is derived from a viral sequence selected from the group consisting of an influenza secretion signal peptide sequence and a non-influenza secretion signal peptide a SARS-CoV-2 secretion signal peptide sequence, a varicella-zoster virus (VZV) secretion signal peptide sequence, a measles secretion signal peptide sequence, a rubella secretion signal peptide sequence, a mumps secretion signal peptide sequence, an Ebola secretion signal peptide sequence, a smallpox secretion signal peptide sequence, and a rabies secretion signal peptide sequence.

[0183] In certain embodiments, the viral secretion signal peptide is selected from the group consisting of influenza hemagglutinin (HA) secretion signal peptide sequences, SARS-CoV-2 spike secretion signal peptide sequences, VZV gB secretion signal peptide sequences, VZV gE secretion signal peptide sequences, VZV gI secretion signal peptide sequences, VZV gK secretion signal peptide sequences, measles F protein secretion signal peptide sequences, rubella E1 protein secretion signal peptide sequences, rubella E2 protein secretion signal peptide sequences, mumps F protein secretion signal peptide sequences, Ebola GP protein secretion signal peptide sequences, smallpox 6kDa IC protein secretion signal peptide sequences, and rabies G protein secretion signal peptide sequences. Preferably, the viral secretion signal peptide includes an HA secretion signal peptide sequence derived from influenza A or influenza B, more preferably an HA secretion signal peptide sequence derived from influenza A.

[0184] In a particular embodiment, the HA secretion signal peptide sequence comprises the amino acid sequence MKX1X2LX3VX4LX5TFX6X7X8X9A (SEQ ID NO: 237), where X1 is selected from A and V, X2 is selected from I and K, X3 is selected from V and L, X4 is selected from L and M, X5 is selected from Y and C, X6 is selected from T and A, X7 is selected from T and A, X8 is selected from A and T, and X9 is selected from N and Y.

[0185] In certain embodiments, the HA secretion signal peptide sequence includes an amino acid sequence selected from MKAKLLVLLCTFTATYA (SEQ ID NO: 210), MKAILVVLLYTFTTANA (SEQ ID NO: 227), MKVKLLVLLCTFTATYA (SEQ ID NO: 228), MKAILVVLLYTFATANA (SEQ ID NO: 211), and MKAILVVMLYTFTTANA (SEQ ID NO: 229).

[0186] In a particular embodiment, the HA secretion signal peptide sequence comprises the amino acid sequence MKX1IIALSX2ILCLVFX3 (SEQ ID NO: 238), where X1 is selected from T and A, X2 is selected from Y, N, C and H, and X3 is selected from T and A.

[0187] In certain embodiments, the HA secretion signal peptide sequence includes an amino acid sequence selected from MKTIIALSYILCLVFT (SEQ ID NO: 230), MKTIIALSYILCLVFA (SEQ ID NO: 212), MKTIIALSNILCLVFA (SEQ ID NO: 231), MKAIIALSNILCLVFA (SEQ ID NO: 232), MKTIIALSCILCLVFA (SEQ ID NO: 233), and MKTIIALSHILCLVFA (SEQ ID NO: 234).

[0188] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MKAIIVLLMVVTSX1A (SEQ ID NO: 239), where X1 is selected from S and N.

[0189] In certain embodiments, the HA secretion signal peptide sequence comprises the amino acid sequence MX1AIIVLLMVVTSNA (SEQ ID NO: 240), where X1 is selected from K and E.

[0190] In certain embodiments, the HA secretion signal peptide sequence includes an amino acid sequence selected from MKAIIVLLMVVTSNA (SEQ ID NO: 213), MKAIIVLLMVVTSSA (SEQ ID NO: 235), and MEAIIVLLMVVTSNA (SEQ ID NO: 236).

[0191] In certain embodiments, the viral secretion signal peptides are MKAKLLVLLCTFTATYA (SEQ ID NO: 210), MKAILVVLLYTFATANA (SEQ ID NO: 211), MKTIIALSYILCLVFA (SEQ ID NO: 212), MKAIIVLLMVVTSNA (SEQ ID NO: 213), MFVFLVLLPLVS (SEQ ID NO: 214), MFLLTTKRTMFVFLVLLPLVS (SEQ ID NO: 215), MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVS (SEQ ID NO: 216), MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 217), M It contains an amino acid sequence selected from the group consisting of FLIQCLISAVIFYIQVTNA (SEQ ID NO: 218), MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 219), MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 220), MGAAAALTAVVLQGYNPPAYG (SEQ ID NO: 221), MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 222), MKVFLVTCLGFAVFSSSVC (SEQ ID NO: 223), MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS (SEQ ID NO: 224), MRSLIIFLLFPSIIYS (SEQ ID NO: 225), and MVPQALLFVPLLVFPLCFG (SEQ ID NO: 226).

[0192] In certain embodiments, the viral secretion signal peptide comprises the amino acid sequence MKAKLLVLLCTFTATYA (SEQ ID NO: 210).

[0193] In certain embodiments, the viral secretion signal peptide is positioned at the N-terminus of the polypeptide disclosed herein.

[0194] In certain embodiments, the viral secretion signal peptide is located at the C-terminus of the polypeptide disclosed herein.

[0195] In certain embodiments, a viral secretion signal peptide is bound to a polypeptide disclosed herein by a linker.

[0196] Transmembrane domain (TMB) The C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein may contain a transmembrane domain (TMB), more specifically heterologous (non-natural) TMB. The inclusion of heterologous TMB can be advantageous because it localizes the antigen to the cell membrane. This reduces intracellular localization of the antigen and can further promote higher immunogenicity compared to antigens that do not contain a TMB sequence.

[0197] C. acnes (C.acnes) CAMP2 polypeptide, C. acnes (C.acnes) DsA1 polypeptide, C. acnes (C.acnes) DsA2 polypeptide, C. acnes (C.acnes) PITP polypeptide, chimeric C. acnes (C.acnes) DsA1 / DsA2 polypeptide, chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide may also contain secretion signal peptide sequences. Typically, nucleic acids described herein that encode chimeric C. acnes (C.acnes) CAMP2 polypeptide containing a heteromembrane domain also contain nucleotide sequences that encode secretion signal peptide sequences. Typically, nucleic acids described herein that encode C. acnes (C.acnes) DsA1 polypeptide containing a heteromembrane domain also contain nucleotide sequences that encode secretion signal peptide sequences. Typically, nucleic acids described herein that encode the C. acnes (C. acnes) DsA2 polypeptide containing a heteromorphic transmembrane domain also include a nucleotide sequence encoding a secretion signal peptide sequence. Typically, nucleic acids described herein that encode the C. acnes (C. acnes) PITP polypeptide containing a heteromorphic transmembrane domain also include a nucleotide sequence encoding a secretion signal peptide sequence. Typically, nucleic acids described herein that encode the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide containing a heteromorphic transmembrane domain also include a nucleotide sequence encoding a secretion signal peptide sequence. Typically, nucleic acids described herein that encode the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide containing a heteromorphic transmembrane domain also include a nucleotide sequence encoding a secretion signal peptide sequence. Typically, nucleic acids described herein that encode the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide containing a heteromorphic transmembrane domain also include a nucleotide sequence encoding a secretion signal peptide sequence.

[0198] TMBs may originate from any known TMBs in the art, including but not limited to TMBs derived from eukaryotic transmembrane proteins (e.g., mammalian transmembrane proteins such as human transmembrane proteins), TMBs derived from prokaryotic transmembrane proteins, and TMBs derived from viral transmembrane proteins. TMBs may be further identified by in silico prediction algorithms in TMHMM prediction methods, for example, Krogh et al. (J Mol Biol. 305(3); 567-580. 2001) and https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / , each of which is incorporated herein by reference in whole. Some characteristics of TMBs are described in more detail in Albers et al. (Chapter 2 - cell membrane structures and functions. Basic Neurochemistry eighth edition. Pages 26-39. 2012), which is incorporated herein by reference. While not exclusive, typically, the TMB consists mainly of nonpolar (hydrophobic) amino acid residues and can traverse the lipid bilayer once or several times. Those skilled in the art are well aware of methods for determining the hydrophobicity of amino acids. See Simm et al. (2016), Biol Res., 49(1):31, Wimlet and White (1996), Nat Struct Biol., 3(10):842-848, https: / / blanco.biomol.uci.edu / hydrophobicity_scales.html and https: / / www.cgl.ucsf.edu / chimera / docs / UsersGuide / midas / hydrophob.html.

[0199] TMBs typically contain α-helices, each helix containing 18-21 amino acids sufficient to span the lipid bilayer. Therefore, in certain embodiments, the transmembrane domain contains one or more α-helices. In certain embodiments, the TMB contains or comprises (a) 15-50 amino acid residues, preferably 15-30 amino acid residues, more preferably 18-25 amino acid residues, and / or (b) at least 50%, at least 55%, or at least 60% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine, and / or (c) at least one α-helix.

[0200] In certain embodiments, the transmembrane domain is derived from an endogenous membrane protein, as further defined herein, where Albers et al. defines "endogenous membrane protein" (also known as endogenous membrane protein) as a membrane protein persistently bound to the lipid membrane. In certain embodiments, the transmembrane domain is derived from an endogenous polytopic protein. An endogenous polytopic protein is a protein that spans the entire membrane. In certain embodiments, the transmembrane domain is derived from a single-pass transmembrane protein, more specifically, for example, a type I or type II bitopic membrane protein. Single-pass transmembrane proteins traverse the membrane only once (i.e., bitopic membrane proteins), while multi-pass transmembrane proteins weave through the membrane and traverse it several times. Single-pass transmembrane proteins can be classified into type I, where their carboxyl terminus is oriented toward the cytosol, or type II, where their amino terminus is oriented toward the cytosol. In certain embodiments, the transmembrane domain is derived from an endogenous monotopic protein. Endogenous monotopic proteins are proteins that bind to only one side of a membrane and do not completely span the lipid bilayer.

[0201] In certain embodiments, the heterologous transmembrane domain is derived from a non-human sequence.

[0202] In certain embodiments, the heterologous transmembrane domain is derived from a viral sequence. The terms “influenza,” “SARS-CoV-2,” “varicella-zoster virus (VZV),” “measles,” “rubella,” “rabies,” “Ebola,” and “smallpox” preceding the phrase “transmembrane domain sequence” indicate that the transmembrane domain sequence is derived from the virus corresponding to that name.

[0203] In certain embodiments, the heterologous transmembrane domain is derived from a viral transmembrane domain sequence selected from the group consisting of influenza transmembrane domain sequences, SARS-CoV-2 transmembrane domain sequences, varicella-zoster virus (VZV) transmembrane domain sequences, measles transmembrane domain sequences, rubella transmembrane domain sequences, mumps transmembrane domain sequences, rabies transmembrane domain sequences, and Ebola transmembrane domain sequences. These specific transmembrane domains are derived from viral sequences in viruses that have been administered to humans as vaccines (attenuated, inactivated, or mRNA) with a proven robust safety profile.

[0204] In certain embodiments, the heterologous transmembrane domain is selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequences, SARS-CoV-2 spike transmembrane domain sequences, VZV gB transmembrane domain sequences, VZV gE transmembrane domain sequences, VZV gI transmembrane domain sequences, VZV gK transmembrane domain sequences, measles F protein transmembrane domain sequences, rubella E1 protein transmembrane domain sequences, rubella E2 protein domain sequences, mumps F protein transmembrane domain sequences, rabies virus glycoprotein (rabies G) transmembrane domain sequences, and Ebola GP protein transmembrane domain sequences.

[0205] In certain embodiments, the heterologous transmembrane domain includes an HA transmembrane domain sequence derived from influenza A or influenza B, preferably influenza A.

[0206] The amino acid sequences of exemplary viral transmembrane domains in this disclosure are shown in Table 4 below.

[0207] [Table 9]

[0208] In certain embodiments, the heterologous TMB sequence is located at the N-terminus or C-terminus (e.g., the C-terminus) of the polypeptide described herein.

[0209] In certain embodiments, the TMB amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0210] In some embodiments, one or more of the C. acnes CAMP2 polypeptide, C. acnes DsA1 polypeptide, C. acnes DsA2 polypeptide, C. acnes PITP polypeptide, chimeric C. acnes DsA1 / DsA2 polypeptide, chimeric C. acnes DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide include heterogeneous transmembrane domains as described herein. In alternative embodiments, the C. acnes (C.acnes)CAMP2 polypeptide, C. acnes (C.acnes)DsA1 polypeptide, C. acnes (C.acnes)DsA2 polypeptide, C. acnes (C.acnes)PITP polypeptide, chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide, chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide and / or chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide do not contain heterologous transmembrane domains. Such polypeptides may contain secretion signal peptide sequences. In further embodiments, the polypeptides of the present invention are secreted. In some embodiments, the C. acnes (C.acnes)CAMP2 polypeptide described herein is a secreted polypeptide. In some embodiments, the C. acnes (C.acnes)DsA1 polypeptide described herein is a secreted polypeptide. In some embodiments, the C. acnes (C. acnes) DsA2 polypeptide described herein is a secreted polypeptide. In some embodiments, the C. acnes (C. acnes) PITP polypeptide described herein is a secreted polypeptide. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide described herein is a secreted polypeptide. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide described herein is a secreted polypeptide.In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide described herein is a secreted polypeptide. The secreted polypeptide described herein includes a secretion signal peptide sequence.

[0211] In certain embodiments, the TMB comprises or consists of (a) 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues, and / or (b) at least 50% hydrophobic amino acid residues, preferably selected from the group consisting of alanine, isoleucine, leucine, valine, phenylalanine, tryptophan, and tyrosine, and / or (c) at least one α-helix.

[0212] In certain embodiments, the TMB is derived from an intrinsic membrane protein, preferably a single-pass transmembrane protein, more preferably a biotopic membrane protein, and even more preferably a type I biotopic membrane protein.

[0213] In certain embodiments, the TMB is derived from a non-human sequence.

[0214] In certain embodiments, the TMB is derived from a viral sequence.

[0215] In certain embodiments, the TMB is derived from an influenza transmembrane domain sequence and a viral transmembrane domain sequence selected from a group consisting of non-influenza transmembrane domain sequences selected from the group consisting of an influenza transmembrane domain sequence, a SARS-CoV-2 transmembrane domain sequence, a varicella-zoster virus (VZV) transmembrane domain sequence, a measles transmembrane domain sequence, a rubella transmembrane domain sequence, a mumps transmembrane domain sequence, an Ebola transmembrane domain sequence, and a rabies transmembrane domain sequence.

[0216] In certain embodiments, the TMB is selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequences, SARS-CoV-2 spike transmembrane domain sequences, VZV gB transmembrane domain sequences, VZV gE transmembrane domain sequences, VZV gI transmembrane domain sequences, VZV gK transmembrane domain sequences, measles F protein transmembrane domain sequences, rubella E1 protein transmembrane domain sequences, rubella E2 protein transmembrane domain sequences, mumps F protein transmembrane domain sequences, Ebola GP protein transmembrane domain sequences, and rabies G protein transmembrane domain sequences. Preferably, the TMB includes an HA transmembrane domain sequence derived from influenza A or influenza B, more preferably an HA transmembrane domain sequence derived from influenza A.

[0217] In certain embodiments, TMB is ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 208), ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 209), ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 241), STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 242), WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 243), FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 244), AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 245), IIIPIVASVMILTAMVI It contains an amino acid sequence selected from the group consisting of VIVI (SEQ ID NO: 246), YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 247), IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 248), LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 249), WWQLTLGAICALLLAGLLACC (SEQ ID NO: 250), IVAALLVLSILSIIISLLFCCW (SEQ ID NO: 251), WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 252), and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 253).

[0218] In certain embodiments, TMB comprises the amino acid sequence ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 208).

[0219] In certain embodiments, the TMB is linked to a polypeptide described herein by a linker.

[0220] In certain embodiments, the TMB is located at the N-terminus of the polypeptide described herein.

[0221] In certain embodiments, the TMB is located at the C-terminus of the polypeptide described herein.

[0222] Linker In some embodiments, the polypeptide of the present invention comprises a linker. In some embodiments, the polypeptide of the present invention comprises two or more antigens linked via a linker.

[0223] In certain embodiments of this disclosure, a secretory signal peptide (SS) sequence or transmembrane domain (TMB) is directly fused to the polypeptide described herein (i.e., there is no linker, such as an amino acid linker, connecting the SS sequence or TMB to the polypeptide described herein).

[0224] In other embodiments, the SS sequences and TMBs of this disclosure are optionally linked to the polypeptides described herein by linkers. In certain embodiments, the linkers are amino acid linkers. In certain embodiments, the amino acid linkers are 1 to 10 amino acid long (for example, the amino acid linkers have lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).

[0225] Exemplary examples of linkers include glycine polymer (Gly)n (wherein n is at least 1, 2, 3, 4, 5, 6, 7, or 8), glycine-serine polymer (GlySer)n (wherein n is at least 1, 2, 3, 4, 5, 6, 7, or 8), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art.

[0226] Glycine and glycine-serine polymers are relatively unstructured and flexible, and therefore may be able to function as neutral tethers between SS sequences and / or TMBs and the polypeptides described herein. In certain embodiments, the linker is SGS or GSG.

[0227] Other exemplary linker sequences include, but are not limited to, the following amino acid sequences: GGG, DGGGS (SEQ ID NO: 254), TGEKP (SEQ ID NO: 255) (Liu et al. Proc. Natl. Acad. Sci. 94, 5525-5530. 1997), GGRR (SEQ ID NO: 256), (GGGGS)n (SEQ ID NO: 257) (wherein n=1,2,3,4 or 5) (Kim et al. Proc. Natl. Acad. Sci. 93, 1156-1160. 1996), EGKSSGSGSESKVD (SEQ ID NO: 258) (Chaudhary et al. Proc. Natl. Acad. Sci. 87, 1066-1070. 1990), KESGSVSSEQLAQFRSLD (SEQ ID NO: 259) (Bird et al. (Cooper et al. Science. 242:423-426. 1988), GGRRGGGS (SEQ ID NO: 260), LQRRDGERP (SEQ ID NO: 261), LRQKDGGGSERP (SEQ ID NO: 262), and GTSTGSGKPGSGEGSTKG (SEQ ID NO: 263) (Cooper et al. Blood. 101(4); 1637-1644. 2003). Preferred linkers are shorter, for example, consisting of 3, 4, or 5 amino acids.

[0228] Further examples of linkers are shown in Chen et al. (Adv Drug Deliv Rev. 65(10): 1357-1369. 2013), which are incorporated herein by reference.

[0229] Compositions - Nucleic acids and polypeptides The present invention provides compositions comprising one or more nucleic acids of the present disclosure. The present invention also provides compositions comprising one or more polypeptides of the present disclosure. The compositions of the present invention may be, for example, pharmaceutical compositions comprising a pharmaceutically acceptable carrier, excipient, or diluent. In certain embodiments, the compositions of the present invention are immunogenic compositions. “Immunogenic composition” means a composition comprising nucleic acids or proteins that, when administered to a subject, induce an immune response, such as an antigen-specific immune response. The immune response may be a humoral (antibody) immune response or a cellular immune response. The compositions of the present invention may be vaccine compositions. An immunogenic composition (e.g., a vaccine composition) may induce immunity (e.g., an antibody response) against C. acnes infection. The antibody response may include antibodies that bind to the surface of C. acnes bacteria and recruit immune effector cells (e.g., effector cells of the immune system such as phagocytes). The antibodies may be capable of inducing opsonin phagocytic injury in vitro. The antibodies may be cross-reactive across various C. acnes strains. Antibodies can reduce or neutralize CAMP2-mediated inflammation.

[0230] As used herein, “protective immunity” or “protective immune response” refers to the induction of immunity or an immune response to an infectious agent indicated by a subject (e.g., C. acnes) that prevents or improves an infection or reduces at least one of its symptoms. Specifically, the induction of protective immunity or a protective immune response by administration of the compositions of the present invention is evident by eliminating or reducing the presence of one or more symptoms of a C. acnes infection. As used herein, the term “immune response” refers to both humoral and cellular immune responses. In preferred embodiments, treatment with the compositions of the present invention as described herein results in protective immunity against an infection caused by C. acnes.

[0231] Nucleic acid composition In one embodiment, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide as described herein.

[0232] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a modified C. acnes CAMP2 polypeptide as described herein.

[0233] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C. acnes) DsA1 polypeptide as described herein.

[0234] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C. acnes) DsA2 polypeptide as described herein.

[0235] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C. acnes)PITP polypeptide as described herein.

[0236] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein.

[0237] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide as described herein.

[0238] In another aspect, the present invention provides a composition comprising nucleic acids as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein.

[0239] Any of the compositions described herein may comprise one or more nucleic acids encoding one or more antigens, as described in International Publication No. 2021 / 165543 (incorporated herein by reference).

[0240] CAMP2 composition In one embodiment, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide as described herein.

[0241] In another aspect, the present invention provides an immunogenic composition comprising a nucleic acid as described herein, wherein the nucleic acid is mRNA, and comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide as described herein.

[0242] In another aspect, the present invention provides a composition comprising a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide as described herein, further comprising an LNP.

[0243] For example, a composition containing the C. acnes CAMP2 polypeptide for use in the present invention, delivered as mRNA and / or in the form of a composition containing LNPs, may induce antibodies in a subject. Such antibodies may neutralize the biological activity of the CAMP2 polypeptide, such as the inflammatory activity of CAMP2. Such antibodies may also neutralize the co-hemolytic activity of the CAMP2 polypeptide.

[0244] In some embodiments, any of the compositions described herein comprising a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes CAMP2 polypeptide further comprises one or more of: (b)(i) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes DsA1 polypeptide, (b)(ii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes DsA2 polypeptide, (b)(iii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes PITP polypeptide, (b)(iv) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 polypeptide, and (b)(v) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 / PITP polypeptide. In some embodiments, the composition comprises the nucleic acid of (b)(i), the nucleic acid of (b)(ii), and the nucleic acid of (b)(iii). In some embodiments, the composition comprises the nucleic acid of (b)(v). In preferred embodiments, the composition comprises the nucleic acid of (b)(iii) and the nucleic acid of (b)(iv).

[0245] In some embodiments, two or more of the nucleic acids of (a) and (b)(i)-(v) are located on the same nucleic acid molecule or on different nucleic acid molecules (e.g., all nucleic acids in the composition are on the same nucleic acid molecule or all nucleic acids in the composition are on individual nucleic acid molecules). In some embodiments, the nucleic acid of (a), the nucleic acid of (b)(iii), and the nucleic acid of (b)(iv) are located on the same nucleic acid molecule or on separate nucleic acid molecules. In some embodiments, the nucleic acid of (a) and the nucleic acid of (b)(v) are located on the same nucleic acid molecule or on separate nucleic acid molecules. In some embodiments, the composition comprises and provides a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein. Typically, the nucleic acid of (a), the nucleic acid of (b)(iii), and the nucleic acid of (b)(iv) are provided by separate nucleic acid molecules.

[0246] Modified CAMP2 composition In another aspect, the present invention provides a composition comprising a nucleic acid as described herein comprising a nucleotide sequence encoding a modified C. acnes CAMP2 polypeptide as described herein.

[0247] In some embodiments, the composition further comprises one or more of: (i) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes DsA1 polypeptide; (ii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes DsA2 polypeptide; (iii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes PITP polypeptide; (iv) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 polypeptide; and (v) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 / PITP polypeptide.

[0248] In some embodiments, the composition comprises nucleic acid (i), nucleic acid (ii), and nucleic acid (iii). In some embodiments, the composition comprises nucleic acid (v). In preferred embodiments, the composition comprises nucleic acid (iii) and nucleic acid (iv).

[0249] DsA1, DsA2, and PITP compositions In another aspect, the present invention provides a composition comprising (i) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes (C. acnes) DsA1 polypeptide, (ii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes (C. acnes) DsA2 polypeptide, (iii) a nucleic acid as described herein comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide, (iv) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide, (v) a nucleic acid as described herein comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide, and (vi) one or more nucleic acids as described herein comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide.

[0250] Combining C. acnes (C. acnes) DsA1, DsA2, and PITP polypeptides may induce an immune response to a greater number of C. acnes (C. acnes) lineages compared to using the individual antigens. Using DsA1 and / or DsA2 in combination with PITP may induce a pool of antibodies that cross-react with a greater number of C. acnes (C. acnes) strains compared to using the individual antigens. Preferably, C. acnes (C. acnes) DsA1 and DsA2 polypeptides are provided as chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptides. C. acnes (C. acnes) PITP polypeptide may be provided as a separate polypeptide molecule, as part of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP polypeptide or as part of the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide. Typically, C. acnes PITP polypeptides are provided as separate polypeptide molecules.

[0251] In some embodiments, the composition comprises nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA1 polypeptide; nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA2 polypeptide; and nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) PITP polypeptide.

[0252] In some embodiments, the composition comprises nucleic acids as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein, and nucleic acids as described herein, comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide as described herein. Typically, a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide contains the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto, and a C. acnes (C. acnes) PITP polypeptide contains the sequence of SEQ ID NO: 73 or a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises the sequence of SEQ ID NO: 70, and the C. acnes (C. acnes) PITP polypeptide comprises the sequence of SEQ ID NO: 73.

[0253] In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide include the nucleotide sequence according to SEQ ID NO: 179 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., 85%) identity therewith. s) A nucleic acid comprising a nucleotide sequence encoding a PITP polypeptide includes the nucleotide sequence according to Sequence ID No. 182 or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., 75%) identity thereto.

[0254] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence described herein) and the sequence according to Sequence ID No. 179 or a sequence having at least 85% identity thereto, and a nucleic acid comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence described herein) and the sequence according to Sequence ID No. 182 or a sequence having at least 75% identity thereto.

[0255] Any one of the compositions of the present invention may comprise a combination of nucleic acids as described herein (for example, they may be formulated in the same composition). Alternatively, a combination of nucleic acids as described herein may comprise two or more separate compositions (for example, as a combination of compositions for simultaneous, separate, or sequential administration (for example, in therapeutic or prophylactic use as described herein)).

[0256] A composition of the Disclosure comprising one or more nucleic acids of the Disclosure may also comprise one or more additional components, such as small molecule immunostimulants (e.g., TLR agonists). A composition of the Disclosure may also comprise a delivery system for nucleic acids (e.g., RNA) described herein, such as liposomes, oil-in-water emulsions, or microparticles. In some embodiments, the composition comprises lipid nanoparticles (LNPs). In certain embodiments, the composition comprises nucleic acid molecules of the Invention encapsulated within LNPs.

[0257] In some embodiments, the compositions described herein are in a frozen liquid form. In some embodiments, the compositions described herein are in a freeze-dried form (e.g., freeze-dried form).

[0258] Polypeptide composition In one embodiment, the present invention provides a composition comprising a C. acnes CAMP2 polypeptide as described herein.

[0259] In another aspect, the present invention provides compositions comprising a modified C. acnes CAMP2 polypeptide as described herein.

[0260] In another embodiment, the present invention provides a composition comprising a C. acnes (C. acnes) DsA1 polypeptide as described herein.

[0261] In another embodiment, the present invention provides a composition comprising a C. acnes (C. acnes) DsA2 polypeptide as described herein.

[0262] In another aspect, the present invention provides a composition comprising a C. acnes PITP polypeptide as described herein.

[0263] In another aspect, the present invention provides a composition comprising a chimeric C. acnes DsA1 / DsA2 polypeptide as described herein.

[0264] In another aspect, the present invention provides a composition comprising a chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein.

[0265] In another aspect, the present invention provides a composition comprising a chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein.

[0266] Any of the compositions described herein may include any of the polypeptide antigens as described in WO 2021 / 165543 pamphlet.

[0267] CAMP2 composition In one aspect, the present invention provides a composition comprising a C. acnes CAMP2 polypeptide as described herein.

[0268] In some embodiments, a composition comprising (a) a C. acnes CAMP2 polypeptide as described herein further comprises (b)(i) a C. acnes DsA1 polypeptide as described herein, (b)(ii) a C. acnes DsA2 polypeptide as described herein, (b)(iii) a C. acnes PITP polypeptide as described herein, (b)(iv) a chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, and (b)(v) a chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein. In some embodiments, the composition comprises the polypeptide of (b)(i), the polypeptide of (b)(ii), and the polypeptide of (b)(iii). In some embodiments, the composition comprises the polypeptide of (b)(v). In a preferred embodiment, the composition comprises the polypeptide of (b)(iii) and the polypeptide of (b)(iv).

[0269] In some embodiments, the polypeptides of (a), (b)(iii) and (b)(iv) are provided as chimeric polypeptides, for example, the chimeric C. acnes (C. acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein. In some embodiments, the polypeptides of (a) and (b)(v) are provided as chimeric polypeptides, for example, the chimeric C. acnes (C. acnes)DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein. Typically, the polypeptides of (a), (b)(iii) and (b)(iv) are provided as separate polypeptides.

[0270] Modified CAMP2 composition In another aspect, the present invention provides compositions comprising a modified C. acnes CAMP2 polypeptide as described herein.

[0271] In some embodiments, a composition comprising a modified C. acnes CAMP2 polypeptide as described herein further comprises one or more of the following: (i) a C. acnes DsA1 polypeptide as described herein, (ii) a C. acnes DsA2 polypeptide as described herein, (iii) a C. acnes PITP polypeptide as described herein, (iv) a chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, and (v) a chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein.

[0272] In some embodiments, the composition comprises polypeptide (i), polypeptide (ii), and polypeptide (iii). In some embodiments, the composition comprises polypeptide (v). In preferred embodiments, the composition comprises polypeptide (iii) and polypeptide (iv).

[0273] DsA1, DsA2, and PITP compositions In another aspect, the present invention provides a composition comprising one or more of the following: (i) C. acnes DsA1 polypeptide as described herein, (ii) C. acnes DsA2 polypeptide as described herein, (iii) C. acnes PITP polypeptide as described herein, (iv) chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, (v) chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein, and (vi) DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein.

[0274] In some embodiments, the composition comprises a C. acnes (C. acnes) DsA1 polypeptide, a C. acnes (C. acnes) DsA2 polypeptide, and a C. acnes (C. acnes) PITP polypeptide, as described herein.

[0275] In some embodiments, the composition comprises a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein and a C. acnes (C. acnes) PITP polypeptide as described herein. Typically, a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide contains the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto, and a C. acnes (C. acnes) PITP polypeptide contains the sequence of SEQ ID NO: 73 or a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises the sequence of SEQ ID NO: 70, and the C. acnes (C. acnes) PITP polypeptide comprises the sequence of SEQ ID NO: 73.

[0276] Any one of the compositions of the present invention may comprise a combination of polypeptides as described herein (for example, they may be formulated in the same composition). Alternatively, a combination of polypeptides as described herein may comprise two or more separate compositions (for example, as a combination of compositions for simultaneous, separate, or sequential administration (for example, in therapeutic or prophylactic use as described herein)).

[0277] A composition of the Disclosure comprising one or more polypeptides of the Disclosure may include adjuvants as described herein. As used herein, “adjuvant” means a substance or vehicle that enhances the immune response to an antigen. Examples of adjuvants include, but are not limited to, suspensions of inorganic substances (e.g., alum, aluminum hydroxide, or phosphate) to which an antigen has been adsorbed, and water-in-oil or oil-in-water emulsions in which an antigen solution is emulsified in mineral oil or water (e.g., Freund’s incomplete adjuvant). Dead mycobacteria may be included to further enhance antigenicity (e.g., Freund’s complete adjuvant). Examples of adjuvants include squalene-based oil-in-water emulsion adjuvants (for example, AF03 as described in International Publication No. 2007006939 and U.S. Patent No. 8,703,095, AS03 as described in International Publication No. 1995017209, International Publication No. 1995017210 and U.S. Patent Nos. 6,623,739, 7,029,678 and 7,510,698, and MF59 as described in International Publication No. 1990014837 and U.S. Patent Nos. 6,299,884 and 6,451,325). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, for example, U.S. Patents No. 6,194,388, 6,207,646, 6,214,806, 6,218,371, 6,239,116, 6,339,068, 6,406,705, and 6,429,199). Adjuvants may also include biological molecules such as Toll-like receptor (TLR) agonists (e.g., AS01 as described in International Publication No. 2007068907 and U.S. Patent Nos. 10,039,823 and 10,143,745, SPA14 as described in International Publication No. 2022090359 and LEQ as described in International Publication No. 2023056089) and co-stimulatory molecules.

[0278] In some embodiments, the adjuvant is selected from the group consisting of aluminum-based adjuvants (e.g., AlOOH), squalene-based oil-in-water emulsion adjuvants (e.g., AF03, AS03, MF59), and liposomal adjuvants containing saponins and TLR4 agonists (e.g., SPA14, LEQ, AS01).

[0279] In a preferred embodiment, the adjuvant is selected from the group consisting of AlOOH, AF03, and SPA14.

[0280] In some embodiments, the compositions described herein are in a freeze-liquid form. In some embodiments, the compositions described herein are in a freeze-dried form.

[0281] Combinations - Nucleic acids and polypeptides Nucleic acid combinations This invention provides a combination comprising two or more nucleic acids.

[0282] CAMP2 combination In some embodiments, the present invention provides nucleic acids comprising a nucleotide sequence encoding the C. acnes (C.acnes)CAMP2 polypeptide, and combinations comprising one or more nucleic acids comprising: (i) a nucleotide sequence encoding the C. acnes (C.acnes)DsA1 polypeptide, (ii) a nucleotide sequence encoding the C. acnes (C.acnes)DsA2 polypeptide, (iii) a nucleotide sequence encoding the C. acnes (C.acnes)PITP polypeptide, (iv) a nucleotide sequence encoding the chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide, and (v) a nucleotide sequence encoding the chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide.

[0283] In some embodiments, the combination comprises nucleic acid (i), nucleic acid (ii), and nucleic acid (iii). In some embodiments, the composition comprises nucleic acid (v). In preferred embodiments, the composition comprises nucleic acid (iii) and nucleic acid (iv). In some embodiments, the combination comprises nucleic acid (v). In preferred embodiments, the combination comprises nucleic acid (iii) and nucleic acid (iv).

[0284] In some embodiments, the combination comprises a nucleic acid containing a nucleotide sequence encoding the C. acnes (C. acnes) CAMP2 polypeptide, the nucleic acid of (iii), and the nucleic acid of (iv). In some embodiments, the combination comprises a nucleic acid containing a nucleotide sequence encoding the C. acnes (C. acnes) CAMP2 polypeptide and the nucleic acid of (v). In some embodiments, such a combination is provided as a nucleic acid containing a nucleotide sequence encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein. Typically, the nucleic acid containing a nucleotide sequence encoding the C. acnes (C. acnes) CAMP2 polypeptide, the nucleic acid of (iii), and the nucleic acid of (iv) are provided by separate nucleic acid molecules.

[0285] Modified CAMP2 combinations In another aspect, the present invention provides nucleic acids as described herein, comprising a nucleotide sequence encoding a modified C. acnes (C.acnes)CAMP2 polypeptide as described herein, and combinations comprising (i) a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C.acnes)DsA1 polypeptide, (ii) a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C.acnes)DsA2 polypeptide, (iii) a nucleic acid as described herein, comprising a nucleotide sequence encoding a C. acnes (C.acnes)PITP polypeptide, (iv) a nucleic acid as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C.acnes)DsA1 / DsA2 polypeptide, and (v) a combination comprising one or more nucleic acids as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C.acnes)DsA1 / DsA2 / PITP polypeptide.

[0286] In some embodiments, the combination includes nucleic acid (i), nucleic acid (ii), and nucleic acid (iii). In some embodiments, the combination includes nucleic acid (v). In preferred embodiments, the combination includes nucleic acid (iii) and nucleic acid (iv).

[0287] Combinations of DsA1, DsA2, and PITP In one embodiment, the combination provides a combination comprising one or more nucleic acids: (i) a nucleic acid comprising a nucleotide sequence encoding the C. acnes (C.acnes) DsA1 polypeptide; (ii) a nucleic acid comprising a nucleotide sequence encoding the C. acnes (C.acnes) DsA2 polypeptide; (iii) a nucleic acid comprising a nucleotide sequence encoding the C. acnes (C.acnes) PITP polypeptide; (iv) a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C.acnes) DsA1 / DsA2 polypeptide; and (v) a nucleic acid comprising a nucleotide sequence encoding the chimeric C. acnes (C.acnes) DsA1 / DsA2 / PITP polypeptide.

[0288] In some embodiments, the combination includes nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA1 polypeptide; nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) DsA2 polypeptide; and nucleic acids as described herein, comprising a nucleotide sequence encoding the C. acnes (C. acnes) PITP polypeptide.

[0289] In some embodiments, the combination includes nucleic acids as described herein, comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein, and nucleic acids as described herein, comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide as described herein. Typically, a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide contains the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto, and a C. acnes (C. acnes) PITP polypeptide contains the sequence of SEQ ID NO: 73 or a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises the sequence of SEQ ID NO: 70, and the C. acnes (C. acnes) PITP polypeptide comprises the sequence of SEQ ID NO: 73.

[0290] In some embodiments, nucleic acids comprising nucleotide sequences encoding the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide include the nucleotide sequence according to SEQ ID NO: 179 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., 85%) identity therewith, C. acnes (C. acnes) P Nucleic acids comprising nucleotide sequences encoding ITP polypeptides include a nucleotide sequence according to any one of SEQ ID NOs: 182 or a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., 75%) identity therewith.

[0291] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence described herein) and the sequence according to Sequence ID No. 179 or a sequence having at least 85% identity thereto, and a nucleic acid comprising a nucleotide sequence encoding a C. acnes (C. acnes) PITP polypeptide comprises a nucleotide sequence encoding a secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence described herein) and the sequence according to Sequence ID No. 182 or a sequence having at least 75% identity thereto.

[0292] Polypeptide combinations This invention provides a combination comprising two or more polypeptides of the present invention.

[0293] CAMP2 combination In some embodiments, the present invention provides a C. acnes CAMP2 polypeptide, as well as combinations comprising one or more of the following: (i) C. acnes DsA1 polypeptide as described herein, (ii) C. acnes DsA2 polypeptide as described herein, (iii) C. acnes PITP polypeptide as described herein, (iv) chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, and (v) chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein.

[0294] In some embodiments, the combination comprises polypeptide (i), polypeptide (ii), and polypeptide (iii). In some embodiments, the combination comprises polypeptide (v). In preferred embodiments, the combination comprises polypeptide (iii) and polypeptide (iv). In some embodiments, such combinations are provided as chimeric polypeptides, for example, the chimeric C. acnes (C. acnes) DsA1 / DsA2 / PITP / CAMP2 polypeptide as described herein. Typically, C. acnes (C. acnes) CAMP2 polypeptide, polypeptide (iii), and polypeptide (iv) are provided as separate polypeptides.

[0295] Modified CAMP2 combinations In another aspect, the present invention provides a modified C. acnes CAMP2 polypeptide as described herein, and a combination comprising one or more of the following: (i) C. acnes DsA1 polypeptide as described herein, (ii) C. acnes DsA2 polypeptide as described herein, (iii) C. acnes PITP polypeptide as described herein, (iv) chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, and (v) chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein.

[0296] In some embodiments, the combination includes polypeptide (i), polypeptide (ii), and polypeptide (iii). In some embodiments, the combination includes polypeptide (v). In preferred embodiments, the combination includes polypeptide (iii) and polypeptide (iv).

[0297] DsA1, DsA2, and PITP compositions In one embodiment, the present invention provides a combination comprising one or more of the following: (i) C. acnes DsA1 polypeptide as described herein, (ii) C. acnes DsA2 polypeptide as described herein, (iii) C. acnes PITP polypeptide as described herein, (iv) chimeric C. acnes DsA1 / DsA2 polypeptide as described herein, and (v) chimeric C. acnes DsA1 / DsA2 / PITP polypeptide as described herein.

[0298] In some embodiments, the combination includes the C.acnes DsA1 polypeptide, the C.acnes DsA2 polypeptide, and the C.acnes PITP polypeptide as described herein.

[0299] In some embodiments, the composition comprises a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein and a C. acnes (C. acnes) PITP polypeptide as described herein. Typically, a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide contains the amino acid sequence of SEQ ID NO: 70 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto, and a C. acnes (C. acnes) PITP polypeptide contains the sequence of SEQ ID NO: 73 or a sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 90% or at least 95%) identity thereto. In some embodiments, the chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide comprises the sequence of SEQ ID NO: 70, and the C. acnes (C. acnes) PITP polypeptide comprises the sequence of SEQ ID NO: 73.

[0300] LNP In certain embodiments, the composition of the present invention (for example, a composition comprising the nucleic acid of the present invention) further comprises lipid nanoparticles (LNPs). In certain embodiments, the nucleic acid of the present invention is encapsulated in the LNPs.

[0301] The LNPs of this disclosure may include lipids from the following four categories: (i) ionized lipids (e.g., cationic lipids), (ii) PEGylated lipids, (iii) cholesterol-based lipids, and (iv) helper lipids.

[0302] A. Ionized lipids Ionized lipids facilitate mRNA encapsulation and may be cationic lipids. Cationic lipids provide a positively charged environment at low pH, making it easier to efficiently encapsulate negatively charged mRNA drug substances.

[0303] In some embodiments, the cationic lipid is OF-02. [ka]

[0304] OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester bond connecting a diketopiperazine core and a biunsaturated tail, while OF-02 contains a non-degradable 1,2-amino-alcohol bond connecting the same diketopiperazine core and biunsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939, U.S. Patent No. 10,201,618). Lipid A, an exemplary LNP formulation as used herein, contains OF-2.

[0305] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60, U.S. Patent No. 9,512,073). [ka]

[0306] Lipid B, an exemplary LNP formulation in this specification, contains cKK-E10.

[0307] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1(2-(4-(2-((3-(bis((Z)-2-hydroxyoctadeca-9-en-1-yl)amino)propyl)disulfanyl(ethyl)piperazine-1-yl)ethyl4-(bis(2-hydroxydecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and having formula III. [ka]

[0308] Lipid C, an exemplary LNP formulation in this specification, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as lipid A or lipid B, except that it contains different cationic lipids.

[0309] Typically, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10(2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanyl)ethyl)piperazine-1-yl)ethyl4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and having formula IV. [ka]

[0310] Lipid D, an exemplary LNP formulation in this specification, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as lipid A or lipid B, except that it contains different cationic lipids.

[0311] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14(2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanyl)ethyl)piperazine-1-yl)ethyl4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid having a piperazine core and having formula V. [ka]

[0312] Lipid E, an exemplary LNP formulation in this specification, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​has the same composition as lipid A or lipid B, except that it contains different cationic lipids.

[0313] The cationic lipids GL-HEPES-E3-E10-DS-3-E18-1(III), GL-HEPES-E3-E12-DS-4-E10(IV), and GL-HEPES-E3-E12-DS-3-E14(V) can be synthesized according to the general procedure presented in Scheme 1. Scheme 1: General synthesis scheme for lipids of formulas (III), (IV), and (V) [ka]

[0314] In some embodiments, the cationic lipid is MC3, which has formula VI. [ka]

[0315] In some embodiments, the cationic lipid is SM-102(9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate), which has formula VII. [ka]

[0316] In some embodiments, the cationic lipid is ALC-0315[(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), which has formula VIII. [ka]

[0317] In some embodiments, the cationic lipid is cOrn-EE1, which has formula IX. [ka]

[0318] In some embodiments, the cationic lipids are cKK-E10, OF-02, [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate (D-Lin-MC3-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (dLin-KC2-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (dLin-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino )Butanoyl)oxy)heptadecanedioate (L319), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), [3-(dimethylamino)-2-[(Z)-octadeca-9-enoyl]oxypropyl](Z)-octadeca-9-enoate (DODAP), 2,5-bis(3-aminopropylamino )-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS), [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Chol), tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazandyl (Iyl)bis(propane-3,1-diyl))bis(azantriyl))tetrapropionate (306Oi10), decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9), ethyl 5,5-di((Z)-heptadeca-8-en-1-yl)-1-(3-(pyrrolidine-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18), bis(2-(dodecyldisulfanyl)ethyl)3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-Diazahexacosyl)azandiyl)dipropionate (BAME-O16B), 1,1'-((2-(4-(2-((2-((bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazin-2,5-dione (cK K-E12), Hexa(octan-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)tris(azandiyl))tris(propane-3,1-diyl))tris(azantriyl))hexanonaate (FTT5), (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azantriyl))tetrakis(ethane-2,1-di (9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-Tetrakis(Octadeca-9,12-dienoate)(OF-Deg-Lin), TT3, N1,N3,N5-Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide) The following can be selected from the group including ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), heptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid5), GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14 and combinations thereof.

[0319] In some embodiments, the cationic lipid is IM-001 and has formula X(EP23306049.0). [ka]

[0320] Lipid F, an exemplary LNP formulation in this specification, contains IM-001. Lipid F has the same composition as lipid A or lipid B, except that it contains different cationic lipids.

[0321] The cationic lipid IM-001(X) can be synthesized according to the general procedure presented in Scheme 2. Scheme 2: General synthesis scheme for lipids of formula (X) [ka]

[0322] Scheme 2 may be carried out as described in Example 12.

[0323] In some embodiments, the cationic lipid is IS-001 and has formula XI(EP23306049.0). [ka]

[0324] Lipid G, an exemplary LNP formulation in this specification, contains IS-001. Lipid G has the same composition as lipid A or lipid B, except that it contains different cationic lipids.

[0325] The cationic lipid IS-001(XI) can be synthesized according to the general procedure presented in Scheme 3. Scheme 3: General synthesis scheme for lipids of formula (XI) [ka]

[0326] Scheme 3 can be carried out as described in Example 14.

[0327] In some embodiments, cationic lipids are biodegradable.

[0328] In some embodiments, cationic lipids are not biodegradable.

[0329] In some embodiments, cationic lipids are cleavable.

[0330] In some embodiments, cationic lipids are not cleavable.

[0331] Cationic lipids are described in more detail in Dong et al. (PNAS.111(11):3955-60.2014), Fenton et al. (Adv Mater.28:2939.2016), U.S. Patent No. 9,512,073, and U.S. Patent No. 10,201,618, each of which is incorporated herein by reference.

[0332] B. PEGylated lipids PEGylated lipid components provide control over the particle size and stability of nanoparticles. Adding such components can prevent complex aggregation, extend circulating life, and provide a means to improve the delivery of lipid-nucleic acid drug compositions to target tissues (Klibanov et al., FEBS Letters 268(1):235-7. 1990). These components may be selected to be rapidly replaced from the drug composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0333] The intended PEGylated lipids are C6-C 20 (For example, C8, C 10 , C 12 , C 14 , C 16 or C 18Examples include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length covalently bonded to lipids having alkyl chains of a certain length, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)](C8 PEG ceramide)). In some embodiments, the PEGylated lipids are 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG), or 1,2-distearoyl-rac-glycero-polyethylene glycol (DSG-PEG), PEG-DAG, PEG-PE, PEG-S-DAG, PEG-S-DMG, PEG-cer, PEG-dialkyloxypropyl carbamate, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), and combinations thereof.

[0334] In certain embodiments, PEG has a high molecular weight, for example, 2000 to 2400 g / mol. In certain embodiments, PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipids herein are DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, C8 PEG2000, or ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000.

[0335] C. Cholesterol lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP contains one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamide cholesterol), l,4-bis(3-N-oleylaminopropyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280, Wolf et al.). al., BioTechniques (1997) 23:139, U.S. Patent No. 5,744,335), Imidazole cholesterol ester ("ICE", International Publication No. 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigma-5,22-dien-3-ol), ergosterol, desmosterol (3β-hydroxy-5,24-cholesterol), lanosterol (8,24-lanostadiene-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24, Examples include 25-dihydrolanosterol), thymosterol (5α-cholesta-8,24-diene-3β-ol), lathosterol (5α-cholesta-7-ene-3β-ol), diosgenin ((3β,25R)-spirosto-5-ene-3-ol), campesterol (campesto-5-ene-3β-ol), campestanol (5α-campestan-3β-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margallate (cholesta-5-ene-3β-ylheptadecanoate), cholesteryl oleate, cholesteryl stearate, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in LNPs is cholesterol.

[0336] D. Helper lipids Helper lipids enhance the structural stability of LNPs and assist in LNP extrusion into endosomes. This improves the uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids with fusion properties to enhance drug payload uptake and release. Examples of helper lipids include 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dieridoyl-sn-glycero-3-phosphoethanolamine (DEPE), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0337] Other exemplary helper lipids include dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramides, cerebrosides, gangliosides, 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0338] In various embodiments, the LNP comprises (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, IM-001, or IS-001, (ii) DMG-PEG2000, (iii) cholesterol, and (iv) DOPE.

[0339] In other embodiments, the LNP comprises (i) SM-102, (ii) DMG-PEG2000, (iii) cholesterol, and (iv) DSPC.

[0340] In other embodiments, the LNP includes (i) ALC-0315, (ii) ALC-0159, (iii) cholesterol, and (iv) DSPC.

[0341] E. Molar ratio of lipid components The molar ratios of the above components are important for the effectiveness of LNPs in mRNA delivery. The molar ratios of cationic lipids, PEGylated lipids, cholesterol lipids, and helper lipids are A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipids (i.e., A) in the LNP to total lipids is 35-55%, for example, 35-50% (e.g., 38-42%, for example, 40%, or 45-50%). In some embodiments, the molar ratio of PEGylated lipid components (i.e., B) to total lipids is 0.25-2.75% (e.g., 1-2%, for example, 1.5%). In some embodiments, the molar ratio of cholesterol lipids (i.e., C) to total lipids is 20-50% (e.g., 27-30%, for example, 28.5%, or 38-43%). In some embodiments, the molar ratio of helper lipids (i.e., D) to total lipids is 5–35% (e.g., 28–32%, e.g., 30%, or 8–12%, e.g., 10%). In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipids. In some embodiments, the molar ratio of cationic lipids to helper lipids in LNP is greater than 1.

[0342] In certain embodiments, the LNP of this disclosure is Cationic lipids in molar ratios of 35% to 55% or 40% to 50% (for example, cationic lipids in molar ratios of 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%), Polyethylene glycol (PEG) conjugate (PEG-conjugated) lipids in molar ratios of 0.25% to 2.75% or 1.00% to 2.00% (e.g., PEG-conjugated lipids in molar ratios of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%), Cholesterol-based lipids in molar ratios of 20%~50%, 25%~45%, or 28.5%~43% (for example, cholesterol-based lipids in molar ratios of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%), and Helper lipids in molar ratios of 5%-35%, 8%-30%, or 10%-30% (for example, helper lipids in molar ratios of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%) The molar ratios are all relative to the total lipid content of LNP.

[0343] In certain embodiments, the LNP comprises a cationic lipid in a molar ratio of 40%, a PEGylated lipid in a molar ratio of 1.5%, a cholesterol-based lipid in a molar ratio of 28.5%, and a helper lipid in a molar ratio of 30%.

[0344] In certain embodiments, the LNPs of this disclosure comprise a cationic lipid in a molar ratio of 45-50%, a PEGylated lipid in a molar ratio of 1.5-1.7%, a cholesterol-based lipid in a molar ratio of 38-43%, and a helper lipid in a molar ratio of 9-10%.

[0345] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0346] In various embodiments, the cholesterol-based lipid is cholesterol.

[0347] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0348] In certain embodiments, the LNP comprises OF-02 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

[0349] In certain embodiments, the LNP comprises cKK-E10 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

[0350] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

[0351] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

[0352] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

[0353] In certain embodiments, the LNP comprises SM-102 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DSPC in a molar ratio of 5% to 35%.

[0354] In certain embodiments, the LNP comprises ALC-0315 in a molar ratio of 35% to 55%, ALC-0159 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DSPC in a molar ratio of 5% to 35%.

[0355] In certain embodiments, the LNP comprises OF-02 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid A”.

[0356] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid B”.

[0357] In certain embodiments, the LNP comprises GL-HEPES-E3-E10-DS-3-E18-1 in a 40% molar ratio, DMG-PEG2000 in a 1.5% molar ratio, cholesterol in a 28.5% molar ratio, and DOPE in a 30% molar ratio. This LNP formulation is referred to herein as "Lipid C".

[0358] Typically, the LNP contains GL-HEPES-E3-E12-DS-4-E10 in a 40% molar ratio, DMG-PEG2000 in a 1.5% molar ratio, cholesterol in a 28.5% molar ratio, and DOPE in a 30% molar ratio. This LNP formulation is referred to herein as "Lipid D".

[0359] In certain embodiments, the LNP comprises GL-HEPES-E3-E12-DS-3-E14 in a 40% molar ratio, DMG-PEG2000 in a 1.5% molar ratio, cholesterol in a 28.5% molar ratio, and DOPE in a 30% molar ratio. This LNP formulation is referred to herein as "Lipid E".

[0360] In certain embodiments, the LNP comprises IM-001 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid F”.

[0361] In certain embodiments, the LNP comprises IS-001 at a molar ratio of 40%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%. This LNP formulation is referred to herein as “Lipid G”.

[0362] In certain embodiments, the LNP comprises 50% molar ratio of 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 38.5% molar ratio of cholesterol, and 1.5% molar ratio of 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0363] In certain embodiments, the LNP comprises (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 46.3%, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 9.4%, cholesterol in a molar ratio of 42.7%, and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.6%.

[0364] In certain embodiments, the LNP comprises (4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) in a molar ratio of 47.4%, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a molar ratio of 10%, cholesterol in a molar ratio of 40.9%, and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) in a molar ratio of 1.7%.

[0365] To calculate the actual amount of each lipid contained in an LNP formulation, first determine the molar amount of the cationic lipid based on the desired N / P ratio (where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA transported by the LNP). Next, calculate the molar amount of each of the other lipids based on the molar amount of the cationic lipid and the selected molar ratio. Then, convert these molar amounts to weight using the molecular weight of each lipid.

[0366] Nucleic acid within F.LNP The LNP compositions described herein may include the nucleic acids (e.g., mRNA) of the present invention.

[0367] If necessary, LNPs may be polyvalent. In some embodiments, LNPs may hold nucleic acids such as mRNA encoding two or more polypeptides (antigens), for example, 2, 3, 4, 5, or 6 polypeptides (antigens). For example, an LNP may hold multiple nucleic acids (e.g., mRNA) as defined herein, each encoding a different polypeptide as defined herein, or it may hold a polycistronic mRNA that can be translated into two or more polypeptides as defined herein (e.g., each antigen-coding sequence is separated by a nucleotide linker encoding a self-cleaving peptide such as a 2A peptide). LNPs holding different nucleic acids (e.g., mRNA) typically contain (encapsulate) multiple copies of each nucleic acid. For example, an LNP holding or encapsulating two different nucleic acids typically holds multiple copies of each of the two different nucleic acids.

[0368] Typically, two or more (e.g., two or three) nucleic acids (e.g., mRNA) encoding different polypeptides as described herein are co-encapsulated in the same LNP. For example, an LNP described herein may co-encapsulate a nucleic acid (e.g., mRNA) encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide and a nucleic acid (e.g., mRNA) encoding a C. acnes (C. acnes) PITP polypeptide. An LNP described herein may co-encapsulate a nucleic acid (e.g., mRNA) encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide and a nucleic acid (e.g., mRNA) encoding a modified C. acnes (C. acnes) CAMP2 polypeptide (or C. acnes) CAMP2 polypeptide). The LNPs described herein may contain a nucleic acid (e.g., mRNA) encoding the C. acnes (C. acnes) PITP polypeptide and a nucleic acid (e.g., mRNA) encoding the modified C. acnes (C. acnes) CAMP2 polypeptide (or C. acnes) CAMP2 polypeptide). Any two nucleic acids (e.g., two mRNAs) as described herein may be present in a 1:1 weight ratio.

[0369] The LNPs described herein may contain co-encapsulation of (i) a nucleic acid (e.g., mRNA) encoding a chimeric C. acnes (C. acnes) DsA1 / DsA2 polypeptide as described herein, (ii) a nucleic acid (e.g., mRNA) encoding a C. acnes (C. acnes) PITP polypeptide as described herein, and (iii) a nucleic acid (e.g., mRNA) encoding a modified C. acnes (C. acnes) CAMP2 polypeptide (or C. acnes) CAMP2 polypeptide) as described herein. The three nucleic acids (e.g., three mRNAs) described herein may be present in a 1:1:1 weight ratio.

[0370] LNPs are as described herein (e.g., lipid D).

[0371] Instead, two or more (e.g., three) nucleic acids (e.g., mRNA) encoding different polypeptides as described herein are encapsulated in separate LNPs. In some embodiments, a single LNP formulation may contain multiple types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of LNPs, each type holding a different nucleic acid (e.g., mRNA).

[0372] If the nucleic acid is mRNA, the mRNA may be unmodified (i.e., containing only native ribonucleotides A, U, C and / or G linked by phosphodiester bonds) or it may be chemically modified (e.g., including nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudridine), 2'-fluororibonucleotide and 2'-methoxyribonucleotide, and / or phosphorothioate bonds). The mRNA molecule may contain a 5' cap and a poly-A tail.

[0373] G. Buffer and other components To stabilize nucleic acids and / or LNPs (for example, to extend the shelf life of a vaccine product), to facilitate the administration of LNP pharmaceutical compositions, and / or to improve the in vivo expression of nucleic acids, nucleic acids and / or LNPs can be formulated in combination with one or more carriers, targeted ligands, stabilizing reagents (e.g., preservatives and antioxidants) and / or other pharmaceutically acceptable excipients. Examples of such excipients include parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0374] The LNP compositions of this disclosure can be provided in a liquid or freeze-dried form. Various cryoprotective agents may be used, but are not limited to sucrose, trehalose, glucose, mannitol, mannose, and dextrose. The cryoprotective agent may constitute 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition contains trehalose, for example, 5-30% (e.g., 10%) (w / v). When formulated with a cryoprotective agent, the LNP composition can be frozen (or freeze-dried and cryopreserved) at -20°C to -80°C.

[0375] The LNP composition may be provided to the patient in an aqueous buffer solution (thawed if pre-frozen, or reconstituted in the aqueous buffer solution at the bedside if pre-lyophilized). The buffer solution is preferably isotonic and suitable for, for example, intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate-buffered saline (PBS).

[0376] nucleic acid The nucleic acid of the present invention may be RNA or DNA. The nucleic acid of the present invention may be single-stranded or double-stranded. In certain embodiments, the nucleic acid is RNA, for example, mRNA.

[0377] mRNA In some embodiments, the nucleic acid of the present invention is messenger RNA (mRNA). The mRNA may be modified or unmodified. The mRNA may contain one or more coding regions and non-coding regions. The coding region is also alternatively referred to as an open reading frame (ORF). Non-coding regions in the mRNA include the 5' cap, the 5' untranslated region (UTR), the 3'UTR, and the poly-A tail. The mRNA may be purified from natural sources, produced using recombinant expression systems (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0378] In certain embodiments, the mRNA includes an ORF encoding the antigen of interest. In certain embodiments, the RNA (e.g., mRNA) further includes at least one 5'UTR, a 3'UTR, a poly(A) tail and / or a 5' cap. In some embodiments, the mRNA includes (i) a 5' cap as defined herein, (ii) a 5' untranslated region (UTR) as defined herein, (iii) a protein-coding region, (iv) a 3'UTR as defined herein, and (v) a poly(A) tail. Typically, the 3' end of (i) directly binds to the 5' end of (ii) via a 3'-to-5' phosphodiester bond, the 3' end of (ii) directly binds to the 5' end of (iii) via a 3'-to-5' phosphodiester bond, the 3' end of (iii) directly binds to the 5' end of (iv) via a 3'-to-5' phosphodiester bond, and the 3' end of (iv) directly binds to the 5' end of (v) via a 3'-to-5' phosphodiester bond.

[0379] In certain embodiments, the mRNA comprises at least one, at least two, at least three or more stop codons, the stop codons may be selected from UAA, UGA, and UAG, and the at least two, at least three or more stop codons may be identical or different. Typically, at least one stop codon comprises UAA or UGA (e.g., UAA). Typically, at least two stop codons comprise at least two identical stop codons such as UAA or UGA (e.g., UAAUAA or UGAUGA) or at least two different stop codons which may be selected in particular from UAA and UGA (e.g., UGAUAA). Typically, at least three stop codons comprise UAA, UGA, and UAG (e.g., UGAUAAUAG).

[0380] mRNA sequences are shown in the 5' to 3' direction unless otherwise specified.

[0381] 5' Cap The 5' cap of mRNA can provide resistance to nucleases found in most eukaryotic cells and can enhance translational efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also called "m7G" or "cap-0") contains guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0382] The 5' cap is typically added as follows: first, one of the terminal phosphate groups is removed from the 5' nucleotide by a phosphatase at the end of the RNA, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyltransferase to form a 5'5'5 triphosphate bond; and then the 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp, (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Further cap structures are described in U.S. Patent Application Publication 2016 / 0032356 and U.S. Patent Application Publication 2018 / 0125989, which are incorporated herein by reference.

[0383] The 5'-cap addition of polynucleotides can be completed simultaneously during an in vitro transcription reaction using the following chemical RNA cap analogues to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G(ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'oMeA)pG, m7G(5')ppp(5')(2'oMeA)pU, m7G(5')ppp(5')(2'oMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies). Using vaccinia virus capaddase, the 5'-cap addition of modified RNA can be completed post-transcriptionally to produce the cap 0 structure: m7G(5')ppp(5')G. Using both vaccinia virus capaddase and 2'-O methyl-transferase, the cap 1 structure can be produced to produce m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure, followed by 2'-O methylation of the third-to-last 5'-nucleotide using 2'-O methyl-transferase. The cap 3 structure can be generated from the cap 2 structure, followed by 2'-O methylation of the fourth-to-last 5'-nucleotide using 2'-O methyl-transferase.

[0384] In certain embodiments, the mRNA of the Disclosure includes a 5' cap selected from the group consisting of 3'-O-Me-m7G(5')ppp(5')G(ARCA cap), G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA)pU, and m7G(5')ppp(5')(2'OMeG)pG.

[0385] In certain embodiments, the mRNA of this disclosure includes the following 5' cap. [ka]

[0386] Untranslated area (UTR) In some embodiments, the mRNA of the present invention includes a 5' and / or 3' untranslated region (UTR). In mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not contain the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0387] In some embodiments, the mRNA disclosed herein may include a 5'UTR containing one or more elements that affect mRNA stability or translation. In some embodiments, the 5'UTR may be about 10 to 5,000 nucleotides long. In some embodiments, the 5'UTR may be about 50 to 500 nucleotides long. In some embodiments, the 5'UTR may be at least about 10 nucleotides long, about 20 nucleotides long, about 30 nucleotides long, about 40 nucleotides long, about 50 nucleotides long, about 100 nucleotides long, about 150 nucleotides long, about 200 nucleotides long, about 250 nucleotides long, about 300 nucleotides long, about 350 nucleotides long, about 400 nucleotides long, about 450 nucleotides long, about 500 nucleotides long, about 550 nucleotides long, about 600 nucleotides long, about The lengths are approximately 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1,000 nucleotides, 1,500 nucleotides, 2,000 nucleotides, 2,500 nucleotides, 3,000 nucleotides, 3,500 nucleotides, 4,000 nucleotides, 4,500 nucleotides, or 5,000 nucleotides.

[0388] In some embodiments, the mRNA disclosed herein may include a 3'UTR comprising one or more polyadenylation signals, protein binding sites affecting the stability of mRNA site in a cell, or one or more binding sites to miRNA. In some embodiments, the 3'UTR may be 50 to 5,000 nucleotides or longer. In some embodiments, the 3'UTR may be 50 to 1,000 nucleotides or longer. In some embodiments, the 3'UTR is at least about 50 nucleotides long, about 100 nucleotides long, about 150 nucleotides long, about 200 nucleotides long, about 250 nucleotides long, about 300 nucleotides long, about 350 nucleotides long, about 400 nucleotides long, about 450 nucleotides long, about 500 nucleotides long, about 550 nucleotides long, about 600 nucleotides long, about 650 nucleotides long, about 700 nucleotides long, about 750 nucleotides long, about 800 nucleotides long, about 850 nucleotides long, about 900 nucleotides long, about 950 nucleotides long, about 1,000 nucleotides long, about 1,500 nucleotides long, about 2,000 nucleotides long, about 2,500 nucleotides long, about 3,000 nucleotides long, about 3,500 nucleotides long, about 4,000 nucleotides long, about 4,500 nucleotides long, or about 5,000 nucleotides long.

[0389] In some embodiments, the mRNA disclosed herein may include a 5' or 3' UTR derived from a gene different from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0390] In certain embodiments, the 5' and / or 3'UTR sequences may be derived from stable mRNA to enhance mRNA stability (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes). For example, the 5'UTR sequence may contain a sub-sequence or fragment thereof of the pre-initial 1 (IE1) gene to improve nuclease resistance and / or improve mRNA half-life. It is also conceivable to include a sequence or fragment thereof encoding human growth hormone (hGH) in the 3' end or untranslated region of the mRNA. Generally, these modifications improve mRNA stability and / or pharmacokinetic properties (e.g., half-life) compared to the unmodified counterpart, and include modifications made to improve such mRNA resistance to nuclease digestion, for example, in vivo.

[0391] Exemplary 5'UTRs include sequences derived from the CMV pre-early 1 (IE1) gene (U.S. Patent Application Publication No. 2014 / 0206753 and U.S. Patent Application Publication No. 2015 / 0157565, each of which is incorporated herein by reference) or sequences GGGAUCCUACC (SEQ ID NO: 264) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference).

[0392] In various embodiments, the 5'UTR may be derived from the 5'UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5'-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-related translational regulation. However, TOP genes with tissue-specific translational regulation are also known. In certain embodiments, the 5'UTR derived from the 5'UTR of a TOP gene lacks the 5'TOP motif (oligopyrimidine) tract (e.g., U.S. Patent Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each incorporated herein by reference).

[0393] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (see U.S. Patent Application Publication No. 2017 / 0029847 above).

[0394] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (see U.S. Patent Application Publication No. 2016 / 0166710 above).

[0395] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (see U.S. Patent Application Publication No. 2016 / 0166710 above).

[0396] In some embodiments, the internal ribosome entry site (IRES) is used instead of the 5'UTR.

[0397] In some embodiments, the 5'UTR includes a nucleic acid sequence presented in SEQ ID NO: 265, which is reproduced below. [ka]

[0398] In some embodiments, the 3'UTR includes a nucleic acid sequence presented in SEQ ID NO: 266, which is reproduced below. CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 266)

[0399] The 5'UTR and 3'UTR are described in further detail in International Publication No. 2012 / 075040, which is incorporated herein by reference.

[0400] Polyadenylated tails As used herein, the terms “poly(A) sequence,” “poly(A) tail,” and “poly(A) region” refer to the sequence of adenosine nucleotides at the 3' end of an mRNA molecule. Poly(A) tails can confer stability to mRNA and protect it from exonuclease degradation. Poly(A) tails can enhance translation. In some embodiments, poly(A) tails are essentially homopolymers. For example, a poly(A) tail of 100 adenosine nucleotides may essentially have a length of 100 nucleotides. In certain embodiments, a poly(A) tail may be interrupted by at least one nucleotide different from adenosine nucleotides (e.g., a nucleotide that is not an adenosine nucleotide). For example, a poly(A) tail of 100 adenosine nucleotides may have a length greater than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide different from adenosine nucleotides or a stretch of nucleotides). In certain embodiments, a poly(A) tail is a sequence [ka] Includes.

[0401] As used herein, “poly(A)tail” typically refers to RNA. However, in relation to this disclosure, the term also refers to the corresponding sequence in a DNA molecule (e.g., “poly(T) sequence”).

[0402] The poly(A) tail may contain about 10 to about 500 adenosine nucleotides, about 10 to about 200 adenosine nucleotides, about 40 to about 200 adenosine nucleotides, or about 40 to about 150 adenosine nucleotides. The length of the poly(A) tail may be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides. In some embodiments, the polyA tail contains at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides). In some embodiments, the polyA tail contains at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides). Typically, the polyA tail contains at least 100 adenosine nucleotides.

[0403] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from the DNA template during in vitro transcription of the RNA. In certain embodiments, the poly(A) tail is obtained in vitro by common chemical synthesis methods without transcription from the DNA template. In various embodiments, the poly(A) tail is produced by enzymatic polyadenylation of RNA (after in vitro transcription of RNA) using commercially available polyadenylation kits and corresponding protocols, or alternatively by using immobilized poly(A) polymerase using methods and means described, for example, in International Publication No. 2016 / 174271.

[0404] Nucleic acids may contain poly(A) tails obtained by enzymatic polyadenylation, and most nucleic acid molecules contain approximately 100 (+ / -20) to approximately 500 (+ / -50) or approximately 250 (+ / -20) adenosine nucleotides.

[0405] In some embodiments, the nucleic acid may include a poly(A) tail obtained from template DNA and may additionally include at least one further poly(A) tail produced by enzymatic polyadenylation, as described, for example, in International Publication No. 2016 / 091391.

[0406] In certain embodiments, the nucleic acid includes at least one polyadenylation signal.

[0407] In various embodiments, the nucleic acid may contain at least one poly(C) sequence.

[0408] As used herein, the term “poly(C) sequence” is intended to be a sequence of cytosine nucleotides up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 10 to about 200 cytosine nucleotides, about 10 to about 100 cytosine nucleotides, about 20 to about 70 cytosine nucleotides, about 20 to about 60 cytosine nucleotides, or about 10 to about 40 cytosine nucleotides. In some embodiments, the poly(C) sequence comprises about 30 cytosine nucleotides.

[0409] chemical modification The mRNA disclosed herein may be modified or unmodified. In some embodiments, the mRNA may contain at least one chemical modification. In some embodiments, the mRNA disclosed herein may typically contain one or more modifications that improve RNA stability. Exemplary modifications include skeletal modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)). In certain embodiments, the disclosed mRNA is a modified nucleotide analog or derivative of purines and pyrimidines, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxymethylaminomethyl-2-thiouracil) It can be synthesized from hydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, keosin, β-D-mannosylkeosin, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0410] In some embodiments, the disclosed mRNA may include, but is not limited to, pseudouridine, N1-methylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudridine, 2-thio-l-methylpseudridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-l-methylpseudridine, 4-thiopseudridine, 5-aza-uridine, dihydropseudridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0411] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0412] In some embodiments, the chemical modification includes N1-methylpseudridine. Typically, the chemical modification includes N1-methylpseudridine in place of any uridine, i.e., 100% of the U residues are N1-methylpseudridine.

[0413] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0414] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0415] Preparations of such analogues are described, for example, in U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642.

[0416] mRNA synthesis The mRNA disclosed herein may be synthesized according to any of the following methods. For example, the mRNA according to this disclosure may be synthesized by in vitro transcription (IVT). Several methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin.Immunol.25(2):152-159 and Brunelle et al. (2013) Methods Enzymol.530:101-14. Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions may vary depending on the specific application. The presence of these reagents is generally undesirable in the final mRNA product, and these reagents may be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. In some embodiments, mRNA obtained from an in vitro transcription reaction may be preferred, but according to this disclosure, other sources of mRNA, including wild-type mRNA produced from bacteria, fungi, plants and / or animals, may be used.

[0417] Process for producing this LNP vaccine This LNP can be prepared by various techniques currently known in the art. For example, multilayer vesicles (MLVs) can be prepared according to the prior art by, for example, dissolving lipids in a suitable solvent to deposit selected lipids on the inner wall of a suitable container or vessel, then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. The MLV can then be formed by adding an aqueous phase to the vessel with vortex motion. The multilayer vesicles can then be homogenized and single-layer vesicles (ULVs) can be formed by ultrasonic treatment or extrusion. In addition, single-layer vesicles can be formed by detergent removal techniques.

[0418] Various methods are described in U.S. Patent Publication No. 2011 / 0244026, U.S. Patent Publication No. 2016 / 0038432, U.S. Patent Publication No. 2018 / 0153822, U.S. Patent Publication No. 2018 / 0125989 and PCT / U.S. Patent Publication No. 2020 / 043223 (filed July 23, 2020) that can be used to carry out the present disclosure. One exemplary process involves encapsulating mRNA by mixing mRNA with a lipid mixture, rather than first pre-forming lipids into lipid nanoparticles, as described in U.S. Patent Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing pre-formed LNPs with mRNA, as described in U.S. Patent Publication No. 2018 / 0153822.

[0419] In some embodiments, the process for preparing mRNA-loaded LNPs includes heating one or more solutions to a temperature higher than ambient temperature, wherein the one or more solutions are a solution containing pre-formed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing mRNA encapsulated in LNPs. In some embodiments, the process includes heating one or both of the mRNA solution and the pre-formed LNP solution before the mixing step. In some embodiments, the process includes heating one or more of the solutions containing the pre-formed LNPs, the mRNA solution, and the mRNA encapsulated in LNPs during the mixing step. In some embodiments, the process includes heating the mRNA encapsulated in LNPs after the mixing step. In some embodiments, the temperature at which one or more solutions are heated is approximately 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C or higher. In some embodiments, the temperature at which one or more solutions are heated is in the range of about 25-70°C, about 30-70°C, about 35-70°C, about 40-70°C, about 45-70°C, about 50-70°C, or about 60-70°C. In some embodiments, the temperature is about 65°C.

[0420] Various methods can be used to prepare mRNA solutions suitable for this disclosure. In some embodiments, mRNA may be dissolved directly in the buffer solution described herein. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with the buffer solution before mixing it with the lipid solution for mounting. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with the buffer solution immediately before mixing it with the lipid solution for mounting. In some embodiments, a suitable mRNA stock solution may contain mRNA in water or buffer solution at concentrations of about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml or 5.0 mg / ml or higher.

[0421] In some embodiments, the mRNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a faster rate than the mRNA stock solution. For example, the buffer solution may be mixed at a rate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times faster than the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of approximately 100–6000 ml / min (e.g., approximately 100–300 ml / min, 300–600 ml / min, 600–1200 ml / min, 1200–2400 ml / min, 2400–3600 ml / min, 3600–4800 ml / min, 4800–6000 ml / min, or 60–420 ml / min). In some embodiments, the buffer solution is mixed at a flow rate of approximately 60 ml / min, 100 ml / min, 140 ml / min, 180 ml / min, 220 ml / min, 260 ml / min, 300 ml / min, 340 ml / min, 380 ml / min, 420 ml / min, 480 ml / min, 540 ml / min, 600 ml / min, 1200 ml / min, 2400 ml / min, 3600 ml / min, 4800 ml / min, or 6000 ml / min or more.

[0422] In some embodiments, the mRNA stock solution is mixed at a flow rate in the range of approximately 10 to 600 ml / min (e.g., approximately 5 to 50 ml / min, approximately 10 to 30 ml / min, approximately 30 to 60 ml / min, approximately 60 to 120 ml / min, approximately 120 to 240 ml / min, approximately 240 to 360 ml / min, approximately 360 to 480 ml / min, or approximately 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of approximately 5 ml / min, 10 ml / min, 15 ml / min, 20 ml / min, 25 ml / min, 30 ml / min, 35 ml / min, 40 ml / min, 45 ml / min, 50 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 200 ml / min, 300 ml / min, 400 ml / min, 500 ml / min, or 600 ml / min or more.

[0423] The process of incorporating a desired mRNA into lipid nanoparticles is referred to as “loading.” Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The nucleic acid incorporated into the LNP may be located entirely or partially within the internal space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the outer surface of the lipid nanoparticle membrane. The incorporation of mRNA into lipid nanoparticles is also referred to herein as “encapsulation,” in which the nucleic acid is contained entirely or substantially within the internal space of the lipid nanoparticle.

[0424] Suitable LNPs can be produced in a variety of sizes. In some embodiments, reducing the size of lipid nanoparticles is associated with more efficient mRNA delivery. By selecting an appropriate LNP size, the site of the target cell or tissue and the application for which the lipid nanoparticles are produced can be considered to some extent.

[0425] Various methods known in the art can be used to size a population of lipid nanoparticles. In the preferred method described herein, LNP particle size is measured using a Zetasizer Nano ZS (Malvern Panalytical). In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is packed into a cuvette and then placed in the Zetasizer instrument. The z-mean diameter (nm) or cumulant mean is considered the average size of the LNPs in the sample. The polydispersity index (PDI) can also be measured using the Zetasizer instrument by employing dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average diameter of the formed LNPs can be reduced by sonication. Intermittent sonication cycles can be alternated with quasi-elastic light scattering (QELS) evaluation to lead to efficient lipid nanoparticle synthesis.

[0426] In some embodiments, the LNP has an average diameter of 30 nm to 200 nm (for example, an average diameter of 80 nm to 150 nm).

[0427] In some embodiments, the majority of the purified LNPs, i.e., more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, have a size of about 70–150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all (e.g., more than 80 or 90%) of the purified lipid nanoparticles have a size of about 70 to 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0428] In some embodiments, the LNPs in the composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0429] In some embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the LNPs in the composition have sizes in the range of about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70 nm (e.g., about 55-65 nm), which are particularly suitable for lung delivery by spraying.

[0430] In some embodiments, the degree of dispersion of LNPs in the pharmaceutical compositions provided herein, i.e., the measure of molecular size heterogeneity (PDI), is less than about 0.5. In some embodiments, the LNPs have a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than 0.08. The PDI can be measured by a Zetasizer instrument such as those described above.

[0431] In some embodiments, about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or more than 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., 80% or more than 90%) of the purified lipid nanoparticles in the pharmaceutical compositions encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99% or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, or more than 99%. Typically, the lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91%, 92%, 93%, 94%, or 95%).

[0432] In some embodiments, the LNPs have an N / P ratio of 1 to 10. In some embodiments, the lipid nanoparticles have an N / P ratio greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, the typical LNPs of this specification have an N / P ratio of 4.

[0433] In some embodiments, the pharmaceutical compositions according to this disclosure contain at least about 0.5 μg, 1 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated mRNA. In some embodiments, the pharmaceutical compositions contain about 0.1 μg to 1000 μg, at least about 0.5 μg, at least about 0.8 μg, at least about 1 μg, at least about 5 μg, at least about 8 μg, at least about 10 μg, at least about 50 μg, at least about 100 μg, at least about 500 μg, at least about 100 μg, or at least about 1000 μg of encapsulated mRNA.

[0434] In some embodiments, mRNA can be produced by chemical synthesis or in vitro transcription (IVT) of a DNA template. In this process, an mRNA transcript is produced using a cDNA template in the IVT process, and the DNA template is degraded by a DNase. The transcript is purified by depth filtration and tangential flow filtration (TFF). The purified transcript is further modified by adding caps and tails, and this modified RNA is purified again by depth filtration and TFF.

[0435] Next, mRNA is prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of LNP. The amphiphilic solution for dissolving the four lipid components of LNP may be an alcoholic solution. In some embodiments, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0 to 7.0, for example, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer may contain other components, such as salts (e.g., sodium salts, potassium salts, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0436] The process for preparing a composition containing LNPs and mRNA involves mixing a buffered mRNA solution with a lipid solution in ethanol in a controlled and homogeneous manner, with the lipid:mRNA ratio maintained throughout the mixing process. In this exemplary embodiment, mRNA is present in an aqueous buffer containing citrate monohydrate, trisodium citrate dihydrate, and sodium chloride. The mRNA solution is added to a solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). A lipid mixture of four lipids (e.g., cationic lipids, PEGylated lipids, cholesterol lipids, and helper lipids) is dissolved in ethanol. The mRNA aqueous solution and the ethanol lipid solution are mixed in a volume ratio of 4:1 in a "T" mixer equipped with a nearly "pulsationless" pump system. The resulting mixture is then subjected to downstream purification and buffer exchange. Buffer exchange can be achieved using a dialysis cassette or a TFF system. Using TFF, the nascent LNPs obtained immediately after the T mixing process can be concentrated and buffer exchanged. The diafiltration process is a continuous operation that maintains a constant volume by adding an appropriate buffer solution at the same rate as the permeate flow.

[0437] vector In one embodiment, a vector comprising an mRNA composition disclosed herein is disclosed herein. An RNA sequence encoding a protein of interest (e.g., mRNA encoding a polypeptide disclosed herein) can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors for specific purposes include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription.

[0438] In certain embodiments, a vector can be used to express mRNA in host cells. In various embodiments, a vector can be used as a template for IVT. Constructions of optimally translated IVT mRNA suitable for therapeutic use are disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780 and Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0439] In some embodiments, the vectors disclosed herein may include, at least, an RNA polymerase promoter, a polynucleotide sequence encoding the 5'UTR, a polynucleotide sequence encoding the ORF, a polynucleotide sequence encoding the 3'UTR, and a polynucleotide sequence encoding at least one RNA aptamer, from 5' to 3'. In some embodiments, the vectors disclosed herein may include a poly(A) sequence and / or a polynucleotide sequence encoding a polyadenylation signal.

[0440] Various RNA polymerase promoters are known. In some embodiments, the promoter may be the T7 RNA polymerase promoter. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of the T7, T3, and SP6 promoters are known.

[0441] Host cells (e.g., mammalian cells, e.g., human cells) containing the vectors or RNA compositions disclosed herein are also disclosed herein. "Host cells" include individual cells or cell cultures that may or may have been recipients of exogenous nucleic acids. Host cells include offspring of a single host cell, which may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or intentional mutations and / or alterations. Host cells include cells that have been transfected or infected with the nucleic acids or vectors disclosed herein in vivo or in vitro.

[0442] Vectors can be introduced into target cells using one of many different methods, including but not limited to commercially available methods such as electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), ECM830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Cologne.), Multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, biological particle delivery systems such as "gene guns" (e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70) or TransIT-RNA transfection kits (Mirus, Madison, WI).

[0443] Chemical means for introducing vectors into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is liposomes (e.g., artificial membrane vesicles).

[0444] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this disclosure, various assays can be performed to confirm the presence of mRNA sequences in host cells.

[0445] Self-replicating RNA, trans-replicating RNA, and non-replicating RNA Typically, the nucleic acid molecules described herein are non-replicating RNA. However, the nucleic acid molecules described herein may alternatively be self-replicating RNA or trans-replicating RNA.

[0446] self-replicating RNA Self-replicating (or self-propagating) RNA can be produced, for example, by using replication elements derived from an alphavirus and substituting a structural viral protein with a nucleotide sequence encoding the protein of interest (e.g., a polypeptide as described herein). Self-replicating RNA is typically a positive-chain molecule that can be directly translated after being delivered to a cell, and this translation provides RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNA transcripts and collinear subgenomic transcripts can themselves be translated to provide in-situ expression of the encoded antigen, or they can be transcribed to provide further transcripts with the same sense as the delivered RNA, which can then be translated to provide in-situ expression of the antigen. The overall result of this series of transcriptions is that many introduced replicon RNAs are widely amplified, so that the encoded antigen becomes the cell's primary polypeptide product.

[0447] One preferred system for achieving self-replication in this manner is to use alphavirus-based replicons. These replicons are positive-sense RNAs that, after delivery to a cell, result in the translation of a replicase (or replicase transcriptase). The replicase is translated into a polyprotein that self-cleaves to provide a replication complex, thereby generating a genomic copy of the delivered positive-sense RNA. These negative-sense transcripts can themselves be transcribed to give further copies of the positive-sense parental RNA and may also give subgenomic transcripts encoding antigens. Translation of these subgenomic transcripts then leads to in-situ expression of the antigen by the infected cell. Suitable alphavirus replicons can be replicases derived from Sindbis virus, Semlik Forest virus, Eastern Equine Encephalitis virus, Venezuelan Encephalitis virus, etc. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 variant of VEEV has been used in replicons. See the following reference: International Publication No. 2005 / 113782 (incorporated herein by reference).

[0448] In one embodiment, each self-replicating RNA described herein encodes (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an influenza protein antigen. The polymerase may be, for example, an alphaviral replicase containing one or more alphaviral proteins nsP1, nsP2, nsP3, and nsP4. While the natural alphaviral genome encodes structural virion proteins in addition to non-structural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphaviral structural proteins. Thus, the self-replicating RNA may result in the production of its own genomic RNA copies in the cell, but not in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphavirus, the self-replicating RNA molecule cannot persist on its own in the infectious form. The alphaviral structural proteins necessary for persistence in wild-type virus are not present in the self-replicating RNA of this disclosure, and their positions are occupied by genes encoding the immunogen of interest; therefore, the subgenome transcript encodes its immunogen, not the structural alphaviral virion protein. Self-replicating RNA is described in more detail in International Publication No. 2011005799, which is incorporated herein by reference.

[0449] Trans-replicated RNA Trans-replicated (or trans-amplified) RNA has elements similar to those of the self-replicating RNA described above. However, trans-replicated RNA uses two distinct RNA molecules. The first RNA molecule encodes the RNA replicase described above (e.g., alphavirus replicase), and the second RNA molecule encodes the protein of interest (e.g., the polypeptide described herein). The RNA replicase replicates one or both of the first and second RNA molecules, thereby significantly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicated RNA is described in more detail in International Publication No. 2017162265, which is incorporated herein by reference.

[0450] non-replicating RNA Non-replicating (or non-amplified) RNA is RNA that is not capable of replicating on its own.

[0451] therapeutic use In another aspect, the present invention provides polypeptides, nucleic acids, combinations, or compositions of the present invention for use as pharmaceuticals. The present invention also provides the use of polypeptides, nucleic acids, combinations, or compositions of the present invention for the manufacture of pharmaceuticals. Pharmaceuticals may be used to treat or prevent diseases such as those described herein. The present invention further provides a method for treating or preventing a disease, comprising administering the polypeptides, nucleic acids, combinations, or compositions of the present invention to a subject in need thereof. The polypeptides, nucleic acids, combinations, or compositions of the present invention may be administered, for example, in an effective amount to treat or prevent a disease of interest. Accordingly, polypeptides, nucleic acids, combinations, or compositions may be administered in an effective amount.

[0452] In another aspect, the present invention provides polypeptides, nucleic acids, combinations, or compositions for use in treating or preventing C. acnes infections in subjects (e.g., humans). The present invention also provides the use of polypeptides, nucleic acids, combinations, or compositions of the present invention for producing pharmaceuticals for treating or preventing C. acnes infections in subjects (e.g., humans). The present invention further provides a method for treating or preventing C. acnes infections in subjects (e.g., humans), the method comprising administering the polypeptides, nucleic acids, combinations, or compositions of the present invention to a subject. The polypeptides, nucleic acids, combinations, or compositions of the present invention can be administered in an effective amount (i.e., an effective amount) for treating or preventing a C. acnes infection in a subject, for example. C. acnes infections can be mild, moderate, or severe (e.g., moderate or severe).

[0453] The polypeptides, nucleic acids, combinations, or compositions of the present invention may be used to induce an immune response in a patient, for example, an immune response to C. acnes infection.

[0454] In another aspect, the present invention provides polypeptides, nucleic acids, combinations, or compositions for use in treating or preventing acne (also known as acne vulgaris) in a subject (e.g., human). The present invention also provides the use of polypeptides, nucleic acids, combinations, or compositions of the present invention for producing pharmaceuticals for treating or preventing acne in a subject (e.g., human). The present invention further provides a method for treating or preventing acne in a subject (e.g., human), the method comprising administering the polypeptides, nucleic acids, combinations, or compositions of the present invention to a subject. The polypeptides, nucleic acids, combinations, or compositions of the present invention can be administered in an effective amount (i.e., an effective amount) for treating or preventing acne in a subject, for example. Acne can be caused by C. acnes. Acne can be mild, moderate, or severe (e.g., moderate or severe).

[0455] In some embodiments, the treatment described herein achieves a reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% (e.g., at least 50%) in the number of inflammatory acne lesions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (e.g., 3 or 6 months) after administration of the treatment (e.g., after the final dose of the treatment), compared to the number of inflammatory acne lesions before treatment. In some embodiments, the inflammatory acne lesions are located on the face of the subject. In some embodiments, the inflammatory acne lesions are located on the face of the subject and one other body area (e.g., the chest or back).

[0456] In some embodiments, the treatment described herein achieves a reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% (e.g., at least 50%) in the number of non-inflammatory acne lesions at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (e.g., 3 or 6 months) after administration of the treatment (e.g., after the final dose of the treatment), compared to the number of non-inflammatory acne lesions before treatment. In some embodiments, the non-inflammatory acne lesions are located on the face of the subject. In some embodiments, the non-inflammatory acne lesions are located on the face of the subject and one other body area (e.g., the chest or back).

[0457] In some embodiments, the treatment described herein achieves a reduction in the Investigator's Global Assessment (IGA) score for the subject compared to the pre-treatment score, for example, at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (e.g., 3 or 6 months) after administration of the treatment. In some embodiments, the treatment described herein achieves an improvement of at least two grades in the Investigator's Global Assessment (IGA) score for the subject compared to the pre-treatment score, for example, at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (e.g., 3 or 6 months) after administration of the treatment (e.g., after the final dose of the treatment). In some embodiments, the improvement in the IGA score results in an IGA score of 0, 1, or 2 (e.g., 0 or 1) at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months (e.g., 3 or 6 months) after administration of the treatment (e.g., after the final dose of the treatment).

[0458] The polypeptides, nucleic acids, combinations, or compositions of the present invention may be used to treat or prevent chronic blepharitis and / or endophthalmitis associated with C. acnes infection.

[0459] The present invention provides polypeptides, nucleic acids, combinations, or compositions for use in a method of providing protective immunity against C. acnes infection in a subject. The present invention also provides the use of polypeptides, nucleic acids, combinations, or compositions for use in a method of providing protective immunity against C. acnes infection in a subject for manufacturing pharmaceuticals. The present invention further provides a method of providing protective immunity against C. acnes infection in a subject, the method comprising administering the polypeptides, nucleic acids, combinations, or compositions of the present invention to the subject. The polypeptides, nucleic acids, combinations, or compositions of the present invention can be administered in an amount effective to provide protective immunity against C. acnes infection in a subject (i.e., an effective amount). Protective immunity can protect against the development of conditions induced by C. acnes infection (e.g., acne). Protective immunity can prevent or mitigate the development of indications related to C. acnes.

[0460] The polypeptides, nucleic acids, combinations, or compositions of the present invention can induce antibodies such as IgG (e.g., antigen-specific antibodies). The antibodies (DsA1, DsA2, and / or PITP-specific antigens) can bind to the surface of C. acnes bacteria. The antibodies (DsA1, DsA2, and / or PITP-specific antigens) can opsonize C. acnes bacteria. This may allow for the recruitment of immune effector cells (e.g., phagocytic cells) to the bacteria. C. acnes bacteria can then be phagocytosed by the recruited immune effector cells. Within the IgG isotype, the antibodies may be IgG1, IgG2, IgG3, or IgG4 subclasses. The antibodies may have κ or λ light chains. In some embodiments, the polypeptides, nucleic acids, combinations, or compositions of the present invention may be cross-reactive to two or more strains or lineages of C. acnes, and may induce an antibody response that is cross-reactive to, for example, two or more lineages (e.g., cross-reactive to lineages IA1, IA2, IB, IC, II, and III).

[0461] The polypeptides, nucleic acids, combinations, or compositions of the present invention may reduce inflammation associated with (e.g., caused by) C. acnes infection. The polypeptides, nucleic acids, combinations, or compositions of the present invention may reduce tissue inflammation mediated by C. acnes.

[0462] The polypeptides, nucleic acids, combinations, or compositions of the present invention can inhibit biofilm formation by C. acnes. In some embodiments, biofilm formation can be hindered. In some embodiments, biofilm formation can be mitigated.

[0463] By administering the polypeptides, nucleic acids, combinations, or compositions of the present invention to a subject, upon exposure to C. acnes bacteria or C. acnes antigen, the subject may be able to produce a population of memory B cells responsive to C. acnes antigen. The polypeptides, nucleic acids, combinations, or compositions of the present invention may be used to induce and / or enhance a primary immune response.

[0464] The polypeptides, nucleic acids, combinations, or compositions of the present invention may be used in a prime-boost vaccination program. Immunity to C. acnes infection according to the present invention may be provided by administering a priming vaccine comprising the polypeptides, nucleic acids, combinations, or compositions of the present invention, followed by a booster vaccine. The booster vaccine may be the same as the primer vaccine.

[0465] In certain embodiments, the subject is a vertebrate, such as a human or a veterinary mammal (e.g., a cat, dog, horse, cow, sheep, cattle, deer, goat, pig, rodent (e.g., mouse)). In preferred embodiments, the subject is a human. The subject (e.g., a human subject) may be male or female. The subject may be a child (0-10 years), adolescent (10-18 years), or adult (over 18 years). In some embodiments, the human subject may be 0-30 years or 0-45 years (e.g., 5-18 years, 12-45 years, 18-45 years, or 9-45 years). For example, the subject may be 9-45 years, 12-45 years, or 18-45 years (e.g., 9-45 years).

[0466] In some embodiments, a subject has at least one of the following: (i) a grade 3 or grade 4 score on the IGA scale, (ii) at least 25 non-inflammatory lesions on the subject's face (e.g., open comedones and / or closed comedones), (iii) at least 20 inflammatory lesions on the subject's face (e.g., papules and / or pustules), or (iv) two or fewer nodular cystic lesions on the subject's face (e.g., nodules and / or cysts). In some embodiments, a subject has at least two of (i) to (iv). In some embodiments, a subject has at least three of (i) to (iv). Typically, a subject has all four of (i) to (iv). A subject having all four of (i) to (iv) may have moderate / severe acne.

[0467] Acne vulgaris (also referred to herein as acne) manifests in varying degrees of severity: mild, moderate, and severe. Moderate and severe acne account for more than one-third of all cases and require medical treatment. In some embodiments, subjects may have mild, moderate, or severe (e.g., moderate or severe acne) C. acnes infection. Subjects may have mild, moderate, or severe acne (e.g., moderate or severe acne). In some embodiments, subjects have mild, moderate, or severe acne (e.g., moderate or severe acne).

[0468] Mode of administration The polypeptides, nucleic acids, combinations, or compositions of the present invention may be administered parenterally (e.g., intramuscularly, intradermally, subcutaneously, intraperitoneally, intravenously, or into the interstitial space of tissue) or by rectal, oral, vaginal, topical, transdermal, intranasal, sublingual, ocular, ear, pulmonary, or other mucosal administration. In some embodiments, delivery is by mucosal administration. Typically, administration is intramuscular.

[0469] In certain embodiments, the nucleic acids, polypeptides, compositions, or combinations (e.g., compositions) of the present invention are provided for use in intramuscular (IM) injection. The nucleic acids, polypeptides, compositions, or combinations (e.g., compositions) can be administered to the thigh or upper arm of a target, for example, the deltoid muscle of the upper arm. In some embodiments, the nucleic acids, polypeptides, compositions, or combinations (e.g., compositions) are provided in pre-filled syringes or syringes (e.g., single-chamber or multi-chamber types). The injection may be via a needle (e.g., a subcutaneous needle), but needle-free injection may be used as an alternative. A typical intramuscular dose is 0.5 ml. In some embodiments, the nucleic acids, polypeptides, compositions, or combinations (e.g., compositions) are provided for use in inhalation and are provided in a pre-filled pump, aerosol generator, or inhaler.

[0470] In certain embodiments, the nucleic acids, polypeptides, compositions, or combinations of the present invention (e.g., compositions) are provided for use in cutaneous injection, for example, in the epidermis, dermis, or subcutaneous tissue of the skin. In some embodiments, the nucleic acids, polypeptides, compositions, or combinations of the present invention (e.g., compositions) are provided in devices suitable for cutaneous injection, such as needles (e.g., epidermal needles, dermal needles, or subcutaneous needles), needle-free devices, microneedle devices, or microprojection array devices. Examples of microneedles or microprojection array devices suitable for skin injection according to the present invention are described in U.S. Patent Publication No. 20230270842A1, U.S. Patent Publication No. 20220339416A1, U.S. Patent Publication No. 20210085598A1, U.S. Patent Publication No. 20200246450A1, U.S. Patent Publication No. 20220143376A1, U.S. Patent Publication No. 20180264244A1, U.S. Patent Publication No. 20180263641A1, and U.S. Patent Publication No. 20110245776A1.

[0471] The nucleic acids, polypeptides, compositions, or combinations (e.g., compositions) of the present invention may be used to induce systemic immunity, cutaneous immunity, and / or mucosal immunity.

[0472] Drug therapy may be a single-dose schedule or a multi-dose schedule. Multi-dose (e.g., two or three doses) may be used in a primary immunization schedule and / or a booster immunization schedule. A booster dose schedule may be performed after the primary dose schedule. Multi-dose (e.g., two or three doses) are typically administered to subjects who need it at intervals of at least one week (e.g., about two weeks, three weeks, four weeks, six weeks, eight weeks, ten weeks, twelve weeks, sixteen weeks, etc.) to achieve the desired therapeutic or prophylactic effect (e.g., two months). Doses (e.g., primary and booster doses) may be spaced apart by intervals of, for example, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, or ten years (e.g., two months). In some embodiments, a single dose is administered intramuscularly to the subject. In some embodiments, the subject receives two intramuscular doses (e.g., at intervals of two months).

[0473] The compositions of the present invention may be in the form of immediate formulations; for example, the compositions of the present invention may be lyophilized. Such compositions may be reconstituted with a physiological buffer (e.g., PBS) immediately before use. The compositions of the present invention may be provided in the form of aqueous solutions or frozen aqueous solutions and may be administered directly to a subject without reconstitution (after thawing if previously frozen).

[0474] In some embodiments of compositions comprising one or more nucleic acids (e.g., mRNA) as described herein, a single dose of the composition contains 1 to 300 (or 1 to 50) μg of mRNA (e.g., monovalent or polyvalent) as described herein. For example, a single dose may contain, for example, intramuscular (IM) injection, one or more nucleic acids (e.g., mRNA) as described herein in amounts of about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, about 15 μg, about 30 μg, about 45 μg, about 60 μg, about 75 μg, about 90 μg, about 105 μg, about 120 μg, about 135 μg, about 150 μg, about 165 μg, about 180 μg, about 195 μg, about 210 μg, about 225 μg, about 240 μg, about 250 μg, about 260 μg, about 275 μg, or about 300 μg. In some embodiments, the composition contains 40-50 μg (e.g., about 45 μg) of one or more nucleic acids (e.g., mRNA). In some embodiments, the composition comprises 110–140 μg (e.g., about 120 μg) of one or more nucleic acids (e.g., mRNA). In some embodiments, the composition comprises 200–250 μg (e.g., about 225 μg) of one or more nucleic acids (e.g., mRNA).

[0475] The composition may comprise three nucleic acids (e.g., three mRNAs) as described herein, encoding different polypeptides as described herein, and the nucleic acids may be present in a 1:1:1 weight ratio. In some embodiments, the composition comprises approximately 45 μg, approximately 120 μg, or approximately 225 μg of nucleic acids in total. Thus, the composition may comprise approximately 15 μg, approximately 40 μg, or approximately 75 μg of each of the three nucleic acids.

[0476] The composition may comprise two nucleic acids (e.g., two mRNAs) as described herein, encoding different polypeptides as described herein, and the nucleic acids may be present in a 1:1 weight ratio.

[0477] In further embodiments, the compositions of the present invention may be provided as polyvalent single doses containing multiple (e.g., two, three, or four) types of LNPs for each different antigen, each type of LNP having an amount of mRNA of, for example, 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, about 15 μg, about 30 μg, about 45 μg, about 60 μg, about 75 μg, about 90 μg, about 105 μg, about 120 μg, about 135 μg, about 150 μg, about 165 μg, about 180 μg, about 195 μg, about 210 μg, about 225 μg, about 250 μg, about 275 μg, or about 300 μg.

[0478] In some embodiments, a subject is administered one or more nucleic acid compositions of the present invention. The nucleic acid compositions may include nucleic acids comprising nucleotide sequences encoding polypeptide antigens as described herein. The nucleic acid compositions may be administered simultaneously, separately, or sequentially. In some embodiments, a subject is administered a combination of nucleic acids of the present invention. The combination of nucleic acids includes a combination of two or more (e.g., three) nucleic acids as described herein. The nucleic acids in the combination may be administered simultaneously, separately, or sequentially.

[0479] In some embodiments, a subject is administered one or more polypeptide compositions of the present invention. The polypeptide compositions may include polypeptide antigens as described herein. The polypeptide compositions may be administered simultaneously, separately, or sequentially. In some embodiments, a subject is administered a combination of polypeptides of the present invention. The polypeptide combination includes a combination of two or more (e.g., three) polypeptides as described herein. The nucleic acids in the combination may be administered simultaneously, separately, or sequentially.

[0480] In some embodiments, one or more nucleic acid compositions and one or more polypeptide compositions of the present invention are administered to a target. The nucleic acid compositions and the one or more polypeptide compositions may be administered simultaneously, separately, or sequentially.

[0481] Compositions administered separately or sequentially may be administered within 12 months, 6 months, or 1 month (e.g., within 10 days) of each other. Compositions may be administered within 7 days, 3 days, 2 days, or 24 hours of each other. Co-administration may involve administering the compositions of the present invention simultaneously. Co-administration may involve administering the compositions of the present invention to a patient within 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour of each other, typically during the same visit to a clinical center.

[0482] The present invention provides a kit comprising one or more compositions described herein in one or more containers, or also provides one or more compositions as described herein in one or more containers and a physiological buffer for reconstitution in another container. The containers may contain single-use doses or multi-use doses. The containers may be pre-treated glass vials or ampoules. The kit may include instructions for use.

[0483] definition The term "includes" encompasses "contains" and "consists of," for example, a composition "includes" X may consist only of X or may include some additional elements (e.g., X + Y).

[0484] The term "approximately" in relation to the numerical value x is optional and can mean, for example, x ± 10%.

[0485] As used herein, the term “effective dose” means a quantity (e.g., nucleic acids, polypeptides, combinations, or compositions as described herein) sufficient to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or dosages and is not intended to be limited to a particular formulation or route of administration. The term “effective dose” includes, for example, therapeutic effective doses and / or prophylactic effective doses. As used herein, the term “effective dose” means a quantity (e.g., nucleic acids, polypeptides, combinations, or compositions as described herein) that is effective in producing some desired therapeutic or prophylactic effect in the treatment or prevention of infection, disease, disorder, and / or condition at a reasonable risk / benefit ratio applicable to any medical treatment.

[0486] Where relating to polypeptides in this disclosure, the terms “fragment” or “variant” include any polypeptide that retains at least some of the characteristics of the reference polypeptide (e.g., the polypeptide’s specific antigenic properties or its ability to contribute to antibody binding induction). Polypeptide fragments include fragments with the N-terminus and / or C-terminus truncated, e.g., C-terminal and N-terminal fragments, and deletion fragments, but do not include naturally occurring full-length polypeptides (or mature polypeptides). A deletion fragment refers to a polypeptide from which one or more internal amino acids have been deleted from a full-length polypeptide. Polypeptide variants include fragments as described above, as well as polypeptides having amino acid sequences modified by amino acid substitution, deletion, or insertion. Variants may be naturally occurring or non-natural. Non-naturally occurring variants may be produced using mutagenesis techniques known in the art. Variant polypeptides may include substitution, deletion, or addition of conserved or non-conserved amino acids. Such mutations (i.e., truncation and / or amino acid substitution, deletion, or insertion) may occur at either the amino acid level or the corresponding nucleic acid level.

[0487] Sequence identity is defined herein as the percentage of nucleic acids or amino acid residues in a candidate sequence that are identical to a reference amino acid sequence, after aligning the sequences to achieve the highest possible percentage of sequence identity, introducing gaps where necessary, and without considering conservative substitutions as part of the sequence identity.

[0488] Sequence identity can be determined by standard methods commonly used to compare the positional similarity of amino acids in two polypeptides or the nucleic acids of two polynucleotides. For example, computer programs such as BLAST or FASTA are used to align two polypeptides so that their respective amino acids are optimally matched (along the entire length of one or both sequences or along a given portion of one or both sequences). These programs provide default open and gap penalties and can be used in combination with score matrices such as PAM250 [standard score matrices; see Dayhoff et al. Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)]. The identity percentage can be calculated as follows: multiply the total number of identical matches by 100, and then divide by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the shorter sequence to align the two sequences.

[0489] As used herein, the term “kit” refers to a packaged set of related components, such as one or more compounds or compositions, and one or more related materials, such as a solvent, solution, buffer, instructions, or desiccant.

[0490] As used herein, the terms “linked” or “bonded” refer to a first amino acid sequence or nucleotide sequence that is covalently bonded to a second amino acid sequence or nucleotide sequence, respectively (e.g., the amino acid sequence of a secretory signaling peptide and / or the amino acid sequence of a heteromorphic transmembrane domain linked to the amino acid sequence of a C. acnes polypeptide). The first amino acid or nucleotide sequence may be directly bonded to the second amino acid or nucleotide sequence, or alternatively, the first sequence may be covalently bonded to the second sequence by an intervening sequence. The term “linked” means not only that the first amino acid sequence is fused to the second amino acid sequence at its C-terminus or N-terminus, but also that the entire first amino acid sequence (or second amino acid sequence) is inserted into any two amino acids in the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence may be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence may be linked to the second nucleotide sequence by a phosphodiester bond or a linker. The linker may be a peptide or polypeptide (in the case of a polypeptide chain), a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical part (in the case of both polypeptide and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0491] Acne lesions can be non-inflammatory or inflammatory. Non-inflammatory lesions of acne include open (blackhead) or closed (whitehead) comedones. These lesions, especially closed comedones, can be precursors to larger inflammatory lesions and are therefore clinically significant. Inflammatory lesions may include papules, pustules, nodules, and nodular cystic lesions, depending on the severity and location of inflammation within the dermis. Papules and pustules may have a surrounding halo of erythema, which can characterize them as inflammatory. Typically, nodules are erythematous and often tender and / or painful. In some embodiments, nodules are located deep within the skin (e.g., concentrated in the dermis and / or subcutaneous tissue). Nodules may exceed 5 mm in diameter.

[0492] Acne can be mild, moderate, or severe based on the number and type of lesions that develop in a specific area of ​​skin. The severity of acne can be determined using the Investigator's Global Assessment (IGA) scale, which grades acne severity from 0 to 4. The IGA scale includes the following classifications: clear (grade 0), nearly clear (grade 1), mild severity (grade 2), moderate severity (grade 3), and severe (grade 4).

[0493] As used herein, the term “mild acne” refers to a grade of acne severity in which several non-inflammatory lesions are present and a small number of inflammatory lesions are present. Typically, the subject has papules and / or pustules, for example, a subject may have only papules and / or pustules. Typically, the subject does not have any nodular or cystic lesions. Mild acne may be a Grade 2 acne according to the IGA scale.

[0494] As used herein, the term “moderate acne” refers to a grade of acne severity in which the subject has many comedones, papules, and / or pustules. The subject may have nodules; for example, a subject may have one or fewer nodules. Moderate acne typically affects more than half of the subject's face. Moderate acne may be grade 3 according to the IGA scale.

[0495] As used herein, the term “severe acne” refers to a grade of acne severity in which the subject has numerous comedones, papules, and pustules. Typically, the subject has one or more nodules and / or cysts. Severe acne typically develops across the entire face of the subject. For example, the entire face of the subject may be covered with comedones, papules, and pustules. Severe acne may be a Grade 4 acne according to the IGA scale.

[0496] The present invention includes at least the embodiments designated by the following numbers.

[0497] 1. A nucleic acid comprising a nucleotide sequence encoding a modified C. acnes (C. acnes) CAMP2 polypeptide, wherein the modified C. acnes (C. acnes) CAMP2 polypeptide comprises an amino acid sequence including a C. acnes (C. acnes) CAMP2 polypeptide sequence and a transmembrane domain sequence.

[0498] 2. The nucleic acid according to Embodiment 1, wherein the transmembrane domain sequence is located at the N-terminus or C-terminus (e.g., C-terminus) of the modified C. acnes CAMP2 polypeptide.

[0499] 3. The nucleic acid according to Embodiment 1 or 2, wherein the transmembrane domain sequence includes a viral transmembrane domain sequence arbitrarily selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequence, SARS-CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles F protein transmembrane domain sequence, rubella E1 protein transmembrane domain sequence, rubella E2 protein transmembrane domain sequence, mumps F protein transmembrane domain sequence, Ebola GP protein transmembrane domain sequence, and rabies transmembrane domain sequence.

[0500] 4. The transmembrane domain sequence is the nucleic acid described in any one of Embodiments 1 to 3, comprising an amino acid sequence based on one of the sequences in Table 4.

[0501] 5. The nucleic acid according to any one of Embodiments 1 to 4, wherein the transmembrane domain sequence comprises an amino acid sequence determined by any one of SEQ ID NOs. 208 to 209 (e.g., SEQ ID NO. 208).

[0502] 6. A modified C. acnes CAMP2 polypeptide is a nucleic acid according to any one of Embodiments 1 to 5, comprising a non-natural (e.g., viral) secretion signal peptide sequence.

[0503] 7. The nucleic acid according to Embodiment 6, wherein the secretory signal peptide sequence is located at the N-terminus or C-terminus (e.g., the N-terminus) of the modified C. acnes CAMP2 polypeptide.

[0504] 8. The nucleic acid according to Embodiment 6 or 7, wherein the secretion signal peptide sequence is a viral secretion signal peptide sequence that optionally includes a sequence selected from the group consisting of influenza hemagglutinin (HA) secretion signal peptide sequence, SARS-CoV-2 spike secretion signal peptide sequence, VZV gB secretion signal peptide sequence, VZV gE secretion signal peptide sequence, VZV gI secretion signal peptide sequence, VZV gK secretion signal peptide sequence, measles F protein secretion signal peptide sequence, rubella E1 protein secretion signal peptide sequence, rubella E2 protein secretion signal peptide sequence, mumps F protein secretion signal peptide sequence, Ebola GP protein secretion signal peptide sequence, and smallpox 6kDa IC protein secretion signal peptide sequence.

[0505] 9. The nucleic acid according to any one of Embodiments 6 to 8, wherein the secretory signal peptide sequence includes a sequence according to either one of the sequences in Table 2 or 3.

[0506] 10. The nucleic acid according to any one of Embodiments 6 to 9, wherein the secretory signal peptide sequence comprises the sequence of any one of SEQ ID NOs. 210 to 211 (e.g., SEQ ID NO. 210).

[0507] 11. A modified C. acnes CAMP2 polypeptide comprises mutations at one or more (e.g., all) positions corresponding to the N-glycosylation sites and / or at one or more (e.g., all) positions corresponding to the O-glycosylation sites of the natural C. acnes CAMP2 polypeptide, wherein the mutations are optionally single amino acid substitutions, as described in any one of Embodiments 1 to 10.

[0508] 12. (i) The C. acnes CAMP2 polypeptide contains a mutation at position N166 relative to SEQ ID NO: 203, and optionally the mutation is a single amino acid substitution, e.g., N166S, and / or (ii) The nucleic acid according to Embodiment 11, wherein the C. acnes CAMP2 polypeptide comprises the substitution of one or more serine (Ser) and / or threonine (Thr) residues.

[0509] 13. A nucleic acid according to any one of Embodiments 1 to 12, which is messenger RNA (mRNA).

[0510] 14. The nucleic acid according to Embodiment 13, wherein the mRNA comprises a 5' cap, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylated (poly(A)) sequence.

[0511] 15. mRNA is either unmodified or (i) mRNA contains at least one chemical modification, which may be optional, for example: 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil Selected from the group consisting of 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methylpseuduracil, keosin, β-D-mannosylkeosin, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine and inosine (for example, chemical modifications include N1-methylpseuduidine): and / or (ii) mRNA is synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, and optionally selected from these modified nucleotide analogs or derivatives of purines and pyrimidines are 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-( Nucleic acids according to Embodiment 13 or 14, selected from the group consisting of carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-pseuduracil, keosin, β-D-mannosylkeosin, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0512] 16. The nucleic acid according to Embodiment 15, wherein the mRNA comprises at least one chemical modification (for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine in the mRNA is chemically modified), and optionally the chemical modification comprises, for example, N1-methylpseudolidine instead of any uridine.

[0513] 17. The nucleic acid according to any one of embodiments 13 to 16, wherein the mRNA is either self-replicating mRNA or non-replicating mRNA.

[0514] 18. The nucleic acid described in Embodiment 17, wherein the mRNA is non-replicating mRNA.

[0515] 19. (i) A modified C. acnes CAMP2 polypeptide includes a sequence such as SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NOs. 43-58, SEQ ID NOs. 1-16, or SEQ ID NOs. 339-363 (e.g., SEQ ID NO: 207), or a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (e.g., at least 85%, 90%, or 95%) identity thereto, and / or (ii) The C. acnes CAMP2 polypeptide contains amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 24, 29, 30, 37, 48, 61, 65, 68, 76, 87, 91, 92, 98, 100, 106, 118, 128, 138, 143, 145, 154, 169, 177, 179, 189, 207, 221 and / or 223 of SEQ ID NO: 203, for example, C. acnes CAMP2 polypeptide contains amino acid substitutions at one or more positions corresponding to residues 6, 9, 11, 18, 19, 21, 21, 21, 223, and / or 223 of SEQ ID NO: 203, for example, C. acnes CAMP2 polypeptide The CAMP2 polypeptide...

Claims

1. A nucleic acid comprising a nucleotide sequence encoding a modified C. acnes CAMP2 polypeptide, wherein the modified C. acnes CAMP2 polypeptide comprises an amino acid sequence including a C. acnes CAMP2 polypeptide sequence and a transmembrane domain sequence.

2. (a) The transmembrane domain sequence is located at the C-terminus of the modified C. acnes CAMP2 polypeptide, and / or (b) The nucleic acid according to claim 1, wherein the transmembrane domain sequence comprises a viral transmembrane domain sequence arbitrarily selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequence, SARS CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles F protein transmembrane domain sequence, rubella E1 protein transmembrane domain sequence, rubella E2 protein transmembrane domain sequence, mumps F protein transmembrane domain sequence, Ebola GP protein transmembrane domain sequence, and rabies transmembrane domain sequence, for example, the transmembrane domain comprises an amino acid sequence of any one of the sequences in Table 4.

3. The modified C. acnes CAMP2 polypeptide contains a non-natural secretion signal peptide sequence (e.g., a viral secretion signal peptide sequence), optionally, (a) The secretion signal peptide sequence is located at the N-terminus of the modified C. acnes CAMP2 polypeptide, and / or (b) The nucleic acid according to claim 1 or 2, wherein the secretion signal peptide sequence is a viral secretion signal peptide sequence arbitrarily selected from the group consisting of influenza hemagglutinin (HA) secretion signal peptide sequence, SARS CoV-2 spike secretion signal peptide sequence, VZV gB secretion signal peptide sequence, VZV gE secretion signal peptide sequence, VZV gI secretion signal peptide sequence, VZV gK secretion signal peptide sequence, measles F protein secretion signal peptide sequence, rubella E1 protein secretion signal peptide sequence, rubella E2 protein secretion signal peptide sequence, mumps F protein secretion signal peptide sequence, Ebola GP protein secretion signal peptide sequence, smallpox 6kDa IC protein secretion signal peptide sequence, and rabies G protein secretion signal peptide sequence, for example, the nucleic acid according to claim 1 or 2, wherein the secretion signal peptide sequence includes a sequence which is any one of the sequences in Table 3.

4. It is messenger RNA (mRNA), and is selectively selected. (a) The mRNA comprises a 5' cap, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylated (poly(A)) sequence, (b) The mRNA is unmodified or comprises at least one chemical modification, optionally comprising at least one chemical modification, for example, the chemical modification comprising N1-methylpseudolidine and / or (c) The nucleic acid according to any one of claims 1 to 3, wherein the mRNA is self-replicating mRNA or non-replicating mRNA, for example, non-replicating mRNA.

5. (a) The modified C. acnes CAMP2 polypeptide comprises the sequence represented by SEQ ID NO: 207 or any one of SEQ ID NOs 5-9 (e.g., SEQ ID NO: 207) or a sequence having at least 60% (e.g., at least 85%, 90%, or 95%) identity therewith, and / or (b) The nucleic acid according to any one of claims 1 to 4, wherein the nucleic acid comprises a nucleotide sequence by any one of SEQ ID NOs: 90 to 94 or SEQ ID NOs: 391 to 392 (for example, SEQ ID NO: 91) or a sequence having at least 50% identity therewith (for example, at least 75%, 80%, or 85%, for example, 75% or 80%).

6. The following structural elements: (i) The following structure: 【Chemistry 1】 A 5' cap with (ii) 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 265, (iii) A protein coding region having a nucleic acid sequence according to one of the following: SEQ ID NOs: 90-91 or SEQ ID NOs: 391-392 (for example, SEQ ID NO: 91), (iv) A 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides. The nucleic acid according to any one of claims 1 to 5, which is mRNA comprising or consisting of (for example, consisting of) the following.

7. A modified C. acnes CAMP2 polypeptide having an amino acid sequence including a C. acnes CAMP2 polypeptide sequence and a transmembrane domain sequence.

8. (A) The transmembrane domain sequence is located at the C-terminus of the modified C. acnes CAMP2 polypeptide, and / or (B) The transmembrane domain comprises a viral transmembrane domain sequence optionally selected from the group consisting of influenza hemagglutinin (HA) transmembrane domain sequence, SARS CoV-2 spike transmembrane domain sequence, VZV gB transmembrane domain sequence, VZV gE transmembrane domain sequence, VZV gI transmembrane domain sequence, VZV gK transmembrane domain sequence, measles F protein transmembrane domain sequence, rubella E1 protein transmembrane domain sequence, rubella E2 protein transmembrane domain sequence, mumps F protein transmembrane domain sequence, Ebola GP protein transmembrane domain sequence, and rabies transmembrane domain sequence, for example, the transmembrane domain comprises an amino acid sequence of any one of the sequences in Table 4, the modified C. acnes CAMP2 polypeptide according to claim 7.

9. A modified C. acnes CAMP2 polypeptide according to claim 7 or 8, comprising the sequence of sequence number 207 or any one of sequence numbers 5 to 9 (for example, sequence number 207) or a sequence having at least 60% (for example, at least 85%, 90%, or 95%) identity therewith.

10. A composition, (a) A nucleic acid (e.g., mRNA) according to any one of claims 1 to 6, wherein the composition is an immunogenic composition, (b) A nucleic acid comprising a nucleotide sequence encoding the C. acnes CAMP2 polypeptide, wherein the composition is an immunogenic composition, or (c) Nucleic acids (e.g., mRNA) containing a nucleotide sequence encoding the C. acnes CAMP2 polypeptide, and (i) Nucleic acids containing a nucleotide sequence encoding the C. acnes DsA1 polypeptide, (ii) Nucleic acids containing a nucleotide sequence encoding the C. acnes DsA2 polypeptide, (iii) C. acnes, nucleic acids containing nucleotide sequences encoding PITP polypeptides. (iv) Nucleic acids containing nucleotide sequences encoding the chimeric C. acnes DsA1 / DsA2 polypeptide, and (v) Nucleic acids containing nucleotide sequences encoding the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide one or more of the Includes, Optionally, the composition according to (c) is an immunogenic composition, Furthermore, optionally, the composition comprising any one of (a) to (c) is in a freeze-liquid form or a freeze-dried form (for example, a freeze-dried form).

11. (c) comprising the nucleic acid of (iii) and / or the nucleic acid of (c)(iv), optionally, (A) The C. acnes CAMP2 polypeptide comprises the sequence specified by Sequence ID No. 203 or a sequence having at least 60% (e.g., at least 85%) identity therewith, (B) The chimeric C. acnes DsA1 / DsA2 polypeptide comprises the sequence specified by Sequence ID No. 70 or a sequence having at least 90% identity thereto, and (C) The composition according to claim 10(c), wherein the C. acnes PITP polypeptide comprises the sequence according to Sequence ID No. 73 or a sequence having at least 75% identity therewith.

12. As described in claim 10(a) or (b), (i) Nucleic acids containing a nucleotide sequence encoding the C. acnes DsA1 polypeptide, (ii) Nucleic acids containing a nucleotide sequence encoding the C. acnes DsA2 polypeptide, (iii) C. acnes, nucleic acids containing nucleotide sequences encoding PITP polypeptides. (iv) Nucleic acids containing nucleotide sequences encoding the chimeric C. acnes DsA1 / DsA2 polypeptide, and (v) Nucleic acids containing nucleotide sequences encoding the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide The composition according to claim 10, further comprising one or more of the above.

13. (iii) comprising the nucleic acid of (iv) and / or the nucleic acid of (iv), optionally, (A) The C. acnes CAMP2 polypeptide comprises the sequence according to SEQ ID NO: 207 or a sequence having at least 60% (e.g., at least 85%) identity thereto, the chimeric C. acnes DsA1 / DsA2 polypeptide comprises the sequence according to SEQ ID NO: 70 or a sequence having at least 90% identity thereto, and the C. acnes PITP polypeptide comprises the sequence according to SEQ ID NO: 73 or a sequence having at least 75% identity thereto, or (B) The composition according to claim 12, wherein the nucleotide sequence encoding the C. acnes CAMP2 polypeptide comprises the sequence according to SEQ ID NO: 91 or a sequence having at least 50% identity thereto, the nucleotide sequence encoding the chimeric C. acnes DsA1 / DsA2 polypeptide comprises the sequence according to SEQ ID NO: 113 or a sequence having at least 75% identity thereto, and the nucleotide sequence encoding the C. acnes PITP polypeptide comprises the sequence according to SEQ ID NO: 115 or a sequence having at least 50% identity thereto.

14. The composition according to claim 10(c) or any one of claims 11 to 13, wherein any two or more nucleic acids are located on the same nucleic acid molecule or on different nucleic acid molecules (for example, all of the nucleic acids in the composition are located on the same nucleic acid molecule, or all of the nucleic acids in the composition are located on individual nucleic acid molecules).

15. (a) The mRNA comprises a 5' cap, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylated (poly(A)) sequence, (b) The mRNA is unmodified or comprises at least one chemical modification, optionally comprising at least one chemical modification, for example, the chemical modification comprising N1-methylpseudolidine and / or (c) The composition according to any one of claims 10 to 14, wherein the mRNA is self-replicating mRNA or non-replicating mRNA, for example, non-replicating mRNA.

16. (A) The following structural elements: (i) 5' cap, (ii) 5'UTR having the nucleic acid sequence according to Sequence ID No. 265, (iii) Protein coding region having nucleic acid sequence according to Sequence ID No. 91, (iv) 3'UTR having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides. A first mRNA containing or consisting of (for example, consisting of) (B) The following structural elements: (i) 5' cap, (ii) 5'UTR having the nucleic acid sequence according to Sequence ID No. 265, (iii) Protein coding region having nucleic acid sequence according to Sequence ID No. 113, (iv) 3'UTR having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides. A second mRNA containing or consisting of (for example, consisting of) (C) The following structural elements: (i) 5' cap, (ii) 5'UTR having the nucleic acid sequence according to Sequence ID No. 265, (iii) Protein coding region having nucleic acid sequence according to Sequence ID No. 115, (iv) 3'UTR having the nucleic acid sequence according to SEQ ID NO: 266, and (v) A polyA tail comprising, optionally, at least 75 adenosine nucleotides (e.g., about 80 adenosine nucleotides) or at least 100 adenosine nucleotides (e.g., about 115 adenosine nucleotides), for example, a polyA tail comprising at least 100 adenosine nucleotides. A third mRNA containing or consisting of (for example, consisting of) The 5' cap includes the following structure: 【Chemistry 2】 The composition according to any one of claims 10 to 15, wherein optionally, one or more (e.g., all three) of the first, second, and third mRNAs comprises at least one chemical modification, for example, the chemical modification comprises N1-methylpseudridine, and preferably all uridines are substituted with N1-methylpseudridine.

17. The composition according to any one of claims 10 to 16, comprising a total of approximately 45 μg, approximately 120 μg, or approximately 225 μg of one or more nucleic acids (e.g., mRNA).

18. It further contains lipid nanoparticles (LNPs), and optionally, (a) One or more nucleic acids are encapsulated in the LNP, (b) Any two or more nucleic acids are co-encapsulated in a single LNP, and / or (c) The composition according to any one of claims 10 to 17, wherein any two or more nucleic acids are encapsulated in separate LNPs.

19. The composition according to claim 18, wherein the LNP comprises at least one cationic lipid, the cationic lipid being selected from the group consisting of OF-02, cKK-E10, IM-001, IS-001 and GL-HEPES-E3-E12-DS-4-E10, and preferably the cationic lipid being GL-HEPES-E3-E12-DS-4-E10.

20. The composition according to claim 18 or 19, wherein the LNP further comprises polyethylene glycol (PEG) conjugate (PEG-modified) lipids, cholesterol-based lipids, and helper lipids.

21. The composition according to any one of claims 18 to 20, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35%.

22. The composition according to any one of claims 18 to 21, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 40%, DMG-PEG2000 in a molar ratio of 1.5%, cholesterol in a molar ratio of 28.5%, and DOPE in a molar ratio of 30%.

23. (a) A modified C. acnes CAMP2 polypeptide according to any one of claims 7 to 9, or (b) C. acnes CAMP2 polypeptide, and (i) C. acnes DsA1 polypeptide, (ii) C. acnes DsA2 polypeptide, (iii) C. acnes PITP polypeptide, (iv) Chimeric C. acnes DsA1 / DsA2 polypeptide, and (v) Chimeric C. acnes DsA1 / DsA2 / PITP polypeptide one or more of the A composition containing, optionally an immunogenic composition.

24. As described in claim 23(a), (A) C. acnes DsA1 polypeptide (B) C. acnes DsA2 polypeptide, (C) C. acnes PITP polypeptide (D) Chimeric C. acnes DsA1 / DsA2 polypeptide, and (E) Chimera C. acnes DsA1 / DsA2 / PITP polypeptide The composition according to claim 23, further comprising one or more of the above.

25. The composition according to any one of claims 10 to 24, wherein the C. acnes CAMP2 polypeptide, the C. acnes DsA1 polypeptide, the C. acnes DsA2 polypeptide, the C. acnes PITP polypeptide, the chimeric C. acnes DsA1 / DsA2 polypeptide, and the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide further comprises a non-natural transmembrane domain sequence (e.g., a viral transmembrane domain sequence) and / or a non-natural secretion signal peptide sequence.

26. (a) One or more of the C. acnes CAMP2 polypeptide, the C. acnes DsA1 polypeptide, the C. acnes DsA2 polypeptide, the C. acnes PITP polypeptide, the chimeric C. acnes DsA1 / DsA2 polypeptide, and the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide contain mutations at one or more (e.g., all) positions corresponding to the N-glycosylation sites and / or one or more (e.g., all) positions corresponding to the O-glycosylation sites of each native C. acnes polypeptide, wherein the mutations are, optionally, single amino acid substitutions and / or (b) The composition according to any one of claims 10 to 25, wherein one or more of the C. acnes DsA1 polypeptide, the C. acnes DsA2 polypeptide, the C. acnes PITP polypeptide, the chimeric C. acnes DsA1 / DsA2 polypeptide, and the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues of each native C. acnes polypeptide, and optionally the single amino acid substitution is a serine substitution of cysteine.

27. (a) The C. acnes CAMP2 polypeptide comprises a sequence represented by any one of SEQ ID NOs. 203, SEQ ID NOs. 43-58, SEQ ID NOs. 1-4, SEQ ID NOs. 10-16, or SEQ ID NOs. 339-363 (for example, SEQ ID NOs. 203), or a sequence having at least 60% (for example, at least 85%, at least 90%, at least 95%) identity therewith, (b) The C. acnes DsA1 polypeptide comprises the sequence represented by SEQ ID NO: 204, or SEQ ID NOs: 17-19, or SEQ ID NOs: 59-61, or a sequence having at least 75% identity thereto. (c) The C. acnes DsA2 polypeptide comprises the sequence represented by SEQ ID NO: 205, or SEQ ID NOs: 20-27, or SEQ ID NOs: 62-69, or a sequence having at least 75% identity thereto. (d) The C. acnes PITP polypeptide comprises the sequence of any one of SEQ ID NOs. 206, SEQ ID NOs. 31-37, or SEQ ID NOs. 73-79, or a sequence having at least 75% (e.g., 90 or 95%) identity therewith. (e) The chimeric C. acnes DsA1 / DsA2 polypeptide comprises a sequence according to any one of SEQ ID NOs. 28-30, SEQ ID NOs. 39, or SEQ ID NOs. 70-72, or SEQ ID NOs. 81, or a sequence having at least 90% identity therewith, and / or (f) The composition according to any one of claims 10 to 26, wherein the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide comprises (i) one sequence of SEQ ID NOs. 28-30, SEQ ID NOs. 39, SEQ ID NOs. 70-72, or SEQ ID NOs. 81, or a sequence having at least 90% identity thereto, and (ii) a PITP polypeptide sequence, wherein the chimeric C. acnes DsA1 / DsA2 / PITP polypeptide comprises one sequence of SEQ ID NOs. 38, SEQ ID NOs. 40-41, SEQ ID NOs. 80, SEQ ID NOs. 82-83, or a sequence having at least 90% identity thereto.

28. (a) C. acnes CAMP2 polypeptide comprising or consisting of the sequence of sequence number 203 (for example, consisting of the sequence), (b) Chimeric C. acnes DsA1 / DsA2 polypeptide comprising or consisting of the sequence of Sequence ID No. 70 (for example, consisting of the sequence of Sequence ID No. 70), and (c) C. acnes PITP polypeptide comprising or consisting of the sequence of Sequence ID No. 73 (for example, consisting of the sequence of Sequence ID No. 73). A composition according to any one of claims 10 to 27, comprising:

29. The composition according to any one of claims 23 to 28, further comprising an adjuvant, wherein the adjuvant is optionally selected from the group consisting of aluminum-based adjuvants (e.g., AlOOH), squalene-based oil-in-water emulsion adjuvants (e.g., AF03, AS03, MF59), and liposome-based adjuvants containing saponins and TLR4 agonists (e.g., SPA14, AS01, LEQ), and further optionally selected from the group consisting of AlOOH, AF03, and SPA14.

30. A nucleic acid comprising a nucleotide sequence encoding a chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide, wherein the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide is (a) Chimeric C. acnes DsA1 / DsA2 polypeptide, (b) Immunogenic fragments of C. acnes PITP polypeptide, wherein the immunogenic fragments optionally contain ENFD of C. acnes PITP polypeptide, and (c) C. acnes CAMP2 polypeptide or its immunogenic fragment Nucleic acids, including

31. (a) Chimeric C. acnes DsA1 / DsA2 polypeptide, (b) Immunogenic fragments of C. acnes PITP polypeptide, wherein the immunogenic fragments optionally contain ENFD of C. acnes PITP polypeptide, and (c) C. acnes CAMP2 polypeptide or its immunogenic fragment A chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide containing [the specified compound].

32. i. The chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises CSD1 of the C. acnes DsA1 polypeptide, CSD2 of the C. acnes DsA2 polypeptide, and CSD3 of the C. acnes DsA1 polypeptide, and optionally, the chimeric C. acnes DsA1 / DsA2 polypeptide of (a) comprises the sequence according to Sequence ID No. 70 or a sequence having at least 90% (e.g., at least 95%) identity thereto. ii. The immunogenic fragment of the C. acnes PITP polypeptide containing the ENFD of the C. acnes PITP polypeptide of (b) comprises a sequence corresponding to amino acid residues 1-133 or 1-146 (e.g., residues 1-146) of SEQ ID NO: 73, or a sequence having at least 90% identity thereto, and / or iii. (c) is (1) a C. acnes CAMP2 polypeptide, wherein (c) optionally comprises sequence number 203 or a sequence having at least 90% identity thereto, or (2) an immunogenic fragment of a C. acnes CAMP2 polypeptide comprising the N-terminal domain of a C. acnes CAMP2 polypeptide, for example, the immunogenic fragment comprising amino acid residues 29-176 of sequence number 202 or a sequence having at least 90% identity with the sequence of amino acid residues 29-176 of sequence number 202, as described in claim 30 or the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide as described in claim 31.

33. The nucleic acid according to claim 30 or 32 or the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide according to claim 31 or 32, wherein the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises a transmembrane domain sequence, and optionally, the transmembrane domain sequence comprises an amino acid sequence according to either one of the sequences in Table 4 or SEQ ID NO: 84 (for example, SEQ ID NO: 208 or 84).

34. (i) The chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide contains the sequence of sequence numbers 374-375, or (ii) The chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises the sequence according to SEQ ID NO: 373 and a transmembrane domain sequence (for example, the transmembrane sequence according to SEQ ID NO: 208 or 84), For example, the nucleic acid according to any one of claims 30, 32, or 33, or the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide according to any one of claims 31 to 33, wherein the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide comprises the sequence according to SEQ ID NO: 374, or comprises the sequence according to SEQ ID NO: 373 and the TMB sequence according to SEQ ID NO:

84.

35. (i) The nucleotide sequence encoding the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence of either SEQ ID NOs. 377-382 or 384-389 (for example, SEQ ID NOs. 384-389), or (ii) The nucleotide sequence encoding the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence given by SEQ ID NO: 384 and the sequence given by SEQ ID NO: 395 or the sequence given by SEQ ID NO: 385 and the sequence given by SEQ ID NO: 396, For example, the nucleic acid according to any one of claims 30 or 32 to 34, wherein the nucleotide sequence encoding the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence according to SEQ ID NO: 387, or the nucleotide sequence encoding the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide includes the sequence according to SEQ ID NO: 385 and the sequence according to SEQ ID NO:

396.

36. A composition comprising a nucleic acid (e.g., mRNA) according to any one of claims 30 or 32 to 35, optionally an immunogenic composition, optionally further comprising lipid nanoparticles (LNPs), and optionally further comprising the nucleic acid (e.g., mRNA) encapsulated in the LNPs, optionally an immunogenic composition.

37. (a) The mRNA comprises a 5' cap, at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylated (poly(A)) sequence, (b) The mRNA is unmodified or comprises at least one chemical modification, optionally comprising at least one chemical modification, for example, the chemical modification comprising N1-methylpseudolidine and / or (c) The composition according to claim 36, wherein the mRNA is non-replicating mRNA.

38. (i) The LNP comprises at least one cationic lipid, the cationic lipid being selected from the group consisting of OF-02, cKK-E10, IM-001, IS-001 and GL-HEPES-E3-E12-DS-4-E10 (for example, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10), and / or (ii) The LNP comprises polyethylene glycol (PEG) conjugate (PEG-conjugated) lipids, cholesterol-based lipids and helper lipids, For example, the composition according to claim 36 or 37, wherein the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 35% to 55%, DMG-PEG2000 in a molar ratio of 0.25% to 2.75%, cholesterol in a molar ratio of 20% to 50%, and DOPE in a molar ratio of 5% to 35% (for example, the LNP comprises GL-HEPES-E3-E12-DS-4-E10 in a molar ratio of 40%, DMG-PEG2000 in a molar ratio of 1.5%, cholesterol in a molar ratio of 28.5%, and DOPE in a molar ratio of 30%).

39. A composition comprising the chimeric C. acnes DsA1 / DsA2 / PITP / CAMP2 polypeptide according to any one of claims 31 to 34, optionally an immunogenic composition.

40. A nucleic acid according to any one of claims 1 to 6, 30, or 32 to 35, a polypeptide according to any one of claims 7 to 9 or 31 to 34, or a composition according to any one of claims 10 to 29 or 36 to 39, for use as a pharmaceutical.

41. A nucleic acid according to any one of claims 1 to 6, 30, or 32 to 35, a polypeptide according to any one of claims 7 to 9, or 31 to 34, or a composition according to any one of claims 10 to 29 or 36 to 39, for use in a method of treating or preventing a target C. acnes infection.

42. For use in a method of treating or preventing a target acne, wherein the acne is mild, moderate or severe (for example, moderate or severe), the nucleic acid according to any one of claims 1 to 6, 30 or 32 to 35, the polypeptide according to any one of claims 7 to 9 or 31 to 34, or the composition according to any one of claims 10 to 29 or 36 to 39.

43. The aforementioned method, (i) administering one dose of the nucleic acid, the polypeptide, or the composition, (ii) Administering two doses of the nucleic acid, polypeptide, or composition, wherein the two doses are administered at intervals of two months. A nucleic acid, polypeptide, or composition for use according to any one of claims 40 to 42, comprising:

44. The nucleic acid, polypeptide, or composition for use according to any one of claims 41 to 43, wherein the subject is a human subject, and the subject is 9 to 45 years old or 12 to 45 years old (for example, 9 to 18 years old or 18 to 45 years old).

45. The method comprises administering the nucleic acid, polypeptide, or composition by intramuscular or intradermal injection, for example, the composition being administered intramuscularly, the nucleic acid, polypeptide, or composition for use according to any one of claims 40 to 44.

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